Synthesis of 2-pyridones as tissue factor VIIa inhibitors

Article abstract of DOI:10.1016/S0040-4020(03)01239-0

2-Pyridones were prepared from 2,6-dibromopyridine via a multi-step synthesis. A variety of chemical transformations, including regioselective nucleophilic addition and selective nitrogen alkylation, afforded the penultimate intermediate 9. A combination

Full text of DOI:10.1016/S0040-4020(03)01239-0

Tetrahedron 59 (2003) 7695–7701
Synthesis of 2-pyridones as tissue factor VIIa inhibitors
*
John J. Parlow and Michael S. South
Department of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh-U2D, St. Louis, MO 63167, USA
Received 19 June 2003; revised 5 August 2003; accepted 5 August 2003
Abstract—2-Pyridones were prepared from 2,6-dibromopyridine via a multi-step synthesis. A variety of chemical transformations,
including regioselective nucleophilic addition and selective nitrogen alkylation, afforded the penultimate intermediate 9. A combination of
two-dimensional NMR techniques to unequivocally assign the structure of 9 is described. Compound 9 was then used in a Suzuki coupling
and further derivatized to afford the targeted tissue Factor VIIa inhibitors. These compounds were tested in several serine protease enzyme
assays with biological activity reported.
q 2003 Elsevier Ltd. All rights reserved.
Cardiovascular disease consisting of unstable angina,
myocardial infarction and sudden death, are the most
frequent cause of mortality in the United States and Western
countries.1 The disease is associated with acute thrombus
formation, often as the result of a plaque rupture.
Thrombosis also occurs in transient ischemic attack, stroke,
peripheral occlusive arterial disease, deep vein thrombosis,
pulmonary embolism, abrupt closure following angioplasty,
and the disseminated intravascular coagulation associated
with sepsis and certain cancers. Effective and safe
antithrombotics are needed to combat these diseases. Most
research has focused on thrombin and Factor Xa inhibitors
as potentially valuable therapeutic agents for these dis-
eases.2 More recently, small molecule inhibitors of tissue
Factor (TF) VIIa have been the point of much research effort
because of their potential to inhibit the coagulation cascade
while lessening the risk of bleeding side effects.3 The
extrinsic coagulation cascade is triggered by the binding of
Factor VIIa to cell surface TF. This cascade is critical in
normal hemostasis, but is also involved in the pathogenesis
of various thrombotic diseases. Under normal conditions,
TF expressed in the sub-endothelium of healthy blood
vessels is not exposed to blood. However, in disease or
injury, vessel wall or plaque TF is exposed and complexes
with its cofactor Factor VIIa, leading to the activation of
Factors IX and X. The complex of Factor Xa and Factor Va
on a membrane surface converts prothrombin to thrombin,
leading to fibrin formation, deposition, and subsequent
thrombus formation.4
analogs as noncovalent tissue Factor VIIa inhibitors.5
These pyrazinone compounds are active-site inhibitors for
Factor VIIa exhibiting potency at the single-digit nanomolar
level (IC50) with excellent selectivity over thrombin (IIa)
and Factor Xa. In an effort to increase the potency and
influence the pharmocokinetic properties, other heterocyclic
ring systems were evaluated. Depicted in Figure 1 is the lead
pyrazinone core structure I. Focusing on the central ring,
one of the exercises was to replace the nitrogen at the
4-position in the pyrazinone ring with carbon, resulting in a
pyridone heterocycle as the core ring.6 Other groups have
disclosed the pyridone template in the design of human
leukocyte elastase (HLC) inhibitors7 and thrombin inhibi-
tors.8 We too were motivated by the attractiveness of the
pyridone template and the fact that it has not been reported
as a TF/VIIa inhibitor. Based on the structure activity
relationship established with the previously prepared
pyrazinone analogs, two targeted pyridone analogs were
selected for preparation. Herein, we describe their synthesis
and biological activity.
Several commercially available substituted pyridines were
evaluated in terms of potential starting material for the
We previously reported the preparation of pyrazinone
Keywords: 2-pyridones; tissue factor VIIa.
*
Corresponding author. Tel.: þ1-314-274-3494; fax: þ1-314-274-1621;
e-mail: john.j.parlow@pfizer.com
Figure 1.
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01239-0

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J. J. Parlow, M. S. South / Tetrahedron 59 (2003) 7695–7701
affording only starting material. However, reacting the
unsubstituted 3-amino-2,6-dibromopyridine 5 with sodium
methoxide in dioxane at reflux afforded exclusively the
desired regioisomer, 3-amino-6-bromo-2-methoxypyridine
6. The proton and carbon NMR of compound 6 was in
agreement with the predicted proton and carbon NMR
calculated by ACD10 with significant differences between
the two regioisomers evident in the proton NMR. It was
decided to fully characterize and conclusively determine the
structure at a later point in the synthesis, after alkylation,
allowing one set of experiments to determine the correct
isomer from a potential set of four isomers.
Thus with the key precursor 6 in hand, the synthesis of the
desired targets was conducted as shown in Scheme 3. Several
reductive amination conditions were surveyed. The pre-
formation of the imine using titanium tetrachloride with
acetone followed by reductionwith sodium cyanoborohydride
afforded cleanly the 6-bromo-2-methoxy-3-isopropylamino-
pyridine 8.11 The methyl ether 8 was cleaved using boron
tribromide to afford the hydroxypyridine 9. Alkylation of the
hydroxypyridine 9 with t-butylbromoacetate using calcium
hydride as the base afforded a single isomer 10. While
alkylation could occur on either the nitrogen or the oxygen,
only one regioisomer was isolated. At this point of the
synthesis, compound 10 could be one offour possible isomers
10, 16, 17, and 18 as shown in Figure 2. Compounds 10 and 16
would result from methoxide displacement of 2,6-dibromo-
pyridine 5 (Scheme 2) at the 2-position, suggested by the
proton and carbon data, but not definitively determined.
Methoxide displacement at the 6-position of 2,6-dibromopyr-
idine 5 would ultimately result in compounds 17 and 18. Each
of these substituted hydroxypyridines could undergo alkyl-
ation of the nitrogen or oxygen, affording the potential
combination of all regioisomers depicted in Figure 2.
Scheme 1.
synthesis. It was decided to proceed with the synthesis using
readily available, inexpensive 2,6-dibromopyridine 1 as the
starting material shown in Scheme 1. The dibromopyridine
1 was nitrated according to the literature procedure to afford
2,6-dibromo-3-nitropyridine 2.9 Several conditions were
attempted to selectively displace the 2-bromo with hydroxide.
At room temperature, incomplete reaction occurred or when
heat was applied, nonregioselective displacement at both the
2- and 6-position occurred. Complete conversion of the
starting material to a mono-substituted pyridine was accom-
plished using two equivalents of potassium hydroxide in
dioxane at room temperature disappointingly resulting in the
wrong regioisomer, 6-hydroxypyridine 3, as the major
product.
In an effort to reduce the potential steric crowding at the
2-position and the electronic nature of the pyridine ring, the
nitro group of compound 2 was reduced with iron to afford
the 3-amino-2,6-dibromopyridine 5 (Scheme 2). Next,
reductive amination of the nitrogen followed by nucleo-
philic displacement of the 2-bromo was attempted. The
reductive amination of 3-amino-2,6-dibromopyridine 5 with
acetone using sodium triacetoxyborohydride afforded the
3-isopropylamino-2,6-dibromopyridine 7. Reaction of 3-
isopropylamino-2,6-dibromopyridine 7 with potassium
hydroxide at reflux in dioxane/water resulted in no reaction,
A considerable amount of data was collected, including
proton, carbon, HSQC, Inadequate, and a HMBC experi-
ment to fully characterize and definitively assign a structure.
The proton NMR spectrum was relatively simple and easy
to assign by inspection. The only minor point was to assign
each aromatic proton and that was determined from a
combination of HSQC and Inadequate data. The various
carbon assignments were determined from the HSQC and
HMBC spectra. One of the key correlations in this spectrum
was the one connecting carbons 2 and 3 (Fig. 3). This piece
of data was critical to provide definitive proof of the
structure. Also important was to identify the other carbonyl
Scheme 2.
Figure 2. All possible regioisomers.

J. J. Parlow, M. S. South / Tetrahedron 59 (2003) 7695–7701
7697
Scheme 3.
carbon as carbon 8 by its correlation to carbon 7. In addition
to the previously mentioned data, there were other key
pieces of information that were critical for determining the
structure. There were two long-range correlations for the
H15/H20 and H14 protons to carbon 3 (136.8 ppm) and in
the Inadequate spectrum there was a correlation from this
same carbon (C3) to the carbonyl carbon (C2). This data
taken together is definitive proof of the structure. The key
correlations and carbon–carbon connections obtained from
the data are shown in Figure 3.
The completion of the targeted pyridones in Scheme 3 was
carried out by performing Suzuki couplings of the
N-alkylated bromopyridone 10 with the boronic acids 11,
affording the biaryl compounds 12. Selective hydrolysis of
the t-butyl ester with hydrochloric acid afforded the
carboxylic acids 13. [Note: Using heat during the evapor-
ation of the solvent in the workup of acid 13a led to
decarboxylation to afford the N-methyl derivative (benzyl 3-
[5-(isopropylamino)-1-methyl-6-oxo-1,6-dihydropyridin-2-
yl]-5-nitrobenzoate). As a result, evaporation of the solvent
was conducted using a stream of nitrogen to afford the acids
Figure 3. Key correlations.

7698
J. J. Parlow, M. S. South / Tetrahedron 59 (2003) 7695–7701
Table 1. IC50 values
potassium hydroxide (1.6 g, 28 mmol) in water (30 mL) and
dioxane (20 mL) was stirred at room temperature for 20 h.
The solution was diluted with 2N hydrochloric acid and
extracted with ether. The organic layer was washed with
water, brine, dried over magnesium sulfate, and filtered. The
solvent was removed by evaporation to afford 2.97 g (97%)
Compound
IC50 (uM)
Xa
VIIa
Thrombin
15a
15b
0.118
0.052
.30
.30
.30
.30
1
of a white solid of product 3; H NMR ppm (deuterio-
methanol): 6.61 (d, 1H, J¼8.4 Hz), 8.10 (d, 1H, J¼8.4 Hz);
13C NMR ppm (deuteriomethanol): 112.8, 133.6, 137.1,
145.7, 163.7; HPLC purity (retention time): 90%
(1.67 min); HRMS calcd for C5H3Br1N2O3 (MþþH)
217.9327, found 217.9324.
13.] The carboxylic acids 13 were coupled with benzyl
amino[4-(aminomethyl)phenyl]methylenecarbamate using
polymer-bound carbodiimide, hydroxybenzotriazole, and
N-methylmorpholine as base to afford the desired products
14. Concomitant deprotection of the Cbz (and benzyl ester
with compound 14b) and reduction of the nitro group was
accomplished using hydrogen with palladium on carbon to
afford the desired target compounds 15.
1.1.2. 3-Amino-2,6-dibromopyridine (5). 2,6-Dibromo-3-
nitropyridine 2 (6.8 g, 24.3 mmol) was stirred in glacial
acetic acid (30 mL). Powdered iron (6.7 g, 119 mmol) was
added and the solution was heated to 808C with vigorous
stirring. The solution was stirred at 808C for 15 min at which
point the iron had turned gray. The reaction mixture was
filtered through celite and the solid was washed with ether
and ethyl acetate. The resultant organic layer was washed
with water, brine, dried over magnesium sulfate, and
filtered. The solvent was removed to afford the crude
product. The product was purified by column chromato-
graphy (20% ethyl acetate–hexane) to afford 5.26 g (87%)
of a white solid of product 5; 1H NMR ppm (deuteriochloro-
form): 3.89 (s, 2H), 6.89 (d, 1H, J¼8.4 Hz), 7.10 (d, 1H,
J¼8.4 Hz); 13C NMR ppm (deuteriochloroform): 124.8,
125.7, 126.9, 127.7, 141.8; HPLC purity (retention time):
.99% (2.95 min); HRMS calcd for C4H4Br2N2 (MþþH)
252.8799, found 252.8779.
The target compounds 15a and 15b were screened for
potency on TF/VIIa and for other enzymes affecting
coagulation to determine specificity (Table 1). Each enzyme
assay consists of the specific enzyme and chromogenic
substrate for that enzyme. Enzyme activity was determined
by monitoring the increase in absorbance at 405 nm caused
by the release of p-nitroaniline when the substrate is
hydrolyzed. Inhibition of the enzyme reduces the change in
absorbance with the data reported as IC50 values. Com-
pounds 15a and 15b are both active against TF/VIIa with an
IC50 of 118 and 52 nM, respectively. Both compounds are
selective over thrombin and Factor Xa. However, both
pyridones 15a and 15b are approximately 5X less potent
against TF/VIIa than their corresponding pyrazinone
analogs.5
1.1.3. 6-Bromo-2-methoxypyridin-3-amine (6). A sol-
ution of 2,6-dibromo-3-aminopyridine
5
(10.4 g,
In summary, targeted pyridones were successfully prepared
via multi-step synthesis using various transformations and
regioselective additions with a pyridine nucleus to afford
novel pyridone derivatives. The analogs were tested against
TF/VIIa but were less potent than their corresponding
pyrazinone analogs. With no improved potency and a lack
of favorable pharmacokinetics, further syntheses using the
pyridone as the core ring were not pursued.
41.5 mmol) and sodium methoxide (15.7 g, 290 mmol) in
dioxane (50 mL) was heated to reflux for 48 h. The brown
reaction was allowed to cool to room temperature and
diluted with saturated solution of ammonium chloride. The
solution was extracted with ether and the organic layer was
washed with water, brine, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation to
afford the crude product. The product was purified by
column chromatography (20% ethyl acetate–hexane) to
afford 7.1 g (84%) of a red solid of product 6; 1H NMR ppm
(deuteriochloroform): 3.94 (s, 3H), 6.71 (d, 1H, J¼6.0 Hz),
6.83 (d, 1H, J¼6.0 Hz); 13C NMR ppm (deuteriochloro-
form): 67.2, 120.4, 122.5, 124.1, 130.3, 152.4; HPLC purity
(retention time): 94% (2.92 min); HRMS calcd for
C6H7Br1N2O (MþþH) 204.9800, found 204.9769.
1. Experimental
1.1. General
1H and 13C NMR spectra were recorded using a 300 and
400 MHz NMR spectrometer. Sample purities were deter-
mined by HPLC analysis equipped with a mass spec
detector using a C18 3.5 mm 30£2.1 mm column, eluting
with a gradient system of 5:95 to 95:5 acetonitrile/water
with a buffer consisting of 0.1% TFA over 6 min at
1 mL/min and detected by UV at 254 and 210 nm using a
diode array detector. Column chromatography was per-
formed on a preparative liquid chromatography instrument
using silica gel columns and on a HPLC system using a
15um 100A, C18 column (25 mm ID£100 mm L). Reported
yields are not optimized with emphasis on purity of products
rather than quantity.
1.1.4.
2,6-Dibromo-3-isopropylaminopyridine
(7).
Sodium triacetoxyborohydride (5.5 g, 25.9 mmol) was
added to a solution of the aminopyridine 5 (1.6 g,
6.5 mmol), acetone (5.0 mL, excess), and a drop of acetic
acid in a tetrahydrofuran-dichloromethane (1:1) solution
(50 mL). After stirring at room temperature for 18 h the
reaction was heated to reflux for 24 h. The solution
was diluted with ether and water. The organic layer was
washed with brine, dried over magnesium sulfate, and
filtered. The solvent was removed by evaporation to afford
the crude product. The product was purified by column
chromatography (20% ethyl acetate–hexane) to afford
1.1.1. 2-Bromo-6-hydroxy-3-nitropyridine (3). A solution
of 2,6-dibromo-3-nitropyridine (2) (3.9 g, 14 mmol) and
1
0.56 g (30%) of a yellow oil of product 7; H NMR ppm

J. J. Parlow, M. S. South / Tetrahedron 59 (2003) 7695–7701
7699
(deuteriochloroform): 1.27 (d, 6H, J¼4.8 Hz), 3.58 (septet,
1H, J¼4.8 Hz), 6.74 (d, 1H, J¼8.4 Hz), 7.26 (d, 1H,
J¼8.4 Hz); 13C NMR ppm (deuteriochloroform): 22.7, 44.7,
120.2, 123.9, 127.6, 128.1, 141.3; LRMS for C10H10N1Br2
(ES, m/z) 293 (MþH).
by column chromatography (10% ethyl acetate–hexane) to
afford 4.1 g (66%) of a white solid of product 10; 1H NMR
ppm (deuteriochloroform): 1.08 (H20,H15) (d, 6H,
J¼6.4 Hz), 1.37 (H11,18,19) (s, 9H), 3.42 (H14) (dsept,
1H, J¼6.4, 8.4 Hz), 4.81 (H7) (s, 2H), 5.05 (NH13) (d, 1H,
J¼8.4 Hz), 6.13 (H4) (d, 1H, J¼7.9 Hz), 6.46 (H5) (d, 1H,
J¼7.9 Hz); 13C NMR ppm (DMSO): 22.3 (C15,C20), 28.2
(C11,18,19), 43.6 (C14), 50.7 (C7), 82.5 (C10), 107.0 (C4),
107.8 (C6), 111.8 (C5), 136.8 (C3), 158.4 (C2), 167.1 (C8);
HPLC purity (retention time): 98% (4.63 min); HRMS calcd
for C14H21Br1N2O3 (MþþH) 345.0814, found 345.0827.
1.1.5. 6-Bromo-N-isopropyl-2-methoxypyridin-3-amine
(8). A solution of titanium tetrachloride 1 M in dichloro-
methane (37 mL, 37 mmol) was added to a solution of
6-bromo-2-methoxypyridin-3-amine 6 (6.8 g, 33.6 mmol)
and acetone (3.3 mL, 44.9 mmol) in dichloromethane
(100 mL). After stirring at room temperature for 3 h,
sodium cyanoborohydride (6.3 g, 100 mmol) was added
and the solution stirred at room temperature for 14 h. The
solution was diluted with ether and water. The organic layer
was washed with brine, dried over magnesium sulfate, and
filtered. The solvent was removed by evaporation to afford
the crude product. The product was purified by column
chromatography (5% ethyl acetate–hexane) to afford 5.7 g
1.3. Benzyl 3-[1-(2-tert-butoxy-2-oxoethyl)-5-
(isopropylamino)-6-oxo-1,6-dihydropyridin-2-yl]-5-
nitrobenzoate (12a)
Tetrakis(triphenylphosphine)palladium(0)
(0.20 g,
0.17 mmol) was added to a solution of compound 10
(0.60 g, 1.7 mmol), boronic acid 11a (0.68 g, 2.3 mmol),
and cesium carbonate (2.2 g, 6.7 mmol) in anhydrous
dioxane (18 mL). The resulting mixture stirred for at 808C
for 16 h. The reaction mixture was allowed to cool to room
temperature and was diluted with water and extracted with
diethyl ether. The organic layer was washed with water,
brine, dried over magnesium sulfate, and filtered. The
solvent was removed by evaporation to afford the crude
product. The product was purified by column chromato-
graphy (25% ethyl acetate–hexane) to afford 0.70 g (78%)
of a red solid of product 12a; 1H NMR ppm (deuteriochloro-
form): 1.24 (d, 6H, J¼4.8 Hz), 1.39 (s, 9H), 3.57 (septet,
1H, J¼4.8 Hz), 4.41 (s, 2H), 5.39 (s, 2H), 6.14 (d, 1H,
J¼5.7 Hz), 6.40 (d, 1H, J¼5.7 Hz), 7.40 (m, 6H), 8.35 (m,
1H), 8.42 (m, 1H), 8.85 (m, 1H); HPLC purity (retention
time): .99% (5.70 min); HRMS calcd for C28H31N3O7
(MþþH) 522.2240, found 522.2242.
1
(69%) of a red oil of product 8; H NMR ppm (deuterio-
chloroform): 1.20 (d, 6H, J¼4.8 Hz), 3.51 (septet, 1H,
J¼4.8 Hz), 3.95 (s, 3H), 6.62 (d, 1H, J¼6.0 Hz), 6.88 (d,
1H, J¼6.0 Hz); HPLC purity (retention time): 95%
(3.97 min); HRMS calcd for C9H13Br1N2O (MþþH)
245.0289, found 245.0240.
1.1.6. 6-Bromo-3-(isopropylamino)pyridin-2-ol (9). A
solution of boron tribromide 1 M in dichloromethane
(46.0 mL, 46 mmol) was added to a solution of 6-bromo-
N-isopropyl-2-methoxypyridin-3-amine
8
(5.6 g,
0.23 mmol) in dichloromethane (150 mL) at 2108C. The
solution warmed to room temperature and stirred for 16 h.
The reaction mixture was diluted with water, neutralized
with a saturated sodium bicarbonate solution, and extracted
with ether. The organic layer was washed with water, brine,
dried over magnesium sulfate, and filtered. The solvent was
removed by evaporation to afford the crude product. The
product was purified by column chromatography (40% ethyl
acetate–hexane) to afford 4.12 g (78%) of a white solid of
product 9; 1H NMR ppm (deuteriochloroform): 1.22 (d, 6H,
J¼4.8 Hz), 3.49 (septet, 1H, J¼4.8 Hz), 6.20 (m, 1H), 6.88
(d, 1H, J¼6.0 Hz); 13C NMR ppm (deuteriochloroform):
22.3, 44.1, 110.0, 128.6, 132.2, 136.4, 160.0; HPLC purity
(retention time): 94% (2.45 min); HRMS calcd for
C8H11Br1N2O (MþþH) 231.0133, found 231.0130.
1.4. tert-Butyl [6-{3-[(tert-butoxycarbonyl)amino]-5-
nitrophenyl}-3-(isopropylamino)-2-oxopyridin-1(2H)-
yl]acetate (12b)
Following the same procedure described for 12a, tetrakis
(triphenylphosphine)palladium(0) (0.33 g, 0.28 mmol),
compound 10 (1.0 g, 2.9 mmol), boronic acid 11b (1.3 g,
4.6 mmol), and cesium carbonate (3.7 g, 11.3 mmol) were
used to give 0.57 g (39%) of an orange solid of product 12b;
1H NMR ppm (deuteriochloroform): 1.24 (d, 6H,
J¼4.8 Hz), 1.40 (s, 9H), 1.49 (s, 9H), 3.67 (septet, 1H,
J¼4.8 Hz), 4.43 (s, 2H), 6.14 (m, 2H), 7.20 (m, 1H), 7.64
(m, 1H), 7.82 (m, 1H), 8.35 (m, 1H); HPLC purity (retention
time): 96% (5.37 min); LRMS for C25H34N4O7 (ES, m/z)
503 (MþþH).
1.2. tert-Butyl [6-bromo-3-(isopropylamino)-2-oxo-
pyridin-1(2H)-yl]acetate (10)
A suspension of calcium hydride (1.7 g, 40.3 mmol) in
tetrahydrofuran (20 mL) was added to 6-bromo-3-(isopro-
pylamino)pyridin-2-ol 9 (4.2 g, 18 mmol) in tetrahydro-
furan (200 mL) dropwise via an addition funnel. The
resulting suspension was heated to reflux for 30 min. To
the mixture was then added a solution of tert-butyl
bromoacetate (2.9 mL, 19.6 mmol) in tetrahydrofuran
(2.3 M). Refluxing of the mixture was continued for 18 h.
The reaction mixture was allowed to cool to room
temperature, and quenched with an ice water mixture. The
aqueous layer was extracted with diethyl ether. The organic
layer was washed with water, brine, dried over magnesium
sulfate, and filtered. The solvent was removed by evapor-
ation to afford the crude product. The product was purified
1.5. [6-{3-[(Benzyloxy)carbonyl]-5-nitrophenyl}-3-
(isopropylamino)-2-oxopyridin-1(2H)-yl]acetic acid 13a
The tert-butyl ester 12a (0.60 g, 1.1 mmol) was dissolved
into 4N hydrochloric acid in dioxane (3 mL) and stirred at
room temperature for 22 h. The solvent was removed under
a stream of nitrogen (NOTE! heating the acid results in
decarboxylation to afford the N-methyl derivative, benzyl 3-
[5-(isopropylamino)-1-methyl-6-oxo-1,6-dihydropyridin-2-
yl]-5-nitrobenzoate) to afford a red gum of product 13a; 1H
NMR ppm (deuteriochloroform): 1.53 (d, 6H, J¼4.8 Hz),

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J. J. Parlow, M. S. South / Tetrahedron 59 (2003) 7695–7701
3.86 (septet, 1H, J¼4.8 Hz), 4.55 (s, 2H), 5.39 (s, 2H), 6.42
(d, 1H, J¼5.4 Hz), 7.39 (m, 5H), 8.29 (d, 1H, J¼5.7 Hz),
8.37 (m, 1H), 8.44 (m, 1H), 8.94 (m, 1H); HPLC purity
(retention time): 78% (4.67 min); HRMS calcd for
C24H23N3O7 (MþþH) 466.1614, found 466.1625.
HPLC purity (retention time): 80% (3.41 min); HRMS calcd
for C32H33N7O6 (MþþH) 612.2565, found 612.2521.
1.9. 3-Amino-5-[1-[2-({4-[amino(imino)methyl]
benzyl}amino)-2-oxoethyl]-5-(isopropylamino)-6-oxo-
1,6-dihydropyridin-2-yl]benzoic acid (15a)
1.6. [6-(3-Amino-5-nitrophenyl)-3-(isopropylamino)-2-
oxopyridin-1(2H)-yl]acetic acid 13b
A catalytic amount of palladium on carbon (10%) in
methanol (5 mL) was added to compound 14a (0.33 g,
0.45 mmol) in methanol (20 mL) and the mixture was
stirred under a balloon of hydrogen at room temperature for
6 h. The mixture was filtered through celite and the solvent
was evaporated to afford the product. The product was
purified by reverse-phase chromatography to afford 0.28 g
Following the same procedure described for 13a, tert-butyl
ester 12b (0.50 g, 0.10 mmol), was used to afford a yellow
1
solid of product 13b; H NMR ppm (deuteriomethanol):
1.38 (d, 6H, J¼4.8 Hz), 3.87 (septet, 1H, J¼4.8 Hz), 4.63 (s,
2H), 6.48 (d, 1H, J¼5.4 Hz), 7.42 (m, 1H), 7.82 (m, 2H),
7.82 (m, 1H), 8.00 (m, 1H); HPLC purity (retention time):
90% (2.72 min); HRMS calcd for C16H18N4O5 (MþþH)
347.1350, found 347.1334.
1
(88%) of a yellow solid of product 15a; H NMR ppm
(deuteriomethanol): 1.26 (d, 6H, J¼5.1 Hz), 3.63 (septet,
1H, J¼5.1 Hz), 4.44 (d, 2H, J¼4.2 Hz), 4.65 (d, 2H,
J¼7.2 Hz), 6.27 (dd, 1H, J¼0.6, 5.7 Hz), 6.78 (dd, 1H,
J¼5.7, 13.8 Hz), 7.62 (m, 9H); HPLC purity (retention
time): .99% (2.39 min); HRMS calcd for C25H28N6O4
(MþþH) 477.2250, found 477.2253.
1.7. Benzyl 3-[1-(2-{[4-((E)-amino{[(benzyloxy)-
carbonyl]imino}methyl)benzyl]amino}-2-oxoethyl)-5-
(isopropylamino)-6-oxo-1,6-dihydropyridin-2-yl]-5-
nitrobenzoate (14a)
1.10. N-{4-[Amino(imino)methyl]benzyl}-2-[6-(3,5-
diaminophenyl)-3-(isopropylamino)-2-oxopyridin-
1(2H)-yl]acetamide (15b)
Polystyrene–carbodiimide (2.3 g, 2.3 mmol) (1.00 mmol/g)
was added to a slurry of the acid 13a (0.53 g, 1.1 mmol), 1-
hydroxybenzotriazole (0.16 g, 0.71 mmol), benzyl amino[4-
(aminomethyl)phenyl]methylcarbamate (0.44 g, 1.3 mmol),
and N-methylmorpholine (1.3 mL, 11.8 mmol) in a dichloro-
methane-N,N-dimethylformamide (3:1) solution (100 mL)
and the suspension was agitated for 13 h. Upon completion
of the reaction, the polyamine resin (2.81 mmol/g)
(5.6 mmol) and polymer-bound aldehyde (2.3 mmol/g)
(2.30 mmol) were added and the suspension was agitated
for 1 h. The solution was filtered and the polymer was rinsed
with N,N-dimethylformamide and dichloromethane until no
more UV activity was seen in the dichloromethane washing.
The solvent was removed by evaporation to afford the crude
product. The product was purified by reverse-phase
chromatography to afford 0.73 g (87%) of an orange solid
Following the same procedure described for 15a, compound
14b (0.20 g, 0.32 mmol) was used to afford the product. The
product was purified by reverse-phase chromatography to
1
afford 0.24 g (39%) of a brown solid of product 15b; H
NMR ppm (deuteriomethanol): 1.26 (d, 6H, J¼4.8 Hz),
3.63 (septet, 1H, J¼4.8 Hz), 4.44 (s, 2H), 4.65 (s, 2H), 6.27
(d, 1H, J¼5.7 Hz), 6.57 (d, 1H, J¼5.7 Hz), 6.65 (m, 3H),
7.55 (d, 2H, J¼6.0 Hz), 7.77 (d, 2H, J¼6.0 Hz); HPLC
purity (retention time): .99% (2.39 min); HRMS calcd for
C24H29N7O2 (MþþH) 448.2456, found 448.2447.
Acknowledgements
1
of product 14a; H NMR ppm (deuterioacetone): 1.19 (d,
6H, J¼4.8 Hz), 3.48 (septet, 1H, J¼4.8 Hz), 4.41 (d, 2H,
J¼9.3 Hz), 4.63 (s, 2H), 5.18 (s, 2H), 5.48 (s, 2H), 6.23 (d,
1H, J¼5.4 Hz), 6.31 (d, 1H, J¼5.4 Hz), 7.40 (m, 12H), 7.99
(m, 2H), 8.18 (bt, 1H), 8.58 (m, 1H), 8.68 (m, 1H), 8.78
(m, 1H); HPLC purity (retention time): 90% (4.50 min);
HRMS calcd for C40H38N6O8 (MþþH) 731.2829, found
731.2853.
The authors thank Rhonda M. LaChance for the biological
evaluation and Claude R. Jones for the NMR experiments
necessary for determination of structure.
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