Synthesis of aryl-heteroaryl ureas (AHUs) based on 4-aminoquinoline and their evaluation against the insulin-like growth factor receptor (IGF-1R)

Article abstract of DOI:10.1016/j.bmc.2010.06.071

The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) involved in all stages of the development and propagation of breast and other cancers. The inhibition of IGF-1R by small molecules remains a promising strategy to treat cancer. Herein, we explore SAR around previously characterized lead compound (1), which is an aryl-heteroaryl urea (AHU) consisting of 4-aminoquinaldine and a substituted aromatic ring system. A library of novel AHU compounds was prepared based on derivatives of the 4-aminoquinoline heterocycle (including various 2-substituted derivatives, and naphthyridines). The compounds were screened for in vitro inhibitory activity against IGF-1R, and several compounds with improved activity (3-5 μM) were identified. Furthermore, a computational docking study was performed, which identifies a fairly consistent lowest energy mode of binding for the more-active set of inhibitors in this series, while the less-active inhibitors do not adopt a consistent mode of binding.

Full text of DOI:10.1016/j.bmc.2010.06.071

Bioorganic & Medicinal Chemistry 18 (2010) 5995–6005
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of aryl-heteroaryl ureas (AHUs) based on 4-aminoquinoline and
their evaluation against the insulin-like growth factor receptor (IGF-1R)
William Engen a, Terrence E. O’Brien a, Brendan Kelly a, Jacinda Do a, Liezel Rillera a, Lance K. Stapleton b,
a,
Jack F. Youngren b, , Marc O. Anderson
*
*
a Department of Chemistry & Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA
b Diabetes and Endocrine Research, University of California, San Francisco/Mt. Zion Medical Center, Box 1616, San Francisco, CA 94143-1616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) involved in all stages
of the development and propagation of breast and other cancers. The inhibition of IGF-1R by small mol-
ecules remains a promising strategy to treat cancer. Herein, we explore SAR around previously character-
ized lead compound (1), which is an aryl-heteroaryl urea (AHU) consisting of 4-aminoquinaldine and a
substituted aromatic ring system. A library of novel AHU compounds was prepared based on derivatives
of the 4-aminoquinoline heterocycle (including various 2-substituted derivatives, and naphthyridines).
The compounds were screened for in vitro inhibitory activity against IGF-1R, and several compounds
Received 17 April 2010
Revised 18 June 2010
Accepted 21 June 2010
Available online 25 June 2010
Keywords:
Quinoline
Naphthyridine
2-Chloroquinoline
2-Quinolone
with improved activity (3–5 lM) were identified. Furthermore, a computational docking study was per-
formed, which identifies a fairly consistent lowest energy mode of binding for the more-active set of
inhibitors in this series, while the less-active inhibitors do not adopt a consistent mode of binding.
Ó 2010 Elsevier Ltd. All rights reserved.
IGF-1R
Diaryl urea
Aryl heteroaryl urea
Insulin like growth factor receptor
Kinase inhibition
Pharmacophore based virtual screening
1. Introduction
small-molecule inhibitors of the IGF-1R kinase.18,19,4,3,20 The devel-
opment of clinically useful inhibitors of IGF-1R kinase is compli-
The insulin-like growth factor (IGF) system is an attractive tar-
get for the development of new anti-cancer drugs.1–4 The IGF sys-
tem is composed of two ligands IGF-1 and IGF-2, as well as two
IGF receptor systems (IGF-1R and IGF-2R), which, along with the
homologous insulin receptor (IR), are members of the superfamily
of transmembrane receptor tyrosine kinases (RTKs). The receptor
IGF-1R is important in the normal growth and development pro-
cesses.5,6 Interestingly, the IGF system also plays a critical role in
the development of breast and other cancers (particularly prostate),
including malignant transformation, mitogenic growth, and anti-
apoptotic activity.7–11 IGF-1R is often over-expressed in a number
of human tumors, including colon, pancreas, prostate, bladder,
and kidney,7,1 although the significance of IGF-1R expression levels
on prognosis is still uncertain.7 Several methods have been studied
to inhibit the IGF system, including monoclonal antibodies target-
ing the receptor12, an approach currently being evaluated in clinical
trials.13,14 Further approaches are the suppression of the IGF-1
ligands and binding proteins,15–17 and also the development of
cated by the high homology (84%) with IR kinase.21 The inability
of small molecule ATP-competitive inhibitors to differentiate be-
tween IR and IGF-1R was a strong initial concern due to potential
hyperglycemic side-effects caused by inhibition of IR.22,23 However,
in vivo studies of dual IGF-1R/IR inhibitors have routinely shown
minimal effects on blood glucose levels, despite some evidence of
systemic insulin resistance.24,25 In fact, the growing evidence that
IR is involved in carcinogenesis itself26,27, in particular due to the
upregulation of its fetal isoform (IR-A), that is activated by IGF-II,
indicates that dual IGF-1R/IR inhibition is ideal in order to fully
block IGF signaling in cancer.22,23
Our overall goal is to explore the aryl-heteroaryl urea (AHU) scaf-
fold in order to identify structurally simple small-molecule inhibi-
tors of IGF-1R. Previously we characterized
a specific AHU
compound (1) as an in vitro and in vivo inhibitor of IGF-1R.28 Subse-
quently we described a parallel synthetic approach to generate li-
braries of AHUs, along with preliminary results attempting to
optimize AHU compounds for inhibitory activity against IGF-1R.29
In our previous work, lead compound 1 was the starting point for
optimization against a cell-based receptor tyrosine autophosphory-
lation assay (Fig. 1). This paper found that AHU compounds with a
* Corresponding authors. Tel.: +1 415 338 6494; fax: +1 415 405 0377 (M.O.A.).
E-mail address: marc@sfsu.edu (M.O. Anderson).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.071

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W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
diverse selection of aromatic and heteroaromatic ring systems (nap-
thalenes, alternatively connected quinolines, benzimidazole, pyri-
dines, and thioimidazole), produced compounds with little or no
activity against IGF-1R, albeit in a cell based autophorphorylation
assay (Eq. 1). Among the structurally diverse heterocycles incorpo-
rated into AHU compounds, the only one that maintained noticeable
activity was 4-aminoquinaldine. Subsequently, we constructed a
library of AHUs based on 4-aminoquinaldine,29 that incorporated
37 different substituted aromatic rings (Eq. 2). A small collection of
Some of the features that were explored were electronically
variable substituents at the 2 position, in 3{2–4}, and additional
heteroaryl nitrogen atoms in the napthyridine 3{5 and 7} and tetra-
hydronapthyridine 3{6} ring systems. The set of substituted aro-
matic ring systems 4{b} evaluated in this series was designed
based on our previous work targeting IGF-1R,29 and we also added
three heterocycle-linked aromatic systems, that is, the aryl-linked
pyrimidine 4{8–9} and 6-methylpyrazine 4{10} frameworks,
due to their presence in commercially-available isocyanates. We
hypothesized that these latter three classes of substituted aromatic
ring systems may enhance aqueous solubility of the final inhibi-
tor structures due to their additional hydrogen-bond accepting
atoms.
The synthesis of aryl-heteroaryl ureas 2{a,b} is accomplished by
the reaction of an amino heterocycle 3{a} with substituted aryl iso-
cyanate 4{b} (Fig. 3). The formation of an undesired symmetrical
urea byproduct 5{b} has been a consistent problem, most likely
due to the transient hydrolysis of aryl isocyanates (and decarbox-
ylation) followed by coupling with another molecule of isocyanate.
This competitive side-reaction seems to be especially problematic
due to the poor nucleophilicity and slower reactivity of the in-
tended heteroaromatic amine nucleophiles. The addition of strong
base to enhance the nucleophilicity of heteroaromatic amines has
been helpful in certain cases, but symmetrical urea formation still
remains a problem. An added complication is the difficulty in puri-
fying desired ureas from symmetrical ureas. Silica-gel chromatog-
raphy is often difficult, even for products well separated by TLC,
due to the tendency of symmetrical ureas to precipitate on silica
columns, causing the impurity to ‘bleed through’ and contaminate
the desired product. Nevertheless, we have found that crystalliza-
tion and preparative RP-HPLC seem to consistently remove most
symmetrical urea contamination from inhibitor samples, generat-
ing compounds of sufficient purity for screening. Yields from the
urea coupling reaction were highly variable, ranging from 5% to
80%. The lower yields were generally accounted for by non-opti-
mized precipitation procedures, designed to recover enough prod-
uct simply for in vitro screening. Purification of the desired product
from undesired symmetrical urea also accounts for lower isolated
yield of compounds.
compounds were identified with noticeable activity below 30 lM,
and four inhibitors were found to be similarly potent as lead com-
pound 1. While this study did not produce a significantly optimized
inhibitor, it did help us establish a tentative pharmacophore model
for aryl-heteroaryl ureas, notably the requirement of the 4-amino-
quinolineringsystem. Interestingly, 4-aminopyridine wasnotfound
to be a satisfactory replacement for 4-aminoquinoline.29 Herein, we
examine inhibitors containing a collection of heterocycles more
closely related to 4-aminoquinoline, notably compounds with alter-
native substitution at the 2 position, and analogous heterocycles
with additional nitrogen atoms in the quinoline ring system (e.g.,
3-aminonaphthyridines).
Since our initial work on AHU compounds as inhibitors of
IGF-1R, several useful inhibitor-bound X-ray co-crystal structures
IGF-1R have been published, facilitating structure-based optimiza-
tion of new inhibitors,30,31 including a recent report of a inhibitor
BMS-754807 which has entered clinical development.32 Particu-
larly useful in our project was the structure of a potent benzimid-
azole scaffold bound to the unactivated IGF-1R kinase.33 As
described herein, we have found that the more active inhibitors
in our library possess a consistent mode of binding when docked
into the unactivated kinase structure, while the less potent inhib-
itors dock in multiple, somewhat arbitrary, binding modes.
2. Results and discussion
Based on our previous work identifying the need of the 4-amino-
quinoline structural framework in this class of inhibitors29, we
chose to investigate new classes of inhibitors that incorporate
analogs of this heterocyclic system (Fig. 2). In contrast to the com-
mercially available heteroaromatic ring systems explored in our
previous work,29 the new classes of ring systems were either highly
expensive or not available. The heteroaryl ring systems 3{a} de-
picted in Figure 2 were chosen due to the presence of the general
4-aminoquinoline framework with additional structural features
which may act to enhance binding with the postulated ATP binding
site of the kinase, particularly nearby polar amino acid residues.
The synthesis of a series of 4-amino-2-trifluoromethylquinoline
aryl-heteroaryl ureas is illustrated in Scheme 1. 4-Chloro-2-trifluo-
romethylquinoline (6) was treated with sodium azide to generate
4-azidoquinoline 7, which was reduced by catalytic hydrogenolysis
to generate aminoquinoline 3{2}, and which was then coupled
with a selection of substituted aromatic isocyanates to generate a
library of aryl-heteroaryl ureas 2{2,b} (see Table 1). In this case,
Figure 1. Design of novel aryl-heteroaryl ureas (AHUs) based on lead scaffold 1. This publication focuses on the approach outlined in Eq. 3.

W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
5997
Figure 2. Overview of aryl-heteroaryl urea library derived from heteroaryl amine chemset 3 and substituted aryl chemset 4. The composition of the library is outlined in
Table 1.
Figure 3. Synthesis of aryl-heteroaryl ureas 2{a,b} and undesired formation of symmetrical ureas 5{b}.
Scheme 1. Synthesis of aryl-heteroaryl ureas derived from 4-amino-2-trifluoromethylquinoline. Reagents and conditions: (a) NaN3, 15-crown-5, 1-butanol; (b) H2, Pd/C,
CH3OH; (c) Ar-NCO, NaOtBu, DMSO.
the 3{2} was poorly nucleophilic, and we found that the coupling
was more effective in the presence of 2 equiv of sodium
tert-butoxide.
The synthesis of AHUs derived from 4-amino-2-methyl-1,8-
napthyridine and 4-amino-2-methyl-1,8-tetrahydronaphthyridine
is shown in Scheme 2. Our first attempt, based on the classical
Knorr and Conrad–Limpach reactions was unsuccessful. Hence, 2-
aminopyridine (8) was transformed to imine 9, while subsequent
cyclization to 10 and then to 11 was unsuccessful. Instead, an alter-
native product (12) was found, as described previously.34 An

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W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
Table 1
In vitro kinase inhibition screening results
Entry
Heterocyclic
amine
Aryl
isocyanate
Product
IGF-1R in vitro
kinase
inhibition (IC50
(error)
c Log P
determined in
MarvinView
(ChemAxon)
lM)
1
2
3
4
5
6
7
8
9
3{1}
3{1}
3{1}
3{1}
3{1}
3{1}
3{1}
3{1}
3{2}
3{2}
3{2}
3{2}
3{2}
3{2}
3{2}
3{2}
3{2}
3{2}
3{3}
3{3}
3{3}
3{3}
3{3}
3{3}
3{3}
3{3}
3{4}
3{5}
3{5}
3{6}
3{7}
3{7}
4{1}
4{2}
4{3}
4{4}
4{5}
4{8}
4{9}
4{10}
4{1}
4{2}
4{3}
4{4}
4{5}
4{6}
4{7}
4{8}
4{9}
4{10}
4{1}
4{2}
4{3}
4{4}
4{5}
4{8}
4{9}
4{10}
4{1}
4{1}
4{10}
4{1}
4{1}
4{6}
2{1,1}
2{1,2}
2{1,3}
2{1,4}
2{1,5}
2{1,8}
2{1,9}
2{1,10}
2{2,1}
2{2,2}
2{2,3}
2{2,4}
2{2,5}
2{2,6}
2{2,7}
2{2,8}
2{2,9}
2{2,10}
2{3,1}
2{3,2}
2{3,3}
2{3,4}
2{3,5}
2{3,8}
2{3,9}
2{3,10}
2{4,1}
2{5,1}
2{5,10}
2{6,1}
2{7,1}
2{7,6}
18.7 (3.0)
>30
13.9 (4.5)
>30
>30
>30
3.85
3.85
3.25
4.01
4.84
4.09
4.09
3.20
4.99
4.99
4.38
5.14
5.97
4.83
4.48
5.16
5.16
4.33
4.55
4.55
3.94
4.70
5.53
4.75
4.75
3.89
2.38
2.96
2.31
2.38
2.64
2.48
>30
5.6 (1.64)
3.5 (0.72)
8.8 (1.5)
>30
28.1 (5.0)
6.7 (1.1)
15.4 (4.1)
27.1 (4.2)
19.3 (5.2)
>30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.2 (2.3)
14.9 (5.3)
>30
>30
21.1 (7.0)
21.4 (11.9)
>30
>30
>30
6.5 (0.8)
24.7 (7.6)
12.5 (3.3)
20.5 (7.1)
>30
>30
Scheme 2. Synthesis of aryl-heteroaryl ureas derived from 4-amino-1,8-napthyridine and 4-amino-1,8-tetrahydronapthyridine. Reagents and conditions: (a) methyl
acetoacetate, POCl3; (b) acetone, POCl3; (c) NaN3, 15-crown-5, CH3OH; (d) H2, cat. PtO2 or Pd/C; (e) NaBH4, CH3OH; (f) Ar-NCO, DMSO.
alternative approach to 4-amino-1,8-naphthyridines begins with
2-aminonicotinic acid (13), which upon treatment with POCl3
forms intermediate 14, which then cyclizes to generate the desired
heterocycle 11. Conversion to the azide (15) was carried out
smoothly. Attempted reduction of the azide through catalytic
hydrogenolysis generated over-reduced tetrahydro-1,8-naphthyri-
dine 3{6} instead. Screening of alternative conditions to perform
this reduction showed that sodium borohydride could carry out
the desired azide reduction selectively to provide the desired 4-
amino-1,8-naphthyridine ring system 3{5}. Coupling of 3{5} and

W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
5999
3{6} to aromatic isocyanates was done smoothly to generate AHUs
2{5,b} and 2{6,b}. The isocyanate coupling of these amines was af-
fected efficiently without the addition of base.
the lead structure was 4-trifluoromethoxyphenyl derivative (entry
13), which is greatly improved over the analogous 4-aminoquinal-
dine-derived inhibitor which was inactive (entry 5). Entry 18, con-
taining the 6-methylpyrazine system 4{10} was noticeably more
potent than the lead structure (entry 1), and similar in potency
to the 4-aminoquinaldine-derived analog (entry 8). As before, the
hydrogen bond accepting 6-methylpiperazine heterocycle in Entry
18 most likely contributed to a decreased c Log P value compared
to other compounds in this series.
The next class of AHU compounds to be examined were the 2-
chloroquinoline AHU inhibitors 2{3,b} (entries 19–26). In general,
this class of inhibitors did not possess notable inhibitory activity
against IGF-1R. Entry 19 was the most potent in the 2-chloroquin-
oline series, which had slightly improved potency when compared
to the lead structure (entry 1), which also contains the 2-methoxy-
5-chlorophenyl ring system.
A single inhibitor containing the 2-quinolone scaffold 3{4} (en-
try 27) did display modestly improved activity in comparison with
the lead structure (entry 1). Unfortunately, elaboration of a larger
library of compounds in this series was complicated by poor nucle-
ophilicity of the heterocycle with isocyanates, combined with
exceptional difficulty in purifying products from symmetrical urea
byproducts 5{b}, by standard methods. The relatively low c Log P
value could indicate improved aqueous solubility for this inhibitor,
relative to the other inhibitors.
The remaining inhibitor scaffolds, based on 1,8-naphthyridine
2{5,b} (entries 28–29), 1,8-tetrahydronaphthyridine 2{6,b} (entry
30), and 1,7-naphthyridine 2{7,b} (entries 31–32), possessed fairly
low inhibitory potency compared to the lead structure (entry 1),
and were not pursued further in this study. Entry 29, based on
the aryl-linked 6-methylpyrazine ring system 4{10} did possess
slightly improved potency when compared to the lead structure
(entry 1).
Overall, the 2-trifluoromethylquinoline scaffold 2{2,b} could
possibly represent more promising quinoline framework for AHU
inhibitors of IGF-1R, given the enhanced potency of this series
compared to 4-aminoquinaldine scaffold 2{1,b}. Interestingly, the
aryl-linked 2-methylpyrazine ring system 4{10} seems to be pres-
ent in a few of the more potent inhibitors (entries 8, 18, 29), and
which is characterized by a relatively low c Log P value. However,
an inhibitor containing a combination of 2-trifluoromethylquino-
line and the 2-methylpyrazine systems (entry 18) did not achieve
enhanced potency compared to the simpler 5-chloro-2-methoxy
system (entry 9), suggesting these two features are not synergistic.
Given this new library of compounds with varying degrees of
inhibitory activity against IGF-1R, we decided to establish a tenta-
tive binding model employing computational docking. We chose to
dock AHU inhibitors into the ATP-binding site of IGF-1R kinase, the
likely site of binding, with the OpenEyes ligand docking utility
After preparing 4-amino-2-methyl-1,8-naphthyridine 3{5}, we
attempted to explore the generality of this method to prepare
other 4-aminonapththyridine-derived AHUs (Scheme 3). Thus we
found that 3-aminoisonicotinic acid (16) could be cleanly con-
verted to 4-chloro-2-methyl-1,7-naphthyridine (17) by treatment
with acetone and POCl3. The 4-chloronaphthyridine was then eas-
ily transformed to azide 18, which was then selectively reduced to
4-amino-2-methyl-1,7-naphthyridine 3{7}, and subsequently cou-
pled to generate AHU compounds 2{7,b}. Extension of this method-
ology to generate 4-chloro-2-methyl-1,6-naphthyridine (20) from
4-aminoisonicotinic acid (19) or from 4-aminopyridine (21) (Con-
rad–Limpach method) was unsuccessful.
In Scheme 4, the synthesis of two final scaffolds related to
4-aminoquinoline are presented. 2,4-dihydroxyquinoline (22)
was readily converted to 2,4-dichloroquinoline (23). This molecule
could be selectively hydrolyzed to the quinolin-2-one species (24)
by treatment with aqueous acid, which was then transformed to
azide 25. The azide could then either be chlorinated again followed
by azide reduction to generate 4-amino-2-chloroquinoline 3{3} or
directly reduced to generate 4-amino-quinolin-2-one 3{4}. These
two species were subsequently coupled to form respective AHU
compounds 2{3,b} or 2{4,b}. In the case of 2{4,b}, separation of
the AHU product from symmetrical urea 5{b} was rather difficult,
and only one library member in this class could successfully be
produced in high purity: 2{4,1}.
Once the library of new AHU compounds was constructed, each
compound was screened to determine in vitro inhibitory potency
against the substrate tyrosine phosphorylation activity of purified
IGF-1R kinase domain proteins. IC50 values were calculated from
ELISA assays that measured tyrosine phosphorylation of immuno-
captured substrates following incubation with purified IGF-1R and
a range of AHU concentrations (Table 1). Additionally c Log P val-
ues were determined for each library member. Entries 2–8 are
based on the lead inhibitor 2{1,1} (entry 1), containing heteroaryl
scaffold 4-aminoquinaldine 3{1}. Entry 8 showed modest improve-
ment in inhibitory potency, compared to the lead structure (entry
1). Entry 8 contains the 6-methylpiperazine heterocycle, which
was incorporated to potentially improve aqueous solubility of the
inhibitor (reflected by a lower c Log P value than other members
in the series).
The first new class of AHU compounds described in this study
were the 2-trifluoromethylquinolines 2{2,b} (entries 9–18). Com-
paring entry 9 with the lead structure (entry 1), which are identical
other than the 2-substituent on the quinoline, shows that entry 9
has obtained a fivefold improvement of inhibitory potency. An-
other entry with a modest improvement in potency compared to
Scheme 3. Synthesis of aryl-heteroaryl ureas derived from 4-amino-1,7-napthyridine. Reagents and conditions: (a) acetone, POCl3; (b) NaN3, 15-crown-5, CH3OH; (c) H2, cat.
PtO2; (d) aryl isocyanate, DMSO; (e) ethyl acetoacetate, POCl3.

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W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
Scheme 4. Synthesis of aryl-heteroaryl ureas derived from 4-amino-2-chloroquinoline and 4-amino-2-oxoquinoline. Reagents and conditions: (a) POCl3; (b) aq HCl,
1,4-dioxane; (c) NaN3, 15-crown-5, CH3OH; (d) H2, Pd/C, CH3OH; (e) NaBH4, CH3OH; (f) Ar-NCO, DMSO.
FRED. Our initial docking studies were carried out with a structure
of the activated IGF-1R kinase, co-crystallized with a substrate
peptide and ATP-mimic (PDB = 1K3A).35 Unfortunately, using this
structure there was little consistency with the mode of docking
and potency of the inhibitors. We then obtained more satisfactory
group and Ser979 (backbone NH) and Thr1127 (sidechain OH). These
interactions could explain the improved activity of 2{2,1} versus
the closely related lead structure 2{1,1}. Additionally, the quinoline
nitrogen is directed toward Lys1003, although the distance to this
residue may be too great to allow an H-bonding interaction. The
3-position of the quinoline is proximal to Val983. In this binding
mode, we do not observe the urea linkage making specific contacts
with residues in the ATP binding site. Our current scaffolds lack use-
ful hydrogen-bond interactions with the Glu1050 and Met1052 polar
backbone atoms in the IGF-1R hinge region, which we hope to tar-
get in a future set of inhibitors.
When the entire set of AHU compounds was docked into the
IGF-1R kinase structure, the binding mode could be classified in
three possible ways (Table 2). The first mode (A) is consistent with
the pose described in Figure 4 for inhibitor 2{2,1}. The second bind-
ing mode (B) has the substituted aromatic ring projecting into the
ATP-binding site, and the quinoline projecting out of the cavity.
The third binding mode (C) is analogous to binding mode A, except
that the orientation of the quinoline is flipped, that is, C5–C8 are
projecting towards Val983, and away from Glu1050 and Met1052. Pre-
sented in Supplementary data are two relatively less potent inhib-
itors: 2{3,8} adopting Binding Mode B; and 2{5,10} adopting
Binding Mode C (Figs. S1 and S2, respectively).
docking results using the co-crystal structure of
a 3-(1H-
benzo[d]imidazol-2-yl)pyridin-2(1H)-one lead compound with
unactivated IGF-1R kinase (PDB = 2OJ9).33
Using the unactivated IGF-1R kinase structure, a number of our
relatively potent AHU compounds produced relatively consistent
poses where the quinoline heteroaryl nitrogen atom is poised to
interact with Lys1003. An example of this binding mode is illustrated
in the lowest-energy pose of the most potent compound in this ser-
ies, 2{2,1}, shown in Figure 4 (‘Binding Mode A’). In this model the
quinoline carbocycle fits into a pocket surrounded by non-polar
residues Val1023 and Val983. A key set of interactions that may
explain the enhanced potency of the 2{2,b} series of inhibitors,
are potential electrostatic contacts between the 2-trifluoromethyl
Interestingly, it appears that when the inhibitors are sorted
from most potent to least potent, the more potent collection of
inhibitors (IC50 <10
mode A, with the exception of inhibitor 2{4,1}. The less potent
inhibitors (IC50 >10 M) appear to adopt a more variable selection
lM) appears to select lowest-energy binding
l
of the binding modes A–C. On the outset, our model suggests that
the most potent AHU inhibitors adopt a consistent binding mode
(A), which will be useful as we plan the rational design of future
inhibitors. Further validation of binding mode A, through X-ray dif-
fraction crystallography, is currently underway.
3. Conclusion
A lead aromatic heteroaryl urea structure 2{1,1} was used as a
starting point for optimization to improve inhibitory activity
against IGF-1R. The general strategy was the replacement of the
4-aminoquinaldine system with other analogous ring systems that
preserve the key heteroaromatic nitrogen atom. An additional
strategy was the incorporation of novel substituted aromatic rings
that contain tethered heterocycles which could improve the drug-
like characteristics of the inhibitors. The results of this study are
summarized in Figure 5. One promising alternative to the 4-amino-
quinaldine heterocycle is 4-amino-2-trifluoromethylquinoline,
which produced a new compound 2{2,1} with fivefold potency
improvement compared to lead structure 2{1,1}. The presence of
the aryl-linked 2-methylpyrazine substituted arene enhanced
Figure 4. Computational docking of the relatively potent AHU inhibitor 2{2,1} into
the active site of IGF-1R kinase. (a) Three-dimensional side-view of postulated
inhibitor–protein complex; (b) two-dimensional diagram showing key interactions
with the ATP-binding site of IGF-1R. This inhibitor orientation will be referred to as
‘Binding Mode A’.

W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
6001
Table 2
Inhibitors sorted by in vitro potency (IC50) along with the computationally-determined lowest-energy binding mode (see text)
Compound
Minimum energy
binding mode
IGF-1R in vitro
kinase inhibition
determined by
docking (see text)
(IC50 lM)
Compounds with IC50 below 10.0
l
M:
M:
2{2,1}
2{2,10}
2{1,10}
2{4,1}
2{2,5}
2{2,2}
A
A
A
B
A
A
3.5
5.2
5.6
6.5
6.7
8.8
Compounds with IC50 above 10.0
l
2{5,10}
2{1,3}
2{3,1}
2{2,6}
2{1,1}
2{2,8}
2{6,1}
2{3,4}
2{3,5}
2{5,1}
2{2,7}
2{2,4}
2{1,2}
2{1,4}
2{1,5}
2{1,8}
2{1,9}
2{2,3}
2{2,9}
2{3,2}
2{3,3}
2{3,8}
2{3,9}
2{3,10}
2{7,1}
C
B
A
A
A
B
B
B
B
C
C
B
A
B
B
C
B
A
C
A
C
B
A
B
A
12.5
13.9
14.9
15.4
18.7
19.3
20.5
21.1
21.4
24.7
27.1
28.1
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
Binding modes: A = quinoline bound into the ATP-binding site with the quinoline N1 atom proximal to Lys1003, C7 near Glu1050, and the C2-substituent (methyl, etc.) proximal
to Ser979 (Fig. 4); B = a backward inhibitor binding mode with the substituted aromatic ring docked into the ATP binding site, and the quinoline is projected toward exposed
solvent (Fig. S1); C = an alternative binding mode to A in which the quinoline is projected into the ATP binding site, but its orientation is flipped, or otherwise noticeably
inconsistent with mode A (Fig. S2).
Figure 5. Summary of most potent compounds identified in this study, with in vitro kinase potency indicated (lM), with error in parenthesis.

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W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
potency in two inhibitors 2{1-2,10}, which provided relatively low
c Log P values, we suspect due to the presence of hydrogen-bond
accepting heteroatoms. Compound 2{4,1}, based on 4-amino-2-
oxoquinoline showed promising inhibitory activity but preparing
additional analogs was prevented due to purification issues.
Inhibitors based on 4-amino-2-chloroquinoline, 4-amino-1,7 and
1,8-napthyridine were not particularly promising, but the novel
syntheses of the parent heterocycles was provided. Lastly, a com-
putational docking model of the more potent series of inhibitors
was presented, showing that these compounds typically adopt a
consistent mode of binding (mode ‘A’), while the less-potent com-
pounds do not adopt a consistent mode of binding. The computa-
tional modeling of these compounds has allowed us to plan the
next step of rational design of inhibitors in this class. Our current
working hypothesis is that by modifying the current quinoline-
based inhibitors to incorporate polar functionalities (e.g., primary
amide) positioned to interact with Glu1050 and Met1052, we will
be able to obtain inhibitors with enhanced potency. Results of
these studies will be presented in due course.
(OpenEyes). FRED was configured to employ consensus scoring, with
its full suite of scoring functions: ChemGauss3, OEChemScore,
ScreenScore, PLP, and ZapBind. Docking of inhibitors was carried
out free of pharmacophore restraints. Each final pose was mini-
mized by FRED by molecular dynamics using the in the MMFF94 force
field within the protein active site. The docking results, for each
inhibitor, were sorted by top consensus score. The final protein–
ligand complexes were visualized using PYMOL (DeLano Scientific).
4.3. In vitro IGF-1R inhibition screening
Inhibition of IGF-1R substrate kinase activity was determined
by ELISA employing recombinant IGF-1R kinase domain proteins
and biotin conjugated IRS-1 peptide substrate (Cell Signaling Tech-
nology, Inc.). IGF-1R (15 ng) were incubated with varying concen-
trations of AHU compounds for 5 min. IRS-1 sequence peptide
(0.3 lM) was then added with 15 lM ATP under final buffer condi-
tions of 60 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3uM
Na3VO4. After 60 minutes the reaction was stopped by the addition
of 50 mM EDTA. Next, the substrate was captured from a 50 lL ali-
quot of the reaction mixture added to a streptavidin-coated 96 well
plate. After the substrate was allowed to bind to the plate, phos-
phorylation was determined by anti-phosphotyrosine antibody
conjugated to HRP.
4. Experimental
4.1. Synthesis
All solvents used in reactions were anhydrous and obtained as
such from commercial sources. All other reagents were used as
supplied unless otherwise stated. Liquid flash column chromatog-
raphy was the procedure described by Still et al.36 1H and 13C NMR
spectra were recorded on either a Bruker DRX 300 MHz or Bruker
Avance 500 MHz spectrometer. 1H NMR chemical shifts are relative
to TMS (d = 0.00 ppm), CDCl3 (d = 7.26 ppm), CD3OD (d = 4.87 and
3.31 ppm), or D2O (d = 4.87). 13C NMR chemical shifts are relative
to CD3OD (d = 49.15 ppm) or CDCl3 (d = 77.23 ppm). Analytical
and semi-preparative HPLC was performed using a Dionex Ulti-
mate 3000 system with either Higgins Analytical Targa C18
150 Â 4.6 mm columns (at 1 ml/min) or a YMC C18 50 Â 20 mm
column (at 20 ml/min), respectively. High resolution mass spectra
were obtained by the University of Notre Dame Mass Spectrometry
and Proteomics Facility, Notre Dame, IN 46556-5670 using ESI
either by direct infusion on a Bruker micrOTOF-II or by LC elution
via an ultra-high pressure Dionex RSLC with C18 column coupled
with a Bruker micrOTOF-Q II.
4.3.1. 4-Azido-2-trifluoromethylquinoline (7)
To a 40 ml scintillation vial was added 4-chloro-2-trifluorometh-
ylquinoline (400 mg, 1.73 mmol) (6), 1-butanol (4 ml), sodium azide
(1.1 g, 17.3 mmol, 10 equiv), and 15-crown-5 (0.342 ml, 1.73 mmol,
1 equiv). The vial was sealed and heated to 150 °C while stirring.
After 4 h, RP-HPLC showed consumption of SM and formation of
product. The reaction mixture was dry-loaded onto Celite, and was
then purified by silica-gel flash column chromatography [hex-
ane?25:1 hexanes/ethyl acetate + 0.05% triethylamine] to generate
the title compound as a colorless film (302.6 mg, 76%). The com-
pound was rapidly advanced to the next step to avoid photochemical
degradation. 1H NMR (CDCl3, 500 MHz): d 7.41 (s, 1 H) 7.64 (t,
J = 7.57 Hz, 1 H), 7.82 (t, J = 5 Hz, 1H), 8.09 (d, J = 8.20 Hz, 1H), 8.16
(d, J = 8.51 Hz, 1H). 13C NMR (CDCl3, 125 MHz): d 105.1, 122.6,
123.0, 129.1, 130.6, 132.4, 148.85 (q, J = 13.75 Hz).
4.3.2. 4-Amino-2-trifluoromethyl-quinoline 3{2}
A
portion of 4-azidotrifluoromethylquinoline (7) (302 mg,
1.27 mmol) was dissolved in MeOH (18 ml), treated with Pd (10%
on carbon) (0.1 equiv), and stirred under argon. The vessel was
subjected twice to evacuation followed by purging with a hydrogen
balloon, and then allowed to stir under hydrogen for 3 h. TLC showed
consumption of starting material and formation of product. The
reaction mixture was filtered through a bed of Celite and then
concentrated in vacuo to yield the product as a slightly yellow solid
4.2. Computational modeling
The collection of inhibitors (Table 1, entries 1–32) was initially
drawn in ChemDraw (Cambridge Software), and converted to a set
of SMILES strings, which was then transformed to a library of single
3D conformations using OMEGA (OpenEyes). The single conformations
were passed through MOLCHARGE (OpenEyes) in order to apply AM1BCC
charges to each compound. Lastly, the charged set of compounds
was passed once again through OMEGA (OpenEyes) to generate mul-
ti-conformational libraries for each compound. The neutral proton-
ationstate oftheinhibitorsgenerated byOMEGA was verifiedusingthe
PKA plugin implemented in MarvinView (ChemAxon). c Log P values
were calculated by the LOGP plugin implemented in JChem/Marvin-
View (ChemAxon). The protein receptor was prepared for docking
with the FRED-RECEPTOR (OpenEyes) utility, employing a 2.00 Å
resolution X-ray crystal structure of the unactivated IGF-1R kinase
domain co-crystallized with an ATP-mimic inhibitor (PDB = 2OJ9).33
To generate the receptor, the co-crystallized inhibitor was removed
using PYMOL (DeLano Scientific), and the active site was then defined
to be a 6 Å box around ATP binding site of the protein.
(269 mg, 73%), which is
a commercially available material.
Mp = 138–140 °C. 1H NMR (CD3CN, 500 MHz): d 5.90 (br s, 1H),
6.88 (s, 1H), 7.49 (t, J = 10 Hz, 1H), 7.66 (t, J = 10 Hz, 1H) 7.87 (d,
J = 8.51 Hz, 1H), 7.92 (d, J = 10 Hz, 1H). 13C NMR (CD3CN, 125
MHz): d 98.7, 118.0, 119.3, 112.2, 126.7, 130.3, 131.2, 148.8, 153.8.
LC-ESI(+)-TOF-HRMS (M+H)+ calcd 213.0639 found 213.0642 for
C10H7F3N2.
4.3.3. General conditions for coupling aminoquinoline
derivatives 3{a} to substituted aromatic isocyanates 4{b} to
generate AHU compounds 2{a,b}
An appropriate 4-aminoquinoline derivative 3{a} was dissolved
in DMSO (0.1 M), treated with sodium tert-butoxide (2.0 equiv only
for aminoquinolines 3{2}, 3{3}, and 3{4}), and stirred 15 min, usually
causing the solution to turn slightly brown. Subsequently, the solu-
tion was treated with an aromatic isocyanate 4{b} (1.0–1.2 equiv)
Upon successful preparation of the library of inhibitors and the
receptor structure, docking was then performed using FRED (v2.2.5)

W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
6003
and stirred 30 min at rt, with the progress of the reaction followed by
RP-HPLC analysis. The products were purified by precipitation by
addition to water/AcOH (1:1), NaOH (0.1 M aq), or directly purified
by semi-preparative RP-HPLC using a Dionex Ultimate 3000
instrument.
in vacuo to yield the product as a white waxy solid film (186 mg,
99%). 1H NMR (MeOH-d4, 500 MHz): d 1.90 (p, J = 5 Hz, 2H), 2.13
(s, 3H), 2.39 (t, J = 10 Hz, 2H), 3.26–3.28 (mult, 2H), 3.31 (mult,
1H), 5.88 (s, 1H). 13C NMR (MeOH-d4, 125 MHz): d 21.5, 22.7,
22.8, 42.1, 96.5, 102.0, 152.9, 155.03, 156.64. LC-ESI(+)-TOF-HRMS
(M+H)+ calcd 164.1187, found 164.1193 for C9H13N3.
4.3.4. 4-Chloro-2-methyl-1,8-naphthyridine (11)
A 60 ml scintillation vial with a polypropylene-lined cap was
charged with 2-aminonicotinic acid (13) (1000 mg) and acetone
(2 ml, 4 equiv), and the reaction mixture was allowed to stand for
ca. 5 min before being treated with POCl3 (35 ml, 0.2 M). The reac-
tion mixture was then heated for 5.5 h at 80 °C. Next, the reaction
mixture was carefully added to 200 ml ice water, which was then
basified to pH 14 with NaOH (6 M aq) and stirred for 40 min (with
additional base added to maintain pH 14). The aqueous material
was split into two portions. Each portion was extracted with DCM
(6 Â 100 ml). The organic layers were combined and concentrated
in vacuo to generate crude product (11) (0.727 g, 56%) which was
employed directly in the next step without further purification or
characterization.
4.3.8. 4-Chloro-2-methyl-1,7-naphthyridine (17)
3-Aminoisonicotinic acid (16) (50 mg, 0.362 mmol) was treated
with acetone (0.133 ml, 1.81 mmol, 5 equiv) and allowed to stand
for 5 min. The reaction mixture was then carefully treated with
POCl3 (20 ml), and then heated at 100 °C under air for 60 min. RP-
HPLC analysis showed consumption of starting material and forma-
tion of suspected product. The reaction mixture was transferred to a
beaker containing 200 ml ice water, and the contents were subse-
quently basified to pH 14 using NaOH solution (5 N aq), and then
treated with NaCl (satd aq) to ‘salt out’ the organic product. The or-
ganic material was extracted with DCM (3 Â 50 ml), dried over
Na2SO4, and concentrated in vacuo to generate the crude product.
The material was then purified by flash column chromatography
(1:1 hexanes/EtOAc) to generate the product (17) (22 mg, 35%) as
an off-color film, which has been previously characterized.37 1H
NMR (MeOH-d4, 500 MHz): d 2.75 (s, 3H), 7.82 (s, 1H), 8.04 (d,
J = 5.67 Hz, 1H), 8.61 (d, J = 5.99 Hz, 1H) 9.27 (s, 1H). 13C NMR
(MeOH-d4, 125 MHz): d 24.3, 117.1, 126.9, 129.1, 142.2, 143.6,
143.9, 152.8, 162.8. LC-ESI(+)-TOF-HRMS (M+H)+ calcd 179.0376,
found 179.0382 for C9H7ClN2.
4.3.5. 4-Azido-2-methyl-1,8-naphthyridine (15)
A solution of crude 4-chloro-2-methyl-1,8-naphthyridine (11)
(727 mg, 4.0 mmol) was dissolved in MeOH (20 ml) and treated
with sodium azide (2.6 g, 40.7 mmol, 10.2 equiv) and 15-crown-5
(0.040 ml, 0.407 mmol, 0.05 equiv). The mixture was heated to
80 °C for 18 h, and then RP-HPLC analysis was used to determine
that the reaction has proceeded quantitatively. The reaction mix-
ture was dry-loaded onto Celite, and was then purified by silica-
gel flash column chromatography (eluted with EtOAc) to generate
the title compound as a white film (548 mg, 73%). The compound
was rapidly advanced to the next step to avoid photochemical deg-
radation. 1H NMR (CDCl3, 500 MHz): d 2.78 (s, 3H), 7.06 (s, 1H) 7.39
(dd, J = 8.20, 4.41 Hz, 1 H), 8.32 (dd, J = 8.20, 1.89 Hz, 1H), 9.06 (d,
J = 5 Hz). 13C NMR (CDCl3, 125 MHz): d 25.8, 109.9, 114.7, 121.0,
131.6, 146.8, 154.2, 156.7, 162.8.
4.3.9. 4-Azido-2-methyl-1,7-naphthyridine (18)
A solution of 4-chloro-2-methyl-1,7-naphthyridine(17) (10.8
mg) in 1-propanol (1.8 ml) was treated with 15-crown-5
(0.024 ml, 0.122 mmol) and sodium azide (40.5 mg, 0.607 mmol,
10 equiv). The reaction mixture was stirred at 120 °C overnight.
RP-HPLC analysis showed conversion of starting material to prod-
uct. The material was purified by flash column chromatography
(40:1 dichloromethane/MeOH) to generate the product (18)
(5.9 mg, 53%) as a slightly yellow film. The compound was rapidly
advanced to the next step to avoid photochemical degradation. IR
(cmÀ1, KBr): 1593, 1665, 2922. 1H NMR (MeOH-d4, 500 MHz): d
2.76 (s, 3H), 7.85 (s, 1H), 8.07 (d, J = 5.7 Hz, 1H), 8.63 (d,
J = 5.67 Hz, 1 H), 9.29 (s, 1H). 13C NMR (MeOH-d4, 125 MHz): d
24.5, 114.2, 115.7, 124.5, 142.9, 143.7, 147.0, 152.0, 162.9.
4.3.6. 4-Amino-2-methyl-1,8-naphthyridine 3{5}
A solution of 4-azido-2-methyl-1,8-naphthyridine (15) (20 mg,
0.108 mmol) was dissolved in MeOH (1 ml) was treated with so-
dium borohydride (8 mg, 0.216 mmol, 2 equiv) and then heated
to 40 °C for 20 min whereupon fairly intense bubbling occurred.
TLC indicated consumption of starting material and formation of
suspected product. To destroy boron complexes, H2O (10 ml) was
added, and then the pH was set to 1 with HCl (1 M aq), and then
the mixture was re-basified to pH 14 with NaOH (6 M aq). The
solution was extracted with DCM (2 Â 20 ml), the organic layer
was dried over Na2SO4, and concentrated in vacuo to yield the
product 3{5} as a slightly orange solid (18 mg, quantitative yield).
Mp = 190–200 °C (dec). IR (cmÀ1, KBr): 1596, 1662, 3060. 1H NMR
(MeOH-d4, 500 MHz): d 2.53 (s, 3H), 6.59 (s, 1H), 7.38 (dd, J = 8.20,
4.41 Hz, 1H), 8.52 (dd, J = 8.35, 1.73 Hz, 1H), 8.84–8.85 (mult, 1H).
13C NMR (MeOH-d4, 125 MHz): d 25.2, 104.3, 113.3, 120.2, 133.7,
153.8, 155.4, 157.8, 163.8. LC-ESI(+)-TOF-HRMS (M+H)+ calcd
160.0874, found 160.0871 for C9H9N3.
4.3.10. 4-Amino-2-methyl-1,7-naphthyridine 3{7}
A solution of 4-azido-2-methyl-1,8-naphthyridine (18) (120
mg, 0.6408 mmol) in methanol (6.5 ml) was treated with platinum
oxide (21.1 mg, 0.093 mmol, 0.15 equiv), and stirred under argon.
The vessel was subjected twice to evacuation followed by purging
with a hydrogen balloon, and then allowed to stir under hydrogen
for 90 min. The reaction mixture was filtered through a bed of
Celite and then concentrated in vacuo to yield the product as a
yellow oil (97.7 mg, 95%). 1H NMR (MeOH-d4, 500 MHz): d 2.54
(s, 3H), 6.69 (s, 1H), 7.95 (d, J = 5.67 Hz, 1H), 8.35 (d, J = 5.67 Hz,
1H), 9.04 (s, 1H). 13C NMR (MeOH-d4, 125 MHz): d 24.07, 105.5,
116.0, 122.1, 140.5, 144.0, 151.4, 152.5, 162.0. LC-ESI(+)-TOF-
HRMS (M+H)+ calcd 160.0874, found 160.0873 for C9H9N3.
4.3.7. 2-Methyl-5,6,7,8-tetrahydro-[1,8]naphthyridin-4-ylamine
3{6}
4.3.11. 2,4-Dichloroquinoline (23)
A solution of crude 4-azido-2-methyl-1,8-naphthyridine (15)
(187 mg, 1.150 mmol) was dissolved in MeOH (11 ml) was treated
with platinum oxide (26 mg, 0.115 mmol, 0.1 equiv), and stirred
under argon. The vessel was subjected twice to evacuation fol-
lowed by purging with a hydrogen balloon, and then allowed to stir
under hydrogen for 18 h. RP-HPLC analysis showed consumption of
starting material and formation of suspected product. The reaction
mixture was filtered through a bed of Celite and then concentrated
To a 20 ml scintillation vial was added 2,4-dihydroxyquinoline
(22) (200 mg, 1.24 mmol), which was then carefully treated with
POCl3 (4.2 ml, 0.3 M) and the reaction was then heated to 100 °C
for 2 h. RP-HPLC analysis showed consumption of starting material
and formation of suspected product. The reaction mixture was
transferred to a beaker containing ice water (100 ml) and NaOH
(6 M, 35 ml), and then stirred to quench unreacted POCl3. The
product was then extracted with diethyl ether (3 Â 50 ml) and

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W. Engen et al. / Bioorg. Med. Chem. 18 (2010) 5995–6005
then the combined organic material was washed with NaCl (satd
aq) (50 ml), dried over Na2SO4, and concentrated in vacuo to yield
the product (23) (221 mg, 90% yield). The compound has been pre-
pared previously38 and is also commercially available. 1H NMR
(CDCl3, 500 MHz): d 7.49 (s, 1H), 7.64 (t, J = 7.72 Hz, 1H), 7.78 (t,
J = 10 Hz, 1H), 8.02 (d, J = 8.51 Hz, 1H), 8.17 (d, J = 10 Hz, 1H). LC-
ESI(+)-TOF-HRMS (M+H)+ calcd 197.9877, found 197.9881 for
C9H5Cl2N.
for 30 min while more base was added to maintain the pH at 14.
The aqueous material was extracted with Et2O/EtOAc (4 Â 40 ml),
and the resultant organic layer was dried over Na2SO4 and concen-
trated in vacuo to yield 4-azido-2-chloro-quinoline (20.0 mg, 91%)
which was used in the next step without further purification. A
solution of 4-azido-2-chloro-quinoline (20 mg, 0.098 mmol) in
MeOH (1 ml) was treated with sodium borohydride (3.7 mg,
0.098 mmol), and stirred at rt for 30 min. HPLC showed conversion
of starting material to tentative product. The mixture was treated
with HCl (1 M aq, 10 ml) to quench borohydride, and was then
basified with to pH 9 with NaOH (1 M), and then extracted with
CH2Cl2 (3 Â 10 ml). The organic layer was concentrated in vacuo
to yield the final product (13.4 mg, 77%). The compound has been
prepared previously38 and is also commercially available. 1H NMR
(CD3OD, 300 MHz): d 6.58 (s, 1H), 7.40–7.46 (mult, 1H), 7.56–7.77
(mult, 2H), 8.03 (d, J = 9 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz): d
100.5, 117.6, 122.5, 124.3, 127.9, 130.3, 148.0, 150.6, 154.1.
LC-ESI(+)-TOF-HRMS (M+H)+ calcd 179.0376, found 179.0381 for
C9H7ClN2.
4.3.12. 4-Chloro-1H-quinolin-2-one (24)
A solution of 2,4-dichloroquinoline (23) (979 mg, 4.94 mmol) in
HCl (6 M):dioxane (1.4:1) was heated to 90 °C for 5 h. RP-HPLC anal-
ysis showed consumption of starting material and formation of
product. Solid K2CO3 was carefully added to the reaction mixture
until the pH was 9. The solution was extracted with EtOAc/Et2O
(1:1) (6 Â 100 ml), and the combined organic layer was washed with
NaCl (satd aq), dried over Na2SO4, and concentrated in vacuo to yield
the product (499 mg, 56%). The compound has been prepared previ-
ously39 and is also commercially available. IR (cmÀ1, KBr): 1664,
2847. 1H NMR (DMSO-d6, 500 MHz): d 6.83 (s, 1H), 7.31 (t, J =
7.57 Hz, 1H), 7.39 (d, J = 8.20 Hz, 1H), 7.63 (t, J = 7.72 Hz, 1H), 7.86
(d, J = 8.20 Hz, 1H), 12.05 (br s, 1H). LC-ESI(+)-TOF-HRMS (M+H)+
calcd 180.0216, found 180.0218 for C9H6ClNO.
Acknowledgments
This work was supported in part by a grant from the National
Institutes of Health (R01 CA-112042-01 A1). Terrance O’Brien re-
ceived funding from the NIH-MARC program (T34-GM08574).
Megumi Tamaki is acknowledged for the re-synthesis and purifica-
tion of several inhibitors. Mass spectroscopy services at SFSU were
funded by grants from the National Institutes of Health (P20
MD00544) and the National Science Foundation (CHE-0619163),
utilizing the services of Dr. Robert Yen. The authors would also like
to extend their gratitude to Wee Tam at the SFSU NMR facility for
his expert assistance. Mass-spectrometry services at University of
Notre Dame were supported by grants from the National Science
Foundation (CHE-0741793). Lastly, the authors would like to
acknowledge OpenEyes Inc. and ChemAxon Inc. for providing free
site licenses to the academic community.
4.3.13. 4-Azido-1H-quinolin-2-one (25)
A solution of 4-chloro-1H-quinolino2-one (24) (480 mg, 2.67
mmol) in DMF (10 ml) was treated with sodium azide (1.7 g,
26.7 mmol, 10 equiv) and 15-crown-5 (0.529 ml, 2.67 mmol,
1.0 equiv), and then the solution was allowed to stir at 100 °C for
20 h. RP-HPLC analysis showed almost complete (ꢀ90%) conver-
sion to product. The reaction mixture was diluted with H2O
(350 ml), and the product was extracted with EtOAc/Et2O (1:1)
(3 Â 120 ml). The organic mixture was dried over Na2SO4, and con-
centrated in vacuo to generate 4-azido-1H-quinolin-2-one, which
was purified by flash column chromatography (hexane/EtOAc
(1:2)?hexane/EtOAc (1:2) + 1% triethylamine) yielding the desired
product (366 mg, 73%) as a white solid. The compound was rapidly
advanced to the next step to avoid photochemical degradation. The
compound has been prepared previously40 and is also commer-
cially available. 1H NMR (DMSO-d6, 500 MHz): d 6.36 (s, 1H),
7.19 (t, J = 10 Hz, 1H), 7.57 (td, J = 7.73, 1.37 Hz, 1H), 7.68 (d,
J = 15 Hz, 1H). LC-ESI(+)-TOF-HRMS (M+H)+ calcd 187.0620, found
187.0622 for C9H6N4O.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.071.
References and notes
4.3.14. 4-Amino-1H-quinolin-2-one 3{4}
A solution of 4-azido-1H-quinolin-2-one (25 mg, 0.134 mmol)
(25) in MeOH (1.5 ml) was treated with palladium (10% on carbon)
(14 mg, 0.013 mmol) and stirred under argon. The vessel was sub-
jected twice to evacuation followed by purging with a hydrogen
balloon, and then allowed to stir under hydrogen for 3 h. The reac-
tion mixture was filtered through a bed of Celite and then concen-
trated in vacuo to yield the final product 3{4} product as a light
grey solid (20 mg, 97%). The compound has been prepared previ-
ously40 and is also commercially available. Mp = 260–265 °C
(dec). IR (cmÀ1, KBr): 1636, 3130. 1H NMR (DMSO-d6, 500 MHz):
d 5.40 (s, 1H), 6.54 (br s, 2H), 7.06 (t, J = 10 Hz, 1H), 7.18 (dd,
J = 8.23, 1.10 Hz, 1H), 7.41 (ddd, J = 8.28, 7.09, 1.28 Hz, 1H), 7.84
(dd, J = 8.14, 1.01 Hz, 1H), 10.71 (br s, 1H). LC-ESI(+)-TOF-HRMS
(M+H)+ calcd 161.0715, found 161.0714 for C9H8N2O.
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