Development of pyrazolone and isoxazol-5-one cambinol analogues as sirtuin inhibitors

Article abstract of DOI:10.1021/jm4018064

Sirtuins are a family of NAD⁺-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC₅₀ ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.

Full text of DOI:10.1021/jm4018064

Article
pubs.acs.org/jmc
Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues
as Sirtuin Inhibitors
Sumit S. Mahajan,† Michele Scian,‡ Smitha Sripathy,† Jeff Posakony,†,# Uyen Lao,† Taylor K. Loe,†
Vid Leko,† Angel Thalhofer,† Aaron D. Schuler,†,⊥ Antonio Bedalov,*,† and Julian A. Simon*,†,§
†Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109,
United States
‡Department of Medicinal Chemistry, University of Washington, Health Sciences Building, Room H-068, Seattle, Washington 98195,
United States
§Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109,
United States
ABSTRACT: Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress
responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as
chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called
cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used
saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that
interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the
identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and
8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these
compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class
may be predominantly due to SIRT2 inhibition.
(SIRT1: human sirtuin isoform 1), was found to be devoid of
chemotherapeutic effect; however, cambinol, tenovin-1, teno-
vin-6, and salermide, nonselective SIRT1/SIRT2 inhibitors,
were found to have significant antitumor activity.1,10−12
Combined use of a nonselective sirtuin inhibitor niacinamide
(nicotinamide) and a pan-type I/II HDAC (i.e., zinc-dependent
histone deacetylases) inhibitor vorinostat yielded encouraging
results in a recent diffuse large B-cell lymphoma phase I clinical
trial further validating sirtuins as antilymphoma drug targets.13
Additionally, SRT1720, a potent direct SIRT1 activator that
was originally developed for its potential in lifespan extension
or antiaging activity, was later found to be beneficial in a rat
diabetes model employing a mechanism which may involve
indirect activation of SIRT1.14 The recent functional character-
ization of other sirtuin isoforms such as SIRT3, SIRT5, SIRT6,
INTRODUCTION
■
Identification of new therapeutic compound classes and
validation of new therapeutic targets remain major hurdles in
drug discovery. In the past decade, human sirtuins (homo-
logues of yeast Silent Information Regulator Two or Sir-2)
have emerged as targets for cancer chemotherapy as well as for
neurodegenerative and aging-related disorders such as
Huntington’s disease, Alzheimer’s disease, and diabetes.2
Although strong evidence exists for sirtuins having a central
role in these debilitating diseases, their validation as targets for
therapeutic intervention using small molecule modulators has
been controversial.3−5 The most publicized efforts at
modulation of sirtuin activity have been with the plant
polyphenol resveratrol.6 This purported sirtuin activator was
shown to have highly beneficial effects in animal models of
metabolic disorders (e.g., diabetes) and lifespan extension using
experimental models that have since been largely shown to be
flawed.7−9 EX-527, a potent and selective SIRT1 inhibitor
Received: November 22, 2013
Published: April 3, 2014
© 2014 American Chemical Society
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Figure 1. Cambinol analogues. (a) cambinol; (b) schematic; (c) pyrazolone analogues (e.g., 8 and 17); and (d) isoxazolone analogues (e.g., 24).
1
Figure 2. H NMR spectrum and STD spectrum of (20) and SIRT1. The chemical structure of (20) using naphthalene numbering for clarity.
Relative saturation transfer (C-7 proton =100%).
and SIRT7 has further complicated the field as it is becoming
increasingly clear that in addition to SIRT1 and 2, these
isoforms may also play major roles in aging (SIRT3, SIRT6) as
well as in cell-proliferation disorders (SIRT7).15 Additional
controversies regarding artifacts of popular in vitro assays to
identify novel small molecule modulators of sirtuin activity have
also hampered the validation of these enzymes for pharmaco-
logical intervention.16
poor solubility. Since the discovery of cambinol, we have
focused on optimization of cambinol’s potency and validation
of specific sirtuin isoforms (e.g., SIRT1 and SIRT2) as targets
for B-cell lymphoma chemotherapy. The cambinol pharmaco-
phore has been well studied (Figure 1). The β-naphthol
nucleus is absolutely essential for activity, and the aryl ring is
also important as replacing it with five-membered or a
heterocyclic ring results in significant loss of potency.1,19−22
The pyrimidinedione (thiouracil) heteroaryl ring requires a
balance of hydrogen bond donor and acceptor groups to
maintain potency.
To further develop the structure activity relationship (SAR),
we investigated substituting the pyrimidinedione ring (hetero-
aryl group, Figure 1b) with five-membered heterocycles
(pyrazolone 1c and isoxazolone 1d) as well as altering the
functional groups on the naphthyl and aryl rings. To guide us
with the specific positioning of the functional group to observe
increased potency, we conducted saturation transfer difference
(STD) NMR experiments using 20 (Figure 2). The data
obtained from STD NMR experiments identified portions of
the ligand that interact with the receptor and suggest sites
available for additional elaboration. Cell-based toxicity assays
show a significant correlation between SIRT2 inhibition and
cytotoxicity in the Namalwa Burkitt’s lymphoma cell line.
Previously, in an attempt to identify isoform selective sirtuin
inhibitors, we carried out a phenotypic screen using an NCI
chemical library that resulted in discovery of cambinol (5-[(2-
hydroxy-1-naphthyl)methyl]-6-phenyl-2-thioxo-2,3-dihydro-
4(1H)-pyrimidinone) 1.1 Titration experiments using SIRT2
and an acetyl-peptide substrate showed that cambinol was
competitive with the peptide substrate but noncompetitive with
NAD+ suggesting that its binding site partially overlaps with
that of the substrate. Cambinol inhibits SIRT1 and SIRT2 with
mid-micromolar IC50 and induces hyperacetylation of p53, a
known SIRT1 target, and α-tubulin, a SIRT2 target, in cell-
based deacetylation assays.17,18 In addition, cambinol was found
to be an effective chemosensitizer for the DNA damaging agent
etoposide both in p53-proficient and p53-deficient cancer cell
lines. As a single agent, cambinol was found to be selectively
toxic to BCL6 expressing B-cell lymphoma cells suggesting that
it could be used as a lead compound for antilymphoma drug
development.1 Cambinol however suffers from major draw-
backs including (1) moderate potency, as it did not induce
tumor regression, but a decrease in tumor growth rate, and (2)
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a
Scheme 1. Synthesis of Naphthyl Pyrimidinedione, Pyrazolone, and 5-Isoxazolone Sirtuin Inhibitors (1−18, 24−26)
a
Reagents and conditions: (a) Cl2CHOMe, TiCl4, CH2Cl2, 0−20 °C, 24 h; (b) aryloyl ethyl acetoacetate, piperidine, EtOH, reflux, 2 h; (c) NaBH4,
pyridine, 20 °C, 2 h; (d) thiourea, NaOEt, EtOH, reflux, 18 h; (e) hydrazine, DMF, 20 °C, 1.5 h; (f) hydroxylamine, DMF, 60 °C, 18 h;
a
Scheme 2. Synthesis of Quinolyl Pyrazolone Sirtuin Inhibitors (19−23)
a
Reagents and conditions: (a) HMTA, TFA, 100 °C, 2 h; (b) POX3, DMF, temp, time X = Cl, Br; (c) aryloyl ethyl acetoacetate, piperidine, EtOH,
reflux, 2 h; (d) NaBH4, pyridine, 20 °C, 24 h; (e) hydrazine, DMF, 20 °C, 1.5 h.
parent of this class of agents, was found to be an nonselective
SIRT1, SIRT2 inhibitor with 54% SIRT1 inhibition, 46%
SIRT2 and 16% SIRT3 inhibition at a single 50 μM
concentration (Table 1), and dose response titration against
these enzymes with cambinol gave IC50 values very similar to
published values (SIRT1 IC50 = 56 μM, SIRT2 IC50 = 51 μM)
and >200 μM SIRT3 IC50.1,19
RESULTS
■
Syntheses on new cambinol analogues closely parallel that of
the parent compound, cambinol (Scheme 1). Target
compounds are prepared by a three-step sequence from 2-
hydroxy-1-naphthaldehyde starting materials. For cambinol,
base-catalyzed Knoevenagel condensation with ethyl-benzoyla-
cetate with 2-hydroxy-1-naphthaldehyde affords the α,β-
unsaturated β-ketoester (Scheme 1). Sodium borohydride
reduction of the α,β-unsaturated olefin followed by base-
promoted 2-thiouracil formation with thiourea yields cambinol.
For naphthyl and quinolyl derivatives of cambinol, hydroxy-
naphthaldehyde and hydroxy-quinaldehyde starting materials
were prepared by titanium-catalyzed Reiche formylation
reaction (1-carbaldehyde-2-hydroxynaphthalene derivatives)
from the corresponding phenols, and the Duff reaction of
hexamethylenetetramine (HMTA) with 6-hydroxy-2-quino-
lones in the presence of trifluoroacetic acid (TFA) afforded
the corresponding 5-carbaldehyde-6-hydroxy-2-quinolones, re-
spectively (Schemes 1 and 2). The pyrazolone analogues were
prepared by reacting the appropriate β-ketoester with
hydrazine, while syntheses of the isoxazolone analogues used
corresponding hydroxylamine compounds as nucleophiles
(Scheme 1).
We carried out an STD NMR experiment using recombinant
SIRT1 and compound 20 (Figure 2).23 The relative saturation
transfer enhancement at positions we were able to evaluate is
shown in Figure 2. The results show that C-7 proton
(naphthalene numbering is used for clarity since most of the
compounds discussed in this section are naphthalene
derivatives) gives the strongest STD signal (100% relative
intensity) and forms a close contact with the protein. The aryl
C-4′ methyl protons (66% signal intensity) and C-8 proton
(74% signal intensity) form contacts with the protein, but these
may be suboptimal. Consistent with this, compounds 1
(cambinol), 2, 6, 13, and 22 that contain a small substituent
at C-4′ (i.e., hydrogen or fluorine) are relatively poor and
nonselective sirtuin inhibitors. The most selective compounds
17 and 24 have methyl groups at C-4′ suggesting that SIRT1
and SIRT2 residues that interact with this group are well
conserved. Likewise, compounds that contain larger than
hydrogen (i.e., methyl or isopropyl) substituents at the C-4′
position of the aryl moiety tend to favor SIRT1 inhibition with
the bulkiest substituent (i.e., isopropyl, compound 23) having
the highest selectivity (>3-fold, Table 4). The STD values for
SIRT1, SIRT2, and SIRT3 enzyme inhibition studies, carried
out using the SIRT-Glo assay (Promega Corp., Madison, WI)
at a single inhibitor concentration (50 μM), revealed a range of
activities against each target (Tables 1, 2, and 3). Cambinol, the
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a
Table 1. Naphthyl Pyrimidinedione (1) and Pyrazolone (2−
Table 3. Naphthyl 5-Isoxazolone Sirtuin Inhibitors
a
18) Sirtuin Inhibitors
compound no.
R2
SIRT1 (% inh) SIRT2 (% inh) SIRT3 (% inh)
24
25
26
H
5
11
2
87
48
45
18
17
24
CH3
SIRT1
(% inh)
SIRT2
(% inh)
SIRT3
(% inh)
compound
R
R′
benzyl
a
cambinol
(1)
cambinol
cambinol
54
46
16
Percentage sirtuin inhibition at 50 μM compound.
2
H
H
48
8
18
25
33
27
29
29
73
53
21
19
23
47
9
9
n.d.
22
21
9
a
Table 4. Isoform-Selective SIRT Inhibitors
3
H
3-Br
SIRT1 IC
SIRT2 IC
SIRT3 IC
a 50
4
H
4-Br
36
58
24
48
66
64
25
39
45
59
62
42
49
78
40
a 50
a 50
compound no.
(μM)
(μM)
(μM)
5
H
4-CH3
4-F
1
56
41
51
32
>200
6
6
H
8
7
H
4-CF3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
H
14
82
35
n.d.
12
7
14
17
23
24
37
>200
>200
68
>200
>200
122
8
6-phenyl
6-CH3O
6-CN
6-CH3
6-C2H5
6-Br
6-Br
7-Br
3-Br
3-CH3O
6-Br
26
9
21
10
11
12
13
14
15
16
17
18
>200
13
>200
a
Concentration giving 50% inhibition of sirtuin activity.
8
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3O
21
13
14
14
18
oxygen) in 17 and 24. The reversal of hydrogen bond
directionality results in a reversal of selectivity with the
hydrogen-bond donor compounds 17 displaying up to a 7.8-
fold SIRT1 selectivity and the hydrogen bond acceptor 24 a
15.4-fold SIRT2 selectivity with approximately similar overall
inhibitory activity against the preferred isoform: 26 μM IC50 for
17 against SIRT1 and 13 μM IC50 for 24 against SIRT2.
Substitution of a hydrophobic aromatic group at C-6 of the
naphthalene group gave the most potent SIRT3 inhibitor
(compound 8, SIRT3 IC50 6 μM). This compound however
was relatively nonselective with SIRT1 and SIRT2 IC50 of 41
and 32 μM, respectively. The potency of C-6 phenyl substituted
naphthalene across the SIRT isoforms suggests a conserved
hydrophobic pocket present in all three proteins.
38
20
19
30
a
Percentage sirtuin inhibition at 50 μM compound; n.d. = not
determined.
a
Table 2. Quinolyl Pyrazolone Sirtuin Inhibitors
Our previous studies with cambinol demonstrated that
SIRT1 inhibition could be quantified in cells by determining
the level of p53 acetylation following induction of DNA
damage by the cytotoxic topoisomerase II poison etoposide.1
We treated NCI-H460 cells with 1 μM etoposide in the
presence or absence of cambinol 1 or 14, a SIRT1-selective
inhibitor (Tables 1 and 4). As expected, the more potent
SIRT1 inhibitor 14 shows a significantly higher level of p53
acetylation at a given concentration (Figure 3). Likewise,
compound
no.
SIRT1
SIRT2
SIRT3
R
R1
R′
CH3
(% inh)
(% inh)
(% inh)
19
20
21
22
23
H
H
39
45
51
27
85
23
17
38
24
37
16
20
42
32
32
Cl
Br
Cl
Cl
H
CH3
H
CH3
CH3
H
H
isopropyl
a
Percentage sirtuin inhibition at 50 μM compound.
the -methylene protons and the NHs of the pyrazolone moiety
could not be determined because of the overlap with the water
signal and the fast exchange with solvent deuterons,
respectively. STD values for the C-3 hydrogen and for protons
in the phenyl ring (C-2′, C-3′, C-5′, C-6′) could not be
accurately determined due to overlapping signals. The most
significant determinant of the selectivity between SIRT1 and
SIRT2 in the present compound series is the H-bond donor or
acceptor at the 2 position of the heteroaryl unit (i.e., N−H or
Figure 3. Inhibition of SIRT1-mediated deacetylation of p53 in the
presence of DNA damage. Determination of p53 acetylation in NCI-
H460 cells by Western blot following 24 h drug treatment.
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SIRT2 inhibition results in hyper-acetylation of α-tubulin. The
SIRT2-selective inhibitor 24 induced a rapid and robust dose-
dependent increase in α-tubulin acetylation in NCI-H460 cells
(Figure 4).
Figure 4. Inhibition of SIRT2-mediated deacetylation of α-tubulin.
Determination of α-tubulin acetylation in NCI-H460 cells by Western
blot following 4 h treatment with trichostatin A (TsA) and (24).
Part of the motivation behind these studies was to determine
whether SIRT1, SIRT2 or SIRT3 inhibition, or possibly a
subset of these enzymes, was responsible for cambinol’s
antilymphoma activity. Cell-based sirtuin activity assays were
carried out by determining the acetylation status of well
established SIRT1 (p53) and SIRT2 (α-tubulin) in the
presence of isoform selective inhibitors. Cytotoxicity assays
were carried out with the human Burkitt’s lymphoma cell line
Namalwa grown under standard conditions.1 Cells were plated
in 96-well plates, treated with the test compound or DMSO
control for 72 h, and viable cells were quantified by
luminescence using CellTiterGlo (Promega Corp., Madison,
WI). Cell viability assays showed that both SIRT1- and SIRT2-
selective series exhibit antiproliferative, cytotoxic activity against
the Namalwa Burkitt’s lymphoma cell line, while the SIRT3-
selective compound was less toxic. In the Namalwa cell line, cell
death occurs by induction of apoptosis as evidenced by dose-
dependent appearance of annexin V-positive cells as indication
of early stage apoptosis (Figure 5). In addition, while there is a
strong correlation between SIRT2 inhibition and Namalwa
cytotoxicity (r = 0.56, p = 0.0014) (Figure 6), neither SIRT1 (r
= −0.11) nor SIRT3 (r = 0.21) (data not shown) inhibition
correlates with Namalwa cytotoxicity. Three compounds, the
SIRT1-selective 17, SIRT2-selective 24 and SIRT3-selective 8,
were tested against an expanded panel of Burkitt’s lymphoma
(Dakiki, Daudi, Mutu, Oku, Ramos and Namalwa), diffuse large
B-cell lymphoma (SU-DHL4 and OCI-Ly8-LAM53), non-
transformed Epstein−Barr virus (EBV) immortalized B-cell
lines (B1 and B2), and epithelial cancer cell lines (HCT116-
colon, MCF7-breast, NCI-H460-nonsmall cell lung cancer and
OVCAR3-ovarian) (Table 5). The SIRT2-selective inhibitor 24
Figure 6. Correlation between sirtuin inhibition and Namalwa
Burkitt’s lymphoma cell line growth inhibition. SIRT1 (red circles)
and SIRT2 (blue triangles) inhibition (y-axis) versus Namalwa growth
inhibition (x-axis).
exhibited potent cytotoxicity in both lymphoma and epithelial
cancer cell lines with IC50 ranging from 3 to 7 μM relative to
the nontransformed B-cell lines (IC50 22−28 μM).
DISCUSSION AND CONCLUSIONS
■
Cambinol, (5-[(2-hydroxy-1-naphthyl)methyl]-6-phenyl-2-thi-
oxo-2,3-dihydro-4(1H)-pyrimidinone) 1, is a nonselective
sirtuin inhibitor with equivalent inhibitory activity against
SIRT1 and SIRT2. It also is a reasonably potent antitumor
agent in vitro and in vivo with particular activity against Burkitt’s
lymphoma cell lines.1 In an effort to delineate the contribution
of SIRT1 and SIRT2 inhibition in this antitumor activity, we
sought to develop cambinol analogues with improved potency
and selectivity. Studies by Medda et al. and Rotili et al. have
partly addressed the structure activity relationships of six-
membered pyrimidinedione-containing (i.e., cambinol-like)
compounds.19,24 In an effort to investigate an alternative
chemical space, we prepared a series of five-membered
pyrazolone- and 5-isoxaolone-containing compounds. The
five-membered ring pyrazolone compounds (i.e., 2−23) exhibit
a range of specificities but generally favor SIRT1 inhibition. The
most selective of these compounds, 14 and 17, exhibit a greater
than 5-fold and 7-fold, respectively, preference for SIRT1 over
SIRT2. The 5-isoxazolone compounds (i.e., 24−26) favor
Figure 5. Induction of apoptosis in Namalwa cells treated with 24. FACS analysis of Namalwa cells treated with DMSO (left), 10 μM (24) (center)
and 25 μM (24) (right) for 16 h. Cells were stained with annexin V-PE (y-axis) and DAPI (x-axis). Cells entering an early phase of apoptosis are
present in the upper left panel.
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a
Table 5. Cell Line Growth Inhibition
compound no.
Dakiki
Daudi
Mutu
Oku
Ramos
Namalwa
SU-DHL4
LAM53
8
68
29
7
n.d.
21
33
15
3
51
11
28
15
7
40
15
4
27
77
5
34
32
7
17
24
5
5
compound no.
B1
B2
HCT116
MCF7
NCIH460
OVCAR3
8
36
41
22
47
58
28
58
>80
8
74
32
5
55
61
3
68
13
10
17
24
a
Concentration giving 50% growth inhibition following 72 h drug treatment. Dakiki, Daudi, Mutu, Oku, Ramos, Namalwa are Burkitt’s lymphoma
cell lines. SU-DHL4 and LAM53(OCI-Ly8-LAM53) are diffuse large B-cell lymphoma lines. B1 and B2 are EBV-immortalized nontransformed B-
cell lines. HCT116 (colon), MCF7 (breast), NCI-H460 (lung), and OVCAR3 (ovarian) are epithelial cancer cell lines.
SIRT2 inhibition with a greater than 15-fold preference shown
by 24.
in our study is consistent with the findings that EX-527, a
potent and selective SIRT1 inhibitor, does not exhibit activity
against B-cell lymphoma cell lines, as reported previously by
others for epithelial tumor cells.25 It is possible, however, that
inhibition of SIRT1 may play a role in other tumor contexts, as
suggested by the findings by Rotili et al.24,26 In addition to
Burkitt’s lymphoma cell lines, we tested 8, 17, and 24 against
normal immortalized lymphoblastic lines (B1 and B2), diffuse
large B-cell lymphoma lines as well as a panel of solid tumor
cell lines (Table 5). Interestingly, colon (HCT116) breast
(MCF7) and nonsmall cell lung carcinoma (NCI-H460) also
showed significant sensitivity to SIRT2 inhibition. The data
presented in this manuscript make a strong case for further
development of sirtuin inhibitors as anticancer agents.
In order to gain a molecular understanding of the observed
selectivity, we sought to identify portions of the ligand involved
in the interaction with SIRT1 protein. The STD NMR
experiment with recombinant SIRT1 and 20, a relatively
nonselective but highly soluble ligand, has identified positions
of the ligand as potential sites for improving selectivity and
potency (Figure 2). The efficiency of saturation transfer was
lowest at C-8 of the quinolone ring system (naphthalene
numbering) and at the 4-methyl group of the aryl substituent
suggesting that these portions of the ligand interact with the
protein surface but in a suboptimal manner. Consistent with
this finding, compounds with large substituents at the C-4
position yielded more potent inhibitors (e.g., 20 versus 23,
Table 2) in agreement with the structural model proposed by
Medda et al.19 Saturation transfer was most efficient at C-4 and
C-7 of the quinoline ring suggesting close ligand/protein
interactions; however, the C-8 proton showed a weaker
interaction. Examination of the structural variation at these
positions suggests a reasonable explanation of the observed
selectivities. The binding site for the C-4 phenyl position
appears to tolerate medium-sized substituents equally well in
SIRT1 and SIRT2, with bulkier groups such as isopropyl
showing an improved preference for SIRT1. The strongest
determinant of selectivity in the current compound series is the
hydrogen bond donor (i.e., N−H at the pyrazolone 2 position
in 17) or hydrogen bond acceptor (i.e., O at the dihydro 1,2-
oxazol-5-one 1 position in 24). Remarkably, the reversal of
directionality of the hydrogen bond donor/acceptor pair leads
to a 7.8-fold SIRT1 selectivity in the case of the former 24 and
a 15.4-fold SIRT2 selectivity for the latter, with approximately
equipotent overall inhibitory activity against the preferred
isoform: 26 μM IC50 for 17 against SIRT1 and 13 μM IC50 for
24 against SIRT2. Future studies will attempt to optimize
interactions at C-4′ and C-8. Placement of a bulky hydrophobic
phenyl group at the naphthalene C-6 position yielded 8, a
potent although relatively nonselective SIRT3 inhibitor.
Elaboration of the C-6 substituent provides a means to increase
overall potency. The SAR trends observed in the current
compound series provide a strong starting point for develop-
ment of potent isoform-selective as well as pan-sirtuin
inhibitors.
EXPERIMENTAL SECTION
■
Protein Expression. Human SIRT1 cDNA was cloned into the
pET-4a vector and transformed into BL21-DE3 Escherichia coli cells.
Expression of SIRT1 protein fusion with hexa-histidine tag (C-
terminal) was induced with IPTG (5 mM). Recombinant protein was
purified from bacterial lysates using the Ni-NTA column (Clontech
Laboratories Inc. Mountain View, CA) according to manufacturer’s
protocols.
Enzyme Inhibition Assays. SIRT1 was expressed and purified as
described above. SIRT2 and SIRT3 were purchased from Cayman
Chemical (Ann Arbor, MI). Enzyme inhibition assays were performed
in 96-well plates using the SIRT-Glo Assay (Promega Corp., Madison
WI) according to the manufacturer’s instructions. Compounds were
dissolved in 100% DMSO and were tested over a five 3-fold dilution
concentration range (final DMSO concentration 0.25%). IC50 values
were determined in triplicate and reported values are averages of two
independent experiments.
Cell Viability Assays. Cell lines were obtained from ATCC
(Manasas, VA) and were grown under standard conditions. For
viability assays, cells were dispensed into 96-well plates and treated
with test compounds in 100% DMSO (final DMSO concentration
0.25%). Cells were incubated with test compounds (or DMSO
controls) for 72 h, and viability was determined using the CellTiterGlo
Assay (Promega Corp., Madison WI) according to manufacturer’s
instructions. Assays were carried out in triplicate reported values are
averages of two independent experiments.
Western Blot. NCI-H460 human lung carcinoma cells (ATCC)
were grown under standard conditions. Drug treatment and Western
blots were carried out as previously described.1 Antibodies for
acetylated α-tubulin (clone 6-11B-1, Sigma, St. Louis, MO), α-tubulin
(clone DM1A, Calbiochem, San Diego, CA), actin (Roche, Indian-
apolis, IN), acetylated p53 (Cell Signaling, Danvers, MA), p53 (Cell
Signaling, Danvers, MA) and acetylated lysine (polyclonal antibody;
Cell Signaling, Danvers, MA) were used at dilutions recommended by
the manufacturer.
While both SIRT1 and SIRT2-selective compounds exhibit
some degree of cytotoxicity, a strong correlation between
SIRT2 inhibition and Namalwa cytotoxicity (r = 0.56, p =
0.0014) suggests that SIRT2 inhibition may be primarily
responsible for the observed antilymphoma activity. Lack of
correlation between SIRT1 inhibition and cytotoxicity observed
Flow Cytometry. Namalwa cells were treated with 10 and 25 μM
(24) or solvent (DMSO) for 16 h, washed in phosphate buffered
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saline, and suspended at approximately 200 000 cells in 200 μL of
binding buffer containing 5 μL of annexin V-PE (BD Biosciences, San
Jose, CA) for measuring phosphatidylserine, and 4′,6-diamidino-2-
phenylindole (DAPI) at a concentration of 2.5 μg/mL, for nucleic
acids. After 15 min incubation at room temperature, cell fluorescence
was analyzed on a FACScanR (Becton Dickinson, Franklin Lakes, NJ).
General Chemistry. Unless specified otherwise, reagents were
obtained from Sigma-Aldrich and used without additional purification.
6-Hydroxy-2(1H)-quinolinone and 4-methylquinoline-2,6-diol were
obtained from Bioblocks Inc. 3-(4-isopropyl-phenyl)-3-oxo-propionic
acid ethyl ester was obtained from Vitas M Laboratories. Thin layer
chromatography was carried out using Merck 60 F254 silica gel plates
using appropriate solvent mixtures. Solvents were ACS reagent grade
and anhydrous solvents (Aldrich and Acros) were used as received.
Medium pressure chromatography was carried out using Biotage
Isolera with Silicycle HP cartridges. LCMS was performed using an
Agilent 1100 HPLC system equipped with a Waters XTerra MS C18 5
um, 4.6 × 50 mm column, an Agilent photodiode array detector and
an in-line Agilent 6130 single quadrupole mass spectrometer. The LC
method involved gradient elution from 0 to 95% acetonitrile in water
(0.1% formic acid) over 6 min. Final purity of compounds was
determined using ACE 3, C8−300, 150 × 3.0 mm column with the
above-mentioned gradient over 15 min. Agilent ChemStation software
was used to develop methods and calculate percentage purity. All
compounds reported are at least 95% pure.
reported by Garcia et al.29 A solution of TiCl4 (18 mmol) and
dichloromethyl ether (9 mmol) in anhydrous dichloromethane (10
mL) was stirred at 0 °C for 15 min. A solution of the corresponding
substituted naphthol (9 mmol) in either CH2Cl2 or 1,2-dichloroethane
(30 mL) was added dropwise, and the reaction was warmed to room
temperature. The reaction was allowed to stir overnight after which it
was quenched by adding 1 N HCl (10 mL). The aqueous layer was
extracted with CH2Cl2 (3 × 10 mL), and the organic layers were then
combined, dried with Na2SO4, and reduced to dryness to afford a
residue which was further purified using medium pressure
chromatography to yield the pure aldehyde.
a. 6-Bromo-2-hydroxynaphthalene-1-carbaldehyde (51). A red-
dish brown residue initially obtained was further purified by medium
pressure chromatography using a gradient EtOAc/hexane solvent
system (1−10% EtOAc) yielding 1.5 g (6 mmol, 67%) as a white
powder. 1H NMR (500 MHz, chloroform-d) δ 13.12 (s, 1H), 10.79 (s,
1H), 8.24 (d, J = 8.8 Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.91 (d, J = 9.3
Hz, 1H), 7.70 (dd, J = 8.8, 2.4 Hz, 1H), 7.19 (d, J = 9.3 Hz, 1H).
LRMS: m/z = 249.1 (M − H)−.
b. 2-Hydroxy-6-phenylnaphthalene-1-carbaldehyde (52). 6-
Bromo-2-hydroxynaphthalene-1-carbaldehyde (1.39 mmol), phenyl
boronic acid (2.09 mmol), bis(dibenzylideneacetone)palladium(0)
(0.0139 mmol), and tricyclohexylphosphine (0.033 mmol) were
suspended in dioxane (7 mL). To this mixture was added potassium
phosphate tribasic (1.27 M), and the solution was heated at 100 °C for
6 h. The reaction was cooled, loaded onto a small silica column, and
eluted with EtOAc (20 mL), and the filtrate was collected. The
extracts were washed with water, dried using Na2SO4, and reduced to
dryness to afford a dark brown residue. The residue was then purified
by medium pressure chromatography using gradient EtOAc/hexane
solvent system (1−10% EtOAc) yielding 140 mg (0.56 mmol, 40%) as
NMR Experiments. All NMR experiments were performed at 25
°C on a Varian Unity Plus 300 spectrometer or Varian Unity-Inova
500 MHz spectrometer equipped with a 5 mm triple-resonance
1H(13C/15N), z-axis pulsed-field gradient probe head. For character-
ization purposes, samples consisted of a ∼5 mM solution of each
compound in chloroform-d (99.8% D, Cambridge Isotopes), dimethyl
sulfoxide-d6 (99.9% D, Cambridge Isotopes), benzene-d6 (99.5% D,
Cambridge Isotopes) or acetone-d6 (99.9% D, Cambridge Isotopes),
and the spectra were referenced to residual solvent peaks at 7.27, 2.50,
1
a white powder. H NMR (500 MHz, chloroform-d) δ 13.16 (s, 1H),
10.85 (s, 1H), 8.42 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.00
(d, J = 2.4 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 7.3 Hz, 2H),
7.51 (t, J = 7.8 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.19 (d, J = 8.8 Hz,
1H), LRMS [ES]+: m/z = 249.1 (M + H)+.
c. 2-Hydroxy-6-methoxynaphthalene-1-carbaldehyde (53). A
black residue was obtained which further purified by medium pressure
chromatography using a gradient EtOAc/hexane solvent system (1−
10% EtOAc) yielding 500 mg (2.47 mmol, 43%). 1H NMR (500 MHz,
chloroform-d) δ 12.90 (s, 1H), 10.79 (s, 1H), 8.27 (d, J = 9.3 Hz, 1H),
7.90 (d, J = 8.8 Hz, 1H), 7.30 (dd, J = 9, 2.7 Hz, 1H), 7.16 (d, J = 8.3
Hz, 1H), 7.13 (d, J = 2.7 Hz, 1H), 3.93 (s, 3H). LRMS [ES]+: m/z =
203.1 (M + H)+.
1
7.16, and 2.05 ppm, respectively. H-1D spectra were acquired at a
resolution of 16k complex points in the time domain with 32
accumulations each (sw = 6000 Hz, d1 = 3 s).
Saturation Transfer Difference (STD) NMR. All STD experi-
ments were performed in D2O solution to eliminate the influence of
exchangeable protein protons. The purified SIRT1 stock solution (12
μM) was dialyzed against deuterated phosphate buffered saline (PBS).
The exchange buffer was prepared by lyophilizing a PBS solution at
pH 7.4 and redissolving it in D2O (99.9% D, Cambridge Isotopes).
NMR samples contained 1.5 μM SIRT1 and 200 μM compound 20 in
PBS in D2O pH 7.4 (uncorrected for D2O) with 1% dimethyl
sulfoxide-d6 as a cosolvent. STD NMR experiments were performed at
25 °C as previously described.27 Briefly, two free induction decay
(FID) data sets were collected in an interleaved manner to minimize
temporal fluctuations with the protein irradiation frequency set on-
resonance (−0.5 ppm) and off-resonance (40 ppm), respectively (sw
=6000 Hz, 16 steady state scans, 2048 transients, 4k complex points,
d1 = 3 s). Protein saturation was obtained using a train of individual 50
ms long, frequency selective Gaussian radio frequency (rf) pulses
separated by an interpulse delay of 1 ms. The number of selective
pulses was set to 50, leading to a total saturation time (τsat) of 2.5 s.
Gradient Tailored Excitation (WATERGATE) scheme was employed
to suppress the residual water signal.28 Suppression of the background
signals arising from the protein was not required. The FID acquired
with off-resonance irradiation generated the reference spectrum (Ioff)
whereas the difference FID (off-resonance−on-resonance) yielded the
STD spectrum (ISTD = Ioff − Ion). Spectra were processed with an
exponential apodization function (1 Hz line-broadening) and zero-
filling to 8k complex points before Fourier transformation and baseline
correction with a third order Bernstein polynomial fit. The STD
measurements were done in duplicate, and all data were processed and
analyzed using MNova 8.1 processing software (Mestrelab Research,
Santiago de Compostela, Spain).
d. 5-Formyl-6-hydroxynaphthalene-2-carbonitrile (54). The
reaction was carried out in 1,2-dichloroethane. The residue obtained
after extraction was purified by medium pressure chromatography
using a gradient EtOAc/hexane solvent system (1−10% EtOAc)
yielding 72 mg (0.365 mmol, 42%). 1H NMR (500 MHz, chloroform-
d) δ 13.36 (s, 1H), 10.82 (s, 1H), 8.45 (d, J = 8.8 Hz, 1H), 8.19 (d, J =
2 Hz, 1H), 8.05 (d, J = 9.3 Hz, 1H), 7.80 (dd, J = 8.8, 2 Hz, 1H), 7.30
(d, J = 9.3 Hz, 1H). LRMS [ES]+: m/z = 198.1 (M + H)+.
e. 2-Hydroxy-6-methylnaphthalene-1-carbaldehyde (55). 6-
Methyl-2-hydroxy-1-naphthalene was synthesized from 6-methoxy-2-
naphthaldehyde as reported previously.30 Formylation was carried
using the above-mentioned general procedure. The reaction residue
was purified using a gradient EtOAc/hexane solvent system (10−25%
1
EtOAc) yielding 386 mg (2.07 mmol, 81%) as a pale yellow solid. H
NMR (500 MHz, chloroform-d) δ 13.03 (s, 1H), 10.81 (s, 1H), 8.26
(d, J = 8.8 Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.59 (d, J= 2.0, 1H), 7.46
(dd, J = 8.8, 2 Hz, 1H), 7.12 (d, J = 9.3 Hz, 1H), 2.51 (s, 3H). LRMS
[ES]+: m/z = 187.1 (M + H)+.
f. 6-Ethyl-2-hydroxynaphthalene-1-carbaldehyde (56). 6-Ethyl-2-
hydroxy-1-naphthalene was synthesized from 6-methoxy-2-aceto-
naphthone as reported previously.30 Formylation was carried using
the above-mentioned general procedure. The residue obtained was
purified using a gradient EtOAc/hexane solvent system (10−25%
1
EtOAc) yielding 322 mg (1.61 mmol, 67%). H NMR (500 MHz,
Chemistry. General Procedure I for Preparation of Substituted
2-Hydroxy-1-Naphthalene Carbaldehydes. The procedure was
adapted from the protocol for formylation of electron rich phenols
chloroform-d) δ 13.07 (s, 1H), 10.85 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H),
7.94 (d, J = 9.3 Hz, 1H), 7.60 (d, J= 2.0, 1H), 7.51 (dd, J = 7.8, 2 Hz,
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1H), 7.12 (d, J = 9.3 Hz, 1H), 2.82 (q, J = 7.8 Hz, 2H), 1.34 (t, J = 7.8
Hz, 3H). LRMS [ES]+: m/z = 201.1 (M + H)+.
f. 2-(4-Methylbenzoyl)-8-phenyl-3H-benzo[f ]chromen-3-one
(35). Yield 204 mg (0.51 mmol, 68%). 1H NMR (500 MHz,
chloroform-d) δ 8.91 (s, 1H), 8.34 (d, J = 8.7 Hz, 1H), 8.19−8.12 (m,
2H), 7.99 (dd, J = 8.7, 1.9 Hz, 1H), 7.88−7.82 (m, 2H), 7.76−7.70
(m, 2H), 7.59−7.48 (m, 3H), 7.47−7.39 (m, 1H), 7.31 (d, J = 7.9 Hz,
2H), 2.45 (s, 3H). LRMS [ES]+: m/z = 391.1 (M + H)+.
g. 7-Bromo-2-hydroxynaphthalene-1-carbaldehyde (57). A red-
dish brown residue was initially obtained which further purified by
medium pressure chromatography using a gradient EtOAc/hexane
solvent system (1−10% EtOAc) yielding 860 mg (3.42 mmol, 76%) as
1
g. 8-Bromo-2-benzoyl-3H-benzo[f ]chromen-3-one (36). Yield
a white powder. H NMR (500 MHz, chloroform-d) δ 13.19 (s, 1H),
1
200 mg (0.52 mmol, 74%) H NMR (500 MHz, chloroform-d) δ
10.74 (s, 1H), 8.50 (s, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.67 (d, J = 8.8
Hz, 1H), 7.54 (dd, J = 8.8, 1.7 Hz, 1H), 7.17 (d, J = 9.3 Hz, 1H).
LRMS: m/z = 249.1 (M − H)−.
10.771 (s, 1H), 8.23 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 2.0 Hz, 1H), 7.89
(d, J = 9.1 Hz, 1H), 7.70−7.71 (m, J = 9.0 Hz, 3H), 7.68 (d, J = 2.0
Hz, 1H), 7.31−7.22 (m, 2H), 7.17 (d, J = 9.1 Hz, 1H). LRMS [ES]+:
m/z = 379.09 (M + H)+.
h. 8-Methoxy-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(37). Yield 95 mg (0.27 mmol, 70%). 1H NMR (500 MHz,
chloroform-d) δ 8.82 (s, 1H), 8.15 (d, J = 9.2 Hz, 1H), 7.99 (d, J =
9.0 Hz, 1H), 7.86−7.80 (m, 2H), 7.50 (dd, J = 9.0, 0.7 Hz, 1H), 7.37
(dd, J = 9.2, 2.6 Hz, 1H), 7.33−7.23 (m, 3H), 3.96 (s, 3H), 2.44 (s,
3H). LRMS [ES]+: m/z = 345.1 (M + H)+.
h. 3-Bromo-2-hydroxynaphthalene-1-carbaldehyde (58). A red-
dish brown residue was initially obtained was further purified by
medium pressure chromatography using a gradient EtOAc/hexane
solvent system (1−10% EtOAc) yielding 850 mg (3.38 mmol, 76%) as
1
a white powder. H NMR (500 MHz, chloroform-d) δ 13.82 (s, 1H),
10.76 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 8.31 (s, 1H), 7.76 (d, J = 8.4
Hz, 1H), 7.66 (ddd, J = 8.8, 7.0, 1.2 Hz, 1H), 7.48 (ddd, J = 8.8, 7.0,
1.2 Hz, 1H). LRMS [ES]+: m/z = 251.0 (M + H)+.
i. 2-(4-Methylbenzoyl)-3-oxo-3H-benzo[f ]chromene-8-carboni-
i. 2-Hydroxy-3-methoxynaphthalene-1-carbaldehyde (59). A
reddish brown residue was initially obtained which further purified
by medium pressure chromatography using a gradient EtOAc/hexane
solvent system (1−10% EtOAc) yielding 75 mg (0.37 mmol, 36%) as a
1
trile (38). Yield 113 mg of light pink solid (0.33 mmol, 73%). H
NMR (500 MHz, benzene-d6) δ 8.01 (s, 1H), 7.91−7.82 (m, 2H),
7.24 (d, J = 1.7 Hz, 1H), 7.03 (dd, J = 8.7, 1.7 Hz, 1H), 6.94 (d, J = 8.7
Hz, 1H), 6.92−6.88 (m, 3H), 6.79−6.75 (m, 1H), 1.96 (s, 3H). LRMS
[ES]+: m/z = 340.1 (M + H)+.
1
white powder. H NMR (500 MHz, chloroform-d) δ 13.46 (s, 1H),
10.74 (s, 1H), 8.23 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.48
(ddd, J = 8.3, 7.6, 1.2 Hz, 1H), 7.41 (ddd, J = 8.3, 7.6, 1.2 Hz, 1H),
7.30 (s, 1H), 4.01 (s, 3H). LRMS [ES]+: m/z = 203.1 (M + H)+.
General Procedure (II) for Preparation of 2-Benzoyl-3H-benzo-
[f]chromen-3-ones. The compounds were synthesized using the
established synthetic route for cambinol and its analogues reported
earlier.19 To a solution of substituted hydroxyl naphthaldehydes (5
mmol) in ethanol (5 mL) were added the corresponding ethyl benzoyl
acetates (5 mmol). Piperidine (5 drops) was added, and the reaction
was heated under reflux for 2 h. The reaction was allowed to cool, and
the yellowish precipitate obtained was collected by filtration and
washed with ethanol several times to get the condensation product.
Synthesis of 2-benzoyl-3H-benzo[f ]chromen-3-one (29) has been
previously reported.19
j. 8-Ethyl-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one (39).
Yield 260 mg (0.76 mmol, 70%). H NMR (500 MHz, chloroform-d)
1
δ 8.87 (s, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H),
7.88−7.80 (m, 2H), 7.73 (d, J = 2.0 Hz, 1H), 7.58 (dd, J = 8.6, 1.8 Hz,
1H), 7.49 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 2.86 (q, J = 7.6
Hz, 2H), 2.44 (s, 3H), 1.35 (t, J = 7.6 Hz, 3H). LRMS [ES]+: m/z =
343.1 (M + H)+.
k. 8-Methyl-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(40). Yield 250 mg (0.76 mmol, 71%). 1H NMR (500 MHz,
chloroform-d) δ 8.87 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 8.02 (d, J = 9.0
Hz, 1H), 7.87−7.80 (m, 2H), 7.72 (d, J = 1.8 Hz, 1H), 7.55 (dd, J =
8.6, 1.7 Hz, 1H), 7.49 (dd, J = 9.0, 0.7 Hz, 1H), 7.33−7.26 (m, 2H),
2.58−2.54 (m, 3H), 2.44 (s, 3H). LRMS [ES]+: m/z = 329.1 (M +
H)+.
a. 2-(3-Bromobenzoyl)-3H-benzo[f]chromen-3-one (30). Yield
700 mg (3.7 mmol, 74%). 1H NMR (500 MHz, chloroform-d) δ
9.00 (d, J = 0.6 Hz, 1H), 8.34−8.28 (m, 1H), 8.18−8.12 (m, 1H),
8.08−8.03 (m, 1H), 8.01−7.95 (m, 1H), 7.86−7.73 (m, 3H), 7.65
(ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.58−7.52 (m, 1H), 7.43−7.35 (m,
1H). LRMS [ES]+: m/z = 379.09 (M + H)+.
l. 8-Bromo-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(41). Yield 650 mg (1.65 mmol, 83%). 1H NMR (500 MHz,
DMSO-d6) δ 9.16 (s, 1H), 8.57 (d, J = 9.0 Hz, 1H), 8.40 (d, J = 2.1
Hz, 1H), 8.28 (d, J = 9.1 Hz, 1H), 7.91−7.81 (m, 3H), 7.71 (d, J = 9.1
Hz, 1H), 7.38−7.32 (m, 2H), 2.40 (s, 3H). LRMS [ES]+: m/z = 393.0
(M + H)+.
m. 9-Bromo-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(42). Yield 180 mg (0.45 mmol, 72%). 1H NMR (500 MHz,
acetone-d6) δ 9.11 (s, 1H), 8.84 (dd, J = 1.8, 0.8 Hz, 1H), 8.34−8.28
(m, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.97−7.90 (m, 2H), 7.78 (dd, J =
8.7, 1.9 Hz, 1H), 7.63 (dd, J = 9.0, 0.7 Hz, 1H), 7.37 (ddt, J = 8.0, 1.5,
0.7 Hz, 2H), 2.44 (s, 3H). LRMS [ES]+: m/z = 393.1 (M + H)+.
n. 5-Bromo-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(43). Yield 80 mg (0.20 mmol, 34%). 1H NMR (500 MHz,
chloroform-d) δ 8.85 (s, 1H), 8.38 (s, 1H), 8.27−8.21 (m, 1H),
7.91−7.81 (m, 3H), 7.73 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.63 (ddd, J =
8.1, 6.9, 1.1 Hz, 1H), 7.34−7.27 (m, 2H), 2.45 (s, 3H). LRMS [ES]+:
m/z = 393.1 (M + H)+.
b. 2-(4-Bromobenzoyl)-3H-benzo[f]chromen-3-one (31). Yield
1
500 mg (1.32 mmol, 72%). H NMR (500 MHz, chloroform-d) δ
8.98 (d, J = 0.7 Hz, 1H), 8.33−8.27 (m, 1H), 8.17−8.11 (m, 1H),
8.00−7.94 (m, 1H), 7.82−7.72 (m, 3H), 7.68−7.59 (m, 3H), 7.57−
7.51 (m, 1H). LRMS [ES]+: m/z = 379.09 (M + H)+.
c. 2-(4-Methylbenzoyl)-3H-benzo[f ]chromen-3-one (32). Yield
1
506 mg (1.59 mmol, 66%). H NMR (500 MHz, chloroform-d) δ
8.89 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H), 7.98−
7.90 (m, 1H), 7.87−7.80 (m, 2H), 7.72 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H),
7.61 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.30 (d, J
= 7.9 Hz, 1H), 7.18 (s, 1H), 2.44 (s, 3H). LRMS [ES]+: m/z = 315.10
(M + H)+.
o. 5-Methoxy-2-(4-methylbenzoyl)-3H-benzo[f ]chromen-3-one
(44). Yield 200 mg (0.58 mmol, 65%). 1H NMR (500 MHz,
chloroform-d) δ 8.86 (s, 1H), 8.20−8.14 (m, 1H), 7.83 (dq, J = 8.3,
2.0 Hz, 3H), 7.61−7.53 (m, 2H), 7.44 (s, 1H), 7.29 (d, J = 7.9 Hz,
2H), 4.10 (s, 3H), 2.44 (s, 3H). LRMS [ES]+: m/z = 345.1 (M + H)+.
p. 8-Bromo-2-(4-methoxybenzoyl)-3H-benzo[f ]chromen-3-one
(45). Yield 95 mg (0.23 mmol, 63%). 1H NMR (500 MHz,
chloroform-d) δ 8.79 (s, 1H), 8.16−8.09 (m, 2H), 8.01 (d, J = 9.1
Hz, 1H), 7.96−7.89 (m, 2H), 7.79 (dd, J = 8.9, 2.1 Hz, 1H), 7.59−
7.53 (m, 1H), 7.01−6.94 (m, 2H), 3.90 (s, 3H). LRMS [ES]+: m/z =
409.0 (M + H)+.
d. 2-(4-Fluorobenzoyl)-3H-benzo[f ]chromen-3-one (33). The
precipitate was recrystallized overnight from ethyl acetate and dried
under vacuum yielding 490 mg (1.57 mmol, H NMR (500 MHz,
chloroform-d) δ 8.95 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 9.0
Hz, 1H), 8.00−7.92 (m, 3H), 7.74 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.63
(ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.22−7.13 (m,
2H). LRMS [ES]+: m/z = 319.1 (M + H)+.
1
e. 2-(4-Trifluoromethylbenzoyl)-3H-benzo[f ]chromen-3-one (34).
The precipitate was recrystallized from ethyl acetate and dried under a
1
vacuum to yield 450 mg (1.21 mmol, 53%). H NMR (500 MHz,
chloroform-d) δ 9.06 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 9.0
Hz, 1H), 7.99 (ddt, J = 13.6, 8.1, 0.8 Hz, 3H), 7.81−7.73 (m, 3H),
7.65 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.54 (dd, J = 9.0, 0.7 Hz, 1H).
LRMS [ES]+: m/z = 369.1 (M + H)+.
General Procedure (III) for Preparation of Pyrazolone-Based
Analogues. The corresponding 2-benzoyl ketocoumarin (0.5 mmol)
was dissolved in dry pyridine (3 mL). To this solution was added
NaBH4 (0.625 mmol), and the reaction was stirred at room temp for 2
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h. The mixture was then poured in cold 2 M hydrochloric acid (10
mL), which resulted in a white precipitate. The precipitate was washed
several times with water, dried under a vacuum to yield the
corresponding 2-benzoyl-1,2-dihydrocoumarin which was taken to
the next step without purification. To a stirring solution of 2-benzoyl-
1,2 dihydrocoumarin (0.1 mmol) in DMF (0.2 mL) was added
hydrazine (0.125 mmol), and the mixture was stirred for 1.5 h at room
temperature. Water (5 mL) was then added, and the aqueous layer was
extracted with EtOAc (15 mL). The organic layer was separated, dried
with Na2SO4, and reduced to dryness to afford a yellowish brown
crude product, which was further purified using medium pressure
chromatography.
i. 6-Hydroxy-5-([5-(4-methylphenyl)-3-oxo-2,3-dihydro-1H-pyra-
zol-4-yl]methyl)naphthalene-2-carbonitrile (10). The crude product
was purified by medium pressure chromatography using a gradient
EtOAc/hexane solvent system (1−10% EtOAc) yielding 10 mg
(0.028, 40%). 1H NMR (500 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.28 (d,
J = 1.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.34 (m, 4H), 7.18 (m, 3H),
4.11 (s, 2H), 2.31 (s, 3H). LRMS [ES]+: m/z = 356.2 (M + H)+.
j. 4-[(2-Hydroxy-6-methylnaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (11). The crude product was
purified by medium pressure chromatography using a gradient EtOAc/
hexane solvent system (10−25% EtOAc) yielding 42 mg (0.12 mmol,
1
80%). H NMR (300 MHz, DMSO-d6) δ 7.52−7.35 (m, 3H), 7.16
(m, 3H), 6.86 (d, J = 8.9 Hz, 1H), 4.07 (s, 2H), 2.62−2.51 (m, 3H),
a. 4-[(2-Hydroxynaphthalen-1-yl)methyl]-5-phenyl-2,3-dihydro-
1H-pyrazol-3-one (2). The solid product was recrystallized from a
mixture of ethyl acetate and n-heptane. Yield 100 mg (0.316 mmol,
2.32 (s, 3H). LRMS [ES]+: m/z = 345.2 (M + H)+.
k. 4-[(6-Ethyl-2-hydroxynaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (12). The crude product was
purified by medium pressure chromatography using a gradient EtOAc/
hexane solvent system (10- 25% EtOAc). Yielding 37 mg (0.10 mmol,
1
64%). H NMR (300 MHz, acetone-d6) δ 7.64 (dd, J = 8.0, 1.4 Hz
1H), 7.53 (d, 8.8 Hz, 1H), 7.51−7.46 (m, 3H), 7.40−7.32, (m, 3H),
7.15−7.01 (m, 3H), 4.11 (s, 2H). LRMS [ES]+: m/z = 317.1 (M +
H)+.
b. 5-(3-Bromophenyl)-4-[(2-hydroxynaphthalen-1-yl)methyl]-2,3-
dihydro-1H-pyrazol-3-one (3). The crude product was purified by
medium pressure chromatography using a gradient MeOH:EtOAc
solvent system (1−10% MeOH). Yield 25 mg (0.063 mmol, 48%). 1H
NMR (500 MHz, acetone-d6) δ 7.83−7.56 (m, 4H), 7.56−7.44 (m,
2H), 7.39 (t, J = 7.9 Hz, 1H), 7.20−7.04 (m, 3H), 4.27 (s, 2H). LRMS
[ES]+: m/z = 395.01 (M + H)+.
c. 5-(4-Bromophenyl)-4-[(2-hydroxynaphthalen-1-yl)methyl]-2,3-
dihydro-1H-pyrazol-3-one (4). The crude product was purified by
medium pressure chromatography using a gradient MeOH−EtOAc
solvent system (1−10% MeOH). Yield 15 mg (0.037 mmol, 49%). 1H
NMR (500 MHz, DMSO-d6) δ 11.67 (s, 1H), 9.88 (s, 1H), 7.68−7.60
(m, 2H), 7.51 (dd, J = 20.3, 8.5 Hz, 3H), 7.35 (d, J = 8.5 Hz, 2H), 7.14
(dt, J = 6.8, 4.0 Hz, 2H), 7.06 (d, J = 8.8 Hz, 1H), 4.11 (s, 2H). LRMS
[ES]+: m/z = 395.01 (M + H)+.
1
71%). H NMR (300 MHz, DMSO-d6) δ 7.54−7.35 (m, 4H), 7.16
(m, 4H), 6.92 (dd, J = 8.9, 1.8 Hz, 1H), 4.08 (s, 2H), 2.60 (q, J = 7.5
Hz, 2H), 2.34 (s, 3H), 1.16 (t, J = 7.5 Hz, 3H). LRMS [ES]+: m/z =
359.2 (M + H)+.
l. 4-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-5-phenyl-2,3-
dihydro-1H-pyrazol-3-one (13). The crude product was purified by
medium pressure chromatography using a gradient MeOH/EtOAc
solvent system (1−10% MeOH) yielding 16 mg (0.045 mmol, 58%).
1H NMR (300 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.53 (d, J = 8.8 Hz,
1H), 7.43 (m, 2H), 7.35 (m, 3H), 7.18 (d, J = 8.8 Hz, 1H), 7.11 (d, J =
8.8 Hz, 1H), 4.10 (s, 2H), 2.32 (s, 3H). LRMS [ES]+: m/z = 394.93
(M + H)+.
m. 4-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (14). The crude product was
purified by medium pressure chromatography using a gradient
MeOH/EtOAc solvent system (1−10% MeOH) yielding 45 mg
1
(0.110 mmol, 62%). H NMR (500 MHz, DMSO-d6) δ 7.89 (d, J =
d. 4-[(2-Hydroxynaphthalen-1-yl)methyl]-5-(4-methylphenyl)-
2,3-dihydro-1H-pyrazol-3-one (5). The crude product was purified
by medium pressure chromatography using a gradient MeOH:EtOAc
solvent system (1−10% MeOH). Yield 22 mg (0.066 mmol, 42%). 1H
NMR (500 MHz, DMSO-d6) δ 11.54 (s, 1H), 10.13 (s, 2H), 7.68−
7.62 (m, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.38
(d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H), 7.16−7.01 (m, 3H), 4.10
(s, 2H), 2.34 (s, 3H). LRMS [ES]+: m/z = 331.20 (M + H)+.
e. 5-(4-Fluorophenyl)-4-[(2-hydroxynaphthalen-1-yl)methyl]-2,3-
2.1 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.46 (s, 1H), 7.33 (d, J = 7.8 Hz,
2H), 7.13 (m, 4H), 4.06 (s, 2H), 2.31 (s, 3H). LRMS [ES]+: m/z =
409.10 (M + H)+.
n. 4-[(7-Bbromo-2-hydroxynaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (15). The crude product was
purified by medium pressure chromatography using a gradient
MeOH/EtOAc solvent system (1−10% MeOH) yielding 16 mg
(0.110 mmol, 52%). 1H NMR (300 MHz, DMSO-d6) δ 7.54−7.35 (m,
4H), 7.16 (m, 4H), 6.92 (dd, J = 8.9, 1.8 Hz, 1H), 4.08 (s, 2H), 2.34
(s, 3H). LRMS [ES]+: m/z = 409.10 (M + H)+.
1
dihydro-1H-pyrazol-3-one (6). Yield 60 mg (0.18 mmol, 58%). H
NMR (500 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.89 (s, 1H), 7.68−7.61
(m, 2H), 7.59−7.46 (m, 2H), 7.41 (dd, J = 8.4, 5.6 Hz, 2H), 7.26−
7.20 (m, 2H), 7.14 (tt, J = 6.3, 2.5 Hz, 2H), 7.05 (d, J = 8.8 Hz, 1H),
4.09 (s, 2H). LRMS [ES]+: m/z = 335.10 (M + H)+.
o. 4-[(3-Bromo-2-hydroxynaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (16). The crude product was
purified by medium pressure chromatography using a gradient
MeOH/EtOAc solvent system (1−10% MeOH) yielding 20 mg
f. 4-[(2-Hydroxynaphthalen-1-yl)methyl]-5-[4-(trifluoromethyl)-
1
(0.110 mmol, 65%). H NMR (300 MHz, DMSO-d6) δ 8.01 (s, 1H),
phenyl]-2,3-dihydro-1H-pyrazol-3-one (7). Yield 60 mg (0.15
7.67 (d, J = 8.1 Hz, 1H), 7.51−7.33 (m, 4H), 7.15 (t, J = 7.5 Hz, 1H),
7.01 (d, J = 9.0 Hz, 1H), 6.87 (t, J = 7.4 Hz, 1H), 4.08 (s, 2H), 2.44 (s,
3H). LRMS [ES]+: m/z = 409.1 (M + H)+.
1
mmol, 57%). H NMR (500 MHz, DMSO-d6) δ 11.82 (s, 1H), 9.78
(s, 1H), 7.67−7.56 (m, 6H), 7.51 (d, J = 8.8 Hz, 1H), 7.14 (td, J = 6.9,
4.6 Hz, 2H), 7.04 (d, J = 8.8 Hz, 1H), 4.16 (s, 2H). LRMS [ES]+: m/z
= 385.10 (M + H)+.
p. 4-[(2-Hydroxy-3-methoxynaphthalen-1-yl)methyl]-5-(4-meth-
ylphenyl)-2,3-dihydro-1H-pyrazol-3-one (17). The crude product was
purified by medium pressure chromatography using a gradient
MeOH/EtOAc solvent system (1−10% MeOH) yielding 25 mg
(0.069 mmol, 48%). 1H NMR (500 MHz, chloroform-d) δ 7.58−7.53
(m, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.19−7.12
(m, 1H), 7.00−6.91 (m, 2H), 6.81 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 4.18
(s, 2H), 2.54 (s, 3H). LRMS [ES]+: m/z = 361.2 (M + H)+.
q. 4-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-5-(4-methox-
yphenyl)-2,3-dihydro-1H-pyrazol-3-one (18). The crude product was
purified by medium pressure chromatography using a gradient
MeOH/EtOAc solvent system (1−10% MeOH) yielding 15 mg
g. 4-[(2-Hydroxy-6-phenylnaphthalen-1-yl)methyl]-5-(4-methyl-
phenyl)-2,3-dihydro-1H-pyrazol-3-one (8). The crude product was
purified by medium pressure chromatography using a gradient EtOAc/
hexane solvent system (1−10% EtOAc) yielding 20 mg (0.05 mmol,
1
50%). H NMR (500 MHz, DMSO-d6) δ 8.28 (d, J = 1.9 Hz, 1H),
8.10−7.97 (m, 4H), 7.93−7.77 (m, 3H), 7.50 (t, J = 7.6 Hz, 2H),
7.44−7.33 (m, 4H), 4.16 (s, 2H), 2.39 (s, 3H). LRMS [ES]+: m/z =
407.1 (M + H)+.
h. 4-[(2-Hydroxy-6-methoxynaphthalen-1-yl)methyl]-5-(4-meth-
ylphenyl)-2,3-dihydro-1H-pyrazol-3-one (9). The crude product was
purified by medium pressure chromatography using a gradient EtOAc/
hexane solvent system (1−10% EtOAc) yielding 10 mg (0.027, 39%).
1H NMR (500 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.62 (s, 1H), 7.43-
1
(0.035 mmol, 48%). H NMR (300 MHz, DMSO-d6) δ 7.92 (d, J =
2.1 Hz, 1H), 7.59−7.43 (m, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.14 (dd, J
= 19.4, 8.9 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 4.06 (s, 2H), 3.78 (s,
3H). LRMS [ES]+: m/z = 427.1 (M + H)+.
General Procedure (IV) for Preparation of 2-Halogenated 6-
Hydroxy-Quinoline Carbaldehydes. The corresponding 6-hydroxy-
7.40 (m, 4H), 7.20 (d, J = 7.5 Hz, 1H), 7.11−7.02 (m, 3H), 6.73−6.67
(m, 1H), 4.06 (s, 2H), 2.34 (s, 3H). LRMS [ES]+: m/z = 361.2 (M +
H)+.
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Article
2(1H)-quinolinone (0.6 mmol) was taken up in trifluoroacetic acid (1
mL). To this solution was added hexamethylenetetramine (1.2 mmol),
and the resulting mixture was then heated at 100 °C for 2 h. The
reaction was allowed to cool, methanol (10 mL) was added, and the
solvent was evaporated to afford a dark residue. Water (15 mL) was
added, and the resulting precipitate was filtered and dried. The
precipitate was dissolved in DMF (0.8 mL), the solution cooled to 0
°C, and phosphorus oxyhalide (POCl3 or POBr3) (1.8 mmol) was
added dropwise. The light gray colored suspension was then stirred at
room temp overnight. Ice-cold water (15 mL) was added, and the
precipitate was filtered and dried under a vacuum to yield the
corresponding aldehyde as a pure product. Synthesis of 6-
hydroxyquinoline-5-carbaldehyde (60) has been reported previously
by using Reimer-Teimann conditions.31
NMR (500 MHz, D2O) δ 11.60 (s, 1H), 8.56 (m, 2H), 8.04 (s, 1H),
7.67 (d, J = 9.0 Hz, 1H), 7.34 (dd, J = 17.7, 8.5 Hz, 4H), 7.17 (d, J =
7.7 Hz, 1H), 4.11 (s, 3H), 2.32 (s, 3H). LRMS [ES]+: m/z = 332.1 (M
+ H)+.
b. 4-[(2-Chloro-6-hydroxyquinolin-5-yl)methyl]-5-(4-methylphen-
yl)-2,3-dihydro-1H-pyrazol-3-one (20). Yield 20 mg (0.054 mmol,
65%). 1H NMR (500 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.25 (s, 1H),
8.08 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.36−7.30 (m, 4H),
7.22 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 4.09 (s, 2H), 2.31
(s, 3H). LRMS [ES]+: m/z = 366.1 (M + H)+.
c. 4-[(2-Bromo-6-hydroxyquinolin-5-yl)methyl]-5-(4-methylphen-
yl)-2,3-dihydro-1H-pyrazol-3-one (21). Yield 15 mg (0.036 mmol,
1
49%). H NMR (300 MHz, DMSO-d6) δ 7.96 (d, J = 8.9 Hz, 1H),
7.59 (d, J = 9.0 Hz, 1H), 7.32 (dd, J = 8.5, 5.2 Hz, 4H), 7.16 (d, J = 7.8
Hz, 1H), 4.08 (s, 2H), 2.31 (s, 3H). LRMS [ES]+: m/z = 411.1 (M +
H)+.
a. 2-Chloro-6-hydroxyquinoline-5-carbaldehyde (61). Yield 80
1
mg (0.38 mmol, 63%). H NMR (500 MHz, DMSO-d6) δ 11.06 (s,
d. 4-[(2-Chloro-6-hydroxy-4-methylquinolin-5-yl)methyl]-5-(4-
methylphenyl)-2,3-dihydro-1H-pyrazol-3-one (22). The crude prod-
uct was purified by medium pressure chromatography using a gradient
EtOAc/hexanes solvent system (10−25% EtOAc). Yield 12 mg (0.031
1H), 8.23 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.39 (d, J =
9.3, 1H), 7.36 (d, J = 9.3, 1H). LRMS [ES]+: m/z = 208.2 (M + H)+.
b. 2-Bromo-6-hydroxyquinoline-5-carbaldehyde (62). POBr3 (3
mmol) was added dropwise to 2,6-dihydroxy-quinaldehyde. The
suspension was heated to 50 °C and stirred for 18 h. Ice-cold water
(15 mL) was then added, and resulting light brown precipitate was
filtered and dried under a vacuum to afford the pure product. Yield 60
1
mmol, 59%). H NMR (500 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.97
(s, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.28 (d, J = 9.0 Hz, 2H), 7.20 (d, J =
7.7 Hz, 2H), 7.08 (d, J = 8.9 Hz, 1H), 4.28 (s, 2H), 2.72 (s, 3H), 2.28
(s, 3H). LRMS [ES]+: m/z = 380.1 (M + H)+.
1
mg (0.23 mmol, 45%). H NMR (500 MHz, DMSO-d6) δ 11.16 (s,
e. 4-[(2-Chloro-6-hydroxyquinolin-5-yl)methyl]-5-[4-(propan-2-
1H), 8.70 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.42 (d, J =
9.3, 1H), 7.39 (d, J = 9.3, 1H). LRMS [ES]+: M + H+ 253.1 (M + H)+.
c. 2-Chloro-6-hydroxy-4-methylquinoline-5-carbaldehyde (63).
Yield 18 mg (0.08 mmol, 66%). 1H NMR (500 MHz, DMSO-d6) δ
12.71 (s, 1H), 10.74 (s, 1H), 8.07 (d, J = 9.3 Hz, 1H), 7.58 (s, 1H),
7.48 (d, J = 9.3, 1H), 2.69 (s, 3H). LRMS [ES]+: m/z = 222.1 (M +
H)+.
Preparation of Quinoline-Based Pyrazolone Compounds. Com-
pounds were prepared using General Procedure (II) Knoevenagel
condensation as described above.
yl)phenyl]-2,3-dihydro-1H-pyrazol-3-one (23). Yield 8 mg (0.020
1
mmol, 52%). H NMR (500 MHz, DMSO-d6) δ 11.48 (s, 1H), 8.03
(d, J = 9.0 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.31 (m, 3H), 7.18 (m,
3H), 4.07 (s, 2H), 2.87 (p, J = 6.9 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H).
LRMS [ES]+: m/z = 394.1 (M + H)+.
General Procedure (IV) for Preparation of Isoxazolone Deriva-
tives. 2-(4-Methylbenzoyl)-3H-benzo[f ]chromen-3-one (0.06 mmol)
was taken in DMF (0.2 mL) to which was added the corresponding
hydroxylamine (0.12 mmol), and the reaction mixture was stirred at 60
°C for 18 h. The solvent was evaporated, and the residue was dissolved
in EtOAc (5 mL). The organic layer was then washed with water, dried
with Na2SO4, and reduced to dryness to yield an oily product. The oil
was further purified by medium pressure chromatography using
gradient EtOAc/hexanes solvent system (0−50% EtOAc) to afford the
pure product.
a. 4-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-methyl-
phenyl)-2,5-dihydro-1,2-oxazol-5-one (24). Yield 13 mg (0.031
mmol, 50%) of 1:1 mixture of imine and enamine tautomers. 1H
NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.04−7.85 (m, 4H), 7.65
(d, J = 8.9 Hz, 1H), 7.53−7.42 (m, 2H), 7.24 (dd, J = 21.9, 8.4 Hz,
3H), 7.05−6.91 (m, 1H), 4.65 (dd, J = 8.6, 5.0 Hz, 1H), 2.35 (s, 3H).
LRMS [ES]+: m/z = 412.1 (M + H)+.
b. 4-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-2-methyl-3-
(4-methylphenyl)-2,5-dihydro-1,2-oxazol-5-one (25). Pyridine (1.5
equiv) was added to the reaction mixture as methyl hydroxylamine was
purchased as a hydrochloride salt. Yield 15 mg (0.035 mmol, 47%). 1H
NMR (300 MHz, DMSO-d6) δ 9.74 (s, 1H), 7.93 (d, J = 2.1 Hz, 1H),
7.54 (dd, J = 9.0, 2.3 Hz, 2H), 7.37 (dd, J = 9.1, 2.2 Hz, 1H), 7.28−
7.15 (m, 4H), 7.03 (d, J = 8.9 Hz, 1H), 3.92 (s, 2H), 2.97 (s, 3H), 2.33
(s, 3H). LRMS [ES]+: m/z = 424.0.
c. 2-Benzyl-4-[(6-bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-
methylphenyl)-2,5-dihydro-1,2-oxazol-5-one (26). Pyridine (1.5
equiv) was added to the reaction mixture as benzyl hydroxylamine
was purchased as a hydrochloride salt. Yield 13 mg (0.018 mmol,
41%). 1H NMR (500 MHz, D2O) δ 9.73 (s, 1H), 7.94 (d, J = 2.1 Hz,
1H), 7.52 (m, 4H), 7.40−7.22 (m, 6H), 7.14−7.07 (m, 2H), 7.04 (d, J
= 8.9 Hz, 1H), 3.90 (s, 2H), 2.51 (s, 2H), 2.36 (s, 3H). LRMS [ES]+:
m/z = 500.1 (M + H)+.
a. 2-(4-Methylbenzoyl)-3H-chromeno[6,5-b]pyridin-3-on (46).
1
Yield 28 mg (0.093 mmol, 54%). H NMR (500 MHz, chloroform-
d) δ 9.06−9.01 (m, 1H), 8.81 (s, 1H), 8.62−8.56 (m, 1H), 8.38 (d, J =
9.3 Hz, 1H), 7.86−7.75 (m, 2H), 7.64 (dd, J = 8.6, 4.2 Hz, 1H), 7.34−
7.24 (m, 2H), 2.45 (s, 3H). LRMS [ES]+: m/z = 316.1 (M + H)+.
b. 8-Chloro-2-(4-methylbenzoyl)-3H-chromeno[6,5-b]pyridin-3-
1
one (47). Yield 65 mg (0.019 mmol, 80%). H NMR (500 MHz,
chloroform-d) δ 8.72 (d, J = 0.6 Hz, 1H), 8.55−8.49 (m, 1H), 8.29
(dd, J = 9.3, 0.8 Hz, 1H), 7.85−7.76 (m, 2H), 7.63 (d, J = 8.8 Hz, 1H),
7.34−7.20 (m, 2H), 2.45 (s, 3H). LRMS [ES]+: m/z = 350.0 (M +
H)+.
c. 8-Bromo-2-(4-methylbenzoyl)-3H-chromeno[6,5-b]pyridin-3-
1
one (48). Yield 65 mg (0.171 mmol, 72%). H NMR (500 MHz,
chloroform-d) δ 8.72 (d, J = 0.7 Hz, 1H), 8.44−8.38 (m, 1H), 8.30
(dd, J = 9.3, 0.7 Hz, 1H), 7.85−7.72 (m, 2H), 7.31 (ddt, J = 7.1, 1.3,
0.7 Hz, 2H), 7.25−7.19 (m, 1H), 2.45 (s, 3H). LRMS [ES]+: m/z =
396.0 (M + H)+.
d. 8-Chloro-10-methyl-2-(4-methylbenzoyl)-3H-chromeno[6,5-
b]pyridin-3-one (49). Yield 55 mg (0.15 mmol, 67%). 1H NMR
(500 MHz, chloroform-d) δ 9.17 (s, 1H), 8.29 (d, J = 9.3 Hz, 1H),
7.80 (dd, J = 23.1, 8.5 Hz, 3H), 7.43 (s, 1H), 7.31 (d, J = 7.9 Hz, 2H),
2.97 (s, 3H), 2.45 (s, 3H). LRMS [ES]+: m/z = 364.1 (M + H)+.
e. 8-Chloro-2-[4-(propan-2-yl)benzoyl]-3H-chromeno[6,5-b]-
pyridin-3-one (50). Yield 25 mg (0.066 mmol, 55%). 1H NMR
(500 MHz, chloroform-d) δ 8.71 (s, 1H), 8.29 (dd, J = 9.3, 0.8 Hz,
1H), 7.89−7.83 (m, 2H), 7.80 (d, J = 9.3 Hz, 1H), 7.63 (d, J = 8.8 Hz,
1H), 7.39−7.33 (m, 2H), 3.00 (p, J = 6.9 Hz, 1H), 1.29 (d, J = 6.9 Hz,
6H). LRMS [ES]+: m/z = 378.1 (M + H)+.
General Procedure (V) for Preparation of Quinoline-Based
Pyrazolones. General procedure (III) for naphthalene pyrazolones
was followed. The compounds were obtained as white precipitates,
which were purified by medium pressure chromatography using a
gradient MeOH/DCM solvent system (1−10% MeOH) unless
otherwise noted.
AUTHOR INFORMATION
Corresponding Authors
*(J.A.S.) E-mail: jsimon@fhcrc.org. Phone (206) 667-6241.
*(A.B.) E-mail: abedalov@fhcrc.org. Phone (206) 667-4863.
■
Present Addresses
a. 4-[(6-Hydroxyquinolin-5-yl)methyl]-5-(4-methylphenyl)-2,3-di-
hydro-1H-pyrazol-3-one (19). Yield 4 mg (0.012 mmol, 48%). H
#(J.P.) Kineta Inc., Seattle, WA 98109.
1
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Article
⊥(A.D.S.) Syntrix Biosystems, Auburn, WA 98001.
Author Contributions
(12) Solomon, J. M.; Pasupuleti, R.; Xu, L.; McDonagh, T.; Curtis,
R.; DiStefano, P. S.; Huber, L. J. Inhibition of SIRT1 catalytic activity
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road ahead. Front. Pharmacol. 2012, 3, 4.
J.A.S. and A.B. conceived this project and designed the
experiments, S.S.M., J.P., A.T, and A.S. contributed to
compound design and carried out the syntheses, T.K.L., Y.L.,
and V.L. carried out the biochemical enzyme and cell-based
assays, and S.S.M. and M.S. conceived and carried out the STD
NMR experiments. S.S.M., A.B., and J.A.S. wrote the manu-
script.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
The research was funded by the National Cancer Institute (CA-
129132). The authors would like to thank Prof. Bill Atkins and
the Department of Medicinal Chemistry, University of
Washington, for providing access to their NMR facility and
resources and Dr. Eric Foss (Fred Hutchinson Cancer Research
Center) for help with statistical analysis.
(16) Pacholec, M.; Bleasdale, J. E.; Chrunyk, B.; Cunningham, D.;
Flynn, D.; Garofalo, R. S.; Griffith, D.; Griffor, M.; Loulakis, P.; Pabst,
B.; Qiu, X.; Stockman, B.; Thanabal, V.; Varghese, A.; Ward, J.;
Withka, J.; Ahn, K. SRT1720, SRT2183, SRT1460, and resveratrol are
not direct activators of SIRT1. J. Biol. Chem. 2010, 285, 8340−8351.
(17) Luo, J.; Nikolaev, A. Y.; Imai, S.; Chen, D.; Su, F.; Shiloh, A.;
Guarente, L.; Gu, W. Negative control of p53 by Sir2alpha promotes
cell survival under stress. Cell 2001, 107, 137−148.
ABBREVIATIONS
SIRT, sirtuin; STD NMR, saturation transfer difference nuclear
magnetic resonance; HMTA, hexamethylenetetramine
■
(18) North, B. J.; Marshall, B. L.; Borra, M. T.; Denu, J. M.; Verdin,
E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin
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