Constrained TRPV1 agonists synthesized via silver-mediated intramolecular azo-methine ylide cycloaddition of α-iminoamides

Article abstract of DOI:10.1016/j.bmcl.2013.12.061

As part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic benzodiazepine. A synthesis effort was initi

Full text of DOI:10.1016/j.bmcl.2013.12.061

Bioorganic & Medicinal Chemistry Letters 24 (2014) 963–968
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Constrained TRPV1 agonists synthesized via silver-mediated
intramolecular azo-methine ylide cycloaddition of a-iminoamides
Thomas O. Painter a, Krisztian Kaszas b, Jacklyn Gross b, Justin T. Douglas c, Victor W. Day c,
Michael J. Iadarola b, Conrad Santini a, ,
⇑
a Center for Chemical Methodologies and Library Development, The University of Kansas, 2034 Becker Drive, Lawrence, KS 66047, United States
b Department of Perioperative Medicine, Clinical Center, National Institutes of Health, Building 10, Room 2C401, 10 Center Drive, MSC 1510, Bethesda, MD 20892-1510, United States
c The University of Kansas Molecular Structures Group, 1251 Wescoe Hall Drive, Lawrence, KS 66045, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput
screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic
benzodiazepine. A synthesis effort was initiated that ultimately afforded racemic seco analogs 12 of the
SMR compound via a silver mediated intramolecular [3+2] cycloaddition of an azo-methine ylide gener-
Received 6 October 2013
Revised 12 December 2013
Accepted 16 December 2013
Available online 21 December 2013
ated from a-iminoamides 11. The cycloaddition set four contiguous stereocenters and, in some cases, also
spontaneously afforded imides 13 from 12. The synthesis of compounds 12, the features that facilitated
the conversion of 12–13, and their partial agonist activity against TRPV1 are discussed.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Chronic pain
Peripheral nerve ion channels
Intramolecular cycloaddition
TRPV1 agonists
One of the most problematic areas in contemporary clinical
practice is the long-term management of chronic pain. There are
numerous analgesics available for this purpose, many of which
act at the opioid receptors in the CNS.1 While these drugs are safe
and effective if administered under rigorously controlled
conditions, long-term use of opiates inevitably leads to tolerance,
physical dependence and sometimes depression.1 More trouble-
some, however, is that the mood altering effects of opiates are such
that, under real life conditions, these medications are often
diverted and abused. The resultant social and economic conse-
quences of these drawbacks are a source of continuing concern.2
As an alternative to opiates, peripherally-acting agents, of
whom NSAIDS are the most familiar examples, have been less use-
ful when pain is in the moderate to severe range.3 Peripheral
agents act by altering molecular and physiological events at the
site where the pain signal is generated instead of where the signal
is received or perceived in the CNS.4 In doing so, peripheral agents
present a major advantage in that they do not alter consciousness,
do not produce other deleterious CNS side effects nor do they inter-
fere with the analgesic actions of centrally-acting agents such as
opiates. Despite these advantages, available peripheral agents have
not demonstrated efficacy as replacements for centrally acting
agents. Currently known peripheral analgesics do not block the
initiation of pain signals with the specificity and to the extent
required for the improvement in the quality of life of chronic pain
patients.
The initial step of pain sensation is the generation of action po-
tential(s) by ion channel mediated depolarization of sensory nerve
terminals in skin or deep tissues.5 Preventing the generation of
these nociceptive signals at their origin by inhibiting the ion
channels responsible for the depolarization would provide highly
effective analgesia since a pain signal that is not generated is one
that requires no blockage further upstream in the CNS. However,
there are a multitude of ion channels in nociceptive nerve endings
that can respond to algesic chemicals released from damaged cells.
For this reason blocking only one channel does not appear to
provide the efficacy necessary for a useful analgesic agent. Alterna-
tively, it may be possible to administer an agent that will
temporarily disable the nociceptive nerve endings but only in the
localized areas directly implicated in the painful condition, essen-
tially acting as a highly specific, activity dependent long-term
analgesic.
TRPV1 is a high-conductance, non-specific cation channel both
restricted to and highly expressed in a subpopulation of nocicep-
tive afferents. The receptor is activated by small-molecule ligands,
noxious heat (>42 °C) and extracellular protons, contributing to
pain sensation under pathological conditions like chronic inflam-
mation (arthritic pain) or chemotherapy-induced and diabetic
neuropathies.6 Initial enthusiasm to develop small-molecule
antagonists of TRPV1 that bind at the capsaicin recognition site
⇑
Corresponding author. Tel.: +1 732 690 4960.
E-mail address: nyprphd@yahoo.com (C. Santini).
Current address: 24780 Chieftain Road, Lawrence, KS 66044, USA.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.061

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T. O. Painter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 963–968
as a novel class of analgesics to ‘block pain at the source’ faded as
side effects stemming from less-studied physiological roles of
TRPV1 emerged during clinical trials.7
As an alternative to antagonists, we have been working on the
development of both potent agonists and positive allosteric modu-
lators (PAMs) of TRPV1 as analgesic agents. Both can temporarily
incapacitate TRPV1 expressing nerve terminals through calcium
overload. Potent agonists like resiniferatoxin (RTX) affect all TRPV1
positive nerve endings, while PAMs act only at nerve terminals
with activated TRPV1, in a state-dependent manner.8 Local admin-
istration of potent TRPV1 agonists or oral administration of TRPV1
PAMs could provide a new class of analgesics and a much sought-
after alternative to opioids in pain control. Designing or discover-
ing such agents could be rendered more facile with increased
understanding of the structure of the relevant ion channels. The
structure of TRPV1 has not yet been solved and, in an effort to ac-
quire understanding of its structure and pharmacology, a screening
program was undertaken to identify either agonists or allosteric
modulators.
Scheme 1. Synthesis of the initially proposed ester scaffolds.
A high-throughput screen of the NIH Small Molecule Repository
(SMR) revealed
a series of geometrically constrained TRPV1
agonists, possessing structural features reminiscent of capsaicin,
with compound MLS002174161 showing highest potency
(Fig. 1).9 In order to better understand the spatial organization of
the TRPV1 agonist pharmacophore responsible for the activity of
MLS002174161, a series of related compounds was proposed for
synthesis and SAR study. Given the structure, we believed that clo-
sely related compounds could be prepared using a method similar
to that of Grigg and used later by Kurth, followed by modifications
at the nitrogen atom and ester (Scheme 1).10 In this sequence phenol
1a was first alkylated with commercially available ethyl trans-4-
bromo-2-butenoate to afford 2a in 49% yield.11 Aldehyde 2a was
next converted to imines 3a and 3b through reaction with the
methyl or benzyl esters of alanine HCl salt in the presence of Hünig’s
base and 4 Å molecular sieves. From here, the imines 3a and 3b were
converted to their corresponding cycladducts 4a and 4b in 55% and
44% yields, respectively. The cycloadducts were obtained as single
diastereomers, with the relative configurations shown, as described
in earlier reports. The cycloadducts 4 were racemic regardless of
whether or not chiral alanine esters were used.10
Scheme 2. Synthesis of hydantoins 6.
our alanine derived case, the presence of the additional methyl
group prevents epimerization.
After encountering more difficulties in diversifying the a-methyl
substituted ester group in 4a and 4b a new strategy was employed
Attempts to acylate the secondary nitrogen atom using aro-
matic acyl chlorides to afford various amides were not successful.
We were, however, able to prepare the related hydantoins 6a and
6b using methyl ester 4a by reaction with aryl isocyantes followed
by intramolecular ring closure in a manner similar to Kurth’s
example (Scheme 2).10b This observation led us to conclude that
only the most reactive and unhindered electrophiles can react with
this particular nitrogen atom. Again, the stereochemistry is implied
from the previous reports, although Kurth notes epimerization at
in which the amide portion was preassembled prior to imine forma-
tion (Scheme 3). The resultant
a-iminoamides 11 could then be
subjected to the 1,3-dipolar cycloaddition conditions to afford the
desired amide compounds in a convergent fashion. To the best of
our knowledge, there have been few reports of
a-iminoamides
being used in this type of azo-methine ylide 1,3-dipolar cycloaddi-
tion, with most examples appearing as part of a larger series that
contain mostly esters.12 Only one report, published in 2012, focuses
exclusively on this class of substrates.13 Of all the 1,3-dipolar cyclo-
the
a-position to the methyl ester occurs under the basic
addition reactions of
a-iminoamides found, we encountered no
conditions employed for ring closure of glycine derivatives. In
intramolecular examples.
Figure 1. High-throughput screen identified a constrained TPRV1 agonist. (A) Structure of capsaicin, archetypical vanilloid TRPV1 agonist. Color coding highlights structural
subdivision according to Walpole, 1993 (red = region A, blue = region B, green = region C) (B) MLS002174161, a fully constrained rigid TRPV1 agonist with proposed alignment
of capsaicin (C) MLS002174161 is a full agonist of TRPV1 with reduced potency compared to capsaicin. This reduction of potency is expected due to the rigidity of the ligand.

T. O. Painter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 963–968
965
Scheme 4. Synthesis a-aminoamides and aldehydes.
12f was isolated in pure form by normal phase chromatography.
However, during reverse phase HPLC analysis or purification of
12f, using an aq ammonia (pH 9.4) modified mobile phase, imide
13f was generated. Imide 13f did not form under acidic conditions.
This apparent ammonia promoted imide formation was not
observed for any of the alanine derived compounds.
To shed some light on the structural features that facilitate the
conversion of cycloadducts 12 to imides 13, alanine derived a-ami-
noamides 9b, 9c and 9d were used to form imines 11g, 11h and 11i
from aldehyde 2a. Imines 11g and 11h were synthesized in 83%
and 75% yields respectively and subjected to the silver-mediated
cyclization conditions (Table 1, entries 6 and 7). 9d failed to form
imine 11i (Table 1, entry 8).
Scheme 3. Synthetic sequence for formation of 12a and 13a.
We first needed to synthesize a suitable a-aminoamide. For rea-
sons related to our previous initial screening study, we wished to
couple aniline derivative 8a to Boc-alanine, affording 9a. This
was accomplished (Scheme 3) with EEDQ being crucial to the
coupling.14 Microwave irradiation accelerated the reaction, conve-
niently providing 9a in 73% yield. These microwave conditions
were adopted for the formation of all subsequent a-aminoamides
described in this report. To finish the sequence, TFA salt 10a was
formed by deprotection of 9a. The conditions shown in Scheme 1
We initially hypothesized that the electron-withdrawing aryl
ortho-carboxymethyl substituent facilitated the conversion of cyc-
loadducts 12 to imides 13 solely by lowering the pKa of the anilide
proton just enough that some polarization of the N–H bond would
occur. The bond polarization would increase the negatively
charged character of the nitrogen and lead to imide formation. In
keeping with this idea, we were not surprised that 11g did not un-
dergo this process, giving only cycloadduct 12g (20% yield; Table 1,
entry 6) with no accompanying formation of imide 13g. We then
predicted that the para-carboxymethyl substituent of 11h would
exert a similar effect, giving first cycloadduct 12h and then imide
13h. This did not occur. Aryl para-carboxymethyl 11h gave only
cycloadduct 12h (44% yield; Table 1, entry 7) without spontane-
ously affording the imide 13h. This did not support our ‘pKa low-
ering’ hypothesis. Another experiment using aryl ortho-ethyl
species 11j was carried out (Table 1, entry 9). The electronically
neutral, but sterically congested, ortho-ethyl 11j also stopped
reacting at the initial cycloadduct stage, giving 12j (28% yield)
without formation of imide 13j. Thus only the electron-withdraw-
ing, sterically congested ortho-esters afforded imides. We con-
cluded that both of the preceding factors are needed to convert
the cycloadducts 12 to the imides 13.
were then used to form
Effecting the cycloaddition reaction as in Scheme 1 was unsuccess-
ful due to the low solubility of -iminoamide 11a in acetonitrile.
a-iminoamide 11a in 76% yield from 9a.
a
The reaction was therefore performed in dichloromethane (DCM)
using silver fluoride and triethylamine.15 These conditions afforded
the desired product 12a in 22% yield, but also provided a second
product which was, by 1H NMR spectroscopy, a mixture of two
other compounds that showed the absence of the ethyl ester group.
LCMS analysis, which showed a single compound, also showed the
loss of ethanol from the mass of the cycloadduct 12a. As a result of
variable temperature 1H NMR spectroscopy coalescence studies
and 15N NMR spectroscopy we assigned the structure of the second
product to be imide 13a (29% yield) as a 2:1 mixture of atropisom-
ers. Using the less basic silver trifluoroacetate in place of silver
fluoride also afforded the same products, but gave 13a in a dimin-
ished 13% yield.
To probe the scope and limitations of this method and provide
some initial SAR, a series of
synthesized using the above conditions (Scheme 4).
Deprotection of N-Boc-protected -aminoamides 9 provided
their -aminoamide TFA salts (corresponding to Scheme 3, struc-
a-aminoamides and aldehydes was
a
a
ture 10). These TFA salts were combined with aldehydes 2 under
the conditions shown in Scheme 3 to provide the desired imines
11 in 58–92% yields in all but one case (Table 1).
Two fully N-substituted
a-iminoamides 11k (from
a-N-Boc-
aminoamide 9f and aldehyde 2a) and 11n (from
a-N-Boc-aminoa-
mide 9g and aldehyde 2d) were also prepared and subjected to AgF
promoted cyclization. Alanine derived imine 11k failed to afford
any cycloadduct or imine products (Table 1, entry 10). Glycine de-
rived imine 11n afforded cycloadduct 12n in 42% yield with no
imide 13n formed (Table 1, entry 11). We believe A1,3 strain that
is present in the formation of the azo-methine intermediate in
Int-11k prevents the cycloaddition reaction from easily occurring.
This strain would not be present in the case of Int-11n or when
Imines 11b, c, d, and e, derived from 9a and aldehydes 2b, c, d
and e, underwent AgF promoted cyclization as shown in Scheme 3
to afford mixtures of cycloadducts 12b–e and their derived imides
13b–e. Cycloadducts 12b–e were formed in 19–22% yield and imi-
des 13b–e in 23–36% yield, respectively (Table 1, entries 1–4).16
Imine 11f (R1 = H), obtained from aldehyde 2a and glycine derived
a-aminoamide 9e, gave cycloadduct 12f (14% yield) and imide 13f
(28% yield; Table 1, entry 5). We observed, qualitatively, that the
conversion of glycine derived 12f to imide 13f was more facile than
the conversion of alanine derived 12a to imide 13a. Cycloadduct
employing
a secondary amide such as shown by Int-11a
(Scheme 5).

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T. O. Painter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 963–968
Table 1
Synthesis of imines 11, cycloadducts 12 and imides 13 (N/A = not applicable)
Entry
1
Ar
R1
R2
H
R3
Imine 11, yield
11b, 77%
Cycloadduct 12, yield
12b, 22%
Imide 13, yield
13b, 36%
Me
5-OMe
2
3
4
5
’’
’’
’’
’’
Me
Me
Me
H
H
H
H
H
4-OMe
5-F
3-F
11c, 91%
11d, 82%
11e, 74%
11f, 92%
12c, 21%
12d, 19%
12e, 19%
12f, 14%
13c, 23%
13d, 30%
13e, 26%
13f, 28%
5-Cl
6a
7
Me
Me
Me
H
H
H
5-Cl
5-Cl
5-Cl
11g, 83%
12g, 20%
12h, 44%
N/A
Not observed
Not observed
N/A
11h, 75%
8
11i, Not observed
9b
Me
H
5-Cl
11j, 90%
12j, 28%
Not observed
10
11
Ph
Ph
Me
H
Me
Me
5-Cl
5-F
11k, 58%
11n, 79%
Not observed
12n, 42%
N/A
Not observed
a
Reaction performed with AgCO2CF3.
36 h reaction time.
b
Finally, to assess the accuracy of our assignments of relative
stereochemistry with respect to the cycloadducts that were ob-
tained using the -iminoamides cycloadduct 12n, purified to
The synthesized compounds 4a, 6a, 6b, 12a–h, 12j, 12n, 13a–f
were assayed for TRPV1 agonist activity. The activity of the above
compounds that showed agonist properties is shown in Fig. 3. Four
compounds, 12a, 12e, 12b and 12d showed partial agonism.
a
>99 area% as analyzed by High Resolution LCMS, was crystallized
from 0.1 N HCl. The crystallization afforded two crystal forms: a
triclinic unit cell as a needle and a monoclinic unit cell as a
plate. X-ray crystallographic data for the plate was obtained
(deposition #CCDC 972332), showing the product as its monohy-
drate with the relative stereochemistry we expected based on
Kurth’s precedent (Fig. 2). The needles were not characterized
by X-ray.
Scheme 5. Proposed A1,3 Strain of 1,3-dipole Int-11k vs. Int-11a and Int-11n.
Figure 2. X-ray crystal structure of 12n as its monohydrate.

T. O. Painter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 963–968
967
Figure 3. Biological activity of synthesized compounds on TRPV1 using a 45Ca2+ uptake assay. (A) Four out of the eighteen compounds exhibited TRPV1 agonist activity.
Compounds 12a, 12e, 12b and 12d all behave as partial TRPV1 agonists with 12e showing the lowest potency and efficacy. (B) Proposed alignment of capsaicin (cyan),
MLS002174161 (salmon) and the four partial agonists (light grey). The proposed alignment shows good overlap of an Aromatic center (orange) and a Hydrophobic region
(green) between all compounds. They also exhibit closely localized hydrogen-bond Donor (pink) and Acceptor (blue) groups and similarly tight grouping of hydrogen-bond
donor and acceptor projections (Don2 (dark cyan) and Acc2 (grey), respectively).
The structures of all the compounds tested can be classified into
three main categories based on the scheme proposed by Walpole
(refer to the above color-coded region structure of capsaicin,
Fig. 1A).8 Compound 4a falls into the first group. This compound
lacks the benzyl group corresponding to region ‘A’ in Walpole’s
scheme, resulting in the expected loss of agonist activity. Group
2 consists of compounds displaying the benzyl moiety together
with an additional methyl-, carboxymethyl group, or both (com-
pounds 12a, 12g, 12e, 12c, 12b, 12h, 12d, 12f and 12n). In addition,
the nitrogen adjacent to the benzyl ring forms an amide bond,
showing good correspondence to the ‘B’ region of capsaicin. The
third group consists of compounds where the nitrogen adjacent
to the benzyl group is incorporated into either a hydantoin ring
or an imide ring and ceases to behave as a hydrogen bond donor
(compounds 13a, 6a, 6b, 13e, 13c, 13b, 13d and 13f), while also
reducing their flexibility.
All TRPV1 agonists identified (12a, 12e, 12b and 12d) belong to
the second group. Their structure relative to capsaicin and their
lower potency align well with observations made by Walpole: (1)
removing the single sp3 carbon ‘spacer’ separating the benzyl
group from the amide group results in a ten-fold reduction of po-
tency {3a–b, Table 1, Walpole region ‘B’ 1993} (2) eliminating
the H-bond donor nitrogen from the amide bond also takes a sig-
nificant toll on potency {3a vs 20, Table 1, Walpole region ‘B’
1993} (3) additional groups on the benzyl ‘A’ region are far from
the optimal 4-Hydroxy 3-methoxy groups found in capsaicin {2b,
Table 1, Walpole region ‘A’, 1993}.
facilitate the development of potent TRPV1 agonists with potential
use in the treatment of localized, chronic pain.
Conflict of interest statement. The authors declare no competing
financial interest.
Acknowledgments
We acknowledge Mr. Patrick Porubsky, KU-CMLD Purification
Chemist and Compound Curator. Financial support for the KU-
CMLD was provided under NIGMS Grant #5P50GM069663 (Prof.
Jeff Aubé, PI). Support for the KU-NMR Center (JTD) was provided
under Grant #NIH 1S10RR024664-01. Support for the KU-X-Ray
facility (VWD) was provided under NSF-MRI Grant CHE-0923449.
Support for the HTS screen was provided by NIMH Grant # 1 R03
MH089480-01 (M. Iadarola); research was also supported by the
Division of Intramural Research, NIDCR, NIH and the Department
of Perioperative Medicine, Clinical Center, NIH. The funding
sources did not participate in the design of the work described in
this report.
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Galeotti, N.; Vivoli, E.; Tortorella, V. J. Med. Chem. 2007, 50, 1907.
15. Ding, Q.; Jiang, N.; Liu, J.-J.; Ross, T. M.; Zhang, J.; Zhang, Z. PCT Int. Appl., WO
2010031713 A1 20100325, 2010.
16. Representative procedure for formation of imine 11c, and adducts 12c and 13c: To
a solution of the TFA salt of 9a (0.169 g, 0.482 mmol) in dichloromethane
(3.40 mL) was added N,N-diisopropylethylamine (0.09 mL, 0.506 mmol). The
reaction mixture was stirred for 15 min then powdered 4 Å mol sieves (0.50 g)
were added followed by a solution of aldehyde 1b (0.127 g, 0.482 mmol) in
DCM (1 mL). The reaction mixture was stirred for 18 h overnight and then
diluted with DCM and washed with water (Â2) and brine (Â1). The organic
layer was dried over MgSO4, filtered and concentrated under vacuum to give
the product 11b (0.179 g, 77%) as a yellow oil that was used in the next step
without further purification. To a solution of imine 11b (0.179 g, 0.371 mmol)
in DCM (4.40 mL) was added triethylamine (0.16 mL, 1.11 mmol)) and silver
fluoride (0.235 g, 1.85 mmol). The reaction vessel was wrapped in foil and
stirred in the dark for 18 h. The reaction was quenched with ammonium
chloride (2 mL) and the mixture filtered through
a phase separator. The
9. For reports on capsaicin SAR, see: (a) Walpole, C. S. J.; Wrigglesworth, R.; Bevan,
S.; Campbell, E. A.; Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.; Winter, J. J.
Med. Chem. 1993, 36, 2362; (b) Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.;
Campbell, E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M. N.; Winter, J. J.
Med. Chem. 1993, 36, 2373; (c) Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.;
Campbell, E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M. N.; Winter, J. J.
Med. Chem. 1993, 36, 2381.
10. (a) Barr, D. A.; Grigg, R.; Gunaratne, H. Q. N.; Kemp, J.; McMeekin, P.; Sridharan,
V. Tetrahedron 1988, 44, 557; (b) Gong, Y.-D.; Najdi, S.; Olmstead, M. M.; Kurth,
M. J. J. Org. Chem. 1998, 63, 3081.
11. Moore, J. L.; Silvestri, A. P.; Read de Alaniz, J.; DiRocco, D. A.; Rovis, T. Org. Lett.
2011, 13, 1742.
12. (a) Dondas, H. A.; Grigg, R.; Kilner, C. Tetrahedron 2003, 59, 8481; (b) Dondas, H.
A.; Soenmez, S. Heterocycl. Commun. 2003, 9, 23; (c) Wu, S.-H.; Sun, W.-Q.;
Zhang, D.-W.; Shu, L.-H.; Wu, H.-M.; Xu, J.-F.; Lao, X.-F. J. Chem. Soc., Perkin
remaining aqueous layer was extracted three times through the phase
separator with DCM and the combined organic layers were dried over
MgSO4, filtered, and concentrated under vacuum. The residue was purified
via silica gel column chromatography (10–50% ethyl acetate in hexanes) to give
12b (0.039 g, 22%) and 13b (0.059 g, 36%) as white, amorphous solids. 12b: 1
H
NMR (500 MHz, CDCl3) d 12.38 (s, 1H), 8.61 (s, 1H), 7.88 (d, J = 8.2 Hz, 1H), 6.89
(m, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.78 (dd, J = 9.0, 3.0 Hz, 1H), 6.44 (d, J = 2.9 Hz,
1H), 4.95 (dd, J = 11.1, 2.2 Hz, 1H), 4.68 (d, J = 2.9 Hz, 1H), 4.16 (q, J = 7.1 Hz,
2H), 4.16 (m, 1H), 3.77 (s, 3H), 3.71 (s, 3H), 2.55–2.46 (m, 3H), 2.44 (s, 3H), 2.40
(s, 3H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 172.1, 167.5, 163.3,
161.0, 153.5, 148.1, 145.2, 140.0, 131.0, 123.7, 121.0, 120.5, 117.7, 115.2, 114.4,
114.0, 66.0, 60.7, 59.2, 55.7, 51.8, 35.3, 33.9, 22.0, 14.2, 13.1; IR: 3234, 2953,
1731, 1694 cmÀ1 HRMS (ESI) m/z calculated for (M+H)+ (C26H31N2O7)+
;
483.2131, found 483.2155; 13b: HRMS (ESI) m/z calculated for (M+H)+
(C24H25N2O6)+ 437.1713, found 437.1718.