Pyridoxine-derived bicyclic amido-, ureido-, and carbamato-pyridinols: Synthesis and antiangiogenic activities

Article abstract of DOI:10.1039/c4ob01221f

We recently developed an efficient and practical synthesis for a novel series of pyridoxine-derived 6-amido-2,4,5-trimethylpyridin-3-ols and found that this novel scaffold has outstanding activity to inhibit angiogenesis measured by the quantitative chick embryo chorioallantoic membrane (CAM) assay. As an effort to extend the scope of the amidopyridinol scaffold, we here report the synthesis and antiangiogenic activities of a series of bicyclic versions of the amidopyridinol including five- and six-membered cyclic amide-, cyclic urea-, and cyclic carbamate-fused pyridinols. The six membered bicyclic derivatives were prepared by the reported procedures, and the five-membered ring-fused ones were synthesized by new synthetic methods developed in this study. CAM assays showed that both six- and five-membered lactam-fused pyridinols have activities comparable to sunitinib malate, the positive control, in inhibition of vascular endothelial growth factor-induced angiogenesis. On the other hand, the urea and the carbamate derivatives showed modest to moderate antiangiogenic activities. In summary, some bicyclic aminopyridinols can provide a good platform for structural exploitation in future medicinal chemistry work.

Full text of DOI:10.1039/c4ob01221f

Organic &
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Pyridoxine-derived bicyclic amido-, ureido-,
and carbamato-pyridinols: synthesis and
antiangiogenic activities†
Cite this: Org. Biomol. Chem., 2014,
12, 8702
Hyunji Lee,a Dong-Guk Kim,a Suhrid Banskota,a You Kyoung Lee,a Tae-gyu Nam,*b
Jung-Ae Kim*a and Byeong-Seon Jeong*a
We recently developed an efficient and practical synthesis for a novel series of pyridoxine-derived
6-amido-2,4,5-trimethylpyridin-3-ols and found that this novel scaffold has outstanding activity to inhibit
angiogenesis measured by the quantitative chick embryo chorioallantoic membrane (CAM) assay. As an
effort to extend the scope of the amidopyridinol scaffold, we here report the synthesis and antiangiogenic
activities of a series of bicyclic versions of the amidopyridinol including five- and six-membered cyclic
amide-, cyclic urea-, and cyclic carbamate-fused pyridinols. The six membered bicyclic derivatives were
prepared by the reported procedures, and the five-membered ring-fused ones were synthesized by new
synthetic methods developed in this study. CAM assays showed that both six- and five-membered
lactam-fused pyridinols have activities comparable to sunitinib malate, the positive control, in inhibition of
vascular endothelial growth factor-induced angiogenesis. On the other hand, the urea and the carbamate
derivatives showed modest to moderate antiangiogenic activities. In summary, some bicyclic aminopyridi-
nols can provide a good platform for structural exploitation in future medicinal chemistry work.
Received 13th June 2014,
Accepted 9th September 2014
DOI: 10.1039/c4ob01221f
www.rsc.org/obc
We recently reported an efficient and practical synthetic
method for a series of 6-amido-2,4,5-trimethylpyridin-3-ols (2)
Introduction
Some cancer cells have the ability to penetrate into blood or starting from pyridoxine hydrochloride (1, vitamin B6) and
lymphatic vessels, circulate through the blood stream, and their antiangiogenic activities (Fig. 1).7 They showed higher
invade other parts of the body, which is called metastasis.1 levels of inhibition against VEGF-induced angiogenesis in the
Angiogenesis, the formation of a new network of blood quantitative chick embryo chorioallantoic membrane (CAM)
vessels,2 is one of the essential events required in metastasis.3 assay8 than two positive controls, SU4312 and sunitinib.
The central role of angiogenesis in tumor growth and meta- SU4312 is well known as a selective and potent VEGF-receptor
stasis is to transport nutrients and metabolites. In order to tyrosine kinase inhibitor.9 Sunitinib, a malate salt, which is
initiate angiogenesis, tumor cells send signals to surrounding currently marketed as Sutent® by Pfizer, is a multi-targeted
normal endothelial cells by releasing proteins or small mole-
cules. Vascular endothelial growth factor (VEGF), one of the
most important endogenous angiogenics, is produced by
many kinds of cancer cells to promote an angiogenic signaling
cascade towards the creation of new blood vessels.4 Because
angiogenesis is necessary for the growth and spread of cancer,
inhibition of the angiogenic process is considered an attractive
cancer target5 and the discovery of angiogenic inhibitors has
been receiving increasing attention for decades.6
aInstitute for Drug Research and College of Pharmacy, Yeungnam University,
Gyeongsan 712-749, Republic of Korea. E-mail: jakim@yu.ac.kr, jeongb@ynu.ac.kr
bDepartment of Pharmacy and Institute of Pharmaceutical Science and Technology,
Hanyang University, Ansan 426-791, Republic of Korea.
E-mail: tnam@hanyang.ac.kr
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of all new compounds. See DOI: 10.1039/c4ob01221f
Fig. 1 Vitamin B6-derived amidopyridinols.
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receptor tyrosine kinase inhibitor for the treatment of renal (4C), were unknown compounds, so we developed new syn-
cell carcinoma and gastrointestinal stromal tumor.10
thetic methods for these novel series (Schemes 1–3).
Remarkably, the majority of 6-amido-2,4,5-trimethylpyridin-
As shown in Scheme 1, pyrrolidinone-fused bicyclic pyridi-
3-ol (2) analogues we have prepared showed good to excellent nol 4A was synthesized from DPN-HCl (5). In an initial syn-
antiangiogenic activities (up to 100-fold better than sunitinib). thetic attempt, we wanted to synthesize 4A starting from
This suggests that the 6-amidopyridin-3-ol skeleton may be a DPN-HCl without any protection/deprotection step in the same
very important core scaffold and could be highly useful for the manner as the synthesis of 3A. We first chlorinated the
treatment of angiogenesis-related diseases.7 As a part of our primary alcohol of 5 using thionyl chloride which afforded the
ongoing efforts to expand the scope of the amidopyridinol corresponding chloro-compound in good yield, but sub-
scaffold, we here report a series of bicyclic versions of the sequent replacement of the chloride with cyanide was not
amidopyridinol. In this study, six basic bicyclic amidopyridi- quite successful (35% yield at best). This low yield is mainly
nol analogues fused with six- or five-membered lactam, cyclic caused by side reactions and by tricky pH control during the
urea, and cyclic carbamate (3 and 4 in Fig. 1) were prepared work-up process. Then, the nitrile compound was subjected to
and their antiangiogenic activities were evaluated.
hot ethanolic sulfuric acid to afford the corresponding ethyl
Results and discussion
Preparation of six-membered bicyclic pyridinols (3A–C)
Bicyclic pyridinols annulated with six-membered rings, i.e.
piperidinone (3A), tetrahydropyrimidinone (3B), and oxazina-
none (3C), were prepared according to the reported synthetic
procedures (Fig. 2).11 Briefly, we first reported the synthesis of
the amido-analogue (3A) in 2010,11a and both the ureido- (3B)
and the carbamato-analogue (3C) were synthesized by the
Porter group in 2013.11b All three analogues (3A–C) share a
common starting material: 4-deoxypyridoxine hydrochloride
(5, DPN-HCl) which was prepared from pyridoxine hydro-
chloride (1, PN-HCl). PN-HCl (1) is readily available at relatively
cheap price from chemical vendors whereas DPN-HCl (5) is
about one hundred times more expensive than PN-HCl (1). We
have already reported an efficient and practical procedure for
the preparation of DPN-HCl (5) from PN-HCl (1).11a In this
study, we have achieved notable improvements, particularly in
terms of ease of purification (see the Experimental section).
Scheme 1 Synthesis of the pyrrolidinone-fused bicyclic pyridinol (4A,
5-hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one). Reagents
and conditions: (a) PhCH2Cl, K2CO3, DMF, r.t., 12 h, 80%; (b) SOCl2, DMF,
ClCH2CH2Cl, reflux, 1 h, 95%; (c) KCN, DMF, 40 °C, 2 d, 96%; (d) H2SO4,
EtOH, reflux, 12 h, 88%; (e) PhNH2, NaNO2, 6 M HCl, 10 M NaOH, H2O–
THF, 0 °C, 1 h → r.t., 2 h, 87%; (f) Zn, AcOH, reflux, 12 h, 87%.
Synthesis of five-membered bicyclic pyridinols (4A–C)
Three bicyclic pyridinols fused with five-membered rings, i.e.
pyrrolidinone (4A), imidazolidinone (4B), and oxazolidinone
Scheme 2 Synthesis of the imidazolidinone-fused bicyclic pyridinol
(4B,
6-hydroxy-5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one).
Reagents and conditions: (a) NaOCl, TEMPO, NaHCO3, CH2Cl2–H2O, r.t.,
10 min, 94%; (b) (i) I2, NH4OH, THF–H2O, r.t., 1 h; (ii) H2O2, NH4OH,
THF–H2O, r.t., 2 h, 90% for 2 steps; (c) m-CPBA, CH2Cl2, r.t., 1 h, 88%;
(d) p-TsCl, phthalimide, i-Pr2NEt, r.t., 1 h, 78%; (e) N2H4, THF–EtOH, r.t.,
12 h, 89%; (f) PhI(OAc)2, KOH, MeOH, 0 °C, 1 h, 87%; (g) H2, Pd/C,
MeOH, r.t., 2 h, 99%.
Fig. 2 Preparation of the six-membered bicyclic pyridinols (3A–C) from
vitamin B6 by known procedures.
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overall yield, which demonstrates the efficiency and practical
usefulness of this synthetic route.
Scheme 2 shows the synthesis of the imidazolidinone-fused
bicyclic pyridinol 4B from the benzyl-protected DPN (6). The
structure of 4B demands that the carbon atom attached at the
C(5)-position of the starting material 6 be converted into a
nitrogen in the end. To this end, we devised a synthetic strat-
egy using Hofmann rearrangement of the corresponding
primary amide 15. First, oxidation of the primary alcohol of 6
to aldehyde 11 was readily carried out under TEMPO-mediated
oxidation conditions. Then, conversion of the aldehyde into
the primary amide was done by a two-step sequence in 90%
overall yield; treatment of the aldehyde 6 with molecular
iodine and aqueous ammonia afforded the corresponding
nitrile, which was then treated with hydrogen peroxide to give
the primary amide 12. We also conducted a one-pot oxidative
conversion of the alcohol 6 directly to the amide 12 with mole-
cular iodine in aqueous ammonia and hydrogen peroxide solu-
tion, which only resulted in moderate yield (68%).12 As for the
introduction of an amino group to the C(6)-position, we
noticed that electrophilic aromatic azo coupling reaction on
compound 12 with a benzenediazonium ion, which was
successful in the transformation of 9 to 10 shown in Scheme 1,
was not very effective when the C(3)–OH group was protected.
Therefore, we envisioned introducing a nitrogen-containing
functionality to the C(6)-position through nucleophilic attack
of a nitrogen nucleophile on a pyridine N-oxide substrate in the
presence of an activator.13 With N-oxide substrate 13 in hand
by oxidation of 12 with m-CPBA, we found the best conditions
for 14: p-TsCl as an activator and phthalimide as a nitrogen
nucleophile in the presence of the Hünig base. The free amino
moiety was finally liberated from the imide 14 under a mild
hydrazinolysis condition to give 15. Hofmann rearrangement of
the primary amide 15 using a hypervalent iodine reagent, dia-
cetoxyiodobenzene, smoothly afforded the corresponding iso-
cyanate, which was trapped intramolecularly by the C(6)–amino
group to form a cyclic urea 16 in good yield.14 The final
product 4B was obtained in almost quantitative yield, after
hydrogenolysis of the benzyl ether of 16. The overall yield from
PN-HCl (1) to the imidazolidinone-annulated bicyclic pyridinol
4B was about 33% in nine steps.
Scheme 3 Synthesis of the oxazolidinone-fused bicyclic pyridinol (4C,
6-hydroxy-5,7-dimethyloxazolo[4,5-b]pyridin-2(3H)-one). Reagents and
conditions: (a) NaOCl, NaOH, THF–H2O, r.t., 1 h → 90 °C, 2 h, 99%; (b)
NaNO2, H2SO4, THF–H2O, 0 °C, 1 h → r.t., 2 h, 81%; (c) PhNH2, NaNO2,
6 M HCl, 2.5 M NaOH, H2O–THF, 0 °C, 1 h → r.t., 1 h, 91%; (d)
Zn, HCO2H, MeOH, reflux, 1 h, 83%; (e) CDI, DMF, r.t., 12 h, 93%; (f) H2,
Pd/C, MeOH, r.t., 2 h, 99%.
ester in 56% yield. The low overall yields (ca. 20%) for these
two steps from DPN-HCl (5) led us to devise a new synthetic
route with higher yields and easier operations. Selective benzyl
protection of the phenolic C(3)–OH over the C(5′)–OH of
DPN-HCl (5) was achieved in 74% yield under mild reaction
conditions (i.e. benzyl chloride with a carbonate base in DMF
at room temperature). It should be noted that stronger reaction
conditions, such as benzyl bromide, hydroxide/alkoxide base,
or elevated reaction temperature, etc., resulted in low yields
due to extra benzylation on the C(5′)–OH and/or on the nitro-
gen atom in the pyridine ring. The primary alcohol in 6 was
then chlorinated using thionyl chloride and a catalytic amount
of DMF in high yield. Displacement of chloride in 7 by nucleo-
philic cyanide was conducted with potassium cyanide in warm
DMF, where a long reaction time was required for the high
yield. It was important that the starting material (7) be clearly
dissolved in DMF, otherwise the reaction was hardly triggered.
In addition, a reaction temperature over 80 °C seemed to lead
to decomposition of the initially formed, desired product.
Overall yield for the three steps from DPN-HCl (5) to the nitrile
compound (8) was 73%.
Next, conversion of the nitrile of 8 into the ethyl ester func-
tional group was accomplished under modified Pinner reac-
tion conditions in which a minimum of 30 equivalents of
sulfuric acid were needed for high yield. Interestingly, the
benzyl protection group on C(3)–OH was quantitatively
removed concomitantly under these acidic ethanolysis con-
ditions, saving us a deprotection step that was otherwise un-
avoidable. Azo coupling of 9 with a benzenediazonium ion
formed in situ from aniline and nitrous acid afforded the C(6)–
nitrogen compound 10. Intramolecular reductive cyclization of
10 with zinc in hot acetic acid finally gave the bicyclic amido-
pyridinol 4A in good yield. In summary, the pyrrolidinone-
fused bicyclic pyridinol (4A) was synthesized through a seven-
step process starting from PN-HCl (1) in approximately 45%
Preparation of the oxazolidinone-annulated bicyclic pyridi-
nol 4C was accomplished in the following order (Scheme 3): (i)
introduction of a hydroxy group at the C(5)-position, (ii) intro-
duction of an amino group at the C(6)-position, and (iii) carb-
amate formation. First we converted the primary amido group
in 12 into a primary amino group by Hofmann rearrangement.
The classical reaction conditions using bromine and hydroxide
only gave moderate yield (66%) and were not reliably reprodu-
cible. A modified method using hypochlorite and hydroxide
was found to be very effective, providing almost quantitative
conversion. Next, the primary amino group of 17 was replaced
with a hydroxyl group via a diazonium ion, prepared in situ
from 17 using sulfuric acid and sodium nitrite, which was
then immediately reacted with water to give the substitution
product 18 in 81% yield. Aqueous tetrafluoroboric acid along
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with sodium nitrite, one of the typical reaction conditions for
this type of transformation, only gave the C(5)–fluoro product
in 65% yield.15 An amino group was then incorporated by the
following two-step sequence: (i) an electrophilic azo coupling
reaction with a benzenediazonium salt under the standard
conditions to afford 19 because an unprotected free hydroxyl
group is present at the ortho-position to the C(6)-position. (ii)
A subsequent reductive cleavage of the azo bond of 19 with
zinc and formic acid in a selective manner in which the benzyl
protection group remained intact. Carbamate formation of the
ortho-aminophenol 20 with 1,1′-carbonyldiimidazole gave the
oxazolidinone-fused bicycle 21, and the finishing deprotection
of the benzyl group afforded the final product 4C. In
summary, the bicyclic pyridinol annulated with oxazolidinone
4C was prepared in ten steps from PN-HCl (1) in 35% overall
yield.
Table 1 Inhibitory effects of the bicyclic amidopyridinols (3 and 4) on
VEGF-induced angiogenesis in vivoa
Treatment
VBP/MVb
Inhibitionc (%)
Inhibitory effects of the bicyclic pyridinols on VEGF-induced
angiogenesis in vivo
PBS
16.8 2.3
48.7 4.3*
19.5 3.6*
21.0 2.3*
28.5 2.7*
37.3 1.2*
21.3 1.1*
26.2 4.2*
38.3 3.1*
—
—
VEGF (20 ng per CAM)
VEGF (20 ng per CAM) +
compound (0.01 nmol per CAM)
22
3A
3B
3C
4A
4B
4C
91.7 11.4*
87.0 7.3*
59.8 9.6*
48.0 12.7*
88.8 4.3*
63.7 15.9*
30.9 8.9*
Investigation of the antiangiogenic activities of six bicyclic pyr-
idinols (3’s and 4’s) was carried out using the quantitative
CAM assay,8 one of the most widely used in vivo models of
angiogenesis.16 Pre-treatment of VEGF to CAMs significantly
increased the number of newly formed blood vessel branch
points compared to the control group treated with phosphate
buffered saline (PBS) (figures in Table 1). Each bicyclic pyridi-
nol (3, 4) and sunitinib malate (22), the positive control, were
treated with the VEGF-pretreated CAMs at a fixed dose
(0.01 nmol per CAM)17 and the results are shown in Table 1.
An obvious correlation between the bicyclic pyridinol structure
and the level of inhibition was observed. The inhibitory activity
was generally increased in the order of carbamato- (3C and 4C)
< ureido- (3B and 4B) < amido-analogues (3A and 4A) regard-
less of the ring size. In particular, the lactam analogues 3A
and 4A showed the best antiangiogenic activities, which were
comparable to that of sunitinib malate.
a Quantification of new branches formed from existing blood vessels
was performed. Photographs were imported into an Image software
program, to measure the new branch points. The data are expressed as
the mean S.E.M. of at least six chick embryos. Dose was 0.01 nmol
per CAM for both the pyridinols (3, 4) and sunitinib malate (22), the
positive control. b Vessel branch points/main vessel. c Inhibition (%) =
[[(the number of vessel branch points induced by VEGF) − (the
number of vessel branch points induced by the test compound and
VEGF)]/(the number of vessel branch points induced by VEGF)] × 100.
*p < 0.05 compared to the VEGF-treated CAM sample.
Dose-dependency and the 50% inhibitory dose (ID50) in the
VEGF-induced angiogenesis were then examined with the
selected compounds: 3A, 4A and sunitinib malate (22). As
shown in Fig. 3, both the lactam-fused bicyclic pyridinols
inhibited angiogenesis in a dose-dependent manner and were
found to have similar ID50 values, which were consistent with
the single-dose activities displayed in Table 1. In addition, we
also confirmed that they have comparable inhibitory abilities
to sunitinib malate, which suggests that the aminopyridinol
skeleton deserves careful investigation as a pivotal starting
point in drug discovery for angiogenesis-related diseases.
It would be interesting to consider compound 4A to share
structural features with sunitinib (i.e. five-membered lactam-
fused bicyclic substructure). Although the commonly shared
substructure covers only a small portion of both molecules, it
might be able to explain the origin of antiangiogenic activities
of both compounds. It would also be interesting to explore the
chemical space around the sunitinib structure and adopt deri-
Fig. 3 Dose–response curves and ID50 values of the selected
vatization strategies. One way to this end would be to prepare compounds.
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hybridized compounds between sunitinib and our pyridinols, were added K2CO3 (37.0 g, 268.93 mmol) and benzyl chloride
i.e. bicyclic pyridinol compounds with sunitinib side chains.
(9.1 mL, 79.09 mmol). The resulting mixture was stirred under
a nitrogen atmosphere for 12 h at room temperature. After
evaporation of DMF, the residue was diluted with EtOAc (1 L)
and the organic layer was washed with water (100 mL × 4). The
organic solution was dried over MgSO4 and concentrated. The
Conclusion
We have developed synthetic methods with high overall yields residue was purified by silica gel column chromatography
and convenient operations for novel pyrrolidinone-, imidazoli- (CHCl3–MeOH = 40 : 1 → 20 : 1) to give 6 (3.08 g, 80%) as a
dinone- and oxazolidinone-fused bicyclic aminopyridinols. brown solid, whose spectroscopic data were identical to those
These bicyclic pyridinols showed highly potent antiangiogenic reported.18
activities. In particular, the pyrrolidinone-fused analogues
3-Benzyloxy-5-chloromethyl-2,4-dimethylpyridine hydrochlo-
(bicyclic amide analogues, 3A and 4A) showed the best activi- ride (7). To a solution of 6 (2.85 g, 11.71 mmol) in 1,2-dichloro-
ties and comparable ID50 values (ID50 = 1.07–1.17 pmol per ethane (35 mL) were added DMF (0.09 mL, 1.17 mmol) and
CAM vs. 0.38 pmol per CAM) to that of a licensed drug, suniti- thionyl chloride (1.7 mL, 23.43 mmol). After stirring for 1 h at
nib, in the inhibition of VEGF-induced angiogenesis.
80 °C, the reaction mixture was cooled to room temperature.
Ethyl ether (150 mL) was added to the mixture, and the result-
ing suspension was stirred for 1 h in an ice-bath. The precipi-
tated solid was collected by filtration and the filter cake was
washed with Et2O to give 7 (3.3 g, 95%) as a brown solid. Rf
0.58 (CHCl3–MeOH = 20 : 1); m.p. 192 °C; 1H-NMR (CHCl3-d)
Experimental
General
Unless noted otherwise, materials were purchased from com- 8.19 (s, 1H), 7.34–7.44 (m, 5H), 4.81 (s, 2H), 4.56 (s, 2H), 2.50
mercial suppliers and used without further purification. Air- (s, 3H), 2.32 (s, 3H) ppm; 13C-NMR (CHCl3-d) δ 153.6, 152.4,
or moisture-sensitive reactions were carried out under an inert 145.1, 139.9, 136.8, 131.1, 128.8, 128.5, 128.1, 75.1, 42.0, 19.9,
gas atmosphere. Progress of reaction was monitored by thin- 11.8 ppm; IR (KBr) ν 3458, 3014, 2399, 2061, 1607, 1529, 1454,
layer-chromatography (TLC) using silica gel F254 plates. Purifi- 1384, 1293, 1265, 1245, 1219, 1095, 949, 916, 839, 770, 749,
cation of the products was performed by flash column chromato- 704, 603, 511 cm−1; HRMS (ESI) m/z calcd for C15H17ClNO
graphy using silica gel 60 (70–230 mesh) or by Biotage ‘Isolera [M + H]+ 262.0993, found 262.0996.
One’ system with indicated solvents. Melting points were deter-
2-(5-Benzyloxy-4,6-dimethylpyridin-3-yl)acetonitrile (8). To a
mined using a Fischer–Jones melting point apparatus and solution of 7 (9.95 g, 38.01 mmol) in anhydrous DMF (500 mL)
were not corrected. NMR spectra were obtained using a was added KCN (7.42 g, 114.04 mmol). The resulting mixture
1
Bruker-250 spectrometer 250 MHz for H-NMR and 62.5 MHz was stirred for 2 days at 40 °C. After evaporation of DMF, the
for 13C-NMR. Chemical shifts (δ) were expressed in ppm using residue was diluted with EtOAc (2 L) and the organic layer was
the solvent as an internal standard and coupling constant (J) washed with water (200 mL × 4). The organic solution was
in hertz. IR spectra were recorded on a Perkin Elmer spectrum dried over MgSO4 and concentrated. The residue was purified
GX FT-IR spectrometer. High-resolution mass spectra (HRMS) by silica gel column chromatography (CHCl3–MeOH = 50 : 1 →
were obtained in Gyeonggi Institute of Science and Technology 20 : 1) to give 8 (8.14 g, 96%) as a brown solid. Rf 0.45 (CHCl3–
1
Promotion (GSTEP) using a Thermo Scientific LTQ Orbitrap XL MeOH = 20 : 1); m.p. 86 °C; H-NMR (CHCl3-d) δ 8.18 (s, 1H),
mass spectrometer, and recorded in positive ion mode with an 7.35–7.43 (m, 5H), 4.80 (s, 2H), 3.61 (s, 2H), 2.51 (s, 3H), 2.26
electrospray (ESI) source.
(s, 3H) ppm; 13C-NMR (CHCl3-d) δ 153.4, 152.2, 144.1, 139.2,
4-Deoxypyridoxine hydrochloride (5). To a suspension of 136.4, 128.9, 128.7, 128.1, 124.1, 116.8, 75.2, 19.8, 19.5,
pyridoxine·HCl (1) (10 g, 48.63 mmol) in glacial AcOH (40 mL) 12.3 ppm; IR (KBr) ν 3446, 2931, 2248, 1593, 1455, 1412, 1369,
was added zinc dust (12.7 g, 194.51 mmol) in small portions. 1211, 1083, 990, 974, 914, 841, 775, 747, 701, 583, 474,
The resulting suspension was refluxed for 12 h and then 423 cm−1; HRMS (ESI) m/z calcd for C16H17N2O [M + H]+
cooled to room temperature. The insoluble solids were filtered 253.1335, found 253.1337.
off through a Celite pad in vacuo and the filter cake was
Ethyl 2-(5-hydroxy-4,6-dimethylpyridin-3-yl)acetate (9). To a
washed with CH3CN (60 mL). After CH3CN in the filtrate was solution of 8 (500 mg, 1.98 mmol) in EtOH (20 mL) was added
evaporated in vacuo, the residual solution was stirred in an ice- H2SO4 (3.7 mL, 69.36 mmol), and the resulting mixture was
bath. In a separate flask, acetyl chloride (10 mL) was slowly refluxed for 12 h. The solution was cooled to room tempera-
added to cold MeOH (30 mL) in an ice-bath to afford a metha- ture and diluted with EtOH (150 mL). Na2CO3 (7.35 g,
nolic hydrogen chloride solution. This solution was added to 69.36 mmol) was added in small portions to the solution and
the above main solution and stirred for 2 h in an ice-bath. The the resulting mixture was stirred for 3 h at room temperature.
precipitated solid was collected by filtration and washed with After evaporation of EtOH, the residue was diluted with EtOAc
Et2O to give 5 (8.7 g, 94%) as a white solid, whose spectro- (150 mL) and the insoluble solids were filtered off through a
scopic data were identical to those reported.11a
(5-Benzyloxy-4,6-dimethylpyridin-3-yl)methanol (6). To
Celite pad and washed with EtOAc. The organic solution was
concentrated and the residue was purified by silica gel column
a
suspension of 5 (3 g, 15.82 mmol) in anhydrous DMF (150 mL) chromatography (CHCl3–MeOH = 70 : 1 → 20 : 1) to give 9
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(366 mg, 88%) as a yellow solid. Rf 0.36 (CHCl3–MeOH = 9 : 1); 4.73 mmol) in H2O (5 mL), and 4% aqueous NaOCl (5 mL).
1
m.p. 79 °C; H-NMR (CHCl3-d) δ 7.80 (s, 1H), 6.96 (br s, 1H), The resulting mixture was stirred for 10 min at room tempera-
4.11 (q, J = 7.1 Hz, 2H), 3.56 (s, 2H), 2.38 (s, 3H), 2.19 (s, 3H), ture, and then diluted with CH2Cl2 (30 mL). The organic layer
1.21 (t, J = 7.1 Hz, 3H) ppm; 13C-NMR (CHCl3-d) δ 171.1, 150.3, was washed with water (20 mL) and brine, then dried over
144.9, 140.6, 134.0, 128.3, 61.3, 36.7, 19.1, 14.3, 12.4 ppm; IR MgSO4 and concentrated. The residue was purified by silica gel
(KBr) ν 3445, 2981, 1732, 1595, 1556, 1445, 1394, 1369, 1280, column chromatography (CH2Cl2–MeOH = 20 : 1) to give 11
1235, 1208, 1148, 1092, 1032, 886, 670, 617, 550 cm−1; HRMS (466 mg, 94%) as a colorless oil. Rf 0.53 (CHCl3–MeOH =
(ESI) m/z calcd for C11H16NO3 [M + H]+ 210.1125, found 10 : 1); 1H-NMR (CHCl3-d) δ 10.19 (s, 1H), 8.63 (s, 1H),
210.1125.
7.34–7.43 (m, 5H), 4.82 (s, 2H), 2.58 (s, 3H), 2.57 (s, 3H) ppm;
Ethyl 2-(5-hydroxy-4,6-dimethyl-2-(phenyldiazenyl)pyridin-3- 13C-NMR (CHCl3-d) δ 197.8, 158.6, 152.7, 149.6, 142.1, 136.3,
yl)acetate (10). In a beaker equipped with a mechanical stirrer 129.6, 129.0, 128.8, 128.3, 75.4, 20.5, 12.3 ppm; IR (KBr) ν
and pH electrode, 9 (1 g, 4.78 mmol) was dissolved in THF– 3432, 1700, 1586, 1432, 1364, 1281, 1225, 1106, 955, 845, 789,
H2O (1 : 1, 30 mL) and cooled in an ice-bath. In a separate 749, 701, 594, 510 cm−1; HRMS (ESI) m/z calcd for C15H16NO2
Erlenmeyer flask, aniline (0.48 mL, 5.26 mmol) was dissolved [M + H]+ 242.1176, found 242.1177.
in 6 M HCl (5 mL) at room temperature, and then cooled in an
5-Benzyloxy-4,6-dimethylnicotinamide (12). To a solution of
ice-bath. A cooled solution of NaNO2 (363 mg, 5.26 mmol) in 11 (466 mg, 1.93 mmol) in THF (10 mL) were added 28%
water (2 mL) was added in small portions to the aniline solu- NH4OH (6 mL) and I2 (736 mg, 2.90 mmol). The resulting
tion. This diazotized aniline solution was then added in small mixture was stirred at room temperature for 1 h. The mixture
portions to the chilled solution of 9. After each addition, the was diluted with CH2Cl2 (50 mL) and the organic layer was
pH of the reaction mixture was quickly adjusted to maintain washed with saturated aqueous Na2S2O3 solution (50 mL). The
pH 8 by the addition of 10 M NaOH. After the addition of the organic solution was washed with brine, dried over MgSO4 and
diazotized aniline solution, the reaction mixture was allowed concentrated. The residue was dissolved in THF (10 mL) and
to warm to room temperature and stirred for 2 h. The reaction 28% NH4OH (6 mL). Then 35% H2O2 (6 mL) was added to the
mixture was extracted with EtOAc (150 mL × 3) and the com- mixture at 0 °C. The mixture was allowed to warm to room
bined extracts were dried over MgSO4 and concentrated. The temperature, stirred for 2 h, diluted with CH2Cl2 (60 mL) and
residue was purified by silica gel column chromatography washed with H2O (60 mL). The organic solution was washed
(CHCl3–MeOH = 80 : 1 → 20 : 1) to give 10 (1.3 g, 87%) as a red with brine, dried over MgSO4 and concentrated. The residue
solid. Rf 0.61 (CHCl3–MeOH = 9 : 1); m.p. 172 °C; 1H-NMR was purified by silica gel column chromatography (CH2Cl2–
(CHCl3-d) δ 7.32–7.37 (m, 4H), 7.08–7.13 (m, 1H), 4.14 (q, J = MeOH = 20 : 1) to give 12 (446 mg, 90%) as a white solid. Rf
1
7.1 Hz, 2H), 3.89 (s, 2H), 2.40 (s, 3H), 2.06 (s, 3H), 1.22 (t, J = 0.38 (CHCl3–MeOH = 10 : 1); m.p. 177 °C; H-NMR (CHCl3-d) δ
7.1 Hz, 3H) ppm; 13C-NMR (CHCl3-d) δ 170.5, 134.7, 129.7, 8.31 (s, 1H), 7.34–7.43 (m, 5H), 4.79 (s, 2H), 2.47 (s, 3H), 2.42
61.3, 33.6, 20.2, 14.4, 11.6 ppm; IR (KBr) ν 3196, 2937, 1730, (s, 3H) ppm; 13C-NMR (CHCl3-d) δ 169.7, 155.1, 152.7, 142.3,
1602, 1567, 1512, 1448, 1410, 1396, 1364, 1329, 1269, 1224, 140.1, 136.5, 132.0, 131.0, 128.9, 128.7, 128.2, 75.0, 19.8,
1186, 1113, 1078, 1059, 1023, 978, 938, 893, 836, 742, 688, 646, 13.3 ppm; IR (KBr) ν 3734, 3366, 3170, 1733, 1716, 1685, 1653,
614, 549, 503, 486, 471, 418 cm−1; HRMS (ESI) m/z calcd for 1635, 1558, 1541, 1507, 1473, 1456, 1436, 1364, 1209, 1054,
C17H20N3O3 [M + H]+ 314.1499, found 314.1501.
985, 736, 700, 602, 448, 418 cm−1; HRMS (ESI) m/z calcd for
5-Hydroxy-4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-2(3H)-one C15H17N2O2 [M + H]+ 257.1285, found 257.1286.
(4A). To a solution of 10 (550 mg, 1.75 mmol) in glacial AcOH
One-pot reaction procedure for the synthesis of the primary
amide 12 from the alcohol 6
(35 mL) was added zinc dust (344 mg, 5.26 mmol) in small
portions. After the reaction mixture was refluxed for 12 h, the
resulting suspension was cooled to room temperature. The To a solution of 6 (200 mg, 0.822 mmol) in THF (0.5 mL) was
insoluble solids in the reaction mixture were filtered through a added 28% ammonium hydroxide solution (3 mL), I2 (626 mg,
Celite pad and washed with CH3CN. After concentration of the 2.47 mmol) and the resulting mixture was stirred at 90 °C for
filtrate, the residue was purified by silica gel column 4 h. After the mixture was cooled to 0 °C, 28% NH4OH (3 mL)
chromatography (CHCl3–MeOH = 80 : 1 → 10 : 1) to give 4A and 35% H2O2 (3 mL) were added at 0 °C. The reaction
(273 mg, 87%) as a brown solid. Rf 0.47 (CHCl3–MeOH = 9 : 1); mixture was stirred for 2 h at room temperature. The reaction
m.p. 314 °C; 1H-NMR (DMSO-d6) δ 10.4 (s, 1H), 8.05 (s, 1H), mixture was diluted with CH2Cl2 (30 mL) and the organic layer
3.38 (s, 2H), 2.27 (s, 3H), 2.07 (s, 3H) ppm; 13C-NMR (DMSO- was neutralized by saturated NH4Cl solution. The aqueous
d6) δ 175.7, 149.5, 144.6, 141.5, 131.9, 117.1, 34.5, 19.0, layer was extracted with CH2Cl2 (30 mL × 3). The combined
12.8 ppm; IR (KBr) ν 3544, 3218, 2919, 2775, 1697, 1621, 1541, organic solution was dried over MgSO4 and concentrated. The
1507, 1485, 1454, 1379, 1285, 1243, 1207, 1117, 1069, 1025, residue was purified by silica gel column chromatography
969, 866, 766, 720, 651, 571, 524, 459, 418 cm−1; HRMS (ESI) (CH2Cl2–MeOH = 20 : 1) to give 12 (143 mg, 68%) as a white
m/z calcd for C9H11N2O2 [M + H]+ 179.0815, found 179.0820.
5-Benzyloxy-4,6-dimethylnicotinaldehyde (11). To a solution
solid.
3-Benzyloxy-5-carbamoyl-2,4-dimethylpyridine-1-oxide (13).
of 6 (500 mg, 2.06 mmol) in CH2Cl2 (10 mL) were added To a solution of 12 (446 mg, 1.74 mmol) in CH2Cl2 (17 mL)
TEMPO (3.2 mg, 0.021 mmol), a solution of NaHCO3 (397 mg, was added m-CPBA (330 mg, 1.91 mmol) and the resulting
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mixture was stirred at room temperature for 1 h. The mixture was filtered and washed with MeOH (1 mL × 3) to give 16
was diluted with CH2Cl2 (40 mL) and the organic layer was (249 mg, 87%) as a white solid. Rf 0.40 (CHCl3–MeOH = 10 : 1);
1
washed with saturated NaHCO3 solution (40 mL). The organic m.p. 295 °C (decomp.); H-NMR (DMSO-d6) δ 11.0 (br s, 1H),
solution was washed with brine, dried over MgSO4 and concen- 7.36–7.50 (m, 4H), 4.77 (s, 2H), 2.33 (s, 3H), 2.20 (s, 3H) ppm;
trated. The residue was purified by silica gel column chromato- 13C-NMR (DMSO-d6) δ 155.3, 146.5, 140.9, 139.8, 137.3, 128.4,
graphy (CH2Cl2–MeOH = 15 : 1) to give 13 (417 mg, 88%) as a 128.2, 128.1, 121.9, 120.3, 74.5, 18.8, 10.6 ppm; IR (KBr)
white solid. Rf 0.35 (CHCl3–MeOH = 10 : 1); m.p. 207 °C; ν 3734, 3649, 3445, 1716, 1646, 1558, 1541, 1507, 1456, 1363,
1H-NMR (CHCl3-d) δ 8.21 (s, 1H), 7.36 (s, 5H), 4.79 (s, 2H), 1260, 1140, 1005, 790, 750, 668, 483, 418 cm−1; HRMS (ESI)
2.39 (s, 3H), 2.38 (s, 3H) ppm; 13C-NMR (CHCl3-d) δ 166.8, m/z calcd for C15H16N3O2 [M + H]+ 270.1237, found 270.1243.
154.1, 146.2, 135.6, 134.2, 132.1, 131.5, 129.1, 128.2, 76.0, 13.2,
12.1 ppm; IR (KBr) ν 3429, 1636, 1541, 1456, 1115, 700 cm−1
6-Hydroxy-5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(4B). To a solution of 16 (249 mg, 0.923 mmol) in MeOH
;
HRMS (ESI) m/z calcd for C15H17N2O3 [M + H]+ 273.1234, (10 mL) was added palladium (10% on activated carbon,
found 273.1241.
50 mg). The mixture was stirred with hydrogen balloon at
5-Benzyloxy-2-(1,3-dioxoisoindolin-2-yl)-4,6-dimethylnicotin- room temperature for 2 h. The solids in the reaction mixture
amide (14). To a solution of 13 (417 mg, 1.53 mmol) in anhy- were filtered through a Celite pad and the filtrate was filtered
drous CH2Cl2 (15 mL) were added phthalimide (228 mg, again with a syringe filter (Advantec® JP050AN). The filtrate
1.55 mmol), N,N-diisopropylethylamine (0.80 mL, 4.59 mmol), was concentrated to give 4B (164 mg, 99%) as a yellow solid. Rf
1
and tosyl chloride (438 mg, 2.30 mmol). The resulting mixture 0.27 (CHCl3–MeOH = 10 : 1); m.p. 227 °C (decomp.); H-NMR
was stirred for 1 h under a nitrogen atmosphere at room temp- (DMSO-d6) δ 2.27 (s, 3H), 2.13 (s, 3H) ppm; 13C-NMR (DMSO-
erature. The mixture was diluted with CH2Cl2 (40 mL) and the d6) δ 155.1, 144.7, 136.6, 135.8, 121.6, 115.8, 19.2, 10.7 ppm;
organic layer was washed with H2O (40 mL). The organic solu- IR (KBr) ν 3445, 1733, 1716, 1699, 1685, 1647, 1558, 1541,
tion was dried over MgSO4 and concentrated. The residual 1522, 1507, 1473, 1457, 1138, 669, 418 cm−1; HRMS (ESI) m/z
solid was filtered and washed with Et2O to give 14 as a white calcd for C8H10N3O2 [M + H]+ 180.0768, found 180.0772.
solid (479 mg, 78%). Rf 0.47 (CHCl3–MeOH
=
10 : 1);
5-Benzyloxy-4,6-dimethylpyridin-3-amine (17). To a solution
m.p. 257 °C; 1H-NMR (CHCl3-d) δ 7.88–7.93 (m, 2H), 7.75–7.80 of NaOH (94 mg, 2.34 mmol) in THF–H2O (1 : 1, 8 mL) were
(m, 2H), 7.37–7.50 (m, 5H), 6.06 (br s, 1H), 5.76 (br s, 1H), 4.88 added 4% aqueous NaOCl solution (2 mL) and 12 (200 mg,
(s, 2H), 2.54 (s, 3H), 2.40 (s, 3H) ppm; 13C-NMR (CHCl3-d) 0.780 mmol) and the resulting mixture was stirred at room
δ 167.9, 167.1, 155.0, 153.5, 141.0, 136.7, 136.6, 135.0, 132.4, temperature for 1 h. Then the reaction was elevated to 90 °C
132.3, 129.2, 129.0, 128.3, 124.5, 75.4, 20.1, 13.9 ppm; IR (KBr) and stirred for 2 h. The mixture was cooled to room tempera-
ν 3391, 1790, 1729, 1661, 1558, 1541, 1507, 1457, 1395, 1285, ture and diluted with CH2Cl2 (40 mL). The organic layer was
1209, 886, 722, 418 cm−1
; HRMS (ESI) m/z calcd for neutralized with saturated NH4Cl solution (40 mL). The
C23H20N3O4 [M + H]+ 402.1448, found 402.1458.
organic solution was washed with brine, dried over MgSO4 and
concentrated. The residue was purified by silica gel column
2-Amino-5-benzyloxy-4,6-dimethylnicotinamide (15). To
a
solution of 14 (479 mg, 1.19 mmol) in THF–EtOH (1 : 1, chromatography (CH2Cl2–MeOH = 10 : 1) to give 17 (176 mg,
1
12 mL) was added hydrazine (4.2 mL) and the resulting 99%) as a yellow oil. Rf 0.27 (CHCl3–MeOH = 10 : 1); H-NMR
mixture was stirred at room temperature for 12 h. The solvent (CHCl3-d) δ 7.79 (s, 1H), 7.33–7.46 (m, 5H), 4.77 (s, 2H), 3.49
was evaporated and the residue was diluted with Et2O. After (br s, 2H), 2.41 (s, 3H), 2.06 (s, 3H) ppm; 13C-NMR (CHCl3-d) δ
the insoluble solids were filtered off, the filtrate was diluted 152.0, 142.6, 140.6, 137.2, 132.2, 128.8, 128.4, 128.2, 124.9,
with CH2Cl2 (40 mL) and the organic layer was washed with 75.1, 19.2, 10.1 ppm; IR (KBr) ν 3347, 3199, 3030, 2919, 1647,
H2O (40 mL). The organic solution was dried over MgSO4 and 1567, 1489, 1454, 1416, 1369, 1330, 1232, 1146, 1028, 990, 896,
concentrated to give 15 (288 mg, 89%) as a white solid. Rf 0.37 842, 776, 747, 698, 586, 477 cm−1; HRMS (ESI) m/z calcd for
(CHCl3–MeOH = 10 : 1); m.p. 194 °C; 1H-NMR (CHCl3-d) C14H17N2O [M + H]+ 229.1335, found 229.1340.
δ 7.24–7.41 (m, 5H), 6.28 (br d, 1H), 4.94 (br s, 1H), 4.68 (s,
5-Benzyloxy-4,6-dimethylpyridin-3-ol (18). To a solution of
2H), 2.31 (s, 3H), 2.27 (s, 3H) ppm; 13C-NMR (CHCl3-d) 17 (176 mg, 0.772 mmol) in THF–H2O (1 : 1, 2 mL) were added
δ 151.9, 151.8, 144.8, 140.0, 136.9, 128.8, 128.5, 128.2, 114.1, 5% aqueous H2SO4 (4 mL) and NaNO2 (59 mg, 0.849 mmol) at
75.3, 19.2, 14.1 ppm; IR (KBr) ν 3853, 3734, 3676, 3649, 3402, 0 °C. The reaction mixture was stirred at 0 °C for 1 h and
1716, 1635, 1558, 1541, 1507, 1455, 1366, 1210, 1051, 733, stirred at room temperature for 2 h. The mixture was diluted
418 cm−1; HRMS (ESI) m/z calcd for C15H18N3O2 [M + H]+ with CH2Cl2 (40 mL) and the organic layer was neutralized
272.1394, found 272.1401.
with saturated NaHCO3 solution (40 mL). The organic solution
6-Benzyloxy-5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one was washed with brine, dried over MgSO4 and concentrated.
(16). To a solution of KOH (179 mg, 3.17 mmol) in MeOH The residue was purified by silica gel column chromatography
(10 mL) was added 15 (288 mg, 1.06 mmol) and the reaction (CH2Cl2–MeOH = 30 : 1) to give 18 (143 mg, 81%) as a white
mixture was cooled to 0 °C. PhI(OAc)2 (342 mg, 1.06 mmol) solid. Rf 0.23 (CHCl3–MeOH = 10 : 1); m.p. 126 °C; 1H-NMR
was added and the resulting mixture was stirred at 0 °C for (CHCl3-d) δ 7.98 (s, 1H) 7.36–7.47 (m, 5H), 4.82 (s, 2H), 2.45
1 h. The mixture was allowed to warm to room temperature (s, 3H), 2.26 (s, 3H) ppm; 13C-NMR (CHCl3-d) δ 153.6, 153.4,
and stirred for one additional hour. The resulting suspension 142.8, 137.1, 130.4, 129.1, 128.8, 128.4, 75.2, 18.0, 9.9 ppm;
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IR (KBr) ν 3444, 3067, 3036, 2999, 2954, 2913, 2869, 1945, 1885, with H2O (30 mL × 2). The organic solution was washed with
1808, 1748, 1578, 1541, 1497, 1454, 1436, 1417, 1399, 1357, brine, dried over MgSO4 and concentrated. The residue was
1260, 1240, 1225, 1212, 1096, 1028, 993, 981, 942, 905, 841, 766, purified by silica gel column chromatography (CH2Cl2–MeOH
748, 703, 687, 626, 585, 547, 524, 487, 419 cm−1; HRMS (ESI) m/z = 20 : 1) to give 21 (156 mg, 93%) as a white solid. Rf 0.41
calcd for C14H16NO2 [M + H]+ 230.1176, found 230.1178.
(CHCl3–MeOH = 10 : 1); m.p. 237 °C; 1H-NMR (CHCl3-d) δ
5-Benzyloxy-4,6-dimethyl-2-(phenyldiazenyl)pyridin-3-ol (19). 7.38–7.41 (m, 5H), 4.82 (s, 2H), 2.55 (s, 3H), 2.30 (s, 3H) ppm;
In a beaker equipped with a mechanical stirrer and pH elec- 13C-NMR (CHCl3-d) δ 154.2, 148.6, 145.7, 140.8, 136.7, 136.2,
trode, 18 (237 mg, 1.04 mmol) was dissolved in THF–H2O 129.1, 129.0, 128.5, 125.1, 76.1, 19.0, 10.0 ppm; IR (KBr) ν
(1 : 1, 7 mL) and cooled in an ice-bath. In a separate Erlen- 3853, 3734, 3675, 3649, 3446, 1792, 1653, 1558, 1541, 1507,
meyer flask, aniline (0.104 mL, 1.14 mmol) was dissolved in 1457, 1097, 749, 668 cm−1
6 M HCl (1.04 mL), and then cooled in an ice-bath. A cooled C15H15N2O3 [M + H]+ 271.1077, found 271.1083.
; HRMS (ESI) m/z calcd for
solution of NaNO2 (79 mg, 1.14 mmol) in water (3 mL) was
6-Hydroxy-5,7-dimethyloxazolo[4,5-b]pyridin-2(3H)-one (4C).
added in small portions to the aniline solution. This diazo- To a solution of 21 (142 mg, 0.525 mmol) in MeOH (5 mL) was
tized aniline solution was then added in small portions to the added palladium (10% on activated carbon, 28 mg). The
chilled solution of 18. After each addition, the pH of the reac- mixture was stirred with hydrogen balloon at room tempera-
tion mixture was quickly adjusted to maintain pH 8 by the ture for 2 h. The solid in the reaction mixture was filtered
addition of 2.5 M NaOH. After the addition of the diazotized through a Celite pad and the filtrate was filtered again with a
aniline solution, the reaction mixture was allowed to warm to syringe filter (Advantec® JP050AN). The filtrate was concen-
room temperature and stirred for 1 h. The mixture was diluted trated to give 4C (94 mg, 99%) as a yellow solid. Rf 0.39
with CH2Cl2, and the organic layer was washed with H2O. The (CHCl3–MeOH = 10 : 1); m.p. 266 °C (decomp.); 1H-NMR
organic solution was washed with brine, dried over MgSO4 and (DMSO-d6) δ 2.29 (s, 3H), 2.16 (s, 3H) ppm; 13C-NMR (DMSO-
concentrated. The residue was purified by silica gel column d6) δ 154.2, 146.0, 145.1, 144.4, 138.6, 137.6, 135.9, 135.0,
chromatography (hexanes–EtOAc = 3 : 1) to give 19 (314 mg, 117.3, 19.1, 9.1 ppm; IR (KBr) ν 3420, 2683, 1771, 1716, 1653,
91%) as a red solid. Rf 0.59 (CHCl3–MeOH = 20 : 1); m.p. 99 °C; 1558, 1541, 1507, 1457, 1436, 1398, 1097, 927, 750, 669,
1H-NMR (CHCl3-d) δ 13.59 (s, 1H) 7.93–7.98 (m, 2H), 7.35–7.55 418 cm−1; HRMS (ESI) m/z calcd for C8H9N2O3 [M + H]+
(m, 8H), 4.91 (s, 2H), 2.55 (s, 3H), 2.23 (s, 3H) ppm; 13C-NMR 181.0613, found 181.0612.
(CHCl3-d) δ 154.9, 150.2, 149.5, 145.6, 143.5, 136.7, 132.1,
130.6, 129.8, 129.1, 129.0, 128.5, 123.0, 75.5, 20.0, 9.6 ppm; IR
Chick chorioallantoic membrane (CAM) model of
angiogenesis
(KBr) ν 3735, 3446, 1653, 1558, 1541, 1507, 1457, 1362, 1145,
975, 761, 695, 418 cm−1; HRMS (ESI) m/z calcd for C20H20N3O2 Angiogenesis was examined using previously published
[M + H]+ 334.1550, found 334.1555.
methods.19 Fertilized chicken eggs were incubated at 37 °C
with 55% relative humidity for ten days. By the 10th day of
2-Amino-5-benzyloxy-4,6-dimethylpyridin-3-ol (20). To
a
solution of 19 (250 mg, 0.750 mmol) in MeOH (7.5 mL) were incubation, the ectoderm of the CAM, one of the three com-
added zinc dust (148 mg, 2.26 mmol) and HCO2H (0.3 mL, prising layers (the ectoderm, mesoderm, and endoderm), con-
7.53 mmol). The resulting mixture was refluxed for 1 h. The tained fully developed capillary plexus, a network of tiny
mixture was cooled to room temperature and filtered through capillaries connecting the arterial and venous blood vessels.
a Celite pad. The filtrate was concentrated and the residue was On the 11th day, a small hole in the shell concealing the air
diluted with CH2Cl2 (40 mL). The organic layer was washed sac was made using a hypodermic needle. A second hole was
with saturated NaHCO3 (40 mL). The organic solution was made on the broad side of the egg directly over the avascular
washed with brine, dried over MgSO4 and concentrated. The portion of the embryonic membrane by candling. A false air
residue was purified by silica gel column chromatography sac was created beneath the second hole by applying negative
(CH2Cl2–MeOH = 15 : 1) to give 20 (153 mg, 83%) as a white pressure through the first hole, causing the CAM to separate
solid. Rf 0.54 (CHCl3–MeOH = 5 : 1); m.p. 160 °C; 1H-NMR from the shell. An approximately 1.0 cm2 window was made in
(CHCl3-d) δ 7.30–7.41 (m, 5H), 4.83 (br s, 3H), 4.69 (s, 2H), the shell over the dropped CAM using a small grinding wheel
2.26 (s, 3H), 2.14 (s, 3H) ppm; 13C-NMR (CHCl3-d) δ 145.9, (Dremel, Racine, WI, USA). Sterile disks of No. 1 filter paper
144.9, 137.1, 128.7, 128.3, 128.1, 75.4, 17.5, 10.0 ppm; IR (KBr) (Whatman International) were pretreated with 3 mg mL−1 of
ν 3392, 2926, 1716, 1698, 1653, 1558, 1507, 1456, 1372, 1154, cortisone acetate and air dried under sterile conditions. Each
1011, 736, 697, 418 cm−1
C14H17N2O2 [M + H]+ 245.1285, found 245.1290.
;
HRMS (ESI) m/z calcd for disk was suspended in 10 μL of PBS containing VEGF (20 ng
per CAM), as a standard proangiogenic agent, or the control
6-Benzyloxy-5,7-dimethyloxazolo[4,5-b]pyridin-2(3H)-one (21). solvent, and the disks were then placed on growing CAMs.
To a solution of 21 (153 mg, 0.623 mmol) in anhydrous DMF Compounds were added to the disk after 30 min. The com-
(6 mL) was added 1,1′-carbonyldiimidazole (CDI) (152 mg, pound-treated CAMs were incubated for 3 days.
0.935 mmol) and the resulting mixture was stirred for 12 h
Microscopic analysis of the CAM sections
under a nitrogen atmosphere at room temperature. The
solvent was removed under reduced pressure. The residue was After the three-day incubation at 37 °C with 55% relative
diluted with CH2Cl2 (40 mL) and the organic layer was washed humidity, the tissue directly beneath each filter disk was
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resected from the control and the drug-treated CAM samples.
Each tissue sample was washed three times with PBS, placed
in a 35 mm Petri dish (Nalge Nunc), and examined under a
stereomicroscope (Zeiss) at 50× magnification. Digital images
of the CAM beneath the filters were collected using a three-
charge-coupled device color video camera system (Toshiba).
The images were then analyzed using the Image-Inside soft-
7 D.-G. Kim, Y. Kang, H. Lee, E. K. Lee, T.-g. Nam, J.-A. Kim
and B.-S. Jeong, Eur. J. Med. Chem., 2014, 78, 126.
8 (a) R. Auerbach, R. Lewis, B. Shinners, L. Kubai and
N. Akhtar, Clin. Chem., 2003, 49, 32; (b) C. Storgard,
D. Mikolon and D. G. Stupack, Mol. Biol., 2005, 294, 123.
9 L. Sun, N. Tran, F. Tang, H. App, P. Hirth, G. McMahon
and C. Tang, J. Med. Chem., 1998, 41, 2588.
ware. The number of vessel branch points contained in a circu- 10 H. K. Gan, B. Seruga and J. J. Knox, Expert Opin. Invest.
lar region (equal to the area of each filter disk) was counted. Drugs, 2009, 18, 821.
One image was counted for each CAM preparation, and the 11 (a) R. Serwa, T.-G. Nam, L. Valgimigli, S. Culbertson,
findings from six to eight CAM preparations were analyzed for
each treatment condition. The resulting angiogenesis index
was expressed as the mean S.E.M. of the new branch points
for each set of samples. Chick embryo experiments were per-
formed according to the institutional guidelines of the Insti-
C. L. Rector, B.-S. Jeong, D. A. Pratt and N. A. Porter, Chem.
– Eur. J., 2010, 16, 14106; (b) R. V. Shchepin, W. Liu, H. Yin,
I. Zagol-Ikapitte, T. Amin, B.-S. Jeong, L. J. Roberts II,
J. A. Oates, N. A. Porter and O. Boutaud, ACS Med. Chem.
Lett., 2013, 4, 710.
tute of Laboratory Animal Resources (1996) and Yeungnam 12 R. Ohmura, M. Takahata and H. Togo, Tetrahedron Lett.,
University for the care and use of laboratory animals (2009).
2010, 51, 4378.
13 R. P. Farrell, M. V. S. Elipe, M. D. Bartberger, J. S. Tedrow
and F. Vounatsos, Org. Lett., 2013, 15, 168.
Acknowledgements
14 (a) A. Casimiro-Garcia, G. F. Filzen, D. Flynn, C. F. Bigge,
J. Chen, J. A. Davis, D. A. Dudley, J. J. Edmunds,
N. Esmaeil, A. Geyer, R. J. Heemstra, M. Jalaie, J. F. Ohren,
R. Ostroski, T. Ellis, R. P. Schaum and C. Stoner, J. Med.
Chem., 2011, 54, 4219; (b) R. M. Moriarty, C. J. Chany II,
R. K. Vaid, O. Prakash and S. M. Tuladhar, J. Org. Chem.,
1993, 58, 2478.
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology (NRF-2012R1A1A2039174).
15 (a) S. Kumar, H. Johansson, T. Kanda, L. Engman,
T. Müller, H. Bergenudd, M. Jonsson, G. F. Pedulli,
R. Amorati and L. Valgimigli, J. Org. Chem., 2010, 75, 716;
(b) Y. Gao, H. T. Ravert, H. Kuwabara, Y. Xiao, C. J. Endres,
J. Hilton, D. P. Holt, A. Kumar, M. Alexander, D. F. Wong,
R. F. Dannals and A. G. Horti, Bioorg. Med. Chem., 2009, 17,
4367.
Notes and references
1 (a) C. A. Klein, Science, 2008, 321, 1785; (b) A. C. Chiang
and J. Massagué, N. Engl. J. Med., 2008, 359, 2814.
2 W. Risau and I. Flamme, Annu. Rev. Cell Dev. Biol., 1995,
11, 73.
3 (a) J. Folkman, Nat. Med., 1995, 1, 27; (b) B. R. Zetter, Annu.
Rev. Med., 1998, 49, 407; (c) P. Carmeliet and R. K. Jain, 16 CAM and rabbit cornea assays are the most widely used
Nature, 2000, 407, 249; (d) J. Folkman, Semin. Oncol., 2002,
29, 15.
4 (a) A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers,
ones as an in vivo model for angiogenesis study. Of these,
CAM provides advantages of being simpler to use, less
expensive, reproducible and reliable.
A. T. Van Oosterom and E. A. De Bruijin, Pharmacol. Rev., 17 Each test compound was prepared in stock solution and
2004, 56, 549; (b) A. M. Byrne, D. J. Bouchier-Hayes and
J. H. Harmey, J. Cell. Mol. Med., 2005, 9, 777;
(c) T. Tammela, B. Enholm, K. Alitalo and K. Paavonen,
Cardiovasc. Res., 2005, 65, 550.
diluted with PBS in a concentration of 1.0 μM. The 10 μL of
this solution was added directly to the disk on top of CAM.
Since we measured the blood vessels in the area under the
disk, the antiangiogenic dose was calculated as 10 μL per
CAM × drug concentration (1.0 μM), i.e. 0.01 nmol per CAM.
5 (a) J. Folkman and D. Ingber, Semin. Cancer Biol., 1992, 3,
89; (b) K. J. Kim, B. Li, J. Winer, M. Armanini, N. Gillett, 18 J. Diakur, W. Haque, W. Zhang, J. Yao and V. Pham, U.S.
H. S. Phillips and N. Ferrara, Nature, 1993, 362, 841. Pat. Appl. Publ., 20040186077 A1, 2003.
6 (a) T. Shin and C. Lindley, Clin. Ther., 2006, 28, 1779; 19 R. W. Colman, R. A. Pixley, I. M. Sainz, J. S. Song, I. Isordia-
(b) K. J. Gotink and H. M. Verheul, Angiogenesis, 2010, 13,
1; (c) D. W. Siemann, Cancer Treat. Rev., 2011, 37, 63.
Salas, S. N. Muhamed, J. A. Powell and S. A. Mousa,
J. Thromb. Haemost., 2003, 1, 164–170.
8710 | Org. Biomol. Chem., 2014, 12, 8702–8710
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