Design, synthesis and characterization of a novel class of coumarin-based inhibitors of inducible nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2005.02.036

Inducible nitric oxide synthase (iNOS) has been implicated in various central and peripheral pathophysiological diseases. Our high throughput screening initially identified a weak inhibitor of iNOS, thiocoumarin 13. From this lead, a number of potent derivatives were prepared that demonstrate favorable potency, selectivity and kinetics. Compound 30 has an IC₅₀ of 60 nM for mouse iNOS and 185-fold and 9-fold selectivity for bovine eNOS and rat nNOS, respectively. In cellular assays for iNOS, this compound has micromolar potency. Furthermore, two compounds (16 and 30) demonstrate a reasonable pharmacokinetic profile in rodents. The synthesis, SAR, and biological activity of this novel class of compounds is described.

Full text of DOI:10.1016/j.bmc.2005.02.036

Bioorganic & Medicinal Chemistry 13 (2005) 2723–2739
Design, synthesis and characterization of a novel class of
coumarin-based inhibitors of inducible nitric oxide synthase
Sharon A. Jackson,a Sukhveen Sahni,a Lan Lee,a Yongyi Luo,a Thaddeus R. Nieduzak,a,*
Guyan Liang,a Yulin Chiang,a Nicola Collar,a David Fink,a Wei He,a Abdelazize Laoui,a
Jean Merrill,a Ray Boffey,b Peter Crackett,b Bryan Rees,b Melanie Wong,b
Jean-Pierre Guilloteau,c Magali Mathieuc and Sam S. Rebelloa
aInternal Medicine, Sanofi-Aventis Pharmaceuticals, Bridgewater, NJ 08807, USA
bArgenta Discovery Ltd, Flex Meadow, Harlow, CM195TR Essex, UK
cDepartment of Structural Biology, Sanofi-Aventis Pharmaceuticals, Vitry/Seine, 94403 cedex, France
Received 28 October 2004; revised 17 February 2005; accepted 17 February 2005
Abstract—Inducible nitric oxide synthase (iNOS) has been implicated in various central and peripheral pathophysiological diseases.
Our high throughput screening initially identified a weak inhibitor of iNOS, thiocoumarin 13. From this lead, a number of potent
derivatives were prepared that demonstrate favorable potency, selectivity and kinetics. Compound 30 has an IC50 of 60 nM for
mouse iNOS and 185-fold and 9-fold selectivity for bovine eNOS and rat nNOS, respectively. In cellular assays for iNOS, this com-
pound has micromolar potency. Furthermore, two compounds (16 and 30) demonstrate a reasonable pharmacokinetic profile in
rodents. The synthesis, SAR, and biological activity of this novel class of compounds is described.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
modification of protein conformation by introduction
of a negative charge, and initiation of autoimmune pro-
cesses because nitrophenols are known to be highly anti-
genic. Myelin protein can be one such target in the CNS
for N-nitrosylation. All these effects of NO may account
for the oligodendrocyte loss that is observed in multiple
sclerosis (MS). In addition to its cytotoxic actions, NO
can also block electrical conduction in demyelinated
and newly remyelinated axons.2 Under ischemic condi-
tions, cessation of blood flow to the brain sets in motion
a cascade of events resulting in neuronal cell death and
enhanced production of NO. The reactive product of
NO (ONOOꢀ) has been proposed to be an important
mediator.1,3
Nitric oxide (NO) is a molecular messenger that is syn-
thesized by nitric oxide synthase (NOS) enzymes. NO is
implicated in a variety of physiological and pathological
states, both in the central and peripheral nervous sys-
tems. In the central nervous system (CNS), activated
parenchymal and perivascular microglia, astrocytes,
neurons, and even endothelial cells (after activation by
proinflammatory cytokines) produce NO. The potential
for pathological consequences as a result of NO over-
production is significant in the CNS. The reaction of
NO with super oxide anion ðOꢀ2 Þ, another free radical
produced by activated macrophages, forms peroxyni-
trite (ONOOꢀ). Peroxynitrite is a footprint of NO pro-
duction and is a strong nitrating agent capable of
producing nitrotyrosines (NT).1
Nitric oxide synthase carries out the oxidation of the
guanidine moiety of L-arginine to deliver citrulline and
NO (Scheme 1). Three quite distinct isoforms of NOS,
the enzymes responsible for the production of NO, have
been identified that are capable of generating NO in a
wide variety of tissues.4 These isoforms are nNOS (type
I or NOS-1) being the isoform first found (and predom-
inating) in neuronal tissue, iNOS (type II or NOS-2)
being the isoform, which is inducible in a wide range
of cells, and eNOS (type III or NOS-3) being the
The reaction which produces NT may also cause alter-
ation of tyrosine phosphorylation dependent signaling,
Keywords: iNOS inhibition; Coumarin.
*
Corresponding author. Tel.: +1 908 231 2234; fax: +1 908 231
3577; e-mail: thaddeus.nieduzak@sanofi-aventis.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.036

2724
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
OH
NH
O
NH
NH
N
H2N
NH
H2N
H2N
NO
+
CO2-
NH+3
CO2-
NH+3
CO2-
NH+3
Arginine
Citrulline
N-hydroxy-Arginine
Scheme 1. NOS oxidation of arginine.
isoform first found in vascular endothelial cells. These
isoforms are also differentiated based on their constitu-
tive (eNOS and nNOS) versus inducible (iNOS) expres-
sion, and their calcium dependence (eNOS and nNOS)
or independence (iNOS). The importance of iNOS and
NO in MS was highlighted by clinical studies that dem-
onstrated increased iNOS mRNA in the brain,5,6 in-
creased NT staining in MS tissue, and NO metabolites
in the spinal fluid.7 Clarification of the role of iNOS in
stroke was provided by the observation that mice lack-
ing the gene for iNOS have significantly reduced infarct
volumes compared with wild-type controls.8 Also, iNOS
mRNA was increased in transient cerebral ischemia in
rats and the expression was mainly localized in vascular
cells of the infarcted region.9
most amidine or guanidine moiety containing com-
pounds were not particularly selective for iNOS relative
to the other isoforms. Based on the structure of L-NIL,
acetamidine derivatives such as S-[2-[(1-imino-
ethyl)amino]ethyl]-4,4-dioxo-L-cysteine (GW273629) 6
and
S-[2-[(1-iminoethyl)amino]ethyl]-L-homocysteine
(GW274150) 7 were designed. However, like L-NIL,
these were time-dependent inhibitors of iNOS.4 This is
undesirable because these inhibitors could lead to irre-
versible inhibition of iNOS, the physiological conse-
quences of which are unknown. AstraZeneca
researchers reported good activity of 3,4-dihydro-1-iso-
quinolinamines 8 (Fig. 2) against iNOS, but poor cellu-
lar potency and recently found 2-aminopyridine
derivatives as highly selective inhibitors of iNOS.16,17
Replacement of the benzo ring with a thieno fused ring
resulted in thienopyridines 9 that showed potent activity
against iNOS but the best compound produced hypo-
tension in vivo.18 Tinker et al. demonstrated potency,
selectivity and efficacy in the rat adjuvant-induced
arthritis model for two representative compounds
(AR-C85016 10 and AR-C102222 11) from the 1,2-dihy-
dro-4-quinazolinamine series.19 Synthesis of dihydropy-
rindin-2(1H)-imines and 1,5,6,7-tetrahydro-2H-azepin-
2-imines 12 has been described, but these lacked suffi-
Initially, the best-known inhibitors of iNOS were ana-
logs of L-arginine (Fig. 1) such as NG-methyl-L-arginine
(L-NMA) 1,10,11 NG-nitro-L-arginine (L-NNA) 2,12 N-
iminoethyl-L-ornithine (L-NIO) 3,13 L-thiocitrulline 4,14
and N-iminoethyl-L-lysine (L-NIL) 5.15 All lack selectiv-
ity over other NOS isoforms and therefore result in
undesirable pharmacological effects in vivo. As such,
NH
O
NH
O
NO2
CH3
N
N
H
HO
N
N
H
HO
H
H
NH2
NH2
O
F
2
1
L-NNA
L-NMA
N
S
N
O
NH
CH3
O
H
NH2
CH3
NH
N
F
NH2
HO
N
HO
H
NH2
NH2
9
8
3
4
O
O
L-NIO
L-NIL
F
F
H
N
N
H
N
N
O
S
O
H
CN
O
S
O
CN
N
CH3
NH
N
N
HO
NH2
N
HO
H
NH2
NH2
NH2
F
NH2
10 AR-C85016
6
5
11 AR-C102222
GW273629
L-thiocitrulline
N
O
O
H
[
]
N
S
CH3
NH
n
HO
R
N
NH2
NH2
S
O
7
12 n = 1,3
GW274150
13
Figure 1. NOS inhibitors based on L-arginine.
Figure 2. Amidine-type NOS inhibitors and thiocoumarin 13.

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
Table 1. Inhibitory activity of compounds 13–21
2725
Y
(CH2)nNR'R"
6
7
R
X
O
Compound
R
R0
R00
n
X
Y
iNOS IC50 (lM)
13
14
15
16
17
18
19
20
21
H
CH3
H
CH3
H
3
3
3
3
3
3
3
3
4
S
O
18 (h)
3.57 (h)
6-Cl
6-Cl
6-Cl
H
O
CH2
CH2
O
H
H
NH
NH
NH
NH
NCH3
NCH3
O
2.15 (h)
0.407 (m)
H
H
CH3
H
CH3
H
O
2.63 (h), 10.4 (m)
3.03 (h), 1.86 (m)
H
O
H
H
H
O
14% I @ 10 lM (m)
6.7% I @ 10 lM (m)
2.6 (m)
H
6-Cl
CH3
H
CH3
H
O
CH2
h = human; m = mouse.
cient selectivity over eNOS (<20-fold) and selectivity
over nNOS is unknown.20
1), were synthesized in a manner analogous to their
respective oxygen counterparts. The all-carbon tether
compounds 14, 15 and 21 (Table 1) were prepared utiliz-
ing the organo-zinc cross-coupling reaction of Yang and
co-workers, which was optimized using Pd(PPh3)2Cl2 as
catalyst (Scheme 3).25,26
Our goal was to discover small molecule inhibitors of
iNOS with good brain penetration and with acceptable
selectivity over the other isozymes. Screening efforts
identified thiocoumarin 13 (Fig. 2) as a weak (IC50
=
18 lM) albeit interesting inhibitor of iNOS. The fact
that it contained neither the amidine nor guanidine
functionality, warranted further investigation and a lim-
ited SAR study was undertaken. This included the prep-
aration of analogs (Tables 1 and 2 and Fig. 3) with
various simple substitutions on the aromatic ring, inves-
tigation of the propyl-tethered amine and changes to the
thiolactone functionality of 13. Here we report on the
design, synthesis and characterization of coumarin-
based inhibitors of iNOS.
3. Results and discussion
As can been seen from the summary of the SAR in Table
3 a lactone or lactam functionality was preferable to the
thiolactone with the former delivering the most potency.
Variation of the propyl tether included chain extension
and contraction (Tables 1 and 2) as well as attempts
to restrict degrees of freedom with cyclic structures
(compounds 53–60, Fig. 3). Acylation of the tethered
amine led to a significant loss of activity. Thus, main-
taining a single basic nitrogen was important with the
primary substituted aminopropyl functionality pre-
ferred. Modeling predictions relative to aromatic substi-
tution of the coumarin were borne out in the fact that
substitution at the 6-position (compound 30) was best
with putative Van der Waals interaction between the
6-chloro of 30 and the side chain (the phenyl ring) of
phenylalanine 363 (F363) in the enzyme (Fig. 4).
2. Chemistry
In undertaking the SAR study of the thiocomarin lead
13 an immediate improvement in activity as well as syn-
thetic adaptability was found by replacement of the sul-
fur of 13 with oxygen. Thus, our efforts focused
primarily on lactones (i.e., coumarins) and no further
thio-containing analogs were prepared. A Mitsunobu
protocol21 or the displacement of an alkyl halide or mes-
ylate by an appropriate hydroxy-substituted intermedi-
ate22,23 were the two methods utilized to prepare
essentially all of the compounds (Scheme 2).
This type of interaction was first discovered in previous
factor Xa work, which led to a breakthrough in trypsin-
like serine protease inhibitor design.27,28 The substitu-
tion at the 4-position (the aminopropyl group) enables
H-bonding interactions with the two carboxylic acids
of HEME, which led us to believe that it is important
to preserve the hydrogens of the terminal amino group.
It is rather difficult to understand the role of oxygen in
the coumarin ring, especially due to its close proximity
to the HEME ring underneath and the backbone oxygen
of tryptophan 366 (W366). It is our best understanding
so far that the oxygen of W366 prefers a H-bonding
group nearby and that the distance in between may
favor a water-mediated H-bond more than a direct one.
Where a tethered primary amine was the product (e.g.,
compounds 14, 16, Table 1) a protecting group was re-
quired in the alkylation step. These could be further
derivatized after deprotection in a straightforward man-
ner (e.g., acylation to deliver amides 26 and 49 and
guanylation24 to afford 27, 45, and 47, Table 2). Mesyl-
ation of the primary amine (compound 25) as well as iso-
thiourea formation (compounds 46 and 48) were
accomplished using standard methods. Aminoquino-
lines 61 and 62 (i.e., the introduction of an amidine-type
functionality ubiquitous in the NOS inhibitors men-
tioned earlier, Scheme 2), and quinolones 16–20 (Table
The best compound, 30, was evaluated further to exam-
ine the kinetics of iNOS inhibition using the human

2726
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
Table 2. Inhibitory activity for coumarin derivatives
O
O
(CH2)nNR'R"
X
R
O
Compound
R
R0
R00
n
X
iNOS IC50 (lM)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
63
H
6-Cl
H
H
2
2
2
3
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
5
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
>10 (h), 73.0 (m)
10.3 (m)
>10 (h,m)
H
H
CH3
H
6-Cl
H
H
SO2CH3
COCH3
15.3 (m)
nt
H
H
H
H
(C@NH)NH2
H
23.5 (h), 10.1 (m)
<5% I @ 50 lM (m)
24% I @ 50 lM (m)
0.095 (h), 0.061 (m)
0.102 (h), 0.143 (m)
1.11 (h), 0.441 (m)
31% I @ 50 lM (m)
0.296 (h), 0.304 (m)
<5% I @ 50 lM (m)
23% I @ 50 lM (m)
7.58 (h), 11.9 (m)
>10 (h), 17.8 (m)
0.111 (h), 0.246 (m)
4.0 (h)
6,7-Di-CH3
7-OCH3
6-Cl
H
H
H
H
H
6-Br
6-F
H
H
H
H
6-OCH3
6-CH3
6,8-Di-Br
5-Cl
H
H
H
H
H
H
H
H
H
H
H
CH3
H
6-Cl
H
H
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
H
7-OCH3
6-Cl
CH3
CH3
24.3% I @ 10 lM (m)
4.1 (h), 1.2 (m)
4.7 (h), 3.0 (m)
6-CH3
6,7-Di-CH3
H
CH3
CH3
13.4% I @ 10 lM (m)
16.5 (h), 9.6 (m)
0% I @ 10 lM (m)
0.509 (h), 0.388 (m)
20% I @ 10 lM (m)
8.1% I @ 10 lM (m)
0.2% I @ 10 lM (m)
1.98 (h), 1.01 (m)
1.33 (m)
(C@NH)NH2
(C@S)NHCH3
(C@NH)NH2
(C@S)NHCH3
COCH3
H
6-Cl
H
H
6-Cl
6-Cl
H
H
5-Cl
6-Cl
H
H
H
H
H
H
6-Cl
6-Cl
H
H
23% I @ 10 lM (m)
h = human; m = mouse; nt = not tested.
enzyme. Compound 30 inhibits human iNOS by com-
peting with arginine at the substrate binding site with
a Ki of 104 nM (Fig. 5). It was also shown that 30 was
not a time-dependent inhibitor of human iNOS (Fig.
5). The enzyme does not appear to lose activity during
the preincubation time as observed by approximately
the same levels of product formation. This feature dis-
tinguishes 30 from other compounds described in the lit-
erature. Furthermore, inhibition of human iNOS by 30
was altered by ꢁ2-fold in the presence of 10% FCS, indi-
cating that protein binding was not significant (Fig. 5).
Despite this observation, the activity in various cell
types was not optimal (3–17 lM for primary cells vs
21 lM to >50 lM in other cells). To rule out a physical
problem with cell penetration, we measured (by LCMS)
the cell and medium concentration of compound 30
after centrifugation. The results (not shown) indicated
that the cell pellet to medium ratio of compound was
ꢁ1. The ratio of 1 means that the compound does not
have a physical problem per se of cell penetration.
Although this fact does not explain the lack of cellular
activity, a difference in mechanism or kinetics of inhibi-
tion may be contributing factors. Our compounds are
reversible and time-independent inhibitors in contrast
to others described in the literature and this may have
influenced the cellular potency. In addition, we have
not ruled out the possibility of compound metabolism
in these specific cell types.
Two compounds, 16 and 30, were selected for pharma-
cokinetic studies (Table 4). Compound 16, the lactam
analog of lactone 30, has an IC50 = 407 nM against
mouse iNOS. After intravenous dosing, the total plasma
clearance (4.71 L/h/kg) of 16 approximated the hepatic
blood flow (5.4 L/h/kg) in mice. Therefore it can be clas-
sified as a high clearance compound. The volume of dis-
tribution of 16 was extensive (10.2 L/kg), such that it
surpassed the total body water (0.72 L/kg) in mouse.
The high volume of distribution contributed to the rela-
tively long half-life. Brain penetration was examined
after intravenous dosing. The compound penetrated
the brain tissue poorly and the extent was lower than
that observed earlier for compound 30.
Compound 30 has an IC50 = 60 nM against mouse
iNOS with 185-fold selectivity over eNOS and 9-fold

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2727
more than the hepatic blood flow in those species
(mice = 5.4 L/h/kg, rats = 3.31 L/h/kg). Therefore, 30
can be classified as a high clearance drug. The volume
of distribution was extensive in both mice (3.58 L/kg)
and rats (5.23 L/kg) in that it surpassed the total body
water in both species (mice = 0.72 L/kg, rats = 0.67 L/
kg). However, since the clearance was much higher than
the volume of distribution, the half-life was short. Oral
administration of the 10 mg/kg dose resulted in rapid
absorption and absolute bioavailability was 31% and
40% in mice and rats, respectively. Brain penetration
of 30 was excellent in mouse compared to that in rats.
In mouse, the brain concentrations paralleled the
plasma concentrations indicating good equilibrium be-
tween the two compartments.
OR
O
X
O
R groups
NH
NH
N
H
53, X = Cl
IC50 = 1.5 µM
54, X = Cl
IC50 = 4 µM
55, X = Br
37% I @ 50 µM
N
N
N
A metabolic study was performed to specifically under-
stand the reason for the high total clearance of 30
in vivo and to determine whether a major hepatic com-
ponent was involved. After 1 h incubation, 99.7% and
12.9% of the parent was metabolized in human and
mouse microsomes, respectively. In human microsomes,
the metabolic pathway was independent of NADPH
(same metabolites observed in microsomal incubations
carried out in the absence of NADPH). In mouse micro-
somes, the metabolic pathway was NADPH dependent
(no metabolites observed in mouse microsomal incuba-
tions carried out in the absence of NADPH). Although
the metabolism profile was quantitatively different, qual-
itatively similar metabolites were observed in both
human and mouse microsomes in the presence of
NADPH. Several metabolites were observed in human
and mouse microsomal incubations. The 4-hydroxy cou-
marin 64 (Scheme 4) (m/z 197, dealkylation of the ether
56, X = Cl
58, X = Cl
56% I @ 50 µM
57, X = Cl
15% I @ 50 µM
49% I @ 50 µM
CO2H
CO2CH3
60, X = Cl
59, X = Cl
not tested
not tested
Figure 3. List of compounds prepared (continued). Activity and %
inhibition are reported for mouse iNOS.
over nNOS. As shown in Table 4, coumarin 30 has a
promising pharmacokinetic profile in mice and rats.
After intravenous dosing, the total plasma clearance
for 30 in mice (13.1 L/h/kg) and rats (25 L/h/kg) was
HO (CH2)n NR'R"
OH
O
(CH2)nNR'R"
O
DIAD, PPh3
THF 0oC
R
OR
R
O
X
X
Br (CH2)n NR'R"
X = N, O
X = N, O
Cs2CO3, DMF
100oC
O
O
N
N
OH
OH
R
Br(CH2)3NPhth
R
R
Ac2O
pyr
O
Cs2CO3, DMF
100 oC
NH
N
NH2
N
NH
O
O
61a R = H
61b R = H
62b R = Cl
62a R = Cl
O
N
NH2
NH2NH2
R
EtOH, reflux
NH2
61 R = H
62 R = Cl
Scheme 2. Synthetic scheme for the preparation of coumarins and quinolines. (Aminoquinolines 61 and 62 had IC50ꢀs greater than 10 lM against
mouse iNOS.)

2728
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
OTs
X
(CH2)nCN
Cl
(CH2)nCH2NH2
Cl
PdP(o-tolyl)3Cl2
NC(CH2)nZnBr
Cl
Raney Ni/H2
O
X
O
X
O
14a X = O
15a X = N
14b X = O, n =3
15b X = N, n = 3
21b X = O, n = 4
14 X = O, n = 3
15 X = N, n = 3
21 X = O, n = 4
Scheme 3. Synthesis of all-carbon tether compounds.
Table 3. SAR summary
NR'R"
O
R
X
O
Compd
X
R
R0
R00
Human iNOS
IC50 (lM)
Mouse iNOS
IC50 (lM)
Bovine eNOS Rat nNOS
IC50 (lM)
A172 cell
Raw cell
IC50 (lM)
IC50 (lM) IC50 (lM)
13
37
42
40
17
39
16
30a
S
H
CH3
H
CH3
H
18
—
—
—
9.11
7.96
>10
>10
0.53
—
—
—
—
—
O
H
7.58
4.08
4.01
2.64
0.11
—
11.9
1.2
69.6
>100
>100
>100
>100
—
O
Cl
H
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
H
>50
—
46
—
O
>10
10.4
0.25
0.41
0.06
NH
O
H
—
—
—
Cl
Cl
Cl
>50
>50
21
NH
O
H
>50
>50
H
H
0.09
11.1
0.56
a IC50 in rat primary astrocytes and microglia was 17 and 3 lM, respectively.
Figure 4. Stereo view of the iNOS binding pocket with thiocoumarin 13 (X-ray coordinates have been deposited in the Brookhaven Protein
Databank with a pdb code of 2BHJ) and coumarin 30 (a computer model based on 2BHJ) superimposed. There are several key interactions between
the inhibitors and the enzyme: (a) H-bonding/charge-charge interaction with backbone W366; (b) hydrophobic interaction with chlorine of the
inhibitors and phenyl ring of F363; (c) H-bonding interaction between the amino group of the inhibitors and the carboxylic acid groups of HEME;
(d) p stacking between the inhibitors and HEME ring.
moiety) was the major metabolite in mouse, whereas
human microsomes indicated the others in significant
amounts. In conclusion, the human microsomal incuba-
tions of 30 carried out in the absence of NADPH indi-
cate the involvement of a CYP P450 independent
pathway.
sible. Unfortunately, these derivatives did not improve
the ADME properties of 30. They were much less active
in the iNOS inhibition assay even though no major geo-
metric changes to the molecule (O and C both sp3 hybrid-
ized) were apparent. Another strategy involved the
introduction of a methyl group into the 3 position (Table
2, compound 63). Since it is known that warfarin-like oral
anticoagulants (3-substituted hydroxycoumarins) have
long half-lives and low clearance,29 compound 63 was
synthesized based upon modeling, which suggested a
The synthesis of the all-carbon tether compounds 14, 15,
and 21 were attempts at thwarting this metabolic pathway
since the hydrolysis to 4-hydroxycoumarin 64 is not pos-

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2729
Time Dependence of Coumarin 30
Competition with L-Arginine
50
40
30
20
10
15
10
5
Buffer
Buffer,
Km = 3.54µM
100 nM30
300 nM30
1000 nM 30
3 nM30
10 nM30
30 nM30
100 nM30
300 nM30
1000 nM30
0
0.1
0
0
1
10
100
10
20
30
40
50
60
70
Log [L-Arginine], µM
Preincubation Time (min)
Double Reciprocal Plot
Effect of Plasma Proteins on
Coumarin 30-mediated iNOS inhibition
7.5
Buffer
100
50
0
no serum,
IC50 75.8 nM
3 nM 30
=
5.0
2.5
0.0
-2.5
10 nM30
30 nM30
100 nM30
300 nM30
1000 nM30
10% FCS,
IC
50 = 141.8 nM
0
1
2
3
4
-9
-8
-7
-6
-5
-4
-10
1/[L-Arginine] (µM-1
)
log [Coumarin 30], M
Figure 5. Competition experiments, double reciprocal plot, time dependence and protein binding of coumarin 30.
Table 4. Pharmacokinetic profile for compounds 30 and 16
Compound Species Dose (mg/kg) Route Matrix AUC 0-inf T1/2 (h)
(ng/h/mL)
CL (L/h/kg) Vss (L/kg) Cmax (ng/mL) Tmax (h)
F
30
Mouse
Rat
2
2
IV
IV
PO
Plasma 153
Brain 209
Plasma 236
0.36
0.47
0.77
13.1
—
3.58
—
—
—
—
—
276
368
0.17
0.25
10
—
—
0.31
2
10
10
IV
Plasma 81.5 5.94 0.18 .01 25.0 1.75
5.23 0.59
—
—
—
0.4
—
PO
PO
Plasma 161a
Brain
43.0a
NC
NC
—
—
—
—
111
56
0.5
0.5
16
Mouse
2
2
IV
IV
Plasma 425
Brain
1.75
0.68
4.71
—
10.2
—
—
31.8
—
0.03
—
—
27.3
a AUC 0-7 h; NC = incalculable result.
R
a respectable human iNOS IC50 = 95 nM. These com-
pounds also demonstrated good selectivity over other
NOS isoforms. Particularly, coumarin 30 was a compet-
itive inhibitor of human iNOS (Ki = 104 nM) and
showed no time-dependency of inhibition. Furthermore,
protein binding did not affect the enzyme inhibitory po-
tency. Although we aimed to design a compound that
possessed nanomolar cellular potency it is not clear
whether the cellular potency of compound 30 is suffi-
cient to demonstrate proof-of-concept in the in vivo
models of disease. Given the fact that the PK profile
was favorable in terms of bioavailability and brain pen-
etration, further testing of these compounds to demon-
strate proof-of-concept in vivo will be undertaken.
OH
O
O
O
NH2
O
O
Cl
Cl
Cl
O
O
O
65 R = CH2OH
66 R = CO2H
64
30
Scheme 4. Metabolites of 30 from liver microsomes.
small alkyl substituent could be tolerated. It was thought
that this substituent might provide steric hindrance to
metabolic attack on the ether functionality. However, this
compound did not improve on 30.
4. Conclusion
5. Experimental
5.1. General
We found and explored the SAR of a novel series of
coumarins as iNOS inhibitors where the usual amidine
or guanidine functionality, ubiquitous in the NOS area
of study, was absent. This gave rise to coumarin 30 with
Commercial reagents and solvents were used as
received. H NMR spectra were recorded on a Varian
1

2730
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
MercuryPlus-300 (300 MHz) or Varian Unity Inova
(400 MHz) spectrometer as indicated. Proton chemical
shifts are reported in d ppm relative to internal tetra-
methylsilane (0.0 ppm). MS (LC–MS) data was
obtained using a Micromass LCT time of flight mass
spectrometer with electrospray ionization and 5 min
data acquisition time for m/z 100–1000. LC (LC–MS)
which was filtered, washed with Et2O, and dried to af-
ford 30 (5.14 g). A second crop (3.1 g) could be obtained
by refrigerating the filtrate overnight with an overall
combined yield of 88%. 1H NMR (DMSO-d6, 300
MHz) d 7.87 (d, 1H, J = 2.75 Hz), 7.80 (br s, 2H), 7.73
(dd, 1H, J = 2.75, 9 Hz), 7.48 (d, 1H, J = 9 Hz), 5.97
(s, 1H), 4.30 (t, 2H, J = 5.75 Hz), 3.04 (br s, 2H), 2.12
(m, 2H); MS (ESI, Pos.) calcd for C12H12ClNO3 m/z
[M+H] = 254.1, found 254.1.
was performed using
a
Hypersil C18 column
(4.6 · 50 mm, 3 lm with mobile phase of 0.1% TFA in
H2O (A) and 0.1% TFA in ACN (B) and a gradient of
5–100% B over 3 min followed by 2 min at 100% B).
Alternatively, a Platform LC–MS with electrospray
source may be used with a HP1100 LC system running
at 2.0 mL/min, 200 lL/min split to the ESI source with
inline HP1100 DAD detection and SEDEX ELS detec-
tion. A Luna C18(2) column (30 · 4.6 mm, 3 lm was
used with a gradient of 5–95% B over 4.5 min with mo-
bile phase of 0.1% formic acid in H20 and 0.1% formic
acid in ACN (B). HPLC purification was performed
5.1.2. Method B. 4-(3-Dimethylamino-propoxy)-1-benzo-
pyran-2-one hydrochloric acid salt (40). 4-Hydroxycoum-
arin (3.0 mmol, 0.487 g) was dissolved in 10 mL of THF.
3-Dimethylamino-propan-1-ol (0.372 mL, 3.15 mmol)
and PPh3 (0.825 g, 3.15 mmol) were added and the solu-
tion was cooled to 0 ꢁC. DIAD (0.647 g, 3.30 mmol) was
added dropwise at 0 ꢁC. The reaction was stirred 30 min
at 0 ꢁC then allowed to warm to rt and stirred overnight.
The solvent was evaporated under reduced pressure, and
a solution of anhydrous HCl/MeOH (prepared by add-
ing ꢁ5 mL acetyl chloride dropwise to 100 mL MeOH)
was added to the residue. Alternatively 4 M HCl in diox-
ane may be used. The solvent was removed under re-
duced pressure and the residue treated with Et2O. The
resulting solid was filtered, washed with Et2O, and dried
to afford 40 (0.485 g, 57%). 1H NMR (DMSO-d6,
300 MHz) d 10.35 (br s, 1H), 7.90 (dd, 1H, J = 1.25,
7.75 Hz), 7.68 (m, 1H), 7.41 (m, 2H), 5.92 (s, 1H), 4.32
(t, 2H, J = 5.75 Hz), 3.27 (m, 2H), 2.80 (s, 6H), 2.25
(m, 2H); MS (ESI, Pos.) calcd for C14H17NO3 m/z
[M+H] = 248.1, found 248.1.
on
a Varian ProStar system using a Dynamax
reversed-phase C18 column with a linear gradient of
ACN/H2O containing 0.1% TFA. Substituted 4-
hydroxycoumarin derivatives were obtained commer-
cially or were synthesized via methods described in the
literature.30 5-Chloro-4-hydroxycoumarin was synthe-
sized using the procedure of Snieckus and co-workers.31
Non-commercial Boc-protected alcohols were prepared
by reaction of the commercially available amino alco-
hols with di-t-butyldicarboxylate using standard
methods.32 trans-2-Methanesulfonyloxymethyl-cyclo-
propane-carboxylic acid methyl ester was prepared as
described previously.33 Toluene-4-sulfonic acid 6-
chloro-2-oxo-2H-1-benzopyran-4-yl ester and toluene-
4-sulfonic acid 2-oxo-2H-1-benzopyran-4-yl ester were
prepared by modification of a published procedure.34
The starting materials for the synthesis of aminoquino-
lines 61 and 62 was prepared according to the procedure
of Kadin and Lamphere.35 Abbreviations used: dichlo-
romethane (DCM), dimethylformamide (DMF) metha-
nol (MeOH), diethylether (Et2O), acetonitrile (ACN),
ethyl acetate (EtOAc), ethanol (EtOH), trifluoroacetic
acid (TFA), tetrahydrofuran (THF), diisopropylazodi-
carboxylate (DIAD), diisopropylethylamine (DIEA), tri-
phen- ylphosphine (PPh3), t-butyloxycarbonyl (Boc);
NMR abbreviations: singlet (s), doublet (d), double
doublet (dd), triplet (t), quartet (q), broad (br).
5.1.3. Method C. 6-Chloro-4-(3-methylamino-propoxy)-1-
benzopyran-2-one trifluoroacetic acid salt (39). 6-Chloro-
4-hydroxycoumarin (0.747 mmol, 0.147 g), PPh3
(0.785 mmol, 0.206 g), and (3-hydroxypropyl)-methyl-
carbamic acid tert-butyl ester (0.785 mmol, 0.148 g)
were suspended in 5 mL THF and the mixture was
cooled to 0 ꢁC. DIAD (0.822 mmol, 0.161 mL) was
added dropwise at 0 ꢁC, and then the mixture
was warmed to rt and stirred for 6 h. The THF was re-
moved under reduced pressure. Neat TFA was added
and the solution was mixed at rt for 30 min to 1 h.
The TFA was evaporated, Et2O was added, and the
resulting precipitate was filtered. (Alternatively the
Et2O trituration step may be omitted.) The crude mate-
rial was further purified by HPLC to afford 39 (54.5 mg,
19%). 1H NMR (DMSO-d6, 300 MHz) d 8.41 (br s, 2H),
7.87 (d, 1H, J = 2.5 Hz), 7.73 (dd, 1H, J = 2.75, 9 Hz),
7.48 (d, 1H, J = 9 Hz), 5.99 (s, 1H), 4.30 (t, 2H,
J = 5.75 Hz), 3.14 (m, 2H), 2.63 (t, 3H, J = 5.5 Hz),
2.14 (m, 2H); MS (ESI, Pos.) calcd for C13H14ClNO3
m/z [M+H] = 268.1, found 268.2.
5.1.1. Method A. 6-Chloro-4-(3-aminopropoxy)-1-benzo-
pyran-2-one trifluoroacetic acid salt (30). 6-Chloro-4-
hydroxycoumarin (5.0 g, 25.4 mmol), PPh3 (6.99 g,
26.67 mmol), and (3-hydroxypropyl)-carbamic acid
tert-butyl ester (4.6 mL, 26.67 mmol) were suspended
in 125 mL of THF. The reaction was cooled to ꢀ78 ꢁC
(smaller scale reactions <5 mmol may be run at 0 ꢁC)
and a solution of DIAD (5.48 mL, 27.94 mmol) in
40 mL of THF was added dropwise via an addition fun-
nel at ꢀ78 ꢁC. The reaction was allowed to warm slowly
to rt and was stirred overnight. The reaction mixture
was concentrated in vacuo and Et2O was added to pre-
cipitate a solid, which was filtered, washed with Et2O,
and dried. The solid was treated with neat TFA for
30 min at rt. The TFA was removed under reduced pres-
sure and Et2O was added to precipitate a white solid,
5.1.4. Toluene-4-sulfonic acid 6-chloro-2-oxo-2H-1-benzo-
pyran-4-yl ester (14a). 6-Chloro-4-hydroxycoumarin
(5.0 g. 25.4 mmol) was suspended in DCM (50 mL)
and pyridine (5.1 mL, 63.3 mmol) was added. The mix-
ture was cooled in an ice bath and p-toluenesulfonyl
chloride (5.1 g, 26.8 mmol) was added. The ice bath
was removed and the compound slowly dissolved and
a solid is deposited (pyridineÆHCl). After 1 h the mixture
was transferred to a separatory funnel, HCl (2 M) was

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2731
added, and a solid formed. The organic layer was re-
moved and the solid-containing aqueous layer was fil-
tered to remove the precipitate. The filtrate was
washed with DCM and the combined organic layers
were washed with H2O, NaHCO3 (satd), H2O, and
brine. The extract was then dried over MgSO4 and
was evaporated. The residue was triturated with Et2O
washed with water and then suspended in Et2O. The so-
lid was filtered and dried in vacuo to afford 15a (6.0 g,
1
68%) as a cream solid. H NMR (400 MHz, DMSO-
d6) d 12.2 (s, 1H), 7.95 (d, 2H, J = 8 Hz), 7.62 (dd, 1H,
J = 2.5, 9 Hz), 7.51 (d, 2H, J = 8 Hz), 7.36 (d, 1H,
J = 2.5 Hz), 7.33 (d, 1H, J = 9 Hz), 6.3 (s, 1H), 2.4 (s,
3H); LCMS (ACN–H2O, Luna 3 lm C18(2) 30 ·
4.6 mm); calcd for C16H12ClNO4S m/z [M+H] = 350.0,
found (ES+) 349 (M+), 699 (2M+), 1049 (3M+).
1
to afford 14a (8.0 g, 90%) as a cream solid. H NMR
(CDCl3, 400 MHz) d 7.91 (d, 2H, J = 8 Hz), 7.54 (d,
1H, J = 2 Hz), 7.51 (dd, 1H, J = 2, 9 Hz), 7.42 (d, 2H,
J = 8 Hz), 7.26 (d, 1H, J = 9 Hz), 6.38 (s, 1H), 2.49 (s,
3H); LCMS (ACN–H2O, Luna 3 lm C18(2)
30 · 4.6 mm); MS (ESI, Pos.) calcd for C16H11ClO5S
m/z [M+H] = 351 found: [no mass ion detected].
5.1.8.
4-(6-Chloro-2-oxo-1,2-dihydro-quinolin-4-yl)-
butyronitrile (15b). Toluene-4-sulfonic acid 6-chloro-2-
oxo-1,2-dihydro-quinolin-4-yl ester (1.7 g, 4.84 mmol)
and [(o-tolyl)3PdCl2 (393 mg, 0.48 mmol) were stirred
in THF (100 mL) under nitrogen at room temperature.
3-Cyanopropylzinc bromide (100 mL of a 0.5 M solu-
tion in THF, 50 mmol) was added and the reaction mix-
ture was refluxed for 16 h. Silica gel was added to the
reaction mixture and concentrated in vacuo. The residue
was purified by flash column chromatography eluting
with CH2Cl2 and gradually increasing polarity to 1%
MeOH–CH2Cl2. The resulting residue was washed with
MeOH and dried in vacuo to afford 15b (812 mg, 68%)
5.1.5. 4-(6-Chloro-2-oxo-2H-1-benzopyran-4-yl)-butyro-
nitrile (14b). Toluene-4-sulfonic acid 6-chloro-2-oxo-
2H-1-benzopyran-4-yl ester (1.05 g, 3 mmol) was
dissolved in THF (30 mL) under nitrogen and [P(o-
tolyl)3]2PdCl2 (0.120 g, 0.15 mmol) was added. 3-
Cyanopropylzinc bromide (9 mL of a 0.5 M solution
in THF, 4.5 mmol) was added and the resulting yellow
solution was stirred at 60 ꢁC for 2 h. Silica gel was added
to the dark red reaction mixture, which was concen-
trated in vacuo and purified by flash column chromato-
graphy eluting with CH2Cl2 and gradually increasing
polarity to 1% MeOH–CH2Cl2 to give a residue, which
was triturated with Et2O to afford 14b (0.54 g, 73%) as
1
as white solid. H NMR (400 MHz, DMSO-d6) d 11.8
(s, 1H), 7.77 (d, 1H, J = 2.5 Hz), 7.50 (dd, 1H, J = 9,
2.5 Hz), 7.28 (d, 1H, J = 9 Hz), 6.39 (s, 1H), 2.83 (t,
2H, J = 7 Hz), 2.57 (t, 2H, J = 7 Hz), 1.86 (m, 2H);
LCMS (ACN–H2O, Luna 3 lm C18(2) 30 · 4.6 mm)
rt = 2.64 min; m/z (ES+) 247 (MH+), 494 (2MH+), 741
(3MH+).
1
a cream solid. H NMR (DMSO-d6, 400 MHz) d 7.92
(d, 1H, J = 2 Hz), 7.68 (dd, 1H, J = 9, 2 Hz), 7.46 (d,
1H, J = 9 Hz), 6.47 (s, 1H), 2.90 (t, 2H, J = 7 Hz), 2.63
(t, 2H, J = 7 Hz), 1.94 (q, 2H, J = 7 Hz). LCMS
(ACN–H2O, Luna 3 lm C18(2) 30 · 4.6 mm); (ESI
Pos.) calcd for C13H10ClNO2 m/z [M+H] = 248; found
[no mass ion detected].
5.1.9. 4-(4-Amino-butyl)-6-chloro-1H-quinolin-2-one HCl
salt (15). 4-(6-Chloro-2-oxo-1,2-dihydro-quinolin-4-yl)-
butyronitrile (0.20 g, 0.81 mmol) was suspended in
THF (3 mL) under nitrogen at room temperature. The
suspension was treated with borane (2 mL, 1 M in
THF), the solid gradually dissolved and the reaction
was stirred overnight. The reaction mixture was concen-
trated in vacuo and the residue dissolved in MeOH
(5 mL), then treated with concd HCl in MeOH (1:1)
and heated at 70 ꢁC for 1 h. The resulting solid was fil-
tered and dried in vacuo to afford 15 (177 mg, 76%) as
5.1.6.
4-(4-Aminobutyl)-6-chloro-1-benzopyran-2-one,
acetic acid salt (14). 4-(6-Chloro-2-oxo-2H-1-benzopy-
ran-4-yl)-butyronitrile (0.20 g, 0.8 mmol) was suspended
in EtOH (20 mL) and a spatula of Raney nickel was
added. The mixture was placed under a hydrogen atmo-
sphere and was stirred vigorously at 60 ꢁC for 1 h. The
resulting suspension was filtered through Hyflo and con-
centrated in vacuo. The residue was dissolved in EtOH
(5 mL) and treated with HCl (1 M in Et2O). The result-
ing solid (128 mg) was purified by flash column chroma-
tography eluting with CH2Cl2–MeOH–AcOH–H2O
(240:60:6:4) to afford 14 (85 mg, 27%) as a white solid.
1H NMR (DMSO-d6, 400 MHz) d 7.93 (d, 1H,
J = 2.5 Hz), 7.68 (dd, 1H, J = 9, 2.5 Hz), 7.46 (d, 1H,
J = 9 Hz), 6.44 (s, 1H), 5.5–4.0 (br s, 3H,), 2.83 (t, 2H,
J = 7 Hz), 2.68 (m, 2H), 1.78 (s, 3H), 1.65 (m, 2H),
1.54 (m, 2H); LCMS ACN–H2O, Luna 3 lm C18(2)
30 · 4.6 mm; calcd for C13H14ClNO2 m/z [M+H] =
252, found [no mass ion detected].
1
a white solid. H NMR (400 MHz, DMSO-d6) d 11.8
(s, 1H), 8.0 (br s, 2H), 7.81 (d, 1H, J = 2.5 Hz), 7.55
(dd, 1H, J = 9, 2.5 Hz), 7.33 (d, 1H, J = 9 Hz), 6.45 (s,
1H), 2.85 (t, 2H, J = 7 Hz), 2.84 (t, 2H, J = 7 Hz), 1.65
(m, 4H); LCMS (ACN–H2O, Luna 3 lm C18(2)
30 · 4.6 mm); m/z (ES+) 251 (MH+), 502 (2MH+), 753
(3MH+).
5.1.10. 4-(3-Amino-propoxy)-6-chloro-1H-quinolin-2-one
TFA salt (16). 6-Chloro-4-hydroxy-1H-quinolin-2-one
(0.513 mmol, 0.100 g), PPh3 (0.539 mmol, 0.141 g),
and (3-hydroxypropyl)-carbamic acid tert-butyl ester
(0.539 mmol, 0.094 g) were dissolved in 5 mL of THF
and the solution cooled to 0 ꢁC. Diazodicarboxylate
(0.564 mmol, 0.110 mL) was added dropwise at 0 ꢁC
and the mixture was stirred at 0 ꢁC for 30 min then
warmed to rt. Added an additional 1 equiv of PPh3,
DIAD and Boc-protected alcohol and stirred at rt for
2 h. Removed the excess solvent. A portion of the mate-
rial (1/3) was purified in the Boc-protected form. To the
remaining 2/3 of the material, added neat TFA and
5.1.7. Toluene-4-sulfonic acid 6-chloro-2-oxo-1,2-dihy-
dro-quinolin-4-yl ester (15a). 6-Chloro-4-hydroxy-1H-
quinolin-2-one (4.89 g, 25 mmol) was dissolved in
DMF (146 mL), tosyl chloride (5.25 g, 27.5 mmol) and
triethylamine (8.7 mL, 62.5 mmol) were added and stir-
red for 1 h to give a precipitate. Water (150 mL) was
added and the reaction mixture filtered, the solid was

2732
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
stirred at rt for 30 min. The reaction mixture was concen-
trated in vacuo and Et2O was added to precipitate a so-
lid, which was further purified by reversed-phase HPLC
amino-propan-1-ol to afford 20 after HPLC purification
(184 mg, 34.4%). 1H NMR (DMSO-d6, 300 MHz) d 9.42
(s, 1H), 7.97 (dd, 1H, J = 1.5, 8 Hz), 7.67 (m, 1H), 7.53
(d, 1H, J = 8.5 Hz), 7.29 (t, 1H, J = 7.5 Hz), 6.06 (s, 1H)
4.21 (t, 2H, J = 6 Hz), 3.57 (s, 3H), 3.30 (m, 2H), 2.85 (s,
6H), 2.21 (m, 2H); MS (ESI, Pos.) calcd for C15H20N2O2
m/z [M+H] = 261.2, found 261.1.
1
to afford 16 (29.9 mg, 26%). H NMR (DMSO-d6) d
11.52 (s, 1H), 7.80 (d, 1H, J = 2.25 Hz), 7.75 (br s, 2H),
7.58 (dd, 1H, J = 2.25 Hz, 8.75 Hz), 7.30 (d, 1H,
J = 8.75 Hz), 5.94 (s,1H), 4.21 (t, 2H, J = 5.75 Hz),
3.05 (m, 2H), 2.11 (m, 2H); MS (ESI, Pos.) calcd for
C12H13ClN2O2 m/z [M+H] = 253.1, found 253.1.
5.1.15. 4-(5-Amino-pentyl)-6-chloro-1-benzopyran-2-one
(21). 5-(6-Chloro-2-oxo-2H-1-benzopyran-4-yl)-pentane-
nitrile (0.76 mmol, 0.200 g) was suspended in EtOH
(20 mL), Raney Nickel (spatula full) was added and
the mixture placed under H2. The mixture was stirred
at 60 ꢁC for 1 h. The resulting mixture was filtered
through Hyflo and evaporated to leave a yellow gum.
The gum was dissolved in EtOH (5 mL) and treated with
1 M HCl in Et2O (5 mL). No solid deposited. The sol-
vent was evaporated in vacuo and the residue purified
by flash column chromatography using CH2Cl2–
MeOH–AcOH–H2O (240:30:3:2) as eluent to afford 21
5.1.11. 4-(3-Dimethylamino-propoxy)-1H-quinolin-2-one
(17). A suspension of 2,4-dihydroxyquinoline (1.0
mmol, 0.161 g), PPh3 (1.0 mmol, 0.262 g), 3-dimethyla-
mino-propan-1-ol (1.0 mmol, 0.103 g), and diisopro-
pylazodicarboxylate (1.0 mmol, 0.202 g) in 10 mL of
THF was stirred vigorously at rt overnight. The mixture
was concentrated under reduced pressure, treated with
1 N HCl (10 mL) followed by extraction with EtOAc
(3 · 50 mL). The aqueous solution was neutralized with
1 N NaOH (15 mL) and extracted with EtOAc
(3 · 100 mL). The organic layer was dried (MgSO4), fil-
tered, and concentrated to afford 17 as a white solid
(0.105 g, 37.2%). 1H NMR (DMSO-d6, 300 MHz) d
11.33 (s, 1H), 7.78 (dd, 1H, J = 1.3, 8.25 Hz), 7.51 (m,
1H), 7.27 (d, 1H, J = 7.75 Hz), 7.16 (m, 1H), 5.85 (s,
1H), 4.14 (t, 2H, J = 6.25 Hz), 2.41 (t, 2H, J = 7 Hz),
2.16 (s, 6H), 1.94 (m, 2H); MS (ESI, Pos.) calcd for
C14H18N2O2 m/z [M+H] = 247.1, found 247.1.
1
as an off-white solid (80 mg, 39%). H NMR (DMSO-
d6) d 7.89 (d, 1H, J = 2.4 Hz), 7.68 (dd, 1H, J = 2.4,
8.8 Hz), 7.46 (d, 1H, J = 8.8 Hz), 6.43 (s, 1H), 2.82 (t,
2H, J = 7.4 Hz), 2.77 (t, 2H, J = 7.4 Hz), 1.67–1.57 (m,
4H), 1.47–1.44 (m, 2H). MS (ESI, Pos.) calcd for
C14H16ClNO2 m/z [M+H] = 266.1, found 266.05.
5.1.16. 4-(2-Amino-ethoxy)-1-benzopyran-2-one trifluoro-
acetic acid salt (22). Prepared via method A at 0 ꢁC from
4-hydroxycoumarin and (2-hydroxyethyl)carbamic acid
5.1.12. 4-(3-Amino-propoxy)-1H-quinolin-2-one trifluoro-
acetic acid salt (18). Suspended quinoline-2,4-diol
(1.34 mmol, 0.215 g), PPh3 (1.41 mmol, 0.369 g), and
(3-hydroxypropyl)-carbamic acid tert-butyl ester
(1.41 mmol, 0.24 mL) in 10 mL of THF. Cooled the
mixture to 0 ꢁC and then added DIAD dropwise
(1.47 mmol, 0.29 mL). Mixed at 0 ꢁC for 10 min then
warmed to rt and stirred overnight. Evaporated the sol-
vent under reduced pressure. Added neat TFA and
mixed at rt for 30 min. Removed the TFA in vacuo
and added Et2O to precipitate a solid, which was filtered
and washed with Et2O. This material was redissolved in
MeOH–H2O and purified by reversed phase HPLC to
afford 18 (0.137 g, 31%). 1H NMR (DMSO-d6,
400 MHz) d 11.40 (s, 1H), 7.86 (br s, 2H), 7.82 (dd,
2H, J = 1.2, 8.1 Hz), 7.52 (m, 1H), 7.28 (d, 1H,
J = 7.8 Hz), 7.17 (m, 1H), 5.88 (s, 1H), 4.20 (t, 2H, J =
5.9 Hz), 3.05 (m, 2H), 2.11 (m, 2H). MS (ESI, Pos.)
calcd for C12H14N2O2 m/z [M+H] = 219.1, found 219.1.
1
tert-butyl ester to afford 22 (250 mg, 59.0%). H NMR
(DMSO-d6, 300 MHz) d 8.13 (dd, 1H, J = 1.5, 8 Hz),
8.09 (br s, 2H), 7.69 (m, 1H), 7.40 (m, 2H), 5.99 (s,
1H), 4.40 (t, 2H, J = 4.75 Hz), 3.36 (2H, buried under
solvent peak); MS (ESI, Pos.) calcd for C11H11NO3
m/z [M+H] = 206.1, found 206.1.
5.1.17. 4-(2-Amino-ethoxy)-6-chloro-1-benzopyran-2-one
trifluoroacetic acid salt (23). Prepared via method A at
0 ꢁC from 6-choro-4-hydroxycoumarin and (2-hydroxy-
ethyl)-carbamic acid tert-butyl ester to afford 23
(35.8 mg, 9.9%). 1H NMR (DMSO-d6, 300 MHz) d
8.21 (d, 1H, J = 2.5 Hz), 8.05 (br s, 2H), 7.72 (dd, 1H
J = 2.5, 9 Hz), 7.47 (d, 1H, J = 8.75 Hz), 6.07 (s, 1H),
4.39 (t, 2H, J = 4.75 Hz), 3.33 (2H, buried under solvent
peak); MS (ESI, Pos.) calcd for C11H10ClNO3 m/z
[M+H] = 240.0, found 240.1.
5.1.18.
4-(2-Methylamino-ethoxy)-1-benzopyran-2-one
5.1.13. 4-(3-Amino-propoxy)-1-methyl-1H-quinolin-2-one
TFA salt (19). Prepared via method C from 4-hydroxy-1-
methyl-2(1H)-quinolone and (3-hydroxypropyl)-car-
bamic acid tert-butyl ester to afford 19 following HPLC
purification (229 mg, 46.3%). 1H NMR (DMSO-d6,
300 MHz) d 7.94 (dd, 1H, J = 1.5, 8 Hz), 7.78 (br s,
2H), 7.67 (m, 1H), 7.52 (d, 1H, J = 8.5 Hz), 7.28 (t, 1H,
J = 7.25 Hz), 6.05 (s, 1H), 4.22 (t, 2H, J = 6 Hz), 3.57
(s, 3H), 2.04 (m, 2H), 2.12 (m, 2H); MS (ESI, Pos.) calcd
for C13H16N2O2 m/z [M+H] = 233.1, found 233.1.
hydrochloric acid salt (24). 4-Hydroxycoumarin
(0.216 g, 1.33 mmol) was dissolved in 10 mL of THF.
PPh3 (0.367 g, 1.4 mmol) and (2-hydroxyethyl)-methyl-
carbamic acid tert-butyl ester (0.250 g, 1.44 mmol) was
added and the solution was cooled to 0 ꢁC. DIAD
(0.287 mL, 1.46 mmol) was added dropwise at 0 ꢁC,
and the solution was stirred 15 min. at 0 ꢁC. The reac-
tion was allowed to warm to rt and was stirred over-
night. Evaporated the solvent under reduced pressure.
Dissolved in neat TFA (10 mL) and mixed at rt for
30 min. The reaction mixture was concentrated in vacuo
and was treated with a HCl/MeOH solution followed by
Et2O precipitation to afford 24, which was filtered,
washed with Et2O, and dried (75 mg, 22.1%). 1H
5.1.14. 4-(3-Dimethylamino-propoxy)-1-methyl-1H-quin-
olin-2-one TFA salt (20). Prepared via method C from 4-
hydroxy-1-methyl-2(1H)-quinolone and 3-dimethyl-

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2733
NMR (DMSO-d6, 300 MHz) d 9.03 (s, 2H), 8.15 (dd,
1H, J = 1.5, 7.75 Hz), 7.69 (m, 1H), 7.40 (m, 2H), 6.00
(s, 1H), 4.49 (t, 2H, J = 4.75 Hz), 3.45 (t, 2H,
J = 4.5 Hz), 2.67 (s, 3H); MS (ESI, Pos.) calcd for
C12H13NO3 m/z [M+H] = 220.1, found 220.1.
(s, 3H), 2.11 (m, 2H); MS (ESI, Pos.) calcd for
C14H17NO3 m/z [M+H] = 248.1, found 248.2.
5.1.23. 4-(3-Amino-propoxy)-7-methoxy-1-benzopyran-2-
one trifluoroacetic acid salt (29). Prepared via method A
at 0 ꢁC from 7-methoxy-4-hydroxycoumarin and (3-
hydroxypropyl)-carbamic acid tert-butyl ester to afford
5.1.19. N-[3-(6-Chloro-2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-methanesulfonamide (25). 4-(3-Aminopropoxy)-
1
29 (167.9 mg, 32%). H NMR (DMSO-d6, 300 MHz) d
6-chloro-1-benzopyran-2-one
trifluoroacetate
salt
7.77 (br s, 2H), 7.75 (d, 1H, J = 8.75 Hz), 7.01 (d, 1H,
J = 2.5 Hz), 6.96 (dd, 1H, J = 2.25, 8.75 Hz), 5.75 (s,
1H), 4.28 (t, 2H, J = 6 Hz), 3.86 (s, 3H), 3.02 (m, 2H),
2.10 (m, 2H); MS (ESI, Pos.) calcd for C13H15NO4
m/z [M+H] = 250.1, found 250.1.
(0.272 mmol, 0.10 g) is suspended in 2 mL of DCM.
DIEA (1.36 mmol, 0.236 mL) is added followed by
methanesulfonyl chloride (0.543 mmol, 0.062 g) and
the reaction is stirred at ambient temperature for 4 h.
The solvent is removed under reduced pressure
and the residue is redissolved and purified by HPLC
to afford 25 (21 mg, 23%). 1H NMR (DMSO-d6,
300 MHz) d 7.80 (d, 1H, J = 2.6 Hz), 7.70 (dd, 1H,
J = 2.5, 8.75 Hz), 7.45 (d, 1H, J = 8.87 Hz), 7.11 (br t,
1H), 5.97 (s, 1H), 4.28 (t, 2H, J = 6 Hz), 3.16 (m, 2H),
2.91 (s, 3H), 2.01 (m, 2H); MS (ESI, Pos.) calcd for
C13H14ClNO5S m/z [M+H] = 332.0, found 331.9.
5.1.24. 4-(3-Amino-propoxy)-6-bromo-1-benzopyran-2-
one trifluoroacetic acid salt (31). Prepared via method
C from 6-bromo-4-hydroxycoumarin and (3-hydroxy-
propyl)-carbamic acid tert-butyl ester to afford 31
(64.6 mg, 25%). 1H NMR (DMSO-d6, 300 MHz) d
7.98 (1H, d, J = 2.5 Hz). 7.84 (1H, dd, J = 2.5, 9 Hz),
7.76 (2H, br s). 7.40 (1H, d, J = 9 Hz), 5.97 (1H, s),
4.30 (2H, t, J = 5.75 Hz), 3.04 (2H, br s), 2.11 (2H, m);
MS (ESI, Pos.) calcd for C12H12BrNO3m/z [M+H] =
298.0, found [no parent obsd].
5.1.20.
N-[2-(2-Oxo-2H-1-benzopyran-4-yloxy)-ethyl]-
acetamide (26). 4-(2-Amino-ethoxy)-1-benzopyran-2-
one trifluoroacetate 22 (0.157 mmol, 0.050 g) was dis-
solved in 5 mL of DCM. Pyridine was added
(0.314 mmol, 25.4 lL) followed by acetic anhydride
(0.314 mmol, 29.6 lL) and the reaction was stirred at
rt for 4 h. The reaction mixture was concentrated
in vacuo and the residue purified by reversed-phase
5.1.25.
4-(3-Amino-propoxy)-6-fluoro-1-benzopyran-2-
one trifluoroacetic acid salt (32). Prepared via method
A at 0 ꢁC from 6-fluoro-4-hydroxycoumarin and (3-
hydroxypropyl)-carbamic acid tert-butyl ester to afford
1
32 (264 mg, 90%). H NMR (DMSO-d6, 300 MHz) d
1
HPLC to afford 26 (31.4 mg, 81%). H NMR (DMSO-
7.79 (br s, 2H), 7.67 (dd, 1H, J = 2.75, 8.75 Hz), 7.52
(m, 2H), 5.97 (s, 1H), 4.30 (t, 2H, J = 5.5 Hz), 3.05 (br
s, 2H), 2.10 (m, 2H); MS (ESI, Pos.) calcd for
C12H12FNO3 m/z [M+H] = 238.1, found 238.2.
d6, 300 MHz) d 8.19 (br t, 1H), 7.91 (dd, 1H, J = 1.5,
7.75 Hz), 7.67 (m, 1H), 7.38 (m, 2H), 5.91 (s, 1H), 4.20
(t, 2H, J = 5.5 Hz), 3.52 (m, 2H), 1.84 (s, 3H); MS
(ESI, Pos.) calcd for C13H13NO4 m/z [M+H] = 248.1,
found 248.0.
5.1.26. 4-(3-Amino-propoxy)-6-methoxy-1-benzopyran-2-
one trifluoroacetic acid salt (33). Prepared via method A
at 0 ꢁC from 6-methoxy-4-hydroxycoumarin and (3-
hydroxypropyl)-carbamic acid tert-butyl ester to afford
5.1.21.
N-[2-(2-Oxo-2H-1-benzopyran-4-yloxy)-ethyl]-
guanidine trifluoroacetic acid salt (27). 4-(2-Amino-eth-
oxy)-1-benzopyran-2-one trifluoroacetate (0.100 g,
0.313 mmol) was placed in a small vial along with
2 mL of acetonitrile. Diisopropylethylamine was added
dropwise until the pH of the solution was ꢁ9 by pH pa-
per. N,N0-Bis(tert-butoxycarbonyl)-1-H-pyrazole-1-car-
boxamidine (0.075 g, 0.24 mmol) was added and the
reaction was mixed at rt for 5 h. The reaction mixture
was concentrated in vacuo, then redissolved in neat
TFA and stirred at rt for 30 min. The TFA was removed
under reduced pressure and the residue purified by re-
versed-phase HPLC to afford 27 (35.0 mg, 31%). 1H
NMR (DMSO-d6, 300 MHz) d 7.88 (dd, 1H, J = 1.5,
8 Hz), 7.81 (br t, 1H), 7.68 (m, 1H), 7.39 (m, 2H), 7.28
(br s, 3H), 5.94 (s, 1H), 4.30 (t, 2H, J = 5 Hz), 3.67 (q,
2H, J = 5.5 Hz); MS (ESI, Pos.) calcd for C12H13N3O3
m/z [M+H] = 248.1, found 248.1.
1
33 (195 mg, 65%). H NMR (DMSO-d6, 300 MHz) d
7.77 (br s, 2H), 7.32 (m, 3H), 5.90 (s, 1H), 4.31 (t, 2H,
J = 6 Hz), 3.83 (s, 3H), 3.04 t, (2H, J = 7.25 Hz), 2.12
(m, 2H); MS (ESI, Pos.) calcd for C13H15NO4 m/z
[M+H] = 250.1, found 250.2.
5.1.27. 4-(3-Amino-propoxy)-6-methyl-1-benzopyran-2-
one trifluoroacetic acid salt (34). Prepared via method
A at 0 ꢁC from 6-methyl-4-hydroxycoumarin and (3-
hydroxypropyl)-carbamic acid tert-butyl ester to afford
1
34 (298 mg, 94%). H NMR (DMSO-d6, 300 MHz) d
7.80 (br s, 2H), 7.65 (br d, 1H), 7.49 (dd, 1H, J = 2,
8.5 Hz), 7.31 (d, 1H, J = 8.5 Hz), 5.87 (s, 1H), 4.30 (t,
2H, J = 6 Hz), 3.04 (m, 2H), 2.39 (s, 3H), 2.10 (m,
2H); MS (ESI, Pos.) calcd for C13H15NO3 m/z [M+H]
234.1, found 234.2.
5.1.22. 4-(3-Amino-propoxy)-6,7-dimethyl-1-benzopyran-
2-one trifluoroacetic acid salt (28). Prepared via method
A at 0 ꢁC from 6,7-dimethyl-4-hydroxycoumarin and (3-
hydroxypropyl)-carbamic acid tert-butyl ester to afford
28 (57.0 mg, 11%). H NMR (DMSO-d6, 300 MHz) d
7.78 (br s, 2H), 7.59 (s, 1H), 7.23 (s, 1H), 5.81 (s, 1H),
4.28 (t, 2H, J = 6 Hz), 3.04 (m, 2H), 2.33 (s, 3H), 2.29
5.1.28. 6,8-Dibromo-4-hydroxy-1-benzopyran-2-one. A
solution was prepared of diethylcarbonate (62.5 mmol,
7.6 mL) in 150 mL of toluene to which was added
NaH (37.5 mmol, 1.5 g of 60% dispersion in mineral
oil) in portions. To this solution was added, dropwise,
30,50-dibromo-20hydroxyacetophenone (12.5 mmol, 3.67
g) in 50 mL of toluene. The reaction was heated to
1

2734
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
1
acid tert-butyl ester to afford 37 (0.210 g, 50.0%). H
105 ꢁC overnight, cooled to rt, 100 mL of 1 N NaOH,
was added and the reaction was stirred vigorously for
5 h at rt. The layers were separated and the aqueous
layer was washed with EtOAc 2·. The combined organic
layers were washed with H2O 1·. The combined aque-
ous layers were acidified to pH 2 with concd HCl to
form a precipitate that was filtered and was washed with
H2O. The solid material was dissolved in ACN/H2O and
speedvacced to dryness to afford 35a as a light tan solid
NMR (DMSO-d6, 300 MHz) d 8.03 (br s, 2H), 7.88
(dd, 1H, J = 1.5, 8 Hz), 7.68 (m, 1H), 7.39 (m, 2H),
5.91 (s, 1H), 4.32 (t, 2H, J = 6 Hz), 3.02 (m, 2H), 2.15
(m, 2H); MS (ESI, Pos.) calcd for C12H13NO3 m/z
[M+H] = 220.1, found 220.1.
5.1.33. 4-(3-Methylamino-propoxy)-1-benzopyran-2-one
trifluoroacetic acid salt (38). Prepared via method A at
0 ꢁC from 4-hydroxycoumarin and (3-hydroxypropyl)-
methyl-carbamic acid tert-butyl ester to afford 38
(0.380 g, 73.0%). 1H NMR (DMSO-d6, 300 MHz) d
8.47 (br s, 2H), 7.89 (dd, 1H, J = 1.5, 8 Hz), 7.68 (m,
1H), 7.40 (m, 2H), 5.91 (s, 1H), 4.31 (t, 2H,
J = 5.75 Hz), 3.14 (t, 2H, J = 7.25 Hz), 2.64 (s, 3H),
2.15 (m, 2H); MS (ESI, Pos.) calcd for C13H15NO3 m/
z [M+H] = 234.1, found 234.1.
1
(0.737 g, 18%). H NMR (DMSO-d6, 300 MHz) d 12.98
(br s, 1H), 8.18 (d, 1H, J = 2.25 Hz), 7.90 (d, 1H,
J = 2.25 Hz), 5.64 (s, 1H); MS (ESI, Pos.) calcd for
C9H4Br2O3 m/z [M+H] = 318.9, found 318.9.
5.1.29. 4-(3-Amino-propoxy)-6,8-dibromo-1-benzopyran-
2-one trifluoroacetic acid salt (35). Prepared via method
A at 0 ꢁC from 6,8-dibromo-4-hydroxy-1-benzopyran-2-
one and (3-hydroxypropyl)-carbamic acid tert-butyl es-
ter. The Boc protected intermediate was HPLC purified
prior to treatment with TFA. The title compound 35
was obtained by reversed-phase HPLC purification
(38.2 mg, 16.0%). 1H NMR (DMSO-d6, 300 MHz) d
8.23 (d, 1H, J = 2.25 Hz), 7.99 (d, 1H, J = 2.25 Hz),
7.75 (br s, 2H), 6.03 (s, 1H), 4.31 (t, 2H, J = 5.75 Hz),
3.04 (m, 2H), 2.11 (m, 2H); MS (ESI, Pos.) calcd for
C12H11Br2NO3 m/z [M+H] = 378.0, found 378.0.
5.1.34. 4-(3-Dimethylamino-propoxy)-7-methoxy-1-benzo-
pyran-2-one hydrochloric acid salt (41). Prepared via
method B from 7-methoxy-4-hydroxycoumarin and
3-dimethylamino-propan-1-ol using 4 M HCl in dioxane
to form 41 (292 mg, 65.0%). 1H NMR (DMSO-d6,
300 MHz) d 10.21 (s, 1H), 7.79 (d, 1H, J = 8.75 Hz),
7.01 (d, 1H, J = 2.25 Hz), 6.95 (dd, 1H, J = 2.25,
8.75 Hz), 5.76 (s, 1H), 4.28 (t, 2H, J = 6 Hz), 3.86 (s,
3H), 3.26 (m, 2H), 2.8 (s, 6H), 2.22 (m, 2H); MS (ESI,
Pos.) calcd for C15H19NO4 m/z [M+H] = 278.1, found
278.2.
5.1.30.
[3-(5-Chloro-2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-carbamic acid tert-butyl ester. A solution of
PPh3 (0.438 g, 1.67 mmol) in 10 mL of THF was cooled
to ꢀ78 ꢁC and DIAD (0.430 g) was added in one por-
tion. (3-Hydroxy-propyl)-carbamic acid tert-butyl ester
(560 mg, 1.3 equiv) in 1 mL of THF was added and
the solution was allowed to warm to rt over the course
of 1 h. The yellow solution was treated with 5-chloro-
4-hydroxycoumarin (0.300 g, 1.5 mmol) and stirred at
rt for 3.5 h. The solvent was removed under reduced
pressure and the crude product purified using an ISCO
4g SiO2 column eluting with ether/heptane gradient to
afford 36a (0.106 g, 20%). 1H NMR (CDCl3, 300
MHz) d 7.27 (m, 3H), 5.71 (s, 1H), 4.73 (br s, 1H),
4.17 (t, 2H, J = 5.75 Hz), 3.41 (q, 2H, J = 6.5 Hz), 2.13
(m, 2H), 1.43 (s, 9H); MS (ESI, Pos.) calcd for
C17H20ClNO5 m/z [M+H+Na] = 376.1, found 376.1.
5.1.35. 6-Chloro-4-(3-dimethylamino-propoxy)-1-benzo-
pyran-2-one hydrochloric acid salt (42). Prepared via
method B from 6-chloro-4-hydroxycoumarin and 3-
dimethylamino-propan-1-ol using 4 M HCl in dioxane
to form 42 (332 mg, 73%). 1H NMR (DMSO-d6,
300 MHz) d 10.23 (s, 1H), 7.88 (d, 1H, J = 2.75 Hz),
7.73 (dd, 1H, J = 2.75, 9 Hz), 7.48 (d, 1H, J = 9 Hz),
6.0 (s, 1H), 4.31 (t, 2H, J = 5.75 Hz), 3.28 (m, 2H),
2.80 (s, 3H), 2.80 (s, 3H), 2.24 (m, 2H); MS (ESI,
Pos.) calcd for C14H16ClNO3 m/z [M+H] = 282.1, found
282.1.
5.1.36. 4-(3-Dimethylamino-propoxy)-6-methyl-1-benzo-
pyran-2-one trifluoroacetic acid salt (43). Prepared via
method A at 0 ꢁC from 6-methyl-4-hydroxycoumarin
and 3-dimethylamino-propan-1-ol. The crude product
was purified by HPLC to afford 43 (151 mg, 28.1%).
1H NMR (DMSO-d6, 300 MHz) d 9.54 (br s, 1H), 7.64
(br s, 1H), 7.49 (dd, 1H, J = 1.75, 8.5 Hz), 7.31 (d, 1H,
J = 8.5 Hz), 5.90 (s, 1H), 4.29 (t, 2H, J = 5.75 Hz),
3.29 (m, 2H), 2.86 (s, 3H), 2.84 (s, 3H), 2.4 (s, 3H),
2.22 (m, 2H); MS (ESI, Pos.) calcd for C15H19NO3
m/z [M+H] = 262.1, found 262.2.
5.1.31. 4-(3-Amino-propoxy)-5-chloro-1-benzopyran-2-
one hydrochloric acid salt (36). [3-(5-Chloro-2-oxo-2H-
1-benzopyran-4-yloxy)-propyl]-carbamic acid tert-butyl
ester (0.079 g, 0.22 mmol) was treated with 4 N HC1/
dioxane for several minutes during which time a precip-
itate formed. The solvent/HCl was removed under re-
duced pressure. The residue was recrystallized from
MeOH–EtOAc followed by a second recrystallization
from EtOH to afford 36 (22.8 mg, 41%). 1H NMR
(DMSO-d6, 300 MHz) d 7.94 (br s, 3H), 7.62 (t, 1H,
J = 8.25 Hz), 7.43 (m, 2H), 5.94 (s, 1H), 4.29 (t, 2H,
J = 5.75 Hz), 3.04 (t, 2H, J = 7 Hz), 2.14 (m, 2H); MS
(ESI, Pos.) calcd for C12H12ClNO3 m/z [M+H] =
254.0, found 254.1.
5.1.37.
4-(3-Dimethylamino-propoxy)-6,7-dimethyl-1-
benzopyran-2-one trifluoroacetic acid salt (44). Prepared
via method A 0 ꢁC from 6,7-dimethy-4-hydroxycouma-
rin and 3-dimethylamino-propan-1-ol. The crude prod-
uct was purified by HPLC to afford 44 (63.6 mg,
15.6%). 1H NMR (DMSO-d6, 300 MHz) d 9.52 (s,
1H), 7.59 (s, 1H), 7.23 (s, 1H), 5.83 (s, 1H), 4.28 (t,
2H, J = 6 Hz), 3.29 (m, 2H), 2.84 (s, 3H), 2.85 (s, 3H)
5.1.32. 4-(3-Amino-propoxy)-1-benzopyran-2-one hydro-
chloric acid salt (37). Prepared via method B from 4-
hydroxycoumarin and (3-hydroxy-propyl)-carbamic

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2735
2.31 (d, 6H), 2.20 (m, 2H); MS (ESI, Pos.) calcd for
C16H21NO3 m/z [M+H] = 276.2, found 276.2.
1H), 7.46 (d, 1H, J = 9 Hz), 5.95 (s, 1H), 4.24 (t, 2H,
J = 5.75 Hz), 3.59 (br s, 2H), 2.79 (br s, 3H), 2.07 (m,
2H); MS (ESI, Pos.) calcd for C14H15ClN2O3S m/z
[M+H] = 327.0, found 327.0.
5.1.38. N-[3-(2-Oxo-2H-1-benzopyran-4-yloxy)-propyl]-
guanidine trifluoroacetic acid salt (45). Prepared accord-
ing to the procedure for N-[2-(2-oxo-2H-1-benzopyran-
4-yloxy)-ethyl]-guanidine from the trifluoroacetic acid
salt of 4-(3-amino-propoxy)-1-benzopyran-2-one to af-
ford 45 (40.0 mg, 35%). 1H NMR (DMSO-d6, 300
MHz) d 7.88 (dd, 1H, J = 1.5, 8 Hz), 7.67 (m, 2H),
7.39 (m, 2H), 7.15 (br s, 3H), 5.92 (s, 1H), 4.26 (t, 2H,
J = 6 Hz), 3.32 (2H, buried under solvent peak), 2.03
(m, 2H); MS (ESI, Pos.) calcd for C13H15N3O3 m/z
[M+H] = 262.1, found 262.1.
5.1.42. N-[3-(6-Chloro-2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-acetamide (49). 6-Chloro-4-(3-methylamino-
propoxy)-1-benzopyran-2-one trifluoroacetic acid salt
(18) (0.272 mmol, 100 mg) was suspended in 3 mL of
DCM. DIEA (0.544 mmol, 95 lL) was added followed
by acetic anhydride (0.816 mmol, 77.1 lL). After stirring
for 1 h at rt, the reaction mix was concentrated in vacuo
and purified by reversed-phase HPLC to afford 49
(39.0 mg, 49%). 1H NMR (DMSO-d6, 300 MHz) d
7.95 (br t, 1H), 7.79 (d, 1H, J = 2.5 Hz), 7.72 (dd, 1H,
J = 2.5, 8.75 Hz), 7.46 (d, 1H, J = 8.75 Hz), 5.95 (s,
1H), 4.23 (t, 2H, J = 6 Hz), 3.24 (q, 2H, J = 6 Hz),
1.95 (m, 2H), 1.80 (s, 3H); MS (ESI, Pos.) calcd for
C14H14ClNO4 m/z [M+H] = 296.1, found 296.2.
5.1.39. 1-Methyl-3-[3-(2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-thiourea (46). Prepared according to the proce-
dure for 1-[3-(6-chloro-2-oxo-2H-1-benzopyran-4-yl-
oxy)-propyl]-3-methyl-thiourea from 4-(3-amino-prop-
oxy)-1-benzopyran-2-one (trifluoroacetic acid salt) to
afford 46 (71 mg, 73%). 1H NMR (DMSO-d6,
300 MHz) d 7.87 (dd, 1H, J = 1.5, 7.75 Hz), 7.67 (m,
1H), 7.56 (br s, 1H), 7.39 (m, 3H), 5.88 (s, 1H), 4.24
(t, 2H, J = 6 Hz), 3.59 (s, 2H), 2.79 (s, 3H), 2.07 (m,
2H); MS (ESI, Pos.) calcd for C14H16N2O3S m/z
[M+H] = 292.1, found 292.4.
5.1.43. 4-(4-Amino-butoxy)-5-chloro-1-benzopyran-2-one
hydrochloric acid salt (50). Prepared using the procedure
for 36 from [3-(5-chloro-2-oxo-2H-1-benzopyran-4-
yloxy)-butyl]-carbamic acid tert-butyl ester to afford 50
1
(52 mg, 92%). H NMR (DMSO-d6, 300 MHz) d 7.86
(br s, 3H), 7.61 (t, 1H, J = 8.25 Hz), 7.42 (t, 2H, J =
9.5, 8.75 Hz), 5.96 (s, 1H), 4.23 (t, 2H, J = 5.75 Hz),
2.85 (m, 2H), 1.83 (m, 4H); MS (ESI, Pos.) calcd for
C13H14ClNO3 m/z [M+H] = 268.1, found 268.1.
5.1.40. N-[3-(6-Chloro-2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-guanidine trifluoroacetic acid salt (47). 6-Chloro-
4-(3-methylamino-propoxy)-1-benzopyran-2-one triflu-
oroacetic acid salt (18) (0.200 g, 0.543 mmol) was sus-
pended in 7 mL of DCM. Diisopropylethylamine was
added dropwise until pH = 9 by pH paper. N,N0-Bis-
(tert-butoxycarbonyl)-1-H-pyrazole-1-carboxamidine (169
mg, 0.543 mmol) was added and the mixture was stirred
at rt overnight. The reaction mixture was concentrated
in vacuo and Et2O was added to form a white precipi-
tate, which was filtered and washed with Et2O. The
crude material was treated with neat TFA (10 mL) for
30 min at rt. The TFA was removed under reduced pres-
sure. Et2O was added to precipitate the product, which
was filtered, washed with Et2O, and dried. The crude
material was further purified by reversed-phase HPLC
to afford 47 (108 mg, 48.6%). 1H NMR (DMSO-d6,
300 MHz) d 7.86 (d, 1H, J = 2.5 Hz), 7.72 (dd, 1H,
J = 2.5, 8.75 Hz), 7.64 (t, 1H, J = 5.5 Hz), 7.47 (d, 1H,
J = 8.75 Hz), 7.12 (br s, 3H), 6.00 (s, 1H), 4.26 (t, 2H,
J = 5.75 Hz), 3.36 (q, 2H, J = 6.5 Hz), 2.04 (m, 2H);
MS (ESI, Pos.) calcd for C13H14ClN3O3 m/z
[M+H] = 296.1, found 296.0.
5.1.44. 4-(4-Amino-butoxy)-6-chloro-1-benzopyran-2-one
trifluoroacetic acid salt (51). Prepared via method A at
0 ꢁC from and 6-chloro-4-hydroxycoumarin and (4-hy-
droxy-butyl)-carbamic acid tert-butyl ester to afford 51
1
(185 mg, 59%). H NMR (DMSO-d6, 300 MHz) d 7.74
(m, 4H), 7.47 (d, 1H, J = 9 Hz), 6.00 (s, 1H), 4.26 (t,
2H, J = 5.75 Hz), 2.89 (m, 2H), 1.87 (m, 2H), 1.74 (m,
2H) MS (ESI, Pos.) calcd for C13H14ClNO3 m/z
[M+H] = 268.1, found 268.2.
5.1.45. 4-(5-Amino-pentyloxy)-6-chloro-1-benzopyran-2-
one trifluoroacetic acid salt (52). Prepared via method
A at 0 ꢁC from 6-chloro-4-hydroxycoumarin and (5-hy-
droxy-pentyl)-carbamic acid tert-butyl ester to afford 52
1
(72.0 mg, 36%). H NMR (DMSO-d6, 300 MHz) d 7.77
(d, 1H, J = 2.75 Hz), 7.72 (dd, 1H, J = 2.5, 8.75 Hz),
7.65 (br s, 2H), 7.47 (d, 1H, J = 8.75 Hz), 5.99 (s, 1H),
4.23 (t, 2H, J = 6.25 Hz), 2.83 (m, 2H), 1.83 (m, 2H),
1.62 (m, 2H), 1.52 (m, 2H); MS (ESI, Pos.) calcd for
C14H16ClNO3 m/z [M+H] = 282.1, found 282.1.
5.1.41. 1-[3-(6-Chloro-2-oxo-2H-1-benzopyran-4-yloxy)-
propyl]-3-methyl-thiourea (48). 6-Chloro-4-(3-methyl-
amino-propoxy)-1-benzopyran-2-one trifluoroacetic acid
salt (18) (0.272 mmol, 100 mg) was suspended in 5 mL
of 1:1 DMF–DCM. DIEA was added dropwise until
pH = 9 by pH paper. Methyl isothiocyanate
(0.299 mmol, 0.022 g) was added and the reaction was
stirred at rt for 3 h. The reaction mixture was concen-
trated in vacuo and the crude material was purified by
reversed-phase HPLC to afford 48 (66.1 mg, 78.7%).
1H NMR (DMSO-d6, 300 MHz) d 7.85 (d, 1H,
J = 2.5 Hz), 7.71 (dd, 1H, J = 2.75, 9 Hz), 7.55 (br m,
5.1.46.
6-Chloro-4-(piperidin-3-ylmethoxy)-1-benzopy-
ran-2-one trifluoroacetic acid salt (53). Prepared via
method A at 0 ꢁC from 6-chloro-4-hydroxycoumarin
and (3-hydroxymethyl-cyclohexyl)-carbamic acid tert-
butyl ester to afford 53 (68.1 mg, 23%). 1H NMR
(DMSO-d6, 300 MHz) d 8.62 (m, 1H), 8.35 (m, 1H),
7.87 (d, 1H, J = 2.75 Hz), 7.73 (dd, 1H, J = 2.5,
8.75 Hz), 7.48 (d, 1H, J = 9 Hz), 6.00 (s, 1H), 4.16 (m,
2H), 3.27 (d, 2H, J = 13 Hz), 2.88 (m, 2H), 2.27 (m,
1H), 1.86 (m, 2H), 1.67 (m, 1H), 1.44 (m, 1H); MS
(ESI,
[M+H] = 294.1, found 294.2.
Pos.)
calcd
for
C15H16ClNO3
m/z

2736
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
5.1.47. 6-Chloro-4-(2-piperidin-2-yl-ethoxy)-1-benzopy-
ran-2-one trifluoroacetic acid salt (54). Prepared via
method C at 0 ꢁC from 6-chloro-4-hydroxycoumarin
and 2-(2-hydroxy-ethyl)-piperidine-1-carboxylic acid
tert-butyl ester to afford 54 (43.7 mg, 20.7%). 1H
NMR (DMSO-d6, 300 MHz) d 8.55 (br s, 1H), 8.36
(br s, 1H), 7.87 (d, 1H, J = 2.5 Hz), 7.73 (dd, 1H,
J = 2.5, 8.75 Hz), 7.48 (d, 1H, J = 8.75 Hz), 5.99 (s,
1H), 4.32 (m, 2H), 3.28 (d, 2H, J = 11.75 Hz), 2.91 (m,
1H), 2.10 (m, 4H), 1.76 (br d, 2H), 1.48 (m, 2H); MS
(ESI, Pos.) calcd for C16H18ClNO3 m/z [M+H] =
308.1, found 308.2.
7.63 mmol), and trans-2-methanesulfonyloxymethyl-
cyclopropanecarboxylic acid methyl ester (1.28 g,
6.14 mmol) were added. The reaction was heated to
100 ꢁC for 5 h, a further 0.2 equiv of the mesylate was
added, and heating was continued for an additional
2 h. After cooling to rt, the DMF was removed in vacuo.
The residue was dissolved in EtOAc and washed with
H2O. The organic layer was dried (MgSO4), filtered,
and the EtOAc was removed under reduced pressure
to afford a yellow solid, which was chromatographed
on SiO2 (35 g) using DCM as the eluent to afford 59
as a white solid (0.98 g, 62%). 1H NMR (DMSO-d6,
300 MHz) d 7.75 (m, 2H), 7.45 (d, 1H), 5.97 (s, 1H),
4.22 (m, 2H), 3.65 (s, 3H), 1.90 (m, 2H), 1.19 (m, 2H);
MS (ESI, Pos.) calcd for C15H13ClO5 m/z
[M+H] = 309.0, found 309.0.
5.1.48. 6-Bromo-4-(piperidin-4-yloxy)-1-benzopyran-2-
one trifluoroacetic acid salt (55). Prepared via method
A at 0 ꢁC from 6-bromo-4-hydroxycoumarin and 4-hy-
droxy-piperidine-1-carboxylic acid tert-butyl ester to af-
ford 55 (47.3 mg, 25.3%). 1H NMR (DMSO-d6,
300 MHz) d 8.46 (br s, 2H), 8.01 (d, 1H, J = 2.25 Hz),
7.84 (dd, 1H, J = 2.5, 8.75 Hz), 7.41 (d, 1H, J = 9 Hz),
6.14 (s, 1H), 4.97 (m, 1H), 3.32 (2H, buried under sol-
vent peak), 3.14 (m, 2H), 2.12 (m, 2H), 2.01 (m, 2H);
MS (ESI, Pos.) calcd for C14H14BrNO3 m/z [M+H] =
324.0, found 324.0.
5.1.53. trans-2-(6-Chloro-2-oxo-2H-1-benzopyran-4-yl-
oxymethyl)-cyclopropanecarboxylic acid (60). trans-2-(6-
Chloro-2-oxo-2H-1-benzopyran-4-yloxymethyl)
cycl-
propanecarboxylic acid methyl ester (0.98 g, 3.19 mmol)
was dissolved in THF (15 mL). A solution of LiOH
(0.134 g, 5.58 mmol) in 5 mL of H2O was added to the
reaction mixture and the mixture was stirred overnight
at rt. An additional 0.5 equiv LiOH was added and
the mixture was stirred for 1 h. The solution was concen-
trated and diluted with H2O, then extracted with Et2O
to remove any unreacted starting material. The aqueous
layer was acidified with 3 N HCl and the resulting pre-
cipitate was extracted into DCM. The DCM layer was
dried (MgSO4), filtered, and evaporated to afford 60 as
a white solid (0.367 g, 40%). 1H NMR (300 MHz,
DMSO-d6) d 12.25 (br s, 1H), 7.74 (m, 1H), 7.70 (d,
1H, J = 2.5 Hz), 7.46 (d, 1H, J = 8.75 Hz), 6.99 (s,
1H), 4.20 (m, 2H), 1.85 (m, 1H), 1.7 (m, 1H), 1.18
(m, 1H), 1.05 (m, 1H); MS (ESI, Pos.) calcd for
C14H11ClO5 m/z [M+H] = 295.0, found 295.1.
5.1.49. 6-Chloro-4-(pyridin-2-ylmethoxy)-1-benzopyran-
2-one trifluoroacetic acid salt (56). Prepared via method
B from 6-chloro-4-hydroxycoumarin and pyridin-2-yl-
methanol to afford 56 after HPLC purification
(36.0 mg, 16.8%). 1H NMR (DMSO-d6, 300 MHz) d
8.63 (m, 1H), 7.94 (m, 1H), 7.83 (d, 1H, J = 2.5 Hz),
7.72 (m, 2H), 7.47 (m, 2H), 6.13 (s, 1H), 5.45 (s, 2H);
MS (ESI, Pos.) calcd for C15H10ClNO3 m/z
[M+H] = 288.0, found 287.9.
5.1.50. 6-Chloro-4-(pyridin-3-ylmethoxy)-1-benzopyran-
2-one Trifluoracetic acid salt (57). Prepared via method
B from 6-chloro-4-hydroxycoumarin and pyridin-3-yl-
methanol using HCl/dioxane to form the hydrochloric
acid salt, which was then purified by HPLC to afford
5.1.54. N-(4-Hydroxy-quinolin-2-yl)-acetamide (61a).
Prepared as for 62a to deliver 0.13 g (0.64 mmol,
51%) of a beige solid mp = 297–299 ꢁC. 1H NMR
(DMSO-d6, 300 MHz) d 10.74 (br s, 1H), 8.017 (d, 1H,
J = 4.2 Hz), 7.82 (br s, 1H), 7.682 (d, 1H, J = 4.2 Hz),
7.611 (t, 1H, J = 3.6 Hz), 7.32 (t, 1H J = 3.6 Hz), 5.79
(br s, 1H) 2.15 (s, 3H); MS (ESI, Pos.) calcd for
C11H10N2O2 m/z [M+H] = 203.2, found 203.0.
1
57 (45.0 mg, 21.0%). H NMR (DMSO-d6, 300 MHz)
d 8.86 (s, 1H), 8.70 (d, 1H, J = 5 Hz), 8.17 (d, 1H,
J = 8 Hz), 7.80 (d, 1H, J = 2.5 Hz), 7.73 (dd, 1H,
J = 2.5, 8.75 Hz), 7.66 (m, 1H), 7.49 (d, 1H, J = 9 Hz),
6.17 (s, 1H), 5.46 (s, 2H); MS (ESI, Pos.) calcd for
C15H10ClNO3 m/z [M+H] = 288.0, found 287.9.
5.1.51. 6-Chloro-4-(pyridin-4-ylmethoxy)-1-benzopyran-
2-one trifluoroacetic acid salt (58). Prepared via method
B from 6-chloro-4-hydroxycoumarin and pyridin-4-yl-
methanol using HCl/dioxane to form the hydrochloric
acid salt, which was then purified by HPLC to afford
5.1.55.
N-{4-[3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-
propoxy]-quinolin-2-yl}-acetamide (61b). To a solution
of 0.20 g (0.99 mmol) of N-(4-Hydroxy-quinolin-2-yl)-
acetamide (61a) in DMF was added 0.26 g (0.99 mmol)
of N-(3-bromopropyl)phthalimide followed by 0.39 g
(1.2 mmol) of cesium carbonate. The mixture was
heated on a steambath under a nitrogen atmosphere
for 4 h. Upon cooling a white solid precipitated out,
which was collected by filtration. Recrystallization from
i-PrOH and a minimal amount of methanol delivered
0.29 g (0.74 mmol, 75%) of a white solid mp = 227–
1
58 (72.6 mg, 33.0%). H NMR (DMSO-d6, 300 MHz)
d 8.77 (d, 2H, J = 5.75 Hz), 7.94 (d, 1H, J = 2.5 Hz),
7.79 (d, 2H, J = 6 Hz), 7.75 (dd, 1H, J = 2.75,
8.75 Hz), 7.50 d, (1H, J = 9 Hz), 6.10 (s, 1H), 5.56 (s,
2H); MS (ESI, Pos.) calcd for C15H10ClNO3 m/z
[M+H] = 288.0, found 288.1.
1
9 ꢁC. H NMR (DMSO-d6, 300 MHz) d 10.67 (s, 1H),
5.1.52. trans-2-(6-Chloro-2-oxo-2H-1-benzopyran-4-yl-
oxymethyl)-cyclopropanecarboxylic acid methyl ester
(59). 6-Chloro-4-hydroxycoumarin (1.0 g, 5.09 mmol)
was dissolved in 80 mL of DMF, and Cs2CO3 (2.49 g,
7.80 (m, 5H), 7.67 (d, 1H, J = 4.2 Hz), 7.61 (m, 1H),
7.22 (m, 1H), 4.26 (t, 2H J = 2.7Hz), 3.88 (t, 2H,
J = 3.3Hz), 2.50 (m, 2H), 2.12 (s, 3H); MS (ESI, Pos.)
calcd for C22H19N3O4 m/z [M+H] = 390.4, found 390.1.

S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
2737
5.1.56. 4-(3-Amino-propoxy)-quinolin-2-ylamine (61).
Prepared as for 62 to deliver an unoptimized 0.40 g
(0.18 mmol, 48%) of a white solid mp = 175–7 ꢁC. H
NMR (DMSO-d6, 300 MHz) d 7.81 (dd, 1H, J =
0.6,4.2 Hz), 7.43 (m, 1H), 7.372 (d, 1H, J = 4.2 Hz),
7.09 (m, 1H), 6.21 (s, 2H), 6.19 (s, 1H), 4.168 (t, 2H,
J = 3.3 Hz), 2.50 (t, 2H, J = 0.90 Hz), 1.91 (m, 2H).
MS (ESI, Pos.) calcd for C12H15N3O m/z [M+H] =
218.27, found 218.1.
rated and the organic layer washed with H2O. The com-
bined aqueous layers were washed with Et2O and the
aqueous layer then acidified to pH 2 with concd HCl.
The resulting off-white precipitate was filtered, washed
with Et2O, and dried to afford the title compound
(0.417 g, 37%). A second crop was obtained by refriger-
1
1
ating the filtrate (0.442 g, 39%). H NMR (DMSO-d6,
300 MHz) d 7.88 (d, 1H, J = 2.5 Hz), 7.61 (dd, 1H,
J = 2.62, 8.87 Hz), 7.40 (d, 1H, J = 8.87 Hz), 2.00 (s,
3H); MS (ESI, Pos.) calcd for C10H7ClO3 m/z
[M+H] = 211.0, found 210.9.
5.1.57. N-(6-Chloro-4-hydroxy-quinolin-2-yl)-acetamide
(62a). To a solution of 0.85 g (4.34 mmol) of 2-amino-
6-chloro-quinolin-4-ol in 30 mL of pyridine is added
1.1 g (10.8 mmol) of acetic anhydride and the mixture
heated on a steambath for 2 h under a nitrogen atmo-
sphere. The mixture is concentrated in vacuo and the
resulting solid is recrystallized from iPrOH to deliver
0.79 g (3.34 mmol, 77%) of a beige solid mp = 365 ꢁC.
1H NMR (DMSO-d6, 300 MHz) d 10.66 (br s, 1H),
7.96 (s, 1H), 7.73 (br s, 1H), 7.636 (d, 1H, J = 1.2 Hz),
7.621 (d, 1H, J = 1.2Hz), 5.78 (br s, 1H) 2.14 (s, 3H);
MS (ESI, Pos.) calcd for C11H9ClN2O2 m/z
[M+H] = 236.7, found 237.0.
5.1.61. 4-(3-Amino-propoxy)-6-chloro-3-methyl-1-benzo-
pyran-2-one (63). 6-Chloro-4-hydroxy-3-methyl-1-benzo-
pyran-2-one (1.0 mmol, 0.210 g) was suspended in 10
mL THF, and PPh3 (1.05 mmol, 0.275 g) and (3-hydro-
xy-propyl)-carbamic acid tert-butyl ester (1.05 mmol,
0.184 g) were added. The reaction was cooled to 0 ꢁC
and DIAD (1.1 mmol, 0.222 g) was added dropwise at
0 ꢁC, and then was warmed to rt and stirred overnight.
The THF was removed under reduced pressure to give
the Boc-protected intermediate. This intermediate was
purified by reversed-phase HPLC and then treated with
neat TFA for 5 min. The TFA was removed in vacuo
and Et2O was added to precipitate the product, which
was purified by reversed-phase HPLC to afford 63
(116 mg, 30%). 1H NMR (DMSO-d6, 300 MHz). d
7.79 (br s, 2H), 7.73 (d, 1H, J = 2.2 Hz), 7.66 (dd, 1H,
J = 2.8, 9 Hz), 7.48 (d, 1H, J = 8.8 Hz), 4.29 (t, 2H,
J = 6.3 Hz), 3.03 (m, 2H), 2.12 (m, 2H), 2.09 (s, 3H);
MS (ESI, Pos.) calcd for C13H14ClNO3 m/z
[M+H] = 268.1, found 268.1.
5.1.58. N-{6-Chloro-4-[3-(1,3-dioxo-1,3-dihydro-isoindol-
2-yl)-propoxy]-quinolin-2-yl}-acetamide (62b). Prepared
as for 61b to deliver an unoptimized 0.10 g (0.23 mmol,
28%) of a beige solid mp = 267–9 ꢁC. 1H NMR (DMSO-
d6, 300 MHz) d 10.74 (s, 1H), 7.85 (s, 1H), 7.80 (m, 4H),
7.73 (d, 1H, J = 0.9 Hz), 7.67 (d, 1H, J = 4.5 Hz), 7.61
(dd, 1H, J = 1.2, 4.5 Hz), 4.27 (t, 2H, J = 2.7Hz), 3.87
(t, 2H, J = 3.3Hz), 2.50 (m, 2H), 2.13 (s, 3H); MS
(ESI, Pos.) calcd for C22H18ClN3O4 m/z [M+H] =
424.8, found 424.1.
5.2. Enzyme assays
5.1.59. 4-(3-Amino-propoxy)-6-chloro-quinolin-2-ylamine
(62). To a solution of 0.16 g (0.38 mmol) of N-{6-
Chloro-4-[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-prop-
oxy]-quinolin-2-yl}-acetamide (62b) in ethanol was
added 0.4 mL of hydrazine (35% aqueous solution)
(4.37 mmol) and the reaction heated on a steambath
for 2 h. The reaction mixture was cooled and filtered
to remove the phthalhydrazide by-product. The mother
liquor was concentrated in vacuo and chromatographed
on silica gel (7 N NH3–MeOH–CH2Cl2: 1:9 as eluant) to
deliver 0.07 g (0.28 mmol, 74%) of a white solid
mp = 167–9 ꢁC. 1H NMR (DMSO-d6, 300 MHz) d
7.74 (d, 1H J = 1.5Hz), 7.43 (dd, 1H, J = 1.2, 4.5 Hz),
7.375 (d, 1H, J = 4.5Hz), 6.38 (s, 2H), 6.24 (s, 1H),
4.17 (t, 2H, J = 3.3 Hz), 2.77 (t, 2H, J = 3.0 Hz), 1.91
(t, 2H, J = 3.3 Hz) MS (ESI, Pos.) calcd for
C12H14ClN3O m/z [M+H] = 252.72, found 252.1.
Nitric Oxide Synthase enzymatic assays are based on the
[3H]-L-Arginine into [3H]-L-Citrulline conversion origi-
nally described by Bredt and Snyder36 using Km concen-
tration of substrate. Recombinant mouse iNOS, bovine
eNOS, and rat nNOS enzymes were purchased from
Cayman Chemical (Cat. No. 60862, 60880, 60870).
Human iNOS was expressed in E. coli at Aventis. The
inhibitory activity of each test compound was evaluated
by measuring the conversion rate from [3H]-L-Arginine
to [3H]-L-Citrulline. Based on this the IC50 values were
computed for each test compound.
5.3. Enzyme kinetics
Lineweaver–Burk analysis of inhibition of human iNOS
by coumarin 30 was performed. Human iNOS activity
was measured under initial velocity conditions. Kinetics
of coumarin 30 were determined using the radioactive
endpoint filtration assay described in the methods. Par-
tially purified human iNOS was incubated in 50 mM
Hepes buffer, pH 7.4, with 1 mM NADPH, 15 lM 6R-
tetrahydrabiopterin, 1 lM FAD, and various concentra-
tions of [3H]-Arginine (0.25, 0.75, 2.5, 7.5, 25, or 75 lM)
at 37 ꢁC for 5–10 min. A buffer control, and six concen-
trations of 30 were used to determine mode of inhibition.
5.1.60.
6-Chloro-4-hydroxy-3-methyl-1-benzopyran-2-
one. Diethylcarbonate (27.2 mmol, 3.3 mL) was dis-
solved in 30 mL toluene, followed by the addition of
NaH (16.3 mmol, 0.65 g of 60% dispersion) in portions.
50-Chloro-20hydroxy-propiophenone (5.4 mmol, 1.0 g)
was dissolved in toluene (10 mL) and added dropwise
via an addition funnel to the above solution. The cloudy
yellow-green mixture was heated to near reflux for 20 h
then cooled to rt. NaOH (1 N, 50 mL) was added and
the mixture stirred at rt for 60 h. The layers were sepa-
Time-dependency of human iNOS inhibition was exam-
ined. Three concentrations of 30 and human iNOS

2738
S. A. Jackson et al. / Bioorg. Med. Chem. 13 (2005) 2723–2739
enzyme were preincubated together for 0, 5, 15, 30, and
60 min prior to initiation of the enzymatic reaction with
the addition of NADPH cofactor and arginine sub-
strate. A control with enzyme and no inhibitor was used
to show that the enzyme is not losing activity by itself
during the preincubation time. Enzyme with 1 mM L-
NMA was used as background control. The standard
30 min endpoint assay with [3H]-arginine was used to
determine if time-dependent decrease in enzyme activity
is observed with coumarin 30.
as vehicle. Blood samples were obtained at T = 0 (pre-
dose), 2, 10, 20, 30 min, and 1, 2, 3, 5, and 7 h post-dose.
The 10 mg/kg (n = 3 for each time point), PO dose was
administered by gavage at 2.5 mL/kg using 0.5% methyl
cellulose/0.2% Tween 80 mixture. Blood and brain sam-
ples were obtained in a non-serial manner at T = 0 (pre-
dose), 5, 15, 30 min, and 1, 2, 3, 4, 5 and 7 h post-dose.
For mouse PK studies, ꢁ30 gm female, SJL/J mice were
used. Intravenous dose of 2.0 mg/kg and oral dose of
10 mg/kg were administered in similar vehicles as de-
scribed above. Blood and brain samples were obtained
in a non-serial manner from IV dosed mice using similar
time schedule. Only plasma samples were obtained from
the PO dosed mice.
5.4. Effect of protein binding on iNOS inhibition
An IC50 determination for coumarin 30 with human
iNOS was conducted in the presence of 10% fetal calf
serum to determine if protein binding has any effect on
the activity of the coumarin series.
Sample analysis was performed using LC/MS/MS
method. Brains were first homogenized with 3.00 mL
of water. A 50.0 lL aliquot of plasma or brain homo-
genate was transferred to a microcentrifuge tube
(1.7 mL), and mixed with 25.0 lL of Internal Standard
solution (250 ng/mL of RPR127963 in water) and
1.0 mL of isopropyl ether. The tubes were vortexed at
setting 10 for 5 min. and centrifuged at 10,000g for
5 min. The organic layer in each tube was transferred
into a glass tube (12 · 75 mm) and dried under nitrogen
at 37 ꢁC. The residue was reconstituted in 75 lL of 50%
acetonitrile in water and transferred into an autosampler
vial for LC/MS/MS analysis. LC/MS/MS analysis was
performed with a PE Sciex API 3000 mass spectrometer
(Analytical column: Inertsil ODS-3, 3 lm, 50 · 2.1 mm
with a 10 · 2.1 mm Guard column, Column tempera-
ture: ambient, Flow rate: 0.2 mL/min, Injection volume:
15 lL, Mobile phase: 70% of 90% ACN in water + 30%
of 0.1% formic acid in water). The lower limit of quan-
titation was 1 ng/mL for plasma and 2 ng/mL for brain
homogenate.
5.5. Whole cell assays
RAW cells (rodent monocytes) and A172 cells (human
glioblastoma) (both available at ATCC) were cultured
in DMEM + 10%FBS. Raw cells were plated at 100,000
cells/well in 96-well plates and incubated at 37 ꢁC over-
night. The cells were pre-activated by LPS (Sigma) at
1 lg/mL and incubated at 37 ꢁC for 24 h (or overnight).
The supernatants were removed. Fresh LPS was added
to the cells at 1 lg/mL; meanwhile the inhibitors (nine dif-
ferent concentrations) were added to the cells. The cells
were incubated for 24 h. Seventy-five microliters of med-
ium from each well were collected for NO measurement.
A172 cells were plated at 30,000 cells/well in 96-well
plates and incubated overnight. The cells were pre-acti-
vated by LPS (1 lg/mL) + interferon-c(400 U/mL) +
tumor necrosis factor-a (40 ng/mL) + IL-1b (4 ng/mL)
and incubated at 37 ꢁC overnight. The supernatant was
removed. Fresh LPS + cytokine mix (same concentration
as described above) were added to the cells; meanwhile
the inhibitors (nine different concentrations) were added
to the cells. The cells were incubated for 24 h. Seventy-
five microliters of medium from each well were collected
for NO measurement. (All cytocykines were ordered
from Roche Molecular Biochemicals.)
5.7. Metabolism studies
Coumarin 30 was incubated at 25 lM concentration in
human and mouse hepatic microsomes for 1 h. Incuba-
tions were done both in the presence and absence of
NADPH. Identification of potential metabolites was
undertaken using LC/MS/MS.
The Griess reaction was used to measure NO. Sulfanil-
amide (1 mM, Sigma) and 0.1 N HCl were added to
the supernatant and cultured at rt for 10 min. Samples
were centrifuged at 1000g for 15 min. Seventy-five
microliters of the supernatant were transferred into a
96-well Elisa plate. The plates were read absorbance at
548 nm (for background). N-(1-Naphthyl)ethylenedi-
amine (1 mM, NEDA, from Sigma) was added to the
supernatant and incubated for 10 min at rt. The plates
were read absorbance at 548 nm. Pre-NEDA was sub-
tracted from post-NEDA for final reading units.
5.8. Docking study
The molecular modeling study was carried out on an
SGI workstation running IRIX 6.5 (http://www.sgi.
scom). InsightII and Cerius2 from Accelrys (http://
www.accelrys.com) was used for most of our molecular
modeling studies. ChemX/DiR from Chemical Design
LLT (later acquired by Accelrys) was used for binding
pose generation and Discover with cff 99 (Accelrys)
was used for all molecular mechanics force field calcula-
tions and binding pose evaluation.
5.6. Pharmacokinetic studies
Intravenous and oral pharmacokinetics of compounds
was characterized in rats and mice. All compounds were
dosed as free base equivalents. For rat PK study, ꢁ300 g
male, Sprague-Dawley rats were used. The 2 mg/kg
(n = 3), IV dose was delivered at 1 mL/kg using saline
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