Identification and synthesis of substituted pyrrolo[2,3-d]pyrimidines as novel firefly luciferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.07.035

A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based NF-κB luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus pyralis with an IC₅₀ value of 0.36 ± 0.05 μM. Kinetic analysis of 3a inhibition showed that it is predominantly competitive with respect to d-luciferin and uncompetitive with respect to ATP. Therefore, several pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relationship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC₅₀ value of 0.06 ± 0.01 μM. In addition, molecular docking studies suggested that both 3a and 4c could be accommodated in the d-luciferin binding pocket, which is expected for a predominantly competitive inhibitor with respect to d-luciferin.

Full text of DOI:10.1016/j.bmc.2012.07.035

Bioorganic & Medicinal Chemistry 20 (2012) 5473–5482
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification and synthesis of substituted pyrrolo[2,3-d]pyrimidines
as novel firefly luciferase inhibitors
Yang Liu a, , Jianping Fang b, , Haiyan Cai c, Fei Xiao b, Kan Ding b, , Youhong Hu a,
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
b Glycochemistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based
NF- B luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus
pyralis with an IC50 value of 0.36 0.05 M. Kinetic analysis of 3a inhibition showed that it is predomi-
nantly competitive with respect to -luciferin and uncompetitive with respect to ATP. Therefore, several
pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relation-
ship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC50
Received 4 February 2012
Revised 21 July 2012
Accepted 21 July 2012
Available online 28 July 2012
j
l
D
Keywords:
Luciferase
Inhibitor
Pyrrolo[2,3-d]pyrimidine
High-throughput screening
Molecular docking
value of 0.06 0.01
accommodated in the
inhibitor with respect to
l
M. In addition, molecular docking studies suggested that both 3a and 4c could be
-luciferin binding pocket, which is expected for a predominantly competitive
-luciferin.
D
D
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
mately 21% of all of the biochemical and cellular HTS assays per-
formed today.9–12
One of the most thoroughly studied oxidative enzymes is firefly
luciferase, which is responsible for bioluminescence in firefly.1–3 It
However, an important consideration in any assay that uses
firefly luciferase as a reporter or a detection component is the inhi-
bition of the enzyme by a variety of low-molecular-weight hetero-
cyclic compounds, which are typically found in screening
collections.13 This will lead to screening artifacts that are not
attributed to modulating the biological activity or signal pathway
of interest but may interfere with the interpretation of assay re-
sults.11 Therefore, it is of utmost importance to eliminate these
artifacts in the early HTS stages of drug discovery to save time,
money, and identify artifacts as false positives.
catalyzes two substrates (
zyme-bound intermediate known as
D
-luciferin and ATP) to produce an en-
-luciferyl-adenylate; this
D
intermediate is oxidized, decarboxylated and converted to a singlet
excited state of oxyluciferin (Scheme 1).2 The spontaneous decay of
oxyluciferin from the excited state to the ground state results in
the release of photons that emit a yellow–green light (562 nm) at
a pH of 7.5–7.8. The complicated reaction kinetics, production inhi-
bition, and side competing dark reactions result in a typical light
flash profile of the in vitro bioluminescence.2,4,5
Due to its high sensitivity and convenience, firefly luciferase
was widely used as the analytical tool for the quantification of
ATP and the monitoring of pyrophosphatase activity in molecular
biology only once after its isolation and purification.1,2,6 The suc-
cessful cloning of the firefly luciferase gene in 1985 allowed firefly
luciferase to be employed as a reporter gene in diverse applica-
tions, especially in the drug discovery area.7,8 Currently, biolumi-
nescence assays based on luciferase enzymes are prevalent in
high-throughput screening (HTS) assays and represent approxi-
Although firefly luciferase has a broad range of applications,
many problems about its bioluminescence, such as the exact
mechanism of luciferase’s action in the reaction, are still not com-
pletely clear until now.2 There is great interest in the identification
and characterization of novel luciferase inhibitors to solve these
problems to aid in the successful application and comprehension
of luciferase. Moreover, a better understanding of luciferase inhib-
itors could shed light on ways to target other oxygenases that may
be relevant to human health, such as cytochrome p450, cyto-
chrome c, and monoamine oxidase.14–17 Furthermore, due to their
structural similarities, firefly luciferase has been proposed as a
model for the l
-opioid receptor,18 and thus some luciferase inhib-
⇑
Corresponding authors. Address: 555 Zuchongzhi Road, Zhangjiang High-Tech
itors might also have analgesic effects. Several different classes of
small molecules that block luciferase activity have been identified,
including substrate-related compounds, intermediates or products
of the luciferase catalyzed reactions,19–21 anesthetics, certain fatty
Park, Shanghai, China. Fax: +86 2150806928 (K.D.); +86 2150805896 (Y.H.).
E-mail addresses: kding@mail.shcnc.ac.cn (K. Ding), yhhu@mail.shcnc.ac.cn (Y.
Hu).
Both authors contributed equally to this study.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.035

5474
Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
ATP-Mg2+
O
P
O
O2
AMP
O
C
O
C
O O
C
Adenosine
HO
O
N
HO
S
N
HO
N
S
N
OH
-
N
S
N
O
S
S
S
D-Luciferyl-AMP
D-Luciferin
PPi-Mg2+
-O
OH
O
*
-O
OH
O
-O
-O
N
N
S
S
N
N
S
S
S
S
S
S
+ CO2 + H+
+ photon
Oxyluciferin
Scheme 1. Schematic representation of luciferase catalyzed reaction.
NH2
H2N
NH
Cl
N
O
N
Cl
O
N
N
(a)
(c)
O
N
N
R1
R1
N
N
N
N
R1
N
1a-1d
N
H
R2
R2
N
N
2a-2i, 2k-2v
Cl
3a-3v
N
(b)
(c)
R1=
1a
H
SMe
R1=
1b
1c
1d
3a
4c
N
R1= SO2Me
R1= NH2
N
N
Figure 1. The novel luciferase inhibitors.
2j
acids, quinolines, benzothiazoles, and other unique compounds
such as naphthoquinone.4,5,13,22–24 Most of these classes of small
molecules are competitive or noncompetitive inhibitors with
respect to the substrates.4 In this paper, we describe luciferase
inhibition by the substituted pyrrolo[2,3-d]pyrimidines that are
structurally unrelated to these previously identified inhibitor clas-
ses with a novel unique mechanism of action.
Scheme 2. Synthetic routes to the compounds. Reagents and conditions: (a) Alkyl
halides, K2CO3, DMF, 60–70 °C, 4–5 h; (b) 2,2-dimethylpropan-1-ol, THF, DEAD,
P(Ph)3, 0 °C -rt, overnight; (c) Amino-phenol, Cs2CO3, DMSO, 80 °C, 6 h.
2.2. Structure–activity relationships
The pyrrolo[2,3-d]pyrimidine scaffold is considered a privileged
structure,25 and it is frequently found in numerous biologically ac-
tive molecules, including protein kinase inhibitors, microtubule
targeting agents, and neurogenesis inducing molecules.26 We
found that the pyrrolo[2,3-d]pyrimidine compound 3a (Fig. 1) is
a novel potent firefly luciferase inhibitor, which emerged from a
The inhibition of NF-jB activation is now widely recognized as
a valid drug-targeting strategy to combat inflammatory diseases. In
an initial cell-based screen, our compound library was tested for
activity on LPS/TLR4/NF-
porter gene system.27 This yielded a hit compound 3a as an ‘active’
NF- B signaling inhibitor. 3a still showed the inhibitory activity
jB signaling using the firefly luciferase re-
j
cell-based LPS/TLR4/NF-
rolo[2,3-d]pyrimidine compound 3a acts as a mixed-type, predom-
inantly competitive inhibitor with respect to -luciferin but an
jB signaling inhibitor screening. The pyr-
when further assessed with cells stably expressing firefly luciferase
(after excluding the possibility of cytotoxicity). However, no
immunosuppressive activities were found when monocytes were
D
uncompetitive inhibitor with respect to ATP. A series of pyrrol-
o[2,3-d]pyrimidine analogues (3b–3v, 4a–4f) were prepared to fur-
ther explore their structure-activity relationships (SAR). We
identified the compound 4c as the most potent luciferase inhibitor.
In addition, 3a and 4c (Fig. 1) were also docked at the D-luciferin
biding site but not the ATP binding site of the crystal structure,
which is suggestive of their partially competitive manner.
applied to study, which a NF-
play. We wondered whether compound 3a is luciferase inhibitor
rather than the NF- B signaling inhibitor, which might result in
false positive hit. Therefore, we examined the ‘active’ compound
3a in an in vitro enzymatic luciferase assay as described in Section
4. This led to discovering that compound 3a displays significant
jB signaling inhibitor should not dis-
j
enzymatic inhibition with an IC50 value of 0.36 0.05 lM (Fig. 2).
Because the luciferase assay is based on the readout of photon
emission at 500–700 nm with a peak at 562 nm, nonspecific inhi-
bition could arise through compound-specific absorbance or light
scattering in this wavelength range. In this case, compounds that
interfere with luminescence detection could appear as false-posi-
tive luciferase inhibitors. The absorbance spectrum (200–800 nm)
was determined for compound 3a, and the result showed that
compound 3a did not show any absorption at a wavelength higher
than 300 nm (data not shown), which eliminated 3a as the possible
quencher of the luciferase signal.
Further studies were performed to investigate the kinetic char-
acteristics of inhibition by 3a because compound 3a exerted a
significant inhibitory activity on firefly luciferase. D-Luciferin was
titrated in the absence and presence of three concentrations of
3a (Fig. 3a), while holding the concentration of ATP constant at
2. Results and discussion
2.1. Chemistry
The general method used for the synthesis of pyrrolo[2,3-
d]pyrimidine derivatives is depicted in Schemes 2 and 3. The
4-chloro-7H-pyrrolo[2,3-d]pyrimidines 1 employed in these routes
are commercially available. Substances 3a–3i, 3k–3v were prepared
from compound 1 by an alkylation reaction with various alkyl ha-
lides and then a substitution with amino-phenol. In a similar man-
ner, substance 3j was obtained from compound 1 by Mitsunobu
reaction with 2-dimethylpropan-1-ol, followed by the substitution
reaction with amino-phenol. Substances 4a–4f were generated from
the corresponding substituted pyrrolo[2,3-d]pyrimidine via a meth-
ylation reaction.
250 lM. Re-plotting these data as a double reciprocal plot

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
5475
H
N
H2N
N
O
N
(a)
O
O
N
N
N
N
N
N
N
N
R2
R2
R2
3a, 3f, 3j
4a-4f
Scheme 3. Synthetic route to the compounds 4a–4f. Reagents and conditions: (a) MeI, NaH, DMF, rt, 6–7 h.
was expected for
regression analysis of the data with a mixed-type inhibition model
gave the dissociation constant (Ki) value of 0.22 0.05 M and Ki
value of 1.43 0.92 M (Table 1). Moreover, the Km and Vmax
values of luciferase towards -luciferin were 12 and
a mixed-type inhibitor. Further nonlinear
l
a
l
D
1 lM
1.11 0.03 Â 105 RLU/ms, consistent with the values obtained
from the analysis of the data in the absence of 3a with Michae-
lis–Menten model, further proof of the rightness of the mixed-type
inhibition model (Table 1).
Because luciferase has a second catalytic site for the other sub-
strate ATP, a similar analysis was carried out to determine the type
of inhibition with respect to ATP.
50 M, while ATP was titrated in the absence and presence of three
concentrations of compound 3a (Fig. 3c). A Lineweaver–Burk plot is
quite different from the one obtained with -luciferin, which
D-Luciferin was held constant at
l
D
Figure 2. Inhibition of luciferase activity by compound 3a. The IC50 value of 3a was
0.36 0.05 M. Representative graph is from one of three independent experiments
performed in triplicate.
showed a series of clearly parallel straight lines (Fig. 3d). This indi-
cated that 3a is uncompetitive with respect to ATP, which implies
that 3a targets the luciferase-ATP complex rather than free
enzyme. As stated above, a nonlinear regression analysis of the data
with Michaelis–Menten model was performed. The result indicated
that the presence of 3a resulted in a significant decrease in both
l
generated a series of straight lines that crossed to the left of the 1/V
axis and above the 1/[D-luciferin] axis (Fig. 3b). This is a typical
form of mixed-type inhibition known as predominantly competi-
tive inhibition. This suggests that 3a can bind both to the free en-
app
app
Km
and Vmax
values in a dose dependent manner (Table 1),
which is expected for an uncompetitive inhibitor. Further nonlinear
regression analysis of the data directly with an uncompetitive inhi-
zyme competing with
D-luciferin for the active site and to the
enzyme-luciferin complex, but with different affinities. Nonlinear
regression analysis of the data with the Michaelis–Menten model
showed that the presence of 3a resulted in an increased apparent
Michaelis constant (Kampp) and a decreased apparent maximum
velocity (Vampapx) in a dose dependent manner (Table 1). This result
bition model showed that the dissociation constant aKi value of 3a
was 0.34 0.03
respect to ATP was 59
result is consistent with the values obtained from the analysis of
lM, while the Km and Vmax values of luciferase with
6
l
M and 1.82 0.06 Â 105 RLU/ms. This
the data in the absence of 3a with the Michaelis–Menten model,
Figure 3. Kinetics of inhibition of luciferase by 3a. The dependence of the initial velocity on the concentration of
of 0.1, 0.2 or 0.4
M 3a. The lines represent a nonlinear least squares fit of the Michaelis–Menten model to the data. The fit parameters, Kmapp and Vmaxapp are represented on
Table 1. (b) A Lineweaver–Burk plot of data in (a). (d) A Lineweaver–Burk plot of data in (c). Representative graphs are from one experiment performed in triplicate.
D-luciferin (a) or ATP (c) in the absence (Control) or presence
l

5476
Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
Table 1
which is further proof of the suitability of the uncompetitive inhibi-
tion model (Table 1). Additionally, Table 1 shows that the obtained
Kinetic parameters for the inhibition of luciferase by 3a with respect to D-luciferin
and ATPa
Km values of luciferase for
M) were in good agreement with that reported by Lembert
et al.28 with a reaction system where albumin was used as an
enzyme stabilization agent (8 M and 48 M, respectively).
Hence, 3a appears to be predominantly competitive with re-
spect to -luciferin and uncompetitive with respect to ATP. These
D-luciferin (12
1
l
M) and ATP (59
6
ATPc
D
-Luciferinb
l
M Vmaxapp/ Â 105 RLU Kmapp/(
l
MVmaxapp/ Â 105 RLU
app
Km
/
l
msÀ1
msÀ1
1
l
2 l
Control
12
17
22
26
2
1.11 0.04
1.04 0.03
0.98 0.04
0.86 0.03
0.22 0.05
6.5 2.7
67 15
1.88 0.12
1.35 0.04
1.15 0.04
0.82 0.02
D
0.1
0.2
0.4
l
l
l
M 3a
M 3a
M 3a
2
3
3
39
35
22
5
5
3
results suggest a model in which the initial steps generating a
luciferase-ATP complex that can predominantly bind either 3a or
Ki/lM
a
D-luciferin. Furthermore, the luciferase-ATP-D-luciferin complex
can also bind 3a at an allosteric site different from the substrates
binding sites. Many inhibitors have been reported to inhibit the
bioluminescent luciferase catalyzed reaction via specific or alloste-
ric mechanisms. For example, oxyluciferin (a luciferin related
substance) is a typical inhibitor with a specific inhibition mecha-
nism,21 while anesthetics are typical examples of allosteric inhibi-
tors.4 Compound 3a differs from the inhibitors discussed above
because it can bind both specific and allosteric sites in luciferase.
a
V
Km
Ki/
l
M
1.43 0.92
1.11 0.03
0.34 0.03
1.82 0.06
max/ Â 105RLU msÀ1
/
l
M
12
1
59
6
a
Values are the means SEM of three independent assays performed in triplicate.
b
The dependence of luciferase activity on the concentration of luciferin at 250
M 3a.
The dependence of luciferase activity on the concentration of ATP at 50
l
M
ATP was assayed in the absence (Control) or presence of 0.1, 0.2 or 0.4
l
c
lM
D
-luciferin was determined in the absence (Control) or presence of 0.1, 0. 2, or
0.4 M 3a, respectively.
l
Table 2
Inhibition of luciferase activity by compounds 3a–3v, expressed as IC50 values or as% inhibition at 10 lM
R3
N
R1
N
N
R2
a
Compound
R1
H
R2
R3
IC50
(
l
M) or % inh.
Compound
R1
R2
R3
IC50(l
M) or % inh.a
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
3a
0.36 0.05
19%
3l
H
H
H
H
H
7.53 0.64
21%
O
O
O
3b
3c
3d
3e
H
H
H
H
H
3m
3n
3o
3p
O
O
O
O
O
O
O
O
NH2
O
48%
33%
OEt
3.51 1.10
0.31 0.10
46%
26%
OMe
CF3
NH2
NH2
NH2
3f
H
0.12 0.02
3q
H
7%
O
O
NH2
NH2
NH2
NH2
NH2
3g
3h
3i
H
H
H
H
H
9.43 2.23
0.29 0.05
2.24 0.91
0.08 0.01
6.46 0.67
3r
3s
3t
H
H
14.71 3.51
7.30 1.18
31%
O
O
O
O
O
O
H2N
O
NH2
NH2
NH2
S
O
O
O
O
S
3j
3u
3v
9%
O
3k
NH2
10.76 3.01
a
Inhibition of luciferase activity (IC50 SEM (lM), n = 3, triplicate) or% inhibition at 10 lM (n = 3, triplicate).

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
5477
Table 3
substituents were increased to four carbons (3k, 3l), the inhibitory
activity started to decrease. The introductions of heteroatoms at
the R2 substituents (3m, 3n) were also detrimental to the activity.
The substituents which contained aryl groups with or without the
substituent (3o, 3p, 3q) led to the complete loss of activity. Sec-
ondly, an amino group at the meta or ortho-position of the phenol
group at the R3 position (3r, 3s) led to the decrease in activity,
which indicated that the 4- aminophenol group at the R3 position
is crucial for inhibitory activity. The introductions of electron
donating or electron withdrawing groups at the R1 position (3t,
3u, 3v) also abolished the inhibitory activity.
Other analogues having substituents at the amino group of the
phenol ring were also synthesized and tested to determine if the
hydrogen of the amino group is crucial for the inhibitory activity
(Table 3). Interestingly, the mono-methyl substitution of the amino
group as found in 4a, 4c and 4e resulted in the highly potent lucif-
erase inhibitors. The most potent luciferase inhibitor 4c had an IC50
Inhibition of luciferase activity by compounds 4a–4f, expressed as IC50 values or as%
inhibition at 10 lM
R3
N
N
R1
N
R2
Compound
R1
H
R2
R3
IC50 (l
M)a
H
N
4a
0.13 0.03
0.18 0.05
0.06 0.01
0.09 0.0
O
N
4b
4c
4d
4e
H
H
H
H
H
value of 0.06 0.01 lM, whereas the di-methyl substitution of the
amino group (4b, 4d, 4f) remained the potency (Table 3).
O
O
Finally, to investigate the molecular basis of these derivatives
interacting with luciferase, a molecular docking approach was per-
formed to analyze the complex structures of 3a and 4c with the en-
zyme. From the crystal structures of firefly luciferase with inhibitor
and AMP, we know that there are two different binding pockets
within the enzyme. The AMP binding pocket is curved while the
flat one is for oxyluciferin.29 As shown in Figure 4 (left), the best
poses obtained (lowest predicted binding free energy of the most
occurring binding mode) for both 3a and 4c are accommodated
well by the oxyluciferin binding pocket, as expected for a predom-
H
N
N
O
O
H
N
inantly competitive inhibitor with respect to D-luciferin. For 3a, the
amino group attached to the phenyl ring provided the hydrophobic
interactions with the carbonyl group of Asn229 and carboxyl group
of Glu311. The nitrogen atom on the pyrimidine formed a hydro-
gen bond with the hydroxyl of residue Ser347. Moreover, the pyr-
role ring of 3a provided an aromatic stacking with residue Phe247,
which might play a major role in ligand binding (Fig. 4, top right).
We also noticed that the oxyluciferin pocket was so flat and narrow
that the substituent at the R1 position was not tolerated. Thus com-
pounds 3t, 3u, and 3v showed little inhibitory activities. Interest-
ingly, based on the conformation obtained of 4c, we observed
that the binding between luciferase and 4c might be a result of
the hydrophobic interactions and well-accommodated space in
the pocket (Fig. 4, lower right). The binding modes of the two li-
gands suggested that hydrophobic residues, such as Phe247 of
the enzyme, might play major roles in ligand binding. Thus, the
analogues with hydrophilic groups near residue Phe247 showed
low activities, such as 3m and 3n. In addition, the pocket space
near residue Phe247 was so narrow that larger groups were not al-
lowed, which might explain why the introduction of a phenyl ring
(3o, 3p, 3q) was not favorable for inhibitory activity.
0.07 0.02
0.12 0.04
N
4f
O
a
Inhibition of luciferase activity (IC50 SEM (
M (n = 3, triplicate).
lM), n = 3, triplicate) or% inhibition
at 10
l
Many inhibitors are reported to be competitive or non-competitive
inhibitors except for -luciferin which acts as a mixed-type non-
competitive-uncompetitive inhibitor19 towards
-luciferin, and
-luciferin
L
D
naphthoquinone which is competitive with respect to
D
and uncompetitive towards ATP.5 In our study, 3a is a luciferase
inhibitor with a new inhibition mechanism.
Subsequently, analogues of pyrrolo[2,3-d]pyrimidine that con-
tain the 4-aminophenol group at the R3 position were designed
and tested for luciferase inhibition to explore the structure–activ-
ity relationships of this series of compounds (Table 2). Initially,
substitutions at the R2 position were investigated firstly, and it
was noted that the activity was completely lost when R2 was a
hydrogen or methyl group (3b, 3c). When R2 was changed to an
ethyl group, which has two carbons in length, compound 3d dis-
3. Conclusions
In summary, we have shown that ‘drug-like’ compounds, such as
3a or 4c, are mixed-type predominantly competitive inhibitors of
firefly luciferase with respect to substrate D-luciferin. Meanwhile,
played an inhibitory activity with an IC50 value of 3.51 1.10 lM,
these compounds are uncompetitive inhibitors with respect to
ATP. The inclusion of these similar compounds would result in a
high number of ‘hits’ in screening campaigns that rely on fire lucif-
erase reporter-gene assays. When these ‘hits’ were further evalu-
ated with other bioactivity assays, they could be found to be ‘false
negatives’ or ‘false positives’. Notably, pyrrolo[2,3-d]pyrimidine
frameworks are ubiquitous structures in a wide variety of naturally
occurring and synthetic compounds that exhibit important biologi-
cal activity, such as a variety of kinase inhibitors30,31 and antivi-
rals.32,33 Thus, they are widely used in the screening of compound
which was less potent than compound 3a. We noted that the inhib-
itory activity could be improved by increasing the length of the
substituents at the R2 position. Next, the length and the steric ef-
fects of the substituents at the R2 position were tested. Interest-
ingly, we found that the substituents which contained bulky
groups with three carbons in length (3e, 3f, 3h, 3j) led to more po-
tent activity. Among them, compound 3j is the best, displaying an
inhibitory activity with an IC50 value of 0.08 0.01 lM. The sub-
stituents that are less steric groups with three carbons in length
(3g, 3i) decreased the inhibitory activity. When the length of the

5478
Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
Figure 4. Predicted conformations of 3a and 4c in the binding pocket of luciferase. 3a is shown in magenta sticks. 4c is shown in green sticks, and PTC124-AMP (conformation
provided by the crystal structure 3IES) is shown in blue sticks. Residues in the binding pocket forming hydrophobic or hydrogen-bond interactions with compounds are
indicated in yellow sticks on the right.
libraries. Hence, the compounds that we found in this paper were
‘flagged’ as firefly luciferase inhibitors, and they could be added into
a counter screen database to exclude the interferences of HTS in
which a luciferase reporter system was increasingly used.11,13 Addi-
tionally, all the novel luciferase inhibitors with a unique mechanism
of action described here should be useful to the scientific commu-
nity for investigating the mechanism of luciferase catalyzed reac-
tions and better understanding the activity of the other oxygenases.
added to a solution of amino-phenol(66 mg, 0.6 mmol) and Cs2CO3
(585 mg, 1.8 mmol) in 10 mL DMSO without further purification.
After stirring for 6 h at 80 °C, the mixture was cooled to room tem-
perature and extracted with ethyl acetate (3 Â 10 mL). The com-
bined organic layers were washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated to give the crude
product, which was further purified by column chromatogra-
phy(petroleum ether/ethyl acetate 2:1) to afford compound 3a as
a brown solid(121 mg, 75% over two steps): 1H NMR (300 MHz,
CDCl3) d 8.44 (s, 1H), 7.16 (d, J = 3.6 Hz, 1H), 7.02 (d, J = 8.9 Hz,
2H), 6.73 (d, J = 8.9 Hz, 2H), 6.39 (d, J = 3.6 Hz, 1H), 5.11 (m, 1H),
3.70 (s, 2H), 1.51 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO)
d 162.5, 151.5, 150.0, 146.3, 142.7, 124.5, 122.2, 114.3, 104.6, 98.0,
46.0, 22.3.; HRMS [M]+ calcd for C15H16N4O268.1324, found
268.1326. The purity of the compound was >98% by HPLC.
4. Experimental section
4.1. Chemistry: material and methods
All reagents used were obtained from commercial sources, and
all solvents were of analytical grade. 1H NMR spectral data were re-
corded in DMSO-d6, CD3OD or CDCl3 on Varian Mercury 400 or 300
NMR spectrometer and 13C NMR were recorded in DMSO-d6 or
CDCl3 on Varian Mercury 100 NMR spectrometer. Chemical shifts
(d) were reported in parts per million (ppm), and the signals were
described as brs (broad singlet), d (doublet), dd (doublet of dou-
blet), m (multiple), q (quarter), s (singlet), and t (triplet). Coupling
constants (J values) were given in Hz. Low-resolution mass spectra
(MS) and high-resolution mass spectra (HRMS) were recorded at
an ionizing voltage of 70 eV on a Finnigan/MAT95 spectrometer.
Column chromatography was carried out on silica gel (200–300
mesh). All reactions were monitored using thin-layer chromatog-
raphy (TLC) on silica gel plates. Yields were of purified compounds
and were not optimized.
4.1.2. 4-(4-Aminophenoxy)-7H-pyrrolo[2,3-d]pyrimidine (3b)
Compound 1a (92 mg, 0.6 mmol) was added to a solution of
amino-phenol (66 mg, 0.6 mmol) and Cs2CO3 (585 mg, 1.8 mmol)
in 10 mL DMSO. After stirring for 9 h at 80 °C, the mixture was
cooled to room temperature and extracted with ethyl acetate
(3 Â 10 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated to
give the crude product, which was further purified by column
chromatography (petroleum ether/ethyl acetate 2:1) to afford
compound 3b as a brown solid (92 mg, 68%): 1H NMR (300 MHz,
d6-DMSO) d 12.12 (s, 1H), 8.27 (s, 1H), 7.38 (d, J = 3.0 Hz, 1H),
6.89 (d, J = 8.7 Hz, 2H), 6.60 (d, J = 8.7 Hz, 2H), 6.24 (d, J = 3.1 Hz,
1H), 5.06 (s, 2H).; 13C NMR (100 MHz, d6-DMSO) d 162.5, 153.4,
150.3, 146.3, 142.8, 124.6, 122.2, 114.3, 104.3, 98.2.; HRMS [M]+
calcd for C12H10N4O 226.0855, found 226.0854. The purity of the
compound was >95% by HPLC.
4.1.1. General procedure for the synthesis of3a, 3e–3i and 3k–3v
from 1
A typical procedure for the preparation of 3a:2-bromopropane
(74 mg, 0.6 mmol) was added to a solution of 1a (92 mg, 0.6 mmol)
and K2CO3 (110 mg, 0.8 mmol) in 5 mL DMF at 70 °C. According to
TLC the reaction went to completion after 4 h. The mixture was ex-
tracted with ethyl acetate (3 Â 10 mL). The combined organic lay-
ers were washed with brine(10 mL), dried over anhydrous Na2SO4,
filtered and concentrated to give the crude product 2a, which was
4.1.3. 7-Methyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3c)
Iodomethane (0.04 ml, 0.6 mmol) was added to a solution of 1a
(92 mg, 0.6 mmol), NaH (24 mg 60%, 0.6 mmol) in 5 mL dry THF at
0 °C. After stirring for 3 h at room temperature, the reaction went

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
5479
to completion according to TLC. Water (10 mL) was added and the
4.1.7. 7-(Propa-1,2-dien-1-yl)-4-(4-aminophenoxy)-7H-
mixturewasextractedwithethylacetate(3 Â 10 mL).Thecombined
organic layers were washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated to give the crude prod-
uct, which was added to a solution of amino-phenol (66 mg,
0.6 mmol) and Cs2CO3 (585 mg, 1.8 mmol) in 10 mL DMSO without
further purification. After stirring for 6 h at 80 °C, the mixture was
cooled to room temperature and extracted with ethyl acetate
(3 Â 10 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated to
give the crude product, which was further purified by column
chromatography (petroleum ether/ethyl acetate 2:1) to afford
compound 3c as a yellow solid (104 mg, 72% over two steps): 1H
NMR (300 MHz, CDCl3) d 8.40 (s, 1H), 7.02–6.91 (m, 3H), 6.67 (d,
pyrrolo[2,3-d]pyrimidine (3g)
Off white solid (70%); 1H NMR (300 MHz, CD3OD) d 8.33 (s, 1H),
7.63 (t, J = 6.5 Hz, 1H), 7.34 (d, J = 3.7 Hz, 1H), 6.96 (d, J = 7.6 Hz,
2H), 6.79 (d, J = 8.5 Hz, 2H), 6.25 (d, J = 3.6 Hz, 1H), 5.70 (d,
J = 7.4 Hz, 3H).; 13C NMR (100 MHz, d6-DMSO) d 201.6, 162.7,
151.3, 151.1, 146.5, 142.5, 124.3, 122.1, 114.3, 105.3, 100.6, 94.5,
89.1.; HRMS [M]+ calcd for C15H12N4O 264.1011, found 264.1016.
The purity of the compound was >97% by HPLC.
4.1.8. 7-Cyclopentyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3h)
Off white solid (77%); 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H),
7.15 (d, J = 3.6 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz,
2H), 6.39 (d, J = 3.6 Hz, 1H), 5.30–5.10 (m, 1H), 3.67 (s, 2H), 2.31–
2.14 (m, 2H), 1.95–1.74 (m, 6H).; 13C NMR (100 MHz, d6-DMSO) d
162.5, 152.1, 150.0, 146.4, 142.8, 125.2, 122.2, 114.3, 104.7, 98.1,
55.3, 32.2, 23.6.; HRMS [M]+ calcd for C17H18N4O 294.1481, found
294.1479. The purity of the compound was >95% by HPLC.
J = 8.6 Hz, 2H), 6.31 (d, J = 3.5 Hz, 1H), 3.80 (s, 3H), 2.82 (s, 2H).; 13
C
NMR (100 MHz, d6-DMSO) d 162.4, 152.3, 150.2, 146.1, 142.8,
128.7, 122.2, 114.4, 104.4, 97.6, 31.1.; HRMS [M]+ calcd for
C13H12N4O 240.1011, found 240.1009. The purity of the compound
was >98% by HPLC.
4.1.4. 7-Ethyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3d)
4.1.9. 7-Allyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3i)
Iodoethane (0.05 ml, 0.6 mmol) was added to a solution of 1a
(92 mg, 0.6 mmol), NaH (24 mg 60%, 0.6 mmol) in 5 mL dry THF at
0 °C. After stirring for 3 h at room temperature, the reaction went
to completion according to TLC. Water (10 mL) was added and the
mixturewasextractedwithethylacetate(3 Â 10 mL).Thecombined
organic layers were washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated to give the crude
product, which was added to a solution of amino-phenol (66 mg,
0.6 mmol) and Cs2CO3 (585 mg, 1.8 mmol) in 10 mL DMSO without
further purification. After stirring for 6 h at 80 °C, the mixture was
cooled to room temperature and extracted with ethyl acetate
(3 Â 10 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated to
give the crude product, which was further purified by column
chromatography (petroleum ether/ethyl acetate 2:1) to afford
compound 3d as a yellow solid (93 mg, 61% over two steps): 1H
NMR (300 MHz, CDCl3) d 8.46 (s, 1H), 7.09 (d, J = 3.5 Hz, 1H), 7.03
(d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 6.38 (d, J = 3.5 Hz, 1H),
4.30 (q, J = 7.3 Hz, 2H), 3.68 (s, 2H), 1.47 (t, J = 7.3 Hz, 3H).; 13C
NMR (100 MHz, d6-DMSO) d 162.5, 151.8, 150.1, 146.3, 142.7,
127.2, 122.24, 114.3, 104.5, 97.8, 39.2, 15.4.; HRMS [M]+ calcd for
Off white solid (64%); 1H NMR (300 MHz, CDCl3) d 8.46 (s, 1H),
7.06 (d, J = 3.5 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 6.73 (d, J = 8.9 Hz,
2H), 6.42 (d, J = 3.5 Hz, 1H), 6.01 (m, 1H), 5.23 (d, J = 10.2 Hz, 1H),
5.11 (d, J = 17.1 Hz, 1H), 4.87 (d, J = 5.5 Hz, 2H), 3.68 (s, 2H).; 13C
NMR (100 MHz, d6-DMSO) d 154.1, 153.2, 151.0, 149.4, 134.3,
131.4, 124.5, 123.2, 116.7, 115.0, 103.0, 98.7, 45.9.; HRMS [M]+
calcd for C15H14N4O 266.1168, found 266.1170. The purity of the
compound was >95% by HPLC.
4.1.10. 7-Neopentyl-4-(4-aminoophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3j)
Diethyl azodicarboxylate (140 mg, 0.8 mmol) was added to a
solution of 1a (92 mg, 0.6 mmol), 2, 2-dimethylpropan-1-ol
(53 mg, 0.6 mmol) and triphenylphosphine (160 mg, 0.6 mmol),
in 10 mL THF at 0 °C under nitrogen atmosphere. After stirring
for 6 h at room temperature, the reaction went to completion
according to TLC. The mixture was extracted with ethyl acetate
(3 Â 10 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated to
give the crude product, which was further purified by column
chromatography(petroleum ether/ethyl acetate 4:1) to afford com-
pound 2j. Compound 2j was added to a solution of amino-phenol
(66 mg, 0.6 mmol) and Cs2CO3 (585 mg, 1.8 mmol) in 10 mL DMSO
at 80 °C. After stirring for 6 h, the mixture was cooled to room tem-
perature and extracted with ethyl acetate (3 Â 10 mL). The com-
bined organic layers were washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated to give the crude
product,which was further purified by column chromatogra-
phy(petroleum ether/ethyl acetate 2:1) to affordcompound 3j as
a white solid (98 mg, 55% over two steps): 1H NMR (300 MHz,
CDCl3) d 8.44 (s, 1H), 7.04 (d, J = 3.5 Hz, 1H), 7.02 (d, J = 8.7 Hz,
2H), 6.71 (d, J = 8.6 Hz, 2H), 6.40 (s, 1H), 4.04 (s, 2H), 3.68 (s, 2H),
0.97 (s, 9H).; 13C NMR (100 MHz, d6-DMSO) d 162.9, 153.2, 150.6,
144.8, 143.9, 127.8, 122.4, 115.8, 104.9, 97.9, 55.9, 33.6, 27.7.;
HRMS [M]+ calcd for C17H20N4O296.1637, found 296.1636. The
purity of the compound was >99% by HPLC
C14H14N4O 254.1168, found 254.1171. The purity of the compound
was >97% by HPLC.
4.1.5. 7-Isobutyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3e)
Brown solid (69%); 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H),
7.03 (dd, J = 6.1, 4.2 Hz, 3H), 6.73 (d, J = 8.9 Hz, 2H), 6.39
(d, J = 3.5 Hz, 1H), 4.04 (d, J = 7.4 Hz, 2H), 3.68 (s, 2H), 2.29–2.14
(m, 1H), 0.92 (d, J = 6.7 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO)
d 154.1, 153.1, 150.8, 149.7, 131.5, 125.1, 123.2, 114.9, 103.0,
98.1, 51.0, 29.0, 19.7.; HRMS [M]+ calcd for C16H18N4O 282.1481,
found 282.1485. The purity of the compound was >95% by HPLC.
4.1.6. 7-(2-Methylallyl)-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3f)
Yellow solid (76%); 1H NMR (300 MHz, CDCl3) d 8.31 (s, 1H),
7.28 (d, J = 3.6 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz,
2H), 6.40 (d, J = 3.6 Hz, 1H), 4.86 (m, 3H), 4.66 (m, 3H), 1.67 (s,
3H).; 13C NMR (100 MHz, d6-DMSO) d 162.6, 152.3, 150.4, 146.4,
142.7, 141.5, 127.9, 122.2, 114.3, 111.8, 104.4, 98.1, 49.5, 19.8.;
HRMS [M]+ calcd for C16H16N4O 280.1324, found 280.1330. The
purity of the compound was >98% by HPLC.
4.1.11. 7-Cyclopentyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3k)
Brown solid (62%); 1H NMR (300 MHz, CDCl3) d 8.30 (s, 1H),
7.36 (d, J = 3.5 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz,
2H), 6.35 (d, J = 3.5 Hz, 1H), 4.67 (s, 2H), 4.31 (s, 2H), 1.74 (q,
J = 7.5 Hz, 2H), 1.51 (m, 1H), 0.94 (d, J = 6.6 Hz, 6H).; 13C NMR

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Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
(100 MHz, d6-DMSO) d 162.5, 152.0, 150.1, 146.3, 142.7, 127.6,
122.2, 114.2, 104.4, 97.8, 42.4, 38.5, 25.1, 22.2.; HRMS [M]+ calcd
for C17H20N4O 296.1637, found 296.1634. The purity of the com-
pound was >99% by HPLC.
105.4, 99.6, 47.7.; HRMS [M]+ calcd for C20H15F3N4O 314.1198,
found 314.1195. The purity of the compound was >96% by HPLC.
4.1.18. 7-Isopropyl-4-(3-aminophenoxy)-7H-pyrrolo[2,3-d]pyrim
idine (3r)
4.1.12. 7-Allyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3l)
White solid (71%); 1H NMR (300 MHz, CDCl3) d 8.47 (s, 1H), 7.20
(dd, J = 12.6, 5.2 Hz, 2H), 6.64–6.56 (m, 3H), 6.42 (d, J = 3.4 Hz, 1H),
5.12 (dt, J = 13.5, 6.8 Hz, 1H), 3.76 (s, 2H), 1.53 (d, J = 6.7 Hz, 6H).;
13C NMR (100 MHz,CDCl3) d 162.3, 154.0, 152.0, 150.5, 147.8,
130.1, 122.8, 112.3, 111.5, 108.4, 105.7, 98.6, 46.1, 22.7.; HRMS
[M]+ calcd for C15H16N4O 268.1324, found 268.1322. The purity
of the compound was >99% by HPLC.
Off white solid (58%); 1H NMR (300 MHz, CDCl3) d 8.46 (s, 1H),
7.06 (d, J = 3.6 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz,
2H), 6.38 (d, J = 3.5 Hz, 1H), 5.41 (t, J = 7.5 Hz, 1H), 4.83 (d,
J = 7.1 Hz, 2H), 3.67 (s, 2H), 1.82 (s, 3H), 1.78 (s, 3H).; 13C NMR
(100 MHz, d6-DMSO) d 162.5, 151.8, 150.2, 146.3, 142.7, 136.3,
127.2, 122.1, 119.7, 114.2, 104.4, 97.9, 41.8, 25.3, 17.8.; HRMS
[M]+ calcd for C17H18N4O 294.1481, found 294.1482. The purity
of the compound was >95% by HPLC.
4.1.19. 7-Isopropyl-4-(2-aminophenoxy)-7H-pyrrolo[2,3-d]pyrim
idine (3s)
Off white solid (66%); 1H NMR (300 MHz, CDCl3) d 8.47 (s, 1H),
7.18 (d, J = 3.6 Hz, 1H), 7.12 (m, 2H), 6.88 (dd, J = 8.3, 1.5 Hz, 1H),
6.83 (td, J = 7.5, 1.5 Hz, 1H), 6.36 (d, J = 3.6 Hz, 1H), 5.12 (dt,
J = 13.5, 6.7 Hz, 1H), 3.76 (s, 2H), 1.53 (d, J = 6.8 Hz, 6H).; 13C
NMR (100 MHz,CDCl3) d 161.8, 152.1, 150.6, 140.1, 139.1, 126.5,
123.0, 122.6, 118.7, 116.8, 105.3, 98.4, 46.2, 22.7.; HRMS [M]+ calcd
for C15H16N4O 268.1324, found 268.1328. The purity of the com-
pound was >98% by HPLC.
4.1.13. 2-(4-(4-Aminophenoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-
yl)acetamide(3m)
Off white solid (56%); 1H NMR (300 MHz, CDCl3) d 8.29 (s, 1H),
7.65 (s, 1H), 7.40 (d, J = 3.3 Hz, 1H), 7.25 (s, 1H), 6.89 (d, J = 8.6 Hz,
2H), 6.61 (d, J = 8.6 Hz, 2H), 6.24 (d, J = 3.3 Hz, 1H), 5.07 (s, 2H),
4.87 (s, 2H).; 13C NMR (100 MHz, d6-DMSO) d 168.9, 162.5, 152.6,
150.2, 146.4, 142.7, 129.2, 122.2, 114.3, 104.5, 97.6, 46.5.; HRMS
[M]+ calcd for C14H13N5O2283.1069, found 283.1071. The purity
of the compound was >96% by HPLC.
4.1.20. 7-Isopropyl-2-(methylthio)-4-(4-aminophenoxy)-7H-pyrr
olo[2,3-d]pyrimidine (3t)
4.1.14. Ethyl 2-(4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)acetate (3n)
Off white solid (63%); 1H NMR (300 MHz, CDCl3) d 7.02 (d,
J = 8.8 Hz, 2H), 6.94 (d, J = 3.6 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H),
6.03 (d, J = 3.6 Hz, 1H), 5.03 (dt, J = 13.6, 6.8 Hz, 1H), 3.69 (s, 2H),
2.48 (s, 3H), 1.47 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO)
d 162.0, 161.9, 152.4, 146.4, 142.6, 123.3, 122.1, 114.2, 101.4, 98.3,
46.1, 22.2, 13.6.; HRMS [M]+ calcd for C16H18N4OS314.1201, found
314.1202. The purity of the compound was >98% by HPLC.
Off white solid (53%); 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H),
7.08 (d, J = 3.6 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 8.7 Hz,
2H), 6.45 (d, J = 3.6 Hz, 1H), 5.01 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H),
3.69 (s, 2H), 1.28 (t, J = 7.1 Hz, 3H).; 13C NMR (100 MHz, d6-DMSO)
d 168.3, 162.5, 152.5, 150.5, 146.4, 142.6, 128.6, 122.2, 114.2,
104.4, 98.1, 61.1, 45.5, 14.0.; HRMS [M]+ calcd for C16H16N4O3
312.1222, found 312.1223. The purity of the compound was
>95% by HPLC.
4.1.21. 7-Allyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-d]pyrimi
dine (3u)
Off white solid (62%); 1H NMR (300 MHz, CDCl3) d 7.33 (d,
J = 3.6 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H),
6.39 (d, J = 3.6 Hz, 1H), 5.23 (dt, J = 13.5, 6.8 Hz, 1H), 3.20 (s, 3H),
1.52 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO) d 162.5,
156.8, 150.3, 146.8, 142.3, 128.0, 121.9, 114.2, 106.0, 99.3, 46.8,
22.3.; HRMS [M]+ calcd for C16H18N4O3S346.1100, found
346.1103. The purity of the compound was >95% by HPLC.
4.1.15. 7-Benzyl-4-(4-aminophenoxy)-7H-pyrrolo[2,3-
d]pyrimidine (3o)
Brown oil (71%); 1H NMR (300 MHz, CDCl3) d 8.34 (s, 1H), 7.54
(d, J = 3.5 Hz, 1H), 7.33–7.29 (m, 2H), 7.25 (t, J = 7.1 Hz, 3H), 6.89
(d, J = 8.7 Hz, 2H), 6.61 (d, J = 8.7 Hz, 2H), 6.30 (d, J = 3.5 Hz, 1H),
5.45 (s, 2H), 5.08 (s, 2H).; 13C NMR (100 MHz, d6-DMSO) d 162.6,
152.1, 150.5, 146.4, 142.6, 137.7, 128.6, 127.8, 127.5, 127.3,
122.2, 114.2, 104.5, 98.3, 47.5.; HRMS [M]+ calcd for C19H16N4O
316.1324, found 316.1326.The purity of the compound was >98%
by HPLC.
4.1.22. 7-Isopropyl-2-amino-4-(4-aminophenoxy)-7H-
pyrrolo[2,3-d]pyrimidine (3v)
White solid (71%); 1H NMR (300 MHz, CDCl3) d 7.01 (d,
J = 8.8 Hz, 2H), 6.77 (d, J = 3.7 Hz, 1H), 6.70 (d, J = 8.8 Hz, 2H),
5.90 (d, J = 3.7 Hz, 1H), 4.88 (dt, J = 13.2, 6.5 Hz, 1H), 4.72 (s, 2H),
3.66 (s, 2H), 1.43 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO)
d 168.3, 164.4, 159.3, 151.2, 148.2, 127.4, 124.6, 119.4, 103.5,
102.2, 49.9, 27.4.; HRMS [M]+ calcd for C15H17N5O 283.1433, found
283.1430. The purity of the compound was >98% by HPLC.
4.1.16. 7-(4-Methoxybenzyl)-4-(4-aminophenoxy)-7H-
pyrrolo[2,3-d]pyrimidine (3p)
Off white solid (75%); 1H NMR (300 MHz, d6-DMSO) d 8.35 (s,
1H), 7.51 (d, J = 3.5 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.88 (t,
J = 9.1 Hz, 4H), 6.61 (d, J = 8.7 Hz, 2H), 6.28 (d, J = 3.5 Hz, 1H),
5.36 (s, 2H), 5.08 (s, 2H), 3.70 (s, 3H).; 13C NMR (100 MHz, d6-
DMSO) d 163.0, 159.2, 152.6, 151.1, 144.9, 144.0, 128.9, 126.0,
122.5, 115.8, 114.1, 105.3, 99.0, 55.2, 47.6.; HRMS [M]+ calcd for
4.1.23. General procedure for the synthesis of 4a–4f
A typical procedure for the preparation of 4e and 4f.Iodometh-
ane (0.065 ml, 1.0 mmol) was added to a solution of 3j (178 mg,
0.6 mmol), NaH (40 mg 60%, 1.0 mmol) in 10 mL dry THF at 0 °C.
After stirring for 8 h at room temperature, the reaction went to
completion according to TLC. Water (10 mL) was added and the
mixture was extracted with ethyl acetate (3 Â 10 mL). The com-
bined organic layers were washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated to give the crude
product, which was further purified by column chromatography
(petroleum ether/ethyl acetate 8:1) to afford compounds4e and
4f (2:3). 4e as a yellow oil(56 mg, 30%): 1H NMR (300 MHz, CDCl3)
C20H18N4O2346.1430, found 346.1426. The purity of the compound
was >98% by HPLC.
4.1.17. 4-(4-Nitrophenoxy)-7-(4-(trifluoromethyl)benzyl)-7H-
pyrrolo[2,3-d]pyrimidine (3q)
Brown solid (80%); 1H NMR (300 MHz, CDCl3) d 8.48 (s, 1H),
7.58 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.08–7.01 (m, 3H),
6.75 (d, J = 8.5 Hz, 2H), 6.47 (d, J = 3.5 Hz, 1H), 5.51 (s, 2H), 3.70
(s, 2H).; 13C NMR (100 MHz,CDCl3) d 163.1, 152.7, 151.3, 144.8,
144.1, 140.8, 130.2, 129.9, 127.5, 126.0, 125.7, 122.4, 115.9,

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
5481
d 8.45 (s, 1H), 7.09–7.05 (m, 3H), 6.66 (d, J = 8.9 Hz, 2H), 6.40 (d,
J = 3.5 Hz, 1H), 4.06 (s, 2H), 3.76 (s, 1H), 2.86 (s, 3H), 0.99 (s,
9H).; 13C NMR (100 MHz, CDCl3) d 163.1, 153.2, 150.8, 147.0,
144.0, 127.8, 122.4, 112.9, 104.9, 98.0, 56.0, 33.7, 31.0, 27.8.; HRMS
[M]+calcd for C18H22N4O 310.1794, found 310.1798. The purity of
the compound was >99% by HPLC.;4f as a light green(88 mg,
45%): 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H), 7.12 (d, J = 9.1 Hz,
2H), 7.05 (d, J = 3.5 Hz, 1H), 6.78 (d, J = 9.1 Hz, 2H), 6.41 (d,
J = 3.5 Hz, 1H), 4.06 (s, 2H), 2.96 (s, 6H), 0.99 (s, 9H).; 13C NMR
(100 MHz, CDCl3) d 163.1, 153.2, 150.8, 148.4, 143.6, 127.8,
122.1, 113.3, 104.9, 98.0, 56.0, 40.9, 33.7, 27.7.; HRMS [M]+ calcd
for C19H24N4O 324.1950, found 324.1952. The purity of the com-
pound was >98% by HPLC.
were prepared in buffer B (50 mM glycine-Tris buffer supple-
mented with 5 mM MgSO4, 0.5 mM EDTA, 0.1% (w/v) BSA, pH
7.8) at 2 mM and 900 lM, respectively, as stock solution and stored
at À80 °C.
All the enzyme reactions took place at room temperature and
were performed at least in triplicate. The bioluminescence tests
were performed in a flexstation 3 Microplate Reader (Molecular
Devices, Sunnyvale, CA, USA) in flex mode according to a slightly
modified protocol from Sigma-Aldrich (EC 1.13.12.7). Briefly, inhi-
bition assays were performed using 32 nM firefly luciferase in buf-
fer A, 50
with either a single concentration of inhibitor (10
concentrations of inhibitor. Inhibition kinetics of luciferase assays
with respect to -luciferin were performed using different concen-
trations of -luciferin (3.125–200 M), where the ATP concentra-
tion was fixed at 250 M. Inhibition kinetics of luciferase assays
with respect to ATP were performed under equal conditions, where
the ATP concentration varied from 25 to 500 M and the concen-
tration of -luciferin was fixed at 50 M. Typically, a well contain-
ing 10 L of buffer B or inhibitor, and 10 L of luciferase solution
were mixed and incubated for 20 min in the dark at room temper-
ature. Then, 80 L of -luciferin/ATP solution was injected; emitted
l
M
D-luciferin and 250
lM ATP in buffer B, and incubated
lM) or different
D
4.1.24. 7-Isopropyl-4-(4-methylaminophenoxy)-7H-pyrrolo[2,3-
d] pyrimidine (4a)
D
l
l
Off white solid (27%); 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H),
7.16 (d, J = 3.6 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.8 Hz,
2H), 6.38 (d, J = 3.6 Hz, 1H), 5.11 (dt, J = 13.5, 6.8 Hz, 1H), 3.74 (s,
1H), 2.86 (s, 3H), 1.52 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz,CDCl3)
d 163.0, 151.9, 150.6, 147.0, 144.0, 122.5, 122.4, 112.9, 105.4, 98.6,
46.1, 30.9, 22.7.; HRMS [M]+ calcd for C16H18N4O 282.1481, found
282.1484. The purity of the compound was >99% by HPLC.
l
D
l
l
l
l
D
light was integrated in 1 ms and recorded in 0.8 s intervals for 46 s.
The peak signal was read as the initial velocity of catalyzed reac-
tion by luciferase. All the indicated concentrations refer to the final
4.1.25. 7-Isopropyl-4-(4-dimethylaminophenoxy)-7H-pyrrolo[2,
3-d]pyrimidine (4b)
volume of 100 lL.
Off white solid (41%); 1H NMR (300 MHz, CDCl3) d 8.45 (s, 1H),
7.16 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz,
2H), 6.39 (d, J = 3.6 Hz, 1H), 5.11 (dt, J = 13.5, 6.8 Hz, 1H), 2.97 (s,
6H), 1.52 (d, J = 6.8 Hz, 6H).; 13C NMR (100 MHz, d6-DMSO) d
163.0, 151.9, 150.6, 148.4, 143.6, 122.5, 122.2, 113.3, 105.5, 98.6,
46.1, 40.9, 22.7.; HRMS [M]+ calcd for C17H20N4O 296.1637, found
296.1635. The purity of the compound was >95% by HPLC.
4.2.1. Data analysis
All data were analyzed using the GraphPad Prism 5.01 software
(GraphPad Software Inc., San Diego, CA, USA). Half of the inhibitory
concentration of compound (IC50) was directly obtained from the
concentration-effect curves fitting with log (inhibitors) vs normal-
app
app
ized response method. The Km
and Vmax
values of luciferase
for -luciferin and ATP in the absence or presence of 3a were ob-
tained from a nonlinear fit analysis of the enzyme kinetics curve
with the Michaelis–Menten model. The dissociation constant value
D
4.1.26. 7-(2-Methylallyl)-4-(4-methylaminophenoxy)-7H-pyrrolo
[2,3-d]pyrimidine (4c)
Yellow solid (22%); 1H NMR (300 MHz, CDCl3) d 8.46 (s, 1H),
7.07 (d, J = 8.9 Hz, 2H), 7.04 (d, J = 3.5 Hz, 1H), 6.65 (d, J = 8.9 Hz,
2H), 6.42 (d, J = 3.5 Hz, 1H), 4.92 (s, 1H), 4.78 (s, 2H), 4.68 (s, 1H),
3.80 (s, 1H), 2.84 (s, 3H), 1.70 (s, 3H).; 13C NMR (100 MHz,CDCl3)
d 163.1, 152.7, 151.1, 147.0, 143.9, 140.9, 126.2, 122.4, 112.9,
105.2, 98.9, 50.1, 30.9, 19.8.; HRMS [M]+ calcd for C17H18N4O
294.1481, found 294.1486. The purity of the compound was
>95% by HPLC.
(Ki or
aKi) of inhibitor and the Km and Vmax values of luciferase
were directly obtained from the nonlinear regression analysis of
the enzyme kinetics curve with a mixed or uncompetitive inhibi-
tion model.
4.2.2. Docking studies
Molecular docking was carried out by software Autodock4.34
The X-ray structure of firefly luciferase (from Photinus pyralis)
inhibitor complex (PDB code: 3IES35) was retrieved from the Pro-
tein Data Bank (http://www.rcsb.org/pdb) for docking calculations.
The active site was defined by a grid box as large as 70 Â 70 Â 70
points with grid spacing of 0.375 Å using AutoGrid4. The box was
centered on the center of the molecule PTC124-AMP in the crystal
structure. The Lamarckian genetic algorithm was applied to ac-
count for protein-ligand interactions. Finally, the conformation
with the lowest predicted binding free energy of the most occur-
ring binding modes in the firefly luciferase active pocket was
selected.
4.1.27. 7-(2-Methylallyl)-4-(4-dimethylaminophenoxy)-7H-pyrr
olo[2,3-d]pyrimidine (4d)
Off white solid (39%); 1H NMR (300 MHz, CDCl3) d 8.47 (s, 1H),
7.12 (d, J = 9.1 Hz, 2H), 7.04 (d, J = 3.5 Hz, 1H), 6.78 (d, J = 9.1 Hz,
2H), 6.43 (d, J = 3.5 Hz, 1H), 4.93 (s, 1H), 4.79 (s, 2H), 4.68 (s, 1H),
2.96 (s, 6H), 1.71 (s, 3H).; 13C NMR (100 MHz, CDCl3) d 163.1,
152.7, 151.1, 148.5, 143.6, 140.9, 126.2, 122.1, 113.3, 112.8, 105.2,
98.9, 50.1, 40.9, 19.9.; HRMS [M]+ calcd for C18H20N4O 308.1637,
found 308.1645. The purity of the compound was >99% by HPLC.
4.2. Biology: Material and methods
Acknowledgments
D
-Luciferin sodium salt (L6882), adenosine 5’-triphosphate
This work was supported by State key Laboratory of Drug Re-
search and National Science Fund for Distinguished Young Scholars
(81125025) in China.
disodium (ATP) (A7699), and luciferase (Photinus pyralis) (L9506)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine
serum albumin (BSA, fraction V) was from Biowest (Miami, Florida,
USA). All other chemicals were obtained from standard commercial
sources. A stock solution of luciferase was prepared by dissolving
the lyophilized powder in buffer A (1 M glycine-Tris buffer con-
taining 10 mM EDTA and 100 mM MgSO4, pH 7.8) (320 nM lucifer-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.035.
ase) and stored in small aliquots at À80 °C.
D-luciferin and ATP

5482
Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 5473–5482
20. Lemasters, J. J.; Hackenbrock, C. R. Biochemistry 1977, 16, 445.
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