Synthesis of novel tricyclic chromenone-based inhibitors of IRE-1 rnase activity

Article abstract of DOI:10.1021/jm5002452

Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase ER stress sensor that is activated in response to excessive accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stress stimuli. Activation of IRE-1 RNase function exerts a cytoprotective effect and has been implicated in the progression of cancer via increased expression of the transcription factor XBP-1s. Here, we describe the synthesis and biological evaluation of novel chromenone-based covalent inhibitors of IRE-1. Preparation of a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was achieved via a Duff formylation that is attended by an unusual cyclization reaction. Biological evaluation in vitro and in whole cells led to the identification of 30 as a potent inhibitor of IRE-1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines.

Full text of DOI:10.1021/jm5002452

Article
pubs.acs.org/jmc
Synthesis of Novel Tricyclic Chromenone-Based Inhibitors of IRE‑1
RNase Activity
Sujeewa Ranatunga,† Chih-Hang Anthony Tang,‡ Chang Won Kang,† Crystina L. Kriss,‡
Bernhard J. Kloppenburg,†,‡ Chih-Chi Andrew Hu,*,‡ and Juan R. Del Valle*,†
‡
†Drug Discovery Department and Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, 12902
Magnolia Drive MRC3E, Tampa, Florida 33612, United States
S
* Supporting Information
ABSTRACT: Inositol-requiring enzyme 1 (IRE-1) is a
kinase/RNase ER stress sensor that is activated in response
to excessive accumulation of unfolded proteins, hypoxic
conditions, calcium imbalance, and other stress stimuli.
Activation of IRE-1 RNase function exerts a cytoprotective
effect and has been implicated in the progression of cancer via
increased expression of the transcription factor XBP-1s. Here,
we describe the synthesis and biological evaluation of novel
chromenone-based covalent inhibitors of IRE-1. Preparation of
a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was
achieved via a Duff formylation that is attended by an unusual
cyclization reaction. Biological evaluation in vitro and in whole
cells led to the identification of 30 as a potent inhibitor of IRE-
1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines.
transcription thus represent useful chemical tools and potential
therapeutic agents.
INTRODUCTION
■
The endoplasmic reticulum (ER) stress response is a
cytoprotective mechanism activated in response to proteotoxic
burden and is crucial for homeostatic regulation.1,2 Disruption
in the stoichiometric balance, transport, or processing of
intracellular proteins leads to the activation of three distinct
pathways mediated by the ER stress sensor proteins IRE-1,
ATF6, and PERK. IRE-1 is unique in that it contains a stress
sensor domain in the lumen of the ER and a cytosolic serine/
threonine kinase domain linked to an RNase domain. Multiple
stress conditions can cause IRE-1 to oligomerize. Oligomeriza-
tion brings the IRE-1 cytoplasmic kinase domains into
proximity, allowing for autophosphorylation and activation
IRE-1 RNase activity. The IRE-1 RNase domain is critical for
the function of IRE-1 because it splices 26 nucleotides from the
mRNA of X-box binding protein 1 (XBP-1), causing a frame
shift in translation.3−5 The spliced XBP-1 mRNA encodes a
functional 54 kDa XBP-1s protein in mammalian cells, which is
a transcription factor that translocates into the nucleus and
regulates ER stress response genes.
Since gene copy number amplifications and aberrant protein
expression are hallmarks of cancer, many human tumors rely on
a robust ER stress response for growth and survival.6,7 As a
result, IRE-1 and related stress sensors have emerged as
potential therapeutic targets for the treatment of cancer.8−10
IRE-1-mediated activation of XBP-1 has also been implicated in
the evasion of virus-induced cytotoxicity11 as well as in the
development of inflammatory arthritis.12,13 Small molecules
capable of modulating IRE-1 RNase activity and XBP-1s
Efforts to identify inhibitors of IRE-1 RNase function have
relied primarily on high-throughput screening of large chemical
libraries. This has led to the discovery of various salicylalde-
hydes with in vitro activity against IRE-1-mediated mRNA
splicing.14−17 A limited number of nonelectrophilic inhibitors
of IRE-1 RNase activity have also been reported (Figure 1).18,19
While aldehydes and related functional groups are generally
considered undesirable with respect to chemical probe
development, the recent FDA approval of various electrophilic
drugs has renewed interest in covalent inhibitors.20 The
importance of the aldehyde moiety for potent IRE-1 RNase
inhibition by 5 (4μ8C)15 and related compounds has been
rationalized by the formation of an unusually stable Schiff base
with lysine 907 in the IRE-1 endonuclease domain.21 Although
IRE-1 contains 25 lysine residues in its cytosolic domain, only
covalent modification at K907 (and in some cases K599) is
observed in vivo.15 This selectivity has been attributed to
specific perturbation of the K907 ε-amino group pKa, resulting
in enhanced nucleophilicity, increased rate of Schiff base
formation with aldehyde inhibitors, and slow off-rate.
Here, we report the synthesis and biological evaluation of
novel chromenone-based inhibitors of IRE-1 RNase activity. A
tandem Duff formylation/annelation reaction en route to
candidate inhibtors gave rise to fused tricyclic chromenopiper-
Received: February 14, 2014
Published: April 21, 2014
© 2014 American Chemical Society
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Figure 1. Selected known inhibitors of IRE-1 RNase activity.
idine, chromenoazepane, and chromenoazecane scaffolds.
Selected analogues based on a tetrahydrochromeno[3,4-
c]pyridine core structure potently inhibit XBP-1 splicing in
vitro and block the expression of XBP-1s in whole cells, making
them useful compounds for interrogating IRE-1 RNase activity
in biological systems.
RESULTS AND DISCUSSION
■
FRET-Suppression Assay of Potential IRE-1 Inhibitors.
To assess the in vitro activity of potential IRE-1 RNase
inhibitors, we carried out the expression and purification of
recombinant human IRE-1 for use in an in vitro FRET-
suppression assay.17 The cytoplasmic kinase/RNase domain (aa
547−977) of human IRE-1 was expressed as a soluble puritin-
His-tagged 59 kDa fusion protein in SF21 cells and purified by
Ni-NTA affinity chromatography. To confirm that hIRE-1
exhibited a functional RNase domain, we evaluated its activity
in vitro using a synthetic mRNA stem-loop corresponding to
the XBP-1 substrate sequence. This stem-loop incorporates a
Cy5 fluorophore on its 5′ end and the black hole quencher
(BHQ) on its 3′ end, resulting in fluorescence only upon site-
specific cleavage by the protein. Protein (5 nM) was incubated
in a 96-well plate at room temperature with different
concentrations of the XPB-1 stem loop for up to 2 h, and
fluorescence was measured upon excitation and emission at 620
and 680 nm, respectively. Recombinant hIRE-1 exhibited
functional RNase activity wth a Km value of 45 nM (see
Supporting Information).
We first evaluated a small set of known IRE-1 inhibitors,
synthetic analogues, and selected commercially available
salicylaldehyde derivatives using the FRET-suppression assay
(Figure 2). We recently reported the in vivo characterization of
naphthaldehyde derivative 2 (A-I06), which was postulated to
be the bioactive breakdown product of the known IRE-1
inhibitor 1 (STF-038010).22 When evaluated in our assay, 1
and 2 exhibit similar IC50 values (9.94 and 9.73 μM,
respectively), while decomposition product 8 and reduced
derivative 9 showed no appreciable inhibition at 20 μM.
Interestingly, the salicylaldehyde moiety alone was not
sufficient for IRE-1 RNase inhibition, as evidenced by the
weak activity (>20 μM IC50) of compounds 10−13.
Modification of the aldehyde or phenol functionalities also
resulted in inactive compounds (14−16). Coumarin derivative
5, recently identified in a high-throughput screening effort,15
Figure 2. Compounds evaluated for anti-IRE-1 RNase activity by
FRET-suppression assay. IC50 and CI values are reported as the mean
of four separate experiments.
exhibited significantly enhanced potency against IRE-1 RNase
function with an IC50 value of 206 nM in our FRET-
suppression assay.
Synthesis of Tricyclic Chromenones. In an effort toward
functionalized derivatives of 5 for use in covalent tagging and
pulldown experiments, we synthesized analogues 20a−d in four
steps from the appropriate amino acids (Figure 3). Installation
of the aldehyde moiety in each case relied on a Duff
formylation carried out using hexamethylenetetramine
(HMTA) in refluxing glacial acetic acid. Interestingly, when
the reaction was carried out in refluxing TFA using
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Scheme 1. Proposed Mechanism of Cyclization during Duff
Formylation
Structure−Activity Relationships. When evaluated in the
FRET-suppression assay, bicyclic derivatives 19a−d exhibited
inhibitory activities in the 100−500 nM range (Figure 4). The
Figure 3. Synthesis of substituted bicyclic and tricyclic 8-formyl
chromenones.
intermediate 19b as a starting material, formylation was
attended by an annelation involving the pendent carbamate
nitrogen to give tetrahydrochromeno[3,4-c]pyridine 21b as the
sole product. The structure and connectivity of this tricyclic
scaffold were confirmed by HMBC NMR. As is typically the
case for Duff formylations,23,24 complete consumption of 19
still resulted in low yields of 20 and 21 due to significant
decomposition. However, the yield of 21b improved to 41%
when the reaction was preceded by acetylation of the o-
hydroxyl group.
A plausible mechanism for the formation of 21b involves
electrophilic aromatic substitution at position 3 of the
chromenone core (Scheme 1). The reaction of electron rich
aromatics with HMTA in organic acid occasionally results in
aminomethylation in addition to formylation via decomposition
of intermediates such as B.23,24 In the case of 21b, this
decomposition is likely precluded by attack of the carbamate
nitrogen onto the electrophilic methylene group in C. The
interrupted Duff reaction at position 3 presumably occurs prior
to formylation at position 8, as the use of only 1 equiv of
HMTA in refluxing TFA afforded intermediate D as the major
product from 19b. The concomitant annelation was not
observed in the case of substrate 19a under any of the
conditions listed in Figure 2. However, hexahydrochromeno-
[3,4-c]azepine 21c and hexahydrochromeno[3,4-c]azocine 21d
were isolated as the sole products from 19c and 19d when TFA
was used as the solvent.
Figure 4. In vitro inhibition of IRE-1 RNase activity by compounds 20
and 21. IC50 and CI values are reported as the mean of four separate
experiments.
constrained tricyclic derivative 21b consistently showed
enhanced activity against IRE-1 RNase activity relative to the
bicyclic compounds 20b and 5 in side-by-side experiments.
Given the optimal in vitro potency and chemical yield of 21b,
we carried out the synthesis of a family of analogues to assess
the importance of the hydroxyl group and the distal N-
substituent (Scheme 2). A potential covalent irreversible
inhibitor 23 was obtained by chlorination of the reduced
derivative 22. Compounds 24 and 25 were prepared by acid-
catalyzed protection of the aldehyde in 21b as the 1,3-dioxane
or dithiane derivative. Analogues 26 and 27 were prepared by
O-alkylation of 24, followed by acidic hydrolysis of the dioxane.
Compounds 29−34 were synthesized by reaction of
intermediate 28 with various acylating or alkylating reagents,
followed by acidolysis.
The presumed importance of the aldehyde functionality for
IRE-1 RNase inhibition also prompted us to explore alternative
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Oxidized variants 40−42 were synthesized via Pinnick
oxidation of 35.
Scheme 2. Synthesis of O- and N-Substituted Analogues
All compounds were evaluated by FRET-suppression assay in
side-by-side experiments using 21b as a control inhibitor
(Table 1). As anticipated, protection of the aldehyde group in
Table 1. In Vitro IRE-1 RNase Inhibition by Analogues of
21b
compd
IC50 (nM)
95% CI (nM)
21b
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
40
41
42
111
>20000
>20000
3051
76−162
2031−4584
12900−20360
16210
>20000
>20000
1230
704−2148
222−439
149−268
62−207
181−500
183−354
35−64
312
200
113
303
255
47
>20000
>20000
1718
1289−2288
>20000
4109
3099−5448
3902−8162
5644
>20000
electrophilic groups at the 8 position of the chromenone core.
Scheme 3 depicts the synthesis of analogues 36−42 from
compound 21b. Formation of the ketone in 36 via oxidation of
the Grignard product required prior protection of the o-
hydroxyl as methoxymethyl ether 35. Olefination of 35 and
acetal hydrolysis afforded electrophilic analogues 37 and 38.
21b as the 1,3-dioxane or dithiane acetal (24 and 25) resulted
in weaker IRE-1 inhibitory activity. Alkylation of the phenol
oxygen (compounds 26, 27, and 35) resulted in a complete loss
of potency below 20 μM. The N-acyl derivative 29 exhibited an
IC50 value of 312 nM, while N-alkyl analogues 30−33 were
found to be slightly more potent. Interestingly, N-benzyl
analogue 31 was almost 3-fold more active than the
corresponding fluorinated derivative 32. Guanidinylation to
give 34 resulted in a notable increase in potency (IC50 = 47
nM) relative to the parent compound, though solubility
significantly decreased. Ketone 36, vinyl sulfone 38, and
Weinreb amide 42 showed no significant IRE-1 RNase
inhibitory activity below 20 μM. However, electrophilic
compounds 37, 40, and 41 displayed moderate potency (1−5
μM) in vitro. Also of note, 1,3-dioxane derivative 24 exhibited
an in vitro IC50 of 3.1 μM, whereas the corresponding 1,3-
dithiane analogue 25 displayed more than 5-fold weaker
activity. To confirm that the enhanced inhibitory activity of 24
is not simply a function of a labile aldehyde masking group, we
carried out stability studies in assay buffer and observed no
significant decomposition of the 1,3-dioxane moiety over 12 h
(see Supporting Information).
Scheme 3. Synthesis of Analogues with Aldehyde Surrogates
Inhibition of XBP-1s Expression in Whole Cells. In
order to determine whether our inhibitors could block the
expression of XBP-1s in whole cells, we incubated LPS-
stimulated B cells from the spleens of wild-type mice with 20
μM selected compounds for 24 h, lysed the cells, and analyzed
the lysates for the expression of XBP-1s by immunoblots.
Compounds 29 and 30 potently suppress the expression of
XBP-1s at 20 μM in wild-type mouse B cells (Figure 5A). In
addition, 5, 21b, and 24 exhibit strong inhibition of XBP-1s, as
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Figure 5. Inhibition of XBP-1s expression in whole cells. (A) B cells were purified from the spleens of wild-type mice, stimulated with LPS for 48 h,
treated with the indicated inhibitors at 20 μM for 24 h, lysed, and analyzed for expression of the indicated proteins by immunoblots. (B) Mino and
(C) Jeko cells were treated with the indicated inhibitors at 20 μM for 24 h, lysed, and analyzed for the expression of indicated proteins by
immunoblots. (D) Mino and (E) Jeko cells were treated with the indicated inhibitors at various doses for 48 h, lysed, and analyzed for the expression
of indicated proteins by immunoblots. (F) Mino and (G) Jeko dose−response curves and IC50 values for inhibition of XBP-1s expression by
indicated inhibitors as determined by immunoblots and densitometry (N = 3).
does treatment with 50 μM 2. Despite their activity in the
FRET-suppression assay, compounds 31−34 did not effectively
inhibit XBP-1s expression in whole cells, presumably because of
poor cell permeability and solubility. Compounds 37, 40, and
41, which feature alternative electrophilic functional groups,
similarly showed little to no inhibitory effect on XBP-1s
expression in B cells at 20 μM. Consistent with previous results
showing up-regulation of IRE-1 in response to XBP-1s
deficiency25 and suppression,22 we observed an inverse
correlation between pharmacological inhibition of XBP-1s
and expression level of IRE-1 (Figure 5A).
The IRE-1/XBP-1 pathway is known to be critical for the
survival multiple myeloma, malignancies derives from plasma
cells.14,26 However, the functional role of the ER stress
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Figure 6. Growth inhibition and induction of apoptosis by 30. (A) Human Mino and Jeko cells were cultured in the presence of 30 at various
concentrations for 48 h and subjected to XTT assay. Percentages of cell growth were calculated relative to DMSO-treated (control) groups. (B)
Human Mino and Jeko cells were cultured for 72 h in the presence of DMSO (control), 30 (50 μM), 21b (50 μM), and 5 (50 μM). Cells were lysed
for the analysis of the indicated proteins by immunoblot.
response in leukemia or lymphoma derived from mature B cells
has been largely overlooked because leukemia and lymphoma
cells do not expand their ER like that of multiple myeloma cells.
We recently showed that chronic lymphocytic leukemia (CLL)
growth and survival is highly dependent on the IRE-1/XBP-1
pathway and is inhibited by small molecules targeting IRE-1
RNase activity.22 Mantle cell lymphoma (MCL) is an incurable
non-Hodgkin’s lymphoma developed from mantle zone-
resident B cells. Since the role of the IRE-1/XBP-1 pathway
in MCL is completely unknown, we examined the MCL cell
lines Mino and Jeko for the expression of XBP-1s and
discovered that XBP-1s is constitutively expressed by both. A
subset of inhibitors was examined for inhibition of XBP-1s in
these human MCL cell lines. As with wild-type mouse B cells,
compounds 21b, 29, and 30 potently suppress the expression
of XBP-1s and induce up-regulation of IRE-1 in Mino and Jeko
cells. N-Isobutenyl derivative 33 also exhibits significant activity
at 20 μM (Figure 5B and Figure 5C).
To establish the dependency of XBP-1s expression on
inhibitor concentration, we used MCL cells to determine the
whole cell IC50 values for 21b, 29, and 30, in comparison to 5,
by immunoblots and densitometry (Figure 5D−G). Com-
pound 30 proved to be the most potent inhibitor of XBP-1s
expression in both Mino and Jeko cell lines (IC50 = 0.57 and
0.98 μM, respectively).
Lastly, we carried out XTT dose−response experiments to
determine approximate GI50 concentrations for 30, our most
potent inhibitor of XBP-1s expression. After a 48 h treatment,
30 exhibited GI50 values of 34 and 19 μM in Mino and Jeko
cells, respectively (Figure 6A). Total growth inhibition by 30
was achieved between 55 and 66 μM for these cell lines. We
confirmed that growth inhibition is the result of apoptosis by
treating Mino and Jeko cells with 30 for 72 h and analyzing cell
lysates for cleaved PARP. Consistent with its superior potency
in the suppression of XBP-1s, compound 30 induced PARP
cleavage more strongly than either 21b or 5 at 50 μM (Figure
6B). We also determined a GI50 value of ∼34 μM in LPS-
stimulated wild-type mouse B cells after treatment with 30 for
72 h (see Supporting Information). As expected, this result
suggests that the growth of antibody-secreting plasma cells is
also sensitive to inhibition of IRE-1 RNase activity.
aldehyde is not sufficient for biological activity. In an effort
toward functionalized derivatives of potent chromenone-based
inhibitors, we prepared a series of carbamate substituted 2H-
chromene-2-ones for further derivatization. Duff formylation of
these substrates resulted in a tandem annelation reaction, giving
rise to novel fused tricyclic scaffolds. Tetrahydrochromeno[3,4-
c]pyridine 21b served as a lead compound for the synthesis of a
family of analogues.
Although replacement of the critical aldehyde group in 21b
with electrophilic surrogates diminished potency, some
compounds retained weak to moderate inhibitory activity in
vitro. Modifications to the phenol group in 21b had a
deleterious effect on potency in the FRET suppression assay,
while changes at the distal N substituent were generally well
tolerated. The ability of selected compounds to inhibit XBP-1s
expression in wild-type B cells and human MCL cell lines
highlights the importance of cell-based assays for this class of
inhibitors, as a number of compounds with low- to mid-
nanomolar activity in the FRET-suppression assay did not
significantly reduce XBP-1s expression in whole cells. The N-
methyl analogue 30 displayed an in vitro IRE-1 RNase IC50
value of 200 nM and potently inhibited the expression of XBP-
1s in Mino and Jeko cells (IC50 = 0.57 and 0.98 μM,
respectively). Compared to 21b, compound 30 is also more
effective at inducing apoptosis in MCL cells. The described
tricyclic chromenones thus represent useful tool compounds for
suppressing IRE-1 RNase activity in whole cells and for probing
the importance of the IRE-1/XBP-1 pathway of the ER stress
response in biological systems.
EXPERIMENTAL SECTION
■
General Synthesis Notes. Unless stated otherwise, reactions were
performed in flame-dried glassware under a positive pressure of argon
or nitrogen gas using dry solvents. Commercial grade reagents and
solvents were used without further purification except where noted.
Diethyl ether, toluene, dimethylformamide dichloromethane, and
tetrahydrofuran were purified by a Glass Contour column-based
solvent purification system. Other anhydrous solvents were purchased
directly from chemical suppliers. Thin-layer chromatography (TLC)
was performed using silica gel 60 F254 precoated plates (0.25 mm).
Flash chromatography was performed using silica gel (60 μm particle
size). The purity of all compounds was judged by TLC analysis (single
spot/two solvent systems) using a UV lamp, CAM (ceric ammonium
molybdate), ninhydrin, or basic KMnO4 stain(s) for detection
purposes. 1D and 2D NMR spectra were recorded on a Varian 400
MHz spectrometer. Proton chemical shifts are reported as δ values
relative to residual signals from deuterated solvents (CDCl3, CD3OD,
or DMSO-d6). The purity of all assayed compounds was determined
by RP-HPLC using an analytical C18 column with MeCN/water (0.1%
CONCLUSION
■
We have described the synthesis and biological characterization
of novel inhibitors of IRE-1. Although various salicylaldehydes
have been reported to inhibit IRE-1 RNase activity in vitro, our
results confirm that the presence of an o-hydroxy aromatic
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formic acid) as eluent (4 mm × 150 mm column, 1 mL/min flow
rate). All final compounds were determined to be between 95% and
98% pure. Compounds 2, 5, 8, 10−12, and 14 were purchased from
commercial sources. Compounds 1 and 9 were synthesized as
described previously.22
102.8, 65.2, 41.0; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C13H14NO5 276.0867, found 276.0863.
Allyl (2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)ethyl)-
carbamate (19b). 19b was obtained in 88% yield from 18b. 1H
NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 7.68 (d, J = 8.8 Hz,
1H), 7.40 (m, 1H), 6.80 (dd, J = 8.7, 2.3 Hz, 1H), 6.71 (d, J = 2.3 Hz,
1H), 6.07 (s, 1H), 5.99−5.78 (m, 1H), 5.24 (m, 1H), 5.15 (m, 1H),
4.45 (m, 2H), 3.29 (m, 2H), 2.87 (t, J = 6.7 Hz, 2H); 13C NMR (101
MHz, DMSO-d6) δ 161.1, 160.3, 156.0, 155.2, 154.2, 133.8, 133.7,
126.3, 116.9, 113.0, 111.3, 110.5, 110.4, 102.5, 102.4, 64.3, 31.5, 23.4;
HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C16H16NO5 302.102 85,
found 302.103 05.
Allyl (2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)propyl)-
carbamate (19c). 19c was obtained in 88% yield yield from 18c.
1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 7.61 (d, J = 8.8 Hz,
1H), 7.33 (t, J = 5.5 Hz, 1H), 6.78 (d, J = 8.7, 1H), 6.69 (d, J = 2.4 Hz,
1H), 6.10 (s, 1H), 5.89 (ddt, J = 17.0, 10.6, 5.4 Hz, 1H), 5.25 (dd, J =
17.2, 1.6 Hz, 1H), 5.15 (d, J = 10.4 Hz, 1H), 4.45 (d, J = 5.3 Hz, 2H),
3.07 (q, J = 6.6 Hz, 2H), 2.72 (t, J = 7.6 Hz, 2H), 1.96−1.63 (m, 2H);
13C NMR (101 MHz, DMSO- d6) δ 161.5, 160.8, 157.0, 156.4, 155.6,
Procedure for Synthesis of β-Ketoesters 18a−d. A solution of
the appropriate (N-Alloc) amino acid 17 (23.9 mmol) in 100 mL of
DCM at 0 °C was treated with 2,2-dimethyl-1,3-dioxane-4,6-dione
(4.47 g, 31.0 mmol), 4-dimethylaminopyridine (2.92 g, 23.9 mmol),
and diisopropylcarbodiimide (3.70 mL, 23.9 mmol). The mixture was
stirred from 0 °C to room temperature over 4 h, then washed with
10% aqueous KHSO4 followed by brine. The organic layer was dried
over Na2SO4 and concentrated. The resulting colorless liquid was
dissolved in 50 mL of a 10:1 MeOH/toluene mixture and stirred at
reflux for 15 h. After cooling, the mixture was concentrated under
reduced pressure. Purification by flash column chromatography over
silica gel (25%−60% EtOAc/hexanes) afforded 18a, 18b, and 18d as
colorless oils. Alkylidene pyrrolidine 18c was obtained as a white solid.
Methyl 5-(((Allyloxy)carbonyl)amino)-3-oxobutanoate (18a).
18a was obtained in 64% yield from 17a. 1H NMR (400 MHz,
CDCl3) δ 5.88 (ddt, J = 16.2, 10.7, 5.6 Hz, 1H), 5.49 (s, 1H), 5.29 (d,
J = 17.2 Hz, 1H), 5.20 (d, J = 10.5 Hz, 1H), 4.56 (d, J = 5.5 Hz, 2H),
4.18 (d, J = 5.1 Hz, 2H), 3.72 (s, 3H), 3.50 (s, 2H); 13C NMR (101
MHz, CDCl3) δ 198.2. 167.0, 156.1, 132.5, 117.9, 66.0, 52.6, 50.8,
46.2; HRMS (ESI-TOF) m/z [M + H]+ calcd for C9H14NO5
216.0867, found 216.0862.
134.3, 126.7, 117.3, 113.3, 111.6, 109.9, 102.9, 64.6, 40.2, 28.7, 28.6;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C16H18NO5 304.1180,
found 304.1172.
Allyl (2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)butyl)-
carbamate (19d). 19d was obtained in 84% yield from 18d. 1H
NMR (400 MHz, DMSO- d6) δ 10.50 (s, 1H), 7.60 (d, J = 8.8 Hz,
1H), 7.21 (t, J = 5.7 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 2.4
Hz, 1H), 6.05 (s, 1H), 5.86 (ddt, J = 17.2, 10.5, 5.3 Hz, 1H), 5.22 (dd,
J = 17.2, 1.7 Hz, 1H), 5.11 (dd, J = 10.4, 1.6 Hz, 1H), 4.42 (d, J = 5.3
Hz, 2H), 3.00 (d, J = 6.1 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H), 1.62−1.51
(m, 2H), 1.51−1.42 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ
161.5, 160.9, 157.5, 156.4, 155.5, 134.3, 126.8, 117.2, 113.3, 111.6,
109.7, 102.8, 64.5, 40.2, 31.0, 29.5, 25.8; HRMS (ESI-TOF) m/z [M +
H]+ calcd for C17H20NO5 318.1336, found 318.1339.
Methyl 5-(((Allyloxy)carbonyl)amino)-3-oxopentanoate
(18b). 18b was obtained in 94% yield from 17b. 1H NMR (400
MHz, CDCl3) δ 5.97−5.82 (m, 1H), 5.37−5.12 (m, 3H), 4.53 (d, J =
5.6 Hz, 2H), 3.73 (s, 3H), 3.50−3.37 (m, 4H), 2.80 (t, J = 5.7 Hz,
2H); 13C NMR (101 MHz, CDCl3) δ 202.2, 167.3, 156.2, 132.8,
132.8, 117.6, 117.5, 65.4, 52.4, 52.4, 48.9, 42.8, 35.3; HRMS (ESI-
TOF) (m/z) [M + H]+ calcd for C10H16NO5 230.10285, found
230.10297.
Duff Reaction Condition A. The appropriate coumarin derivative
19 (0.73 mmol) in 9 mL of AcOH was treated with HMTA (255 mg,
1.82 mmol) and stirred for 18 h at 95 °C. The reaction mixture was
concentrated, and the resulting slurry was dissolved in 12 mL of a 1:1
1 M aqueous HCl/EtOAc solution and stirred at 60 °C for 2 h. The
organic layer was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layers were washed with water, dried
with MgSO4, and concentrated. Purification by silica gel flash column
chromatography (EtOAc/hexane) afforded the desired bicyclic formyl
derivatives 20a−d.
Duff Reaction Condition B. The appropriate coumarin derivative
19 (0.73 mmol) in 3 mL of TFA was treated with HMTA (255 mg,
1.82 mmol) and stirred for 18 h at 75 °C. The reaction mixture was
concentrated, and the resulting slurry was dissolved in 12 mL of a 1:1
1 M aqueous HCl/EtOAc solution and stirred at 60 °C for 2 h. The
organic layer was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layers were washed with water, dried
with MgSO4, and concentrated. Purification by silica gel flash column
chromatography (EtOAc/hexane) afforded the desired bicyclic and
tricyclic formyl derivatives.
Duff Reaction Condition C. The appropriate coumarin derivative
19 (0.47 mmol) in 15 mL of MeCN was treated with pyridine (18.5
mg, 0.23 mmol) and acetic anhydride (239 mg, 2.35 mmol). After
being stirred for 6 h at room temperature, the mixture was diluted with
brine and extracted with EtOAc. The organic layer was dried with
MgSO4 and concentrated. The resulting crude product dissolved in 2
mL of TFA was treated with HMTA (164 mg, 1.17 mmol) and stirred
for 18 h at 95 °C. The reaction mixture was concentrated, and the
resulting slurry was dissolved in 12 mL of a 1:1 1 M aqueous HCl/
EtOAc solution and stirred at 60 °C for 2 h. The organic layer was
separated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with water, dried with MgSO4,
and concentrated. Purification by silica gel flash column chromatog-
raphy (EtOAc/hexane) afforded the desired bicyclic and tricyclic
formyl derivatives.
Allyl 2-(2-Methoxy-2-oxoethylidene)pyrrolidine-1-carboxy-
1
late (18c). 18c was obtained in 56% yield from 17c. H NMR (400
MHz, CDCl3) δ 6.52 (s, 1H), 5.94 (ddt, J = 17.2, 10.5, 5.7 Hz, 1H),
5.33 (d, J = 17.2 Hz, 1H), 5.25 (d, J = 10.4 Hz, 1H), 4.66 (d, J = 5.7
Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 3.65 (s, 3H), 3.17 (t, J = 7.7 Hz,
2H), 1.91 (p, J = 7.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 169.2,
157.3, 152.6, 131.9, 118.5, 96.4, 66.6, 50.8, 49.5, 31.6, 21.1; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C11H16NO4 226.1074, found
226.1068.
Methyl 7-(((Allyloxy)carbonyl)amino)-3-oxoheptanoate
(18d). 18d was obtained in 65% yield from 17d. 1H NMR (400
MHz, CDCl3) δ 5.89 (ddt, J = 16.2, 10.7, 5.4 Hz, 1H), 5.28 (dd, J =
17.2, 1.5 Hz, 1H), 5.19 (dd, J = 10.4, 1.1 Hz, 1H), 4.82 (s, 1H), 4.53
(d, J = 5.5 Hz, 2H), 3.72 (s, 3H), 3.43 (s, 2H), 3.16 (dd, J = 12.9, 6.5
Hz, 2H), 2.56 (t, J = 7.1 Hz, 2H), 1.68−1.57 (m, 2H), 1.56−1.43 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 202.4, 167.6, 156.3, 132.9,
117.6, 65.4, 52.4, 49.0, 42.4, 40.5, 29.1, 20.2; HRMS (ESI-TOF) m/z
[M + H]+ calcd for C12H20NO5 258.1336, found 258.1326.
General Procedure for Synthesis of Coumarins 19a−d. A
solution of the appropriate β-keto ester 18 (10.1 mmol) in 50 mL of
methanesulfonic acid at 0 °C was treated with resorcinol (1.11 g, 10.1
mmol) and stirred for 3.5 h. The mixture was poured into ice cold
water, and the resulting yellow mixture was filtered. The filtrate was
extracted with EtOAc and combined with the solids. The combined
organic layer was concentrated and purified by flash chromatography
over silica gel (0−20% MeOH/CHCl3) to afford the pure coumarin
derivatives 19a−d.
Allyl (2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-
carbamate (19a). 19a was obtained in 36% yield from 18a. 1H
NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 7.88 (t, J = 5.9 Hz, 1H),
7.64 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 2.3 Hz,
1H), 5.99 (s, 1H), 5.92 (ddt, J = 17.0, 10.6, 5.4 Hz, 1H), 5.29 (dd, J =
17.2, 1.6 Hz, 1H), 5.18 (d, J = 10.5 Hz, 1H), 4.52 (d, J = 5.3 Hz, 2H),
4.37 (d, J = 5.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 161.7,
160.8, 156.6, 155.4, 154.2, 134.0, 126.2, 117.6, 113.4, 110.3, 107.9,
4295
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Allyl (2-(8-Formyl-7-hydroxy-2-oxo-2H-chromen-4-yl)-
methyl)carbamate (20a). 20a was obtained in 4% yield (methods
A, B, and C) from 19a. 1H NMR (400 MHz, CDCl3) δ 12.24 (s, 1H),
10.60 (s, 1H), 7.73 (d, J = 9.0 Hz, 1H), 6.91 (d, J = 9.0 Hz, 1H), 6.32
(s, 1H), 5.94 (ddt, J = 16.5, 11.1, 5.8 Hz, 1H), 5.34 (d, J = 17.2 Hz,
1H), 5.27 (d, J = 10.3 Hz, 1H), 5.19 (t, J = 5.6 Hz, 1H), 4.64 (dt, J =
5.7, 1.4 Hz, 2H), 4.54 (d, J = 6.3 Hz, 2H); 13C NMR (101 MHz,
CDCl3) δ 193.3, 165.4, 159.1, 156.3, 156.1, 152.1, 132.2, 131.8, 118.5,
114.7, 109.74, 109.71, 108.8, 66.4, 41.3; HRMS (ESI-TOF) m/z [M +
H]+ calcd for C15H14NO6 304.0816, found 304.0820.
Allyl (2-(8-Formyl-7-hydroxy-2-oxo-2H-chromen-4-yl)ethyl)-
carbamate (20b). 20b was obtained in 10% yield (method A) from
19b. 1H NMR (400 MHz, CDCl3) δ 12.24 (s, 1H), 10.60 (s, 1H), 7.92
(d, J = 9.1 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.19 (s, 1H), 5.90 (m,
1H), 5.39−5.15 (m, 2H), 5.03 (bs, 1H), 4.58 (m, 2H), 3.49 (m, 2H),
2.99 (t, J = 7.2 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 193.5, 193.4,
165.5, 159.2, 156.6, 156.5, 153.4, 133.1, 132.6, 118.2, 114.8, 112.2,
112.1, 111.1, 109.0, 66.0, 40.1, 32.8; HRMS (ESI-TOF) (m/z) [M +
H]+ calcd for C16H16NO6 318.097 77, found 318.097 46.
Allyl (2-(8-Formyl-7-hydroxy-2-oxo-2H-chromen-4-yl)-
propyl)carbamate (20c). 20c was obtained in 13% yield (method
A) from 19c. 1H NMR (400 MHz, CDCl3) δ 12.20 (s, 1H), 10.58 (s,
1H), 7.72 (d, J = 9.0 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H), 6.19 (s, 1H),
5.90 (ddt, J = 16.8, 11.1, 5.6 Hz, 1H), 5.29 (dd, J = 17.2, 1.5 Hz, 1H),
5.20 (dd, J = 10.4, 1.2 Hz, 1H), 4.98 (t, J = 5.2 Hz, 1H), 4.56 (d, J =
5.4 Hz, 2H), 3.33 (q, J = 6.5 Hz, 2H), 2.98−2.59 (m, 2H), 1.90 (tt, J =
13.7, 6.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 193.4, 165.2, 159.3,
156.4, 156.3, 155.6, 132.7, 132.5, 117.9, 114.4, 111.0, 110.9, 108.8,
65.7, 40.4, 29.1, 28.5; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C17H18NO6 332.1129, found 332.1128.
Allyl (2-(8-Formyl-7-hydroxy-2-oxo-2H-chromen-4-yl)butyl)-
carbamate (20d). 20d was obtained in 15% yield (method A) from
19d. 1H NMR (400 MHz, CDCl3) δ 12.22 (s, 1H), 10.60 (s, 1H), 7.74
(d, J = 9.0 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.17 (s, 1H), 5.90 (ddt, J
= 16.1, 10.8, 5.7 Hz, 1H), 5.29 (dd, J = 17.2, 1.6 Hz, 1H), 5.20 (dd, J =
10.4, 1.3 Hz, 1H), 4.80 (s, 1H), 4.55 (d, J = 5.6 Hz, 2H), 3.26 (q, J =
6.4 Hz, 2H), 2.93−2.56 (m, 2H), 1.78−1.56 (m, 4H); 13C NMR (101
MHz, CDCl3) δ 139.4, 165.2, 159.4, 156.38, 156.37, 156.1, 132.8,
132.7, 117.8, 114.4, 111.1, 111.0, 109.8, 65.6, 40.3, 31.5, 29.9, 25.2;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C18H20NO6 346.1285,
found 346.1288.
2H), 1.92−1.80 (m, 2H), 1.80−1.69 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 139.3, 164.8, 159.1, 155.8, 155.5, 152.0, 132.9, 132.6, 119.5,
117.6, 114.5, 111.3, 108.7, 66.5, 46.2, 44.4, 26.1, 25.7, 25.1; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C19H20NO6 358.1285, found
358.1290.
Allyl 8-Hydroxy-7-(hydroxymethyl)-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3(2H)-carboxylate (22). Compound
21b (24 mg, 73 μmol) in 2 mL of MeOH at 0 °C was treated with
sodium borohydride (3.0 mg, 73 μmol) and stirred for 40 min. The
reaction was quenched with 1 M aqueous HCl and extracted with
EtOAc. The organic layers were dried over Na2SO4 and concentrated.
Purification by flash column chromatography over silica gel (40%
−50% EtOAc/hexane) afforded 22 as a white foam (16 mg, 66%). 1H
NMR (400 MHz, CDCl3) δ 9.65 (bs, 1H), 7.35 (d, J = 8.4 Hz, 1H),
6.85 (d, J = 8.7 Hz, 1H), 5.94 (m, J = 11.1, 5.6 Hz, 1H), 5.33 (m, 3H),
5.24 (m, 1H), 4.64 (m, 2H), 4.40 (s, 2H), 3.77 (t, J = 5.7 Hz, 2H),
2.84 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 180.6, 160.4, 149.8,
147.8, 132.7, 123.5, 118.2, 114.7, 111.8, 111.1, 66.7, 59.0, 41.8, 39.3,
24.9; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C17H18NO6
332.113 42, found 332.114 73.
Allyl 8-Hydroxy-7-chloro-5-oxo-4,5-dihydro-1H-chromeno-
[3,4-c]pyridine-3(2H)-carboxylate (23). Compound 22 (17 mg,
51 μmol) in 2 mL of DCM at room temperature was treated with
thionyl chloride (19 μL, 257 μmol) and stirred for 5.5 h. The reaction
was diluted with DCM and washed with saturated aqueous NH4Cl,
dried over Na2SO4, and concentrated under reduced pressure. The
resulting white solid 23 was suffiently pure by NMR and HPLC
analysis for further use (12 mg, 67%). 1H NMR (400 MHz, CDCl3) δ
9.42 (m, 0.5H), 7.69 (m, 0.5H), 7.38 (m, 1H), 6.89 (m, 1H), 5.95 (m,
1H), 5.30 (m, 3H), 4.91 (s, 1H), 4.65 (m, 2H), 4.44 (d, J = 17.7 Hz,
2H), 3.79 (m, 2H), 2.85 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
160.3, 157.9, 151.5, 149.9, 132.7, 124.7, 123.6, 118.3, 114.5, 113.1,
112.7, 112.3, 111.9, 111.2, 66.8, 58.9, 42.0, 39.4, 34.3, 29.8, 24.8;
HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C17H16ClNO5 350.078
98, found 346.128 50 (observed mass corresponds to the 7-
methoxymethyl derivative, resulting from displacement of the chloride
with methanol during LCMS).
Allyl 7-(1,3-Dioxan-2-yl)-8-hydroxy-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3(2H)-carboxylate (24). A solution of
21b in (150 mg, 455 μmol) in 4 mL of benzene was treated with 1,3-
propanediol (99.0 μL, 1.40 mmol) and p-toluenesulfonic acid
monohydrate (4.3 mg, 23 μmol) and stirred for 2 h. The reaction
was quenched with 2 drops of NEt3, diluted with EtOAc, and washed
with brine. The organic layer was dried over Na2SO4 and concentrated.
Purification by flash column chromatography over silica gel (30%−
50% EtOAc/hexanes eluent) afforded 24 as a yellow solid (157 mg,
Allyl 7-Formyl-8-hydroxy-5-oxo-4,5-dihydro-1H-chromeno-
[3,4-c]pyridine-3(2H)-carboxylate (21b). 21b was obtained in 22%
1
(method B) and 41% (method C) yield from 19b. H NMR (400
MHz, CDCl3) δ 12.15 (s, 1H), 10.61 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H),
6.92 (d, J = 9.0 Hz, 1H), 5.94 (m, 1H), 5.33 (m, 1H), 5.24 (m, 1H),
4.64 (d, J = 5.7 Hz, 2H), 4.47 (m, 2H), 3.81 (t, J = 5.8 Hz, 2H), 2.86
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 193.3, 164.9, 158.4, 155.2,
154.7, 146.4, 132.7, 131.8, 118.3, 117.2, 114.8, 111.2, 108.7, 66.7, 41.9,
39.2, 24.9; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C17H16NO6
330.097 21, found 330.096 24.
1
89%). H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 7.36 (d, J = 8.2
Hz, 1H), 6.79 (d, J = 8.8 Hz, 1H), 6.28 (s, 1H), 5.91 (m, 1H), 5.30
(m, 1H), 5.20 (m, 1H), 4.61 (d, J = 5.6 Hz, 2H), 4.39 (s, 2H), 4.28
(dd, J = 11.6, J = 4.6 Hz, 2H), 4.09 (m, 2H), 3.74 (t, J = 5.8 Hz, 2H),
2.79 (m, 2H), 2.26 (m, 1H), 1.53 (m, 1H); 13C NMR (101 MHz,
CDCl3) δ 159.5, 159.3, 155.2, 150.5, 146.6, 132.8, 125.3, 118.0, 116.3,
114.5, 111.8, 109.9, 98.1, 67.9, 66.5, 41.8, 39.3, 25.8, 24.7; HRMS
(ESI-TOF) (m/z) [M + H]+ calcd for C20H22NO7 388.139 08, found
388.138 10.
A l l y l 8 - F o r m y l - 9 - h y d r o x y - 6 - o x o - 2 , 3 , 5 , 6 -
tetrahydrochromeno[3,4-c]azepine-4(1H)-carboxylate (21c).
21c was obtained in 18% (method B) and 17% (method C) yield
1
from 19c. H NMR (400 MHz, CDCl3) δ 12.17 (s, 1H), 10.61 (s,
1H), 7.79 (d, J = 9.2 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 5.87 (ddt, J =
16.3, 10.8, 5.3 Hz, 1H), 5.28 (dd, J = 17.2, 1.5 Hz, 1H), 5.16 (d, J =
10.8 Hz, 1H), 4.65 (s, 2H), 4.55 (d, J = 5.1 Hz, 2H), 3.98−3.57 (m,
2H), 3.08−2.96 (m, 2H), 2.13−2.00 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 193.4, 164.8, 159.3, 155.7, 155.0, 152.2, 132.54, 132.50,
122.0, 117.3, 114.4, 111.9, 108.6, 66.3, 47.8, 42.9, 27.6, 24.6; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C18H18NO6 344.1129, found
344.1137.
Allyl 9-Formyl-10-hydroxy-7-oxo-2,3,5,6-tetrahydro-1H-
chromeno[3,4-d]azocine-4(7H)-carboxylate (21d). 21d was
obtained in 3% (method B) and 9% (method C) yield from 19d.
1H NMR (400 MHz, CDCl3) δ 12.18 (s, 1H), 10.61 (s, 1H), 7.78 (d, J
Allyl 7-(1,3-Dithian-2-yl)-8-hydroxy-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3(2H)-carboxylate (25). Compound
21b (39.0 mg, 118 μmol) and 1,3-propanedithiol (13.0 μL, 130
μmol) in 2.5 mL of DCM at room temperature were treated with BF3·
OEt2 (6.0 μL, 47 μmol) and stirred for 17 h. The reaction was
quenched with saturated aqueous NaHCO3, and the aqueous layer was
extracted with EtOAc. The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure. Purification by flash
column chromatography over silica gel (25−50% EtOAc/hexane)
1
afforded 25 as a white foam (39 mg, 79%). H NMR (400 MHz,
CDCl3) δ 7.55 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.9 Hz,
1H), 6.26 (s, 1H), 5.95 (m, 1H), 5.33 (m, 1H), 5.24 (m, 1H), 4.64 (m,
2H), 4.47 (m, 2H), 3.78 (t, J = 5.8 Hz, 2H), 3.17 (m, 2H), 2.91 (m,
4H), 2.24 (m, 1H), 1.94 (m, 1H); 13C NMR (101 MHz, CDCl3) δ
159.5, 155.3, 149.8, 146.9, 132.8, 124.8, 118.1, 116.7, 114.9, 112.5,
= 8.6 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 5.95 (ddt, J = 16.9, 10.8, 5.6
Hz, 1H), 5.32 (d, J = 17.1 Hz, 1H), 5.21 (d, J = 10.3 Hz, 1H), 4.66 (s,
2H), 4.64 (d, J = 4.7 Hz, 2H), 3.62−3.49 (m, 2H), 3.07−2.96 (m,
4296
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Article
111.1, 110.6, 77.5, 77.2, 76.8, 66.6, 42.0, 39.2, 37.4, 31.3, 24.9, 24.7,
23.0, 14.3, 14.3. HRMS (ESI-TOF) (m/z) [M + H]+ calcd for
C20H22NO5S2 420.093 99, found 420.092 48.
(17 mg, 90%). 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 10.46
(s, 1H), 7.90 (m (rotomer), 1H), 7.00 (d, J = 8.9 Hz, 1H), 4.32 (m,
2H), 3.73 (t, J = 5.7 Hz, 2H), 2.96 (m, 2H), 2.83 (m, 1H), 2.10 (m
(rotomer), 3H); 13C NMR (101 MHz, DMSO-d6) δ 191.1, 191.0,
168.9, 163.2, 163.1, 158.2, 153.5, 147.0, 146.8, 132.2, 116.7, 116.5,
113.9, 111.1, 109.1, 104.6, 43.2, 41.4, 36.3, 25.0, 24.3, 21.8, 21.3;
HRMS (ESI-TOF) (m/z) [M + H]+ calcd C15H14NO5 288.087 20,
found 288.086 54.
Allyl 7-Formyl-8-methoxy-5-oxo-4,5-dihydro-1H-chromeno-
[3,4-c]pyridine-3(2H)-carboxylate (26). A solution of 24 (20 mg,
52 μmol) in 1 mL of DMF was treated with K2CO3 (36 mg, 258
μmol) followed by iodomethane (10 μL, 155 μmol). After being
stirred at room temperature for 18 h, the mixture was diluted with
saturated aqueous NH4Cl, extracted with DCM, and concentrated to
dryness. The residue was taken up in 500 μL of dioxane, treated with 2
mL of 4 M aqueous HCl, and stirred at room temperature for 30 min.
The mixture was diluted with water and extracted with DCM. The
combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. Purification by flash column chromatography
over silica gel (0−10% MeOH/CHCl3) afforded 26 as a white powder
(12 mg, 67%). 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 7.71 (d,
J = 8.7 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 5.95 (m, 1H), 5.33 (m, 1H),
5.24 (m, 1H), 4.65 (m, 2H), 4.48 (s, 2H), 4.01 (s, 3H), 3.82 (t, J = 5.8
Hz, 2H), 2.87 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 187.2, 162.6,
158.6, 157.2, 155.3, 145.7, 132.8, 132.7, 132.7, 129.7, 118.3, 118.2,
112.9, 112.7, 108.2, 66.7, 56.8, 42.0, 39.3, 29.9, 24.9; HRMS (ESI-
TOF) (m/z) [M + H]+ calcd for C18H18NO6 344.113 41, found
344.114 32.
8-Hydroxy-3-methyl-5-oxo-2,3,4,5-tetrahydro-1H-
chromeno[3,4-c]pyridine-7-carbaldehyde (30). A solution of 28
(50.0 mg, 165 μM) in 2 mL of 1:1 dioxane/THF was treated with 37%
aqueous formaldehyde (27.0 μL, 330 μM), 10% Pd/C (40 mg), placed
under H2 atmosphere, and stirred at room temperature for 3 h. The
mixture was filtered through Celite with MeOH rinsing and
concentrated to afford the crude methylamine. The residue was
taken up in 500 μL of dioxane, treated with 2 mL of 4 M aqueous HCl,
and stirred at room temperature 30 min. The mixture was diluted with
water and extracted with DCM. The combined organic layers were
dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash column chromatography over silica gel (0−10%
1
MeOH/CHCl3) afforded 30 as a white powder (35 mg, 67%). H
NMR (400 MHz, CDCl3) δ 12.15 (s, 1H), 10.61 (s, 1H), 7.66 (d, J =
9.0 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 3.59 (s, 2H), 3.03−2.97 (m,
2H), 2.97−2.90 (m, 2H), 2.64 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ 193.2, 164.8, 158.4, 154.6, 145.5, 131.7, 117.3, 114.6, 111.0, 108.6,
51.6, 50.2, 45.0, 25.3; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C14H14NO4 260.0917, found 260.0915.
Allyl 7-Formyl-8-benzyloxy-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3(2H)-carboxylate (27). A solution of
24 (20 mg, 52 μmol) in 1 mL of DMF was treated with K2CO3 (36
mg, 260 μmol) followed by benzyl bromide (9.0 μL, 78 μmol). After
being stirred at room temperature for 18 h, the mixture was diluted
with saturated aqueous NH4Cl, extracted with DCM, and concen-
trated to dryness. The residue was taken up in 500 μL of dioxane,
treated with 2 mL of 4 N aqueous HCl, and stirred at room
temperature 30 min. The mixture was diluted with water and extracted
with DCM. The combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure. Purification by flash column
chromatography over silica gel (0−10% MeOH/CHCl3) afforded 27
3-Benzyl-8-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-
chromeno[3,4-c]pyridine-7-carbaldehyde (31). A solution of 28
(20 mg, 66 μmol) in 1.5 mL of DMF at room temperature was treated
with NEt3 (10 mg, 99 μmol) and benzyl bromide (12 mg, 73 μmol).
After being stirred for 5 h, the mixture was concentrated and treated
with 4 mL of 4 M aqueous HCl and stirred for 1 h. The mixture was
adjusted to pH 7 with 10% aqueous Na2CO3, extracted with DCM,
dried over MgSO4, and concentrated. Purification by silica gel flash
column chromatography (MeOH/CHCl3) gave 31 as a white solid
1
as a white powder (18 mg, 72%). H NMR (400 MHz, CDCl3) δ
1
10.72 (d, J = 5.4 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.58−7.31 (m,
5H), 7.01 (d, J = 9.0 Hz, 1H), 5.95 (m, 1H), 5.35 (m, 0.5H), 5.30 (m,
2.5H), 5.24 (m, 1H), 4.65 (m, 2H), 4.47 (s, 2H), 3.81 (t, J = 5.8 Hz,
2H), 2.87 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 187.1, 161.7,
158.7, 154.0, 145.6, 135.4, 132.7, 129.5, 128.9, 128.5, 127.0, 118.3,
113.2, 113.1, 109.6, 71.2, 66.7, 51.3, 42.0, 39.2, 29.8, 24.8; HRMS
(ESI-TOF) (m/z) [M + H]+ calcd for C24H22NO6 420.144 71, found
420.145 29.
(15.4 mg, 70%). H NMR (400 MHz, CDCl3) δ 12.15 (s, 1H), 10.61
(s, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.42−7.30 (m, 5H), 6.90 (d, J = 9.0
Hz, 1H), 3.87 (s, 2H), 3.59 (s, 2H), 2.94 (s, 4H); 13C NMR (101
MHz, CDCl3) δ 193.3, 164.5, 158.8, 154.5, 146.2, 137.1, 131.7, 129.2,
128.5, 127.6, 119.0, 114.3, 111.5, 108.5, 62.3, 50.1, 48.0, 26.0; HRMS
(ESI-TOF) m/z [M + H]+ calcd for C20H18NO4 336.1230, found
336.1224.
3-(4-Fluorobenzyl)-8-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-
chromeno[3,4-c]pyridine-7-carbaldehyde (32). A solution of 28
(20 mg, 66 μmol) in 1.5 mL of DMF at room temperature was treated
with NEt3 (10 mg, 99 μmol) and 4-fluorobenzyl bromide (12 mg, 73
μmol). After being stirred for 5 h, the mixture was concentrated and
treated with 4 mL of 4 M aqueous HCl and stirred for 1 h. The
mixture was adjusted to pH 7 with 10% aqueous Na2CO3, extracted
with DCM, dried over MgSO4, and concentrated. Purification by silica
gel flash column chromatography (MeOH/CHCl3) gave 32 as a pale
7-(1,3-Dioxan-2-yl)-8-hydroxy-3,4-dihydro-1H-chromeno-
[3,4-c]pyridin-5(2H)-one (28). A solution of 24 (70 mg, 180 μmol)
in 4 mL of DCM at room temperature was treated with phenylsilane
(67 mg, 540 μmol) and tetrakis(triphenylphosphine)palladium(0) (10
mg, 9.0 μmol) and stirred at room temperature for 25 min. The
mixture was concentrated and the residue purified by flash
chromatography over silica gel (0%−10% MeOH/CHCl3) to afford
1
28 as a yellow solid (54 mg, 98%). H NMR (400 MHz, CDCl3) δ
1
7.35 (d, J = 8.8 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 6.28 (s, 1H), 4.24
(m, 2H), 4.06 (m, 2H), 3.75 (m, 2H), 3.11 (t, J = 5.8 Hz, 2H), 2.70
(m, 2H), 2.36−2.11 (m, 1H), 1.92 (bs, 1H), 1.50 (m, 1H); 13C NMR
(101 MHz, CDCl3) δ 160.2, 159.0, 150.6, 146.8, 135.0, 125.1, 119.0,
114.3, 112.5, 109.9, 98.3, 68.0, 43.4, 42.0, 25.9, 25.3; HRMS (ESI-
TOF) (m/z) [M + H]+ calcd for C16H18NO5 304.117 95, found
304.117 82.
3-Acetyl-8-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-chromeno-
[3,4-c]pyridine-7-carbaldehyde (29). A solution of 28 (20 mg, 66
μmol) in 1 mL of DCM was treated with pyridine (11 μL, 130 μmol)
and acetyl chloride (7.0 μL, 99 μmol), then stirred at room
temperature for 20 min. After concentration under reduced pressure,
the residue was taken up in 500 μL of dioxane, treated with 2 mL of 4
M aqueous HCl, and stirred at room temperature for 30 min. The
mixture was diluted with water and extracted with DCM. The
combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. Purification by flash column chromatography
over silica gel (0−10% MeOH/CHCl3) afforded 29 as a white powder
yellow solid (12 mg, 49%). H NMR (400 MHz, CDCl3) δ 12.15 (s,
1H), 10.62 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.36 (m, 2H), 7.06 (m,
2H), 6.86 (m, 1H), 3.70 (bs, 2H), 3.51 (m, 2H), 2.86 (m, 4H); 13C
NMR (101 MHz, CDCl3) δ 193.4, 164.7, 158.8, 156.3, 154.7, 146.2,
143.8, 131.9, 131.0, 127.9, 125.4, 115.7, 115.5, 114.6, 114.0, 111.5,
108.7, 68.7, 68.0, 61.5, 50.4, 48.1, 31.2, 29.9, 26.0; HRMS (ESI-TOF)
m/z [M + H]+ calcd for C20H17FNO4 354.114 16, found 354.114 38.
8-Hydroxy-3-(2-methylallyl)-5-oxo-2,3,4,5-tetrahydro-1H-
chromeno[3,4-c]pyridine-7-carbaldehyde (33). A solution of 28
(20 mg, 67 μmol) in 1.5 mL of DMF at room temperature was treated
with NEt3 (10 mg, 99 μmol) and 3-bromo-2-methylpropene (9.9 mg,
74 μmol). After being stirred for 5 h, the mixture was concentrated
and treated with 4 mL of 4 M aqueous HCl and stirred for 1 h. The
mixture was adjusted to pH 7 with 10% aqueous Na2CO3, extracted
with DCM, dried over MgSO4, and concentrated. Purification by silica
gel flash column chromatography (MeOH/CHCl3) gave 33 as a
1
yellow solid (15 mg, 75%). H NMR (400 MHz, CDCl3) δ 12.14 (s,
1H), 10.61 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H),
4297
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Journal of Medicinal Chemistry
Article
1
5.01 (s, 2H), 3.52 (s, 2H), 3.22 (s, 2H), 2.94 (s, 2H), 2.87 (s, 2H),
1.81 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 193.2, 164.6, 158.6,
154.6, 146.0, 140.5, 131.7, 115.3, 114.5, 111.3, 108.5, 105.0, 64.4, 50.2,
48.0, 25.6, 20.8; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C17H18NO4 300.1230, found 300.1223.
hexane) to afford 36 as a white foam (6.0 mg, 75%). H NMR (400
MHz, CDCl3) δ 13.54 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 6.95 (d, J =
9.0 Hz, 1H), 5.96 (m, 1H), 5.33 (m, 2H), 5.24 (ddd, J = 10.4, 2.5, 1.2
Hz, 1H), 4.65 (dt, J = 5.7, 1.3 Hz, 2H), 4.47 (m, 2H), 3.81 (t, J = 5.8
Hz, 2H), 2.98 (s, 3H), 2.87 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
204.4, 166.3, 158.6, 155.3, 153.8, 132.8, 130.2, 118.2, 116.6, 115.7,
111.3, 109.5, 66.7, 41.8, 39.3, 34.2, 25.1; HRMS (ESI-TOF) (m/z) [M
+ H]+ calcd for C18H18NO6 344.112 86, found 344.111 16.
7-Formyl-8-hydroxy-5-oxo-4,5-dihydro-1H-chromeno[3,4-
c]pyridine-3(2H)-carboximidamide (34). A solution of 28 (20 mg,
66 μmol) in 1 mL of DCM was treated with NEt3 (28 μL, 198 μmol)
followed by 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (58 mg,
146 μmol) and stirred at room temperature for 18 h. The mixture was
diluted with saturated aqueous NH4Cl and extracted with DCM. The
combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. Purification by flash column chromatography
over silica gel (40% EtOAc/hexanes) gave the guanidinylated
intermediate as a glassy solid. The material was then treated with 2
mL of a 1:1 TFA/DCM solution and stirred at room temperature for 4
h. The mixture was concentrated to remove TFA, and the resulting
solid was washed with three portions of DCM. Drying of the solid
under vacuum afforded 34 (12 mg, 63%), which was pure by NMR. 1H
NMR (400 MHz, DMSO-d6) δ 11.93 (s, 1H), 10.46 (s, 1H), 7.94 (d, J
= 9.0 Hz, 1H), 7.64 (m, 3H), 7.02 (d, J = 9.0 Hz, 1H), 4.31 (s, 2H),
3.71 (t, J = 5.7 Hz, 2H), 2.99 (m, 2H); 13C NMR (101 MHz, DMSO-
d6) δ 190.8, 163.5, 158.1, 156.3, 153.5, 146.7, 132.3, 115.3, 114.1,
110.9, 109.3, 104.7, 43.2, 41.0, 24.2; HRMS (ESI-TOF) m/z [M + H]+
calcd for C14H14N3O4 288.098 43, found 288.098 81.
Allyl 7-(3-Ethoxy-3-oxoprop-1-en-1-yl)-8-hydroxy-5-oxo-
4,5-dihydro-1H-chromeno[3,4-c]pyridine-3(2H)-carboxylate
(37). Compound 35 (40.0 mg, 107 μmol) in 2 mL of DCM at room
temperature was treated with triethylphosphonoacetate (48.0 mg, 139
μmol) and stirred for 20 h. The mixture was concentrated under
reduced pressure. Purification by flash column chromatography over
silica gel (20%−40% EtOAc/hexanes) afforded the intermediate ethyl
enoate as a white solid (46 mg, 96%). 1H NMR (400 MHz, CDCl3) δ
8.11 (d, J = 16.4 Hz, 1H), 7.46 (m, 1H), 7.16 (d, J = 9.0 Hz, 1H), 7.05
(d, J = 16.4 Hz, 1H), 5.96 (m, 1H), 5.34 (m, 2.5H), 5.30 (m, 0.5H),
5.23 (m, 1H), 4.64 (dt, J = 5.6, 1.2 Hz, 2H), 4.46 (s, 2H), 4.28 (q, J =
7.1 Hz, 2H), 3.79 (t, J = 5.8 Hz, 2H), 3.50 (s, 3H), 2.85 (m, 2H), 1.35
(t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 167.8, 158.5,
151.8, 132.8, 132.7, 132.2, 132.1, 132.1, 132.0, 128.7, 128.5, 125.2,
124.4, 118.1, 113.8, 112.4, 110.9, 94.8, 66.6, 60.7, 56.8, 56.8, 42.0, 39.3,
24.9, 14.5; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C23H26NO8
444.165 29, found 444.165 76.
The above ethyl enoate (20 mg, 45 μmol) in 2 mL of MeOH/
CHCl3 (3:1) at room temperature was treated with 2 mL of 4 N
aqueous HCl and stirred for 18 h. The reaction was extracted with
EtOAc, dried over Na2SO4, and concentrated under reduced pressure.
Purification by flash column chromatography over silica gel (40%−
Allyl 7-Formyl-8-(methoxymethoxy)-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3(2H)-carboxylate (35). Compound
21b (301 mg, 914 μmol) in 5 mL of DCM at 0 °C was treated
with DIEA (790 μL, 4.57 mmol) and chloromethyl methyl ether (347
μL, 4.57 mmol). The mixture was stirred for 30 h, quenched with
saturated aqueous NH4Cl, and the organic layer was washed with
saturated aqueous NH4Cl. The organic layer was dried over Na2SO4
and concentrated. Purification by flash column chromatography over
silica gel (35%−70% EtOAc/hexanes) afforded 35 as a white solid
(226 mg, 67%). 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 7.67 (d,
J = 8.9 Hz, 1H), 7.20 (d, J = 9.0 Hz, 1H), 5.95 (m, 1H), 5.35 (m,
2.5H), 5.30 (m, 0.5H), 5.24 (m, 1H), 4.64 (d, J = 5.7 Hz, 2H), 4.48 (s,
2H), 3.81 (t, J = 5.8 Hz, 2H), 3.53 (s, 3H), 2.87 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ 186.8, 160.2, 158.4, 155.0, 153.4, 145.7, 132.6,
129.4, 118.2, 117.9, 113.5, 113.3, 111.4, 94.9, 66.4, 56.8, 41.7, 39.1,
24.7; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C19H20NO7
374.123 43, found 374.123 10.
Allyl 7-Acetyl-8-hydroxy-5-oxo-4,5-dihydro-1H-chromeno-
[3,4-c]pyridine-3(2H)-carboxylate (36). Compound 35 (50.0 mg,
134 μmol) in 2 mL of THF at −78 °C under Ar was treated with 3 M
MeMgBr in Et2O (134 μL, 402 μmol). After 3 h at −78 °C, the
reaction was carefully quenched. Then the mixture was diluted with
saturated aqueous NH4Cl, warmed to room temperature, and
partitioned with EtOAc. The organic layer was dried over Na2SO4
and concentrated under reduced pressure to give the crude alcohol as
an oil.
The above alcohol was dissolved in 3 mL of DCM and treated with
Dess−Martin periodinane (123 mg, 291 μmol) and stirred at room
temperature for 3 h. The reaction was quenched with 10% aqueous
Na2S2O3 and washed with brine. The organic layer was dried over
Na2SO4, concentrated, and the residue was purified by flash column
chromatography over silica gel (35−70% EtOAc/hexane) to give the
intermediate ketone as a gum (34 mg, 66%, two steps). 1H NMR (400
MHz, CDCl3) δ 7.50 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 8.9 Hz, 1H),
5.95 (m, 1H), 5.34 (m, 1H), 5.30 (m, 1H), 5.25 (m, 2.5H), 5.21 (m,
0.5H), 4.64 (d, J = 5.7 Hz, 2H), 4.45 (m, 2H), 3.80 (t, J = 5.8 Hz, 2H),
3.48 (s, 3H), 2.86 (m, 2H), 2.61 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 199.1, 158.9, 155.6, 149.3, 145.8, 133.5, 132.7, 125.0, 120.6,
118.2, 114.0, 111.2, 108.6, 94.8, 66.7, 56.7, 42.0, 39.3, 32.7, 29.8, 24.8;
HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C20H22NO7 388.139 08,
found 388.139 45.
1
70% EtOAc/hexanes) afforded 37 as a white solid (15 mg, 83%). H
NMR (400 MHz, DMSO-d6) δ 11.57 (m, 1H), 8.01 (d, J = 16.3 Hz,
1H), 7.62 (d, J = 8.8 Hz, 1H), 6.99 (m, 2H), 5.96 (ddt, J = 17.2, 10.6,
5.3 Hz, 1H), 5.31 (m, 1H), 5.22 (m, 1H), 4.59 (dt, J = 5.3, 1.4 Hz,
2H), 4.34−4.12 (m, 4H), 3.68 (m, 2H), 2.87 (m, 2H), 1.27 (t, J = 7.1
Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 167.0, 160.3, 158.7,
154.3, 151.7, 146.9, 133.3, 133.2, 126.7, 121.5, 117.4, 112.9, 111.3,
108.2, 65.6, 60.1, 41.3, 24.4, 14.3; HRMS (ESI-TOF) (m/z) [M + H]+
calcd for C21H22NO7 400.139 08, found 400.139 92.
Allyl 8-Hydroxy-7-(2-(methylsulfonyl)vinyl)-5-oxo-4,5-dihy-
dro-1H-chromeno[3,4-c]pyridine-3(2H)-carboxylate (38). LiCl
(8.0 mg, 20 μmol) and diethyl(methylsulfonylmethyl) phosphonate
(54.0 g, 236 μmol) in 2.5 mL of acetonitrile at room temperature was
treated with DBU (24.0 μL, 157 μmol) and stirred for 10 min.
Compound 35 (43.0 mg, 131 μmol) in 2 mL of acetonitrile was
cannulated into the mixture and stirred for 2 h. The reaction was
quenched with saturated aqueous NH4Cl and extracted with EtOAc.
Purification by flash column chromatography over silica gel (0−5%
MeOH/CHCl3) afforded the intermediate vinyl sulfone as a white
1
solid (41 mg, 79%). H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 15.8
Hz, 1H), 7.68 (d, J = 15.8 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.20 (d, J
= 9.0 Hz, 1H), 5.95 (m, 1H), 5.34 (m, 2.5H), 5.30 (m, 0.5H), 5.23 (m,
1H), 4.64 (m, 2H), 4.46 (s, 2H), 3.80 (t, J = 5.8 Hz, 2H), 3.51 (s, 3H),
3.06 (s, 3H), 2.86 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 158.8,
158.6, 155.3, 152.2, 146.1, 132.7, 131.7, 131.5, 126.8, 118.2, 113.8,
110.9, 109.9, 95.1, 66.6, 57.0, 43.3, 41.9, 39.3, 24.9; HRMS (ESI-TOF)
(m/z) [M + H]+ calcd for C21H24NO8S 450.121 72, found 450.123 90.
The above vinyl sulfone (38 mg, 84 μmol) in 2.5 mL of acetonitrile/
CHCl3 (2:1) was treated with 2.5 mL of 4 N aqueous HCl and stirred
for 20 h at room temperature. The mixture was concentrated under
reduced pressure. The resulting white solid was washed with DCM/
Et2O and the resulting solid dried to afford pure 38 (32 mg, 92%). 1H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 7.87 (d, J = 15.7 Hz,
1H), 7.68 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 15.7 Hz, 2H), 7.01 (d, J =
8.9 Hz, 1H), 5.97 (m, 1H), 5.31 (m, 1H), 5.21 (m, 1H), 4.59 (dt, J =
5.3, 1.5 Hz, 2H), 4.29 (s, 2H), 3.84 (s, 3H), 3.15 (s, 3H), 2.88 (s, 2H);
13C NMR (101 MHz, DMSO-d6) δ 160.5, 158.6, 154.3, 151.8, 146.9,
133.3, 130.8, 130.1, 127.6, 117.4, 112.9, 111.3, 106.5, 65.6, 42.6, 41.3,
24.2; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C19H20NO7S
406.095 50, found 406.095 00.
The ketone above (9.0 mg, 23 μmol) in 1.5 mL of 33% TFA/DCM
solution was stirred for 1.5 h at room temperature. The mixture was
concentrated under reduced pressure and the resulting residue was
purified by flash column chromatography over silica gel (30% EtOAc/
4298
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3-((Allyloxy)carbonyl)-8-(methoxymethoxy)-5-oxo-2,3,4,5-
tetrahydro-1H-chromeno[3,4-c]pyridine-7-carboxylic Acid
(39). Compound 35 (80.0 mg, 214 μmol) and 2-methyl-2-butene
(272 μL, 2.57 mmol) in 3.5 mL of t-BuOH/H2O/CH3CN (3:3:1) at 0
°C were treated with a solution of sodium chlorite (145 mg, 1.29
mmol) and sodium monophosphate (265 mg, 1.93 mmol) in water,
dropwise. After 30 min the reaction was quenched with 5% aqueous
Na2S2O3. The pH of the solution was adjusted to 6, and the aqueous
portion was extracted with EtOAc. The organic layer was dried over
Na2SO4 and concentrated. Purification by flash column chromatog-
raphy over silica gel (70−100% EtOAc/hexanes) afforded 39 as a thick
oil (64 mg, 77%). 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.2 Hz,
1H), 7.19 (d, J = 9.0 Hz, 1H), 6.07 (bs,12H), 5.96 (m, 1H), 5.33 (m,
2.5H), 5.24 (m, 1H), 4.65 (m, 2H), 4.48 (s, 2H), 3.82 (t, J = 5.7 Hz,
2H), 3.52 (s, 3H), 2.90 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
166.5, 163.5, 162.5, 159.9, 156.9, 156.4, 155.4, 150.3, 149.7, 146.7,
132.7, 125.5, 118.3, 113.7, 113.5, 111.5, 111.1, 99.9, 95.0, 94.7, 91.8,
66.8, 56.9, 56.7, 41.9, 41.7, 39.3, 24.8; HRMS (ESI-TOF) (m/z) [M +
H]+ calcd for C19H20NO8 390.118 35, found 390.117 50.
The resulting thick oil was dissolved in 4 mL of DCM and treated
with 4-N-methylmorpholine (60 μL, 540 μmol), N,O-dimethylhy-
droxylamine hydrochloride (27 mg, 280 μmol), and EDC (53 mg, 280
μmol). The mixture was stirred for 20 h at room temperature, diluted
with DCM, and washed with 1 M aqueous HCl. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash column chromatography over silica gel (3−6%
MeOH/CHCl3) gave the intermediate Weinreb amide as a gum (61
1
mg, 51%, two steps). H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.6
Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 5.94 (m, 1H), 5.28 (m, 4H), 4.63 (d,
J = 5.6 Hz, 2H), 4.42 (m, 2H), 3.96 (s, 0.5H), 3.73 (m, 2H), 3.48 (m,
5.5H), 3.43 (m, 2.5H), 3.14 (s, 0.5H), 2.87 (s, 2H); 13C NMR (101
MHz, CDCl3) δ 164.3, 159.1, 155.8, 155.3, 149.5, 145.8, 132.8, 125.4,
124.7, 118.2, 115.0, 113.8, 111.1, 94.7, 66.6, 61.8, 61.2, 56.7, 42.1, 39.3,
35.8, 32.4, 24.8; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for
C21H25N2O8 433.160 54, found 433.158 86.
The above amide (15 mg, 35 μmol) was treated with 1.5 mL of 33%
TFA/DCM at room temperature and stirred for 2 h. The excess TFA
was removed under reduced pressure to afford pure 42 as a semisolid
(13 mg, 96%). 1H NMR (400 MHz, CDCl3) δ 7.49 (bs, 1H), 6.96 (d,
J = 7.2 Hz, 1H), 6.88−6.35 (bs 1H), 5.94 (m, 1H), 5.33 (m, 1H), 5.24
(m, 1H), 4.65 (m, 2H), 4.45 (m, 2H), 3.80 (m, 2H), 3.75−3.50 (bs,
3H), 3.39 (s, 3H), 2.87 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
159.2, 158.9, 155.4, 150.0, 146.6, 132.6, 126.4, 118.3, 116.9, 114.2,
112.0, 108.8, 76.6, 76.5, 66.8, 61.8, 41.9, 39.4, 24.9; HRMS (ESI-TOF)
(m/z) [M + H]+ calcd for C19H21N2O7 389.134 33, found 389.133 65.
Recombinant Human IRE-1 Expression and Purification.
Expression of 59.2 kDa polyhistidine-tagged puritin-hIRE-1 fusion
protein was carried out in SF21 cells using the Bac to Bac expression
system (Invitrogen) according to manufacturer’s specifications. An 8×-
His-puritin sequence was fused to the N-terminal end of the
cytoplasmic kinase/RNase domain of human IRE-1 (aa 547−977) in
the pFastbacDual-PBL expression vector and included a PreScission
protease cleavage site in the linker. Frozen insect cell paste (1 g) was
suspended in 8 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM
NaCl, 5 mM βME, 10 mM imidazole) containing one protease
inhibitor tablet and lysed using sonication. After removal of the cell
debris via centrifugation, the supernatant was applied to a Ni(NTA)
column (5 mL). After the untagged protein was washed by flushing
with 10 column volumes of lysis buffer, the target protein was eluted
using a linear imidazole gradient (15 column volumes, 10−300 mM).
Fractions were analyzed via SDS−PAGE. Pooled protein-containing
fractions were concentrated and rebuffered into 50 mM Tris, pH 8.0,
150 mM NaCl, 1 mM DTT via ultrafiltration. Typically, 1 L of insect
cell culture yielded 3 mg of recombinant 8×-His-puritin-hIRE-1
following Ni(NTA) column purification.
In Vitro IRE-1 RNase FRET-Suppression Assay. The endor-
ibonuclease activity of recombinant hIRE-1 was assayed by incubation
of 50 μL of 10 nM hIRE-1 and 50 μL of various concentrations (0.01−
1 μM) of fluorescently tagged XBP-1 RNA stem loop (5′-Cy5-
CAGUCCGCAGCACUG-BHQ-3′, obtained from Sigma-Aldrich
Co.) in assay buffer (20 mM HEPES, pH 7.5, 50 mM KOAc, 0.5
mM MgCl2, 3 mM DTT, 0.4% PEG, and 5% DMSO) for up to 2 h at
room temperature in a black 96-well plate. Fluorescence was read at
various time points using a Biotek Synergy H1 plate reader with
excitation and emission at 620 and 680 nm, respectively. The Km of
purified recombinant hIRE-1 was determined to be 45 nM using the
Michaelis−Menten kinetic model. Inhibition of RNA cleavage by small
molecules was determined by preincubation of 40 μL of 15 nM hIRE-1
with various concentrations of compounds (40 μL) in assay buffer for
30 min at room temperature. A 150 nM solution of fluorescent XBP-1
RNA (40 μL) was then added to each well and the reaction allowed to
proceed for 2 h before reading fluorescence as described above. Final
concentrations of hIRE-1 and XBP-1 RNA were 5 and 50 nM,
respectively. All fluorescence readings were corrected using back-
ground values from wells containing only 120 μL of 50 nM XBP-1
RNA. Dose−response experiments were carried out a minimum of 4
times on different days and IC50 values calculated from the mean
inhibition value at each concentration.
3-((Allyloxy)carbonyl)-8-hydroxy-5-oxo-2,3,4,5-tetrahydro-
1H-chromeno[3,4-c]pyridine-7-carboxylic Acid (40). Compound
39 (32 mg, 82 μmol) in 1 mL of MeOH was treated with 2 mL of 4 N
aqueous HCl and stirred for 20 h at room temperature. The mixture
was concentrated under reduced pressure. Purification by semi-
preparative RP-HPLC (C18 column, 0−70% MeCN/H2O gradient
over 20 min) and subsequent lyophilization afforded compound 40 as
1
a white solid (12 mg, 56%). H NMR (400 MHz, CD3CN) δ 12.39−
11.57 (m, 1H), 7.75 (d, J = 9.0 Hz, 0.7H), 7.69 (rotamer: d, J = 8.9 Hz,
0.3H), 6.95 (dd, J = 9.0, 1.0 Hz, 0.7H), 6.85 (rotamer: d, J = 8.9 Hz,
0.3H), 5.99 (m, 1H), 5.32 (m, 1H), 5.21 (m, 1H), 4.61 (m, 2H), 4.31
(s, 2H), 3.73 (m, 2H), 2.86 (m, 2H); 13C NMR (101 MHz, CD3CN)
δ 171.4, 165.9, 159.7, 153.7, 148.9, 147.7, 134.3, 131.4, 130.4, 117.6,
116.1, 115.0, 112.9, 102.5, 66.8, 42.4, 42.3, 25.6; HRMS (ESI-TOF)
(m/z) [M + H]+ calcd for C17H16NO7 346.092 13, found 346.091 98.
3-Allyl 7-Methyl-8-hydroxy-5-oxo-4,5-dihydro-1H-
chromeno[3,4-c]pyridine-3,7(2H)-dicarboxylate (41). Com-
pound 39 (20 mg, 51 μmol) in 2 mL of acetone at room temperature
was treated with potassium carbonate (10 mg, 77 μmol) and methyl
iodide (5.0 μL, 77 μmol) and stirred for 24 h. The mixture was diluted
with EtOAc, washed with brine, and dried over Na2SO4. Purification
by flash column chromatography over silica gel (50−70% EtOAc/
hexanes) afforded the intermediate methyl ester as a thick oil (10 mg,
1
48%). H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.2 Hz, 1H), 7.13
(d, J = 9.0 Hz, 1H), 5.94 (m, 1H), 5.37−5.18 (m, 4H), 4.63 (m, 2H),
4.44 (s, 2H), 3.98 (s, 3H), 3.78 (t, J = 5.8 Hz, 2H), 3.48 (m, 3H), 2.85
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 164.4, 163.5, 158.8, 156.3,
150.1, 145.6, 132.8, 125.6, 125.4, 118.2, 113.9, 113.4, 111.2, 94.8, 91.9,
66.7, 56.7, 53.2, 42.2, 39.3, 24.8; HRMS (ESI-TOF) (m/z) [M + H]+
calcd for C20H22NO8 404.133 99, found 404.134 65.
The above ester (10 mg, 25 μmol) was treated with 1.5 mL of 33%
TFA/DCM at room temperature and stirred for 1 h. The excess TFA
was removed under reduced pressure to afford 41 as a semisolid (8.0
mg, 90%). 1H NMR (400 MHz, CDCl3) δ 11.96 (bs, 1H), 7.63 (d, J =
8.5 Hz, 1H), 7.01 (d, 8.9 Hz, 1H), 5.95 (m, 1H), 5.33 (m, 1H), 5.25
(m, 1H), 4.66 (m, 2H), 4.49 (s, 2H), 4.08 (s, 3H), 3.80 (m, 2H), 2.88
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 170.4, 165.3, 152.8, 152.6,
146.8, 132.5, 129.5, 118.5, 115.1, 111.9, 102.2, 101.0, 67.0, 53.5, 41.8,
39.5, 25.0; HRMS (ESI-TOF) (m/z) [M + H]+ calcd for C18H18NO7
360.107 78, found 360.107 59.
Allyl 8-Hydroxy-7-(methoxy(methyl)carbamoyl)-5-oxo-4,5-
dihydro-1H-chromeno[3,4-c]pyridine-3(2H)-carboxylate (42).
Compound 39 (91.0 mg, 276 μmol) and 2-methyl-2-butene (350
μL, 3.31 mmol) in 3.5 mL of CH3CN/H2O (1:1) at 0 °C was treated
with a solution of sodium chlorite (187 mg, 1.66 mmol) and sodium
monophosphate (343 mg, 2.48 mmol) in water, dropwise. After the
mixture was stirred for 1 h, the reaction was quenched with 5%
aqueous Na2S2O3 solution in water. The pH of the solution was
adjusted to 6 and the aqueous portion extracted with EtOAc. The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure.
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Article
Antibodies and Reagents. Antibodies against IRE-1 (Cell
Signaling), PARP (Cell Signaling), XBP-1s (Santa Cruz), p97
(Fitzgerald), and actin (Sigma), were obtained commercially.
Cell Culture. Primary B cells were purified from wild-type mouse
spleens by negative selection using anti-CD43 magnetic beads
(Miltenyi Biotech). These cells as well as the human mantle cell
lymphoma (MCL) cell lines Mino and Jeko were cultured in RPMI
1640 medium (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin G
sodium, 100 μg/mL streptomycin sulfate, 1 mM sodium pyruvate, 0.1
mM nonessential amino acids, and 0.1 mM β-mercaptoethanol (β-
ME).
Protein Isolation and Immunoblotting. Cells were lysed using
RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40;
0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented
with protease inhibitors (Roche). Protein concentrations were
determined by BCA assays (Pierce). Samples were boiled in SDS−
PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10%
glycerol; 0.1% bromophenol blue) with β-ME and analyzed by SDS−
PAGE. Proteins were transferred to nitrocellulose membranes, blocked
in 5% nonfat milk (wt/vol in PBS), and immunoblotted with indicated
primary antibodies and appropriate horseradish peroxidase-conjugated
secondary antibodies. Immunoblots were developed using Western
Lighting chemiluminescence reagent (PerkinElmer).
Cell Proliferation XTT Assays. Appropriate numbers of cells were
suspended in phenol red-free culture medium, seeded in 96-well cell
culture plates, and treated with indicated IRE-1 inhibitors. After 48 h,
cells were spun down and proliferation was assessed by XTT assays
(Roche) according to the manufacturer’s instructions. Briefly, 50 μL of
XTT labeling reagent, 1 μL of electron-coupling reagent, and 100 μL
of phenol red-free culture medium were combined and applied to each
well of the 96-well plates. Cells were then incubated for 4 h in a CO2
incubator to allow for the yellow tetrazolium salt XTT to be cleaved by
mitochondrial dehydrogenases of metabolic active cells to form the
orange formazan dye, which can be quantified at 492 nm using a
BioTek Synergy H1 microplate reader.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra for assayed inhibitors, kinetic RNase activity
data for recombinant IRE-1, FRET-suppression assay dose−
response curves for all active inhibitors, stability data for
compound 24, and XTT assay with 30 in the presence of
mouse B cells. This material is available free of charge via the
Internet at http://pubs.acs.org.
(14) Papandreou, I.; Denko, N. C.; Olson, M.; Van Melckebeke, H.;
Lust, S.; Tam, A.; Solow-Cordero, D. E.; Bouley, D. M.; Offner, F.;
Niwa, M.; Koong, A. C. Identification of an Ire1alpha endonuclease
specific inhibitor with cytotoxic activity against human multiple
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Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.;
Ron, D.; Harding, H. P. The molecular basis for selective inhibition of
unconventional mRNA splicing by an IRE1-binding small molecule.
Proc. Nat. Acad. Sci. U.S.A 2012, 109, E869−E878.
AUTHOR INFORMATION
■
Corresponding Authors
*J.R.D.V.: phone, 813-745-6142; fax, (813) 745-6817; e-mail,
juan.delvalle@moffitt.org.
*C.-C.A.H.: phone, 813-745-4167; e-mail, chih-chi.hu@moffitt.
org.
Notes
The authors declare no competing financial interest.
(16) Mimura, N.; Fulciniti, M.; Gorgun, G.; Tai, Y. T.; Cirstea, D.;
Santo, L.; Hu, Y.; Fabre, C.; Minami, J.; Ohguchi, H.; Kiziltepe, T.;
Ikeda, H.; Kawano, Y.; French, M.; Blumenthal, M.; Tam, V.; Kertesz,
N. L.; Malyankar, U. M.; Hokenson, M.; Pham, T.; Zeng, Q.;
Patterson, J. B.; Richardson, P. G.; Munshi, N. C.; Anderson, K. C.
Blockade of XBP1 splicing by inhibition of IRE1alpha is a promising
therapeutic option in multiple myeloma. Blood 2012, 119, 5772−5781.
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ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(Grant R01CA163910), the American Cancer Society (Grants
IRG9309214 and IRG9303216), a Moffitt Team Science award,
and the Miles for Moffitt Foundation.
ABBREVIATIONS USED
■
XBP-1s, X-box binding protein 1 spliced form; FRET,
fluorescence resonance energy transfer; HMBC, heteronuclear
multiple bond correlation; CI, confidence interval
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NOTE ADDED AFTER ASAP PUBLICATION
■
Due to a production error, the IC50 curve and Western Blot gel
in the abstract graphic were omitted from the version published
on May 2, 2014. The revised version was reposted on May 2,
2014.
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