Discovery of ML326: The first sub-micromolar, selective M5 PAM

Article abstract of DOI:10.1016/j.bmcl.2013.03.032

This Letter describes the further chemical optimization of the M ₅ PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M₅ PAMs (M₅ EC ₅₀ >10 μM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M₅ PAM, ML326 (VU0467903), (human and rat M₅ EC₅₀s of 409 nM and 500 nM, respectively) with excellent mAChR selectivity (M ₁-M₄ EC₅₀s >30 μM) and a robust 20-fold leftward shift of the ACh CRC.

Full text of DOI:10.1016/j.bmcl.2013.03.032

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2996–3000
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of ML326: The first sub-micromolar, selective M5 PAM
Patrick R. Gentry b,c,d, Thomas M. Bridges b,c, Atin Lamsal e, Paige N. Vinson b,c, Emery Smith e,
Peter Chase e, Peter S. Hodder e,f, Julie L. Engers b,c, Colleen M. Niswender a,b,c, J. Scott Daniels a,b,c
,
P. Jeffrey Conn a,b,c, Michael R. Wood a,b,c,d, Craig W. Lindsley a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
e Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA
f Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172.
A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number
of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign
Received 21 January 2013
Revised 28 February 2013
Accepted 7 March 2013
Available online 16 March 2013
identified several weak M5 PAMs (M5 EC50 >10 lM) with a structurally related isatin core that possessed
a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be
optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micro-
molar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409 nM and 500 nM, respectively) with
Keywords:
Muscarinic acetylcholine receptors
M5
Positive allosteric modulator (PAM)
ML326
excellent mAChR selectivity (M1–M4 EC50s >30
l
M) and a robust 20-fold leftward shift of the ACh CRC.
Ó 2013 Elsevier Ltd. All rights reserved.
There are five muscarinic acetylcholine receptor (mAChR) sub-
types (M1–M5) widely expressed in both the central nervous sys-
tem (CNS) and periphery of mammals.1–5 These receptors, whose
endogenous agonist is acetylcholine (ACh), play critical roles in
regulating a variety of diverse physiological processes. Within
the CNS, the M1, M4 and M5 subtypes are believed to be the most
important with respect to normal neuronal functioning.1–5 Of these
three, M5 is the least studied as a combined result of its lower
expression levels6 (<2% of total muscarinic receptor population
within the brain) and, until recently, a near absence of highly selec-
tive M5 receptor ligands. Much of our current understanding sur-
rounding the function of M5 has come from M5 receptor
localization, M5 knock-out mice7 and experiments conducted with
non-selective muscarinic ligands.8 It is intriguing to note that the
be critical in the ACh-induced dilation necessary for normal blood
perfusion.12 In a broader sense, M5 KO mice show decreased pre-
pulse inhibition,13 CNS neuronal abnormalities and cognitive defi-
cits,14 thus supporting the potential for M5 ligands in the treatment
of numerous CNS disorders including schizophrenia, Alzheimer’s
disease, ischemia and migraine.1–5
To date, few M5 PAMs have been reported,15–17 and none of
these display the potency and efficacy necessary to definitively
probe the role of selective M5 activation (Fig. 1); therefore, in this
Letter, we detail the further optimization of these ligand, and the
discovery of the first submicromolar and selective M5 PAMs.
Within the isatin scaffold, alternate substitution afforded a
highly selective M1 PAM, ML137, 4.18 We recently reported on a
number of replacements (tertiary hydroxyls and spirocyclic
replacements, etc.) for the isatin moiety that maintained M1 PAM
activity (Fig. 2).19,20 Therefore, our optimization plan for ML129
consisted of evaluating both spirocyclic and other replacements
for the isatin moiety, while continuing to investigate alternatives
for the southern p-OMe benzyl motif in an effort to improve M5
M5 receptor is the only muscarinic receptor observed the substan-
tia nigra pars compacta (based on mRNA detection),9,10 leading to
the prediction that M5 functions in addiction/reward mechanisms.
This hypothesis was supported in man through the clinically ob-
served correlation between a specific M5 gene mutation and an in-
crease in cigarette consumption (+27%), as well as, an increased
risk for cannabis dependence (+290%).11 Additionally, M5 receptors
have been localized on the cerebrovascular system and shown to
PAM potency to below 1 lM (Fig. 3).
First, we explored alternatives to the 5-OCF3 moiety on the isat-
in core, and despite evaluating other positions and alternate func-
tionalities, all were devoid of M5 PAM activity. Employing the
chemistry previously reported for analogs 5 of ML137,19 we pre-
pared the corresponding 3° hydroxyl and dioxalane analogs of
⇑
Corresponding author.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.032

P. R. Gentry et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2996–3000
2997
O
O
O
F3CO
F3CO
F3CO
O
O
O
N
N
F
N
N
OMe
O
O
1 ML129
,
2 ML172
,
3 VU0414747
,
hM5 EC50 = 1.1 µM (91% AChmax
hM5 ACh fold shift (@30 µM) = 14-fold hM5 ACh fold shift (@30 µM) = 5-fold
hM1 - hM4 EC50 > 30 µM hM1 - hM4 EC50 >> 30 µM
)
hM5 EC50 = 1.9 µM (75% AChmax)
hM5 EC50 = 1.5 µM (62% AChmax
)
hM5 ACh fold shift (@30 µM) = 6-fold
hM1 - hM4 EC50 >> 30 µM
Figure 1. Structures and activities of the reported M5 PAMs 1–3. All three were derived from a pan-M1, M3, M5-PAM that afforded both M1 selective PAMs and, by virtue of the
5-OCF3 moiety, M5 selective PAMs.
X
Y
R1
O
R2
O
O
O
N
N
N
Ar
Ar
F
5
6
NMe
R1, R2 = H or O
1 = OH, R2 = CH3 or CF3
X = CH2,Y = NR
Y = CH2, X = O or NR
N
R
4 ML137
,
hM1 EC50 = 0.6 µM (45% AChmax
hM2 - hM5 EC50 > 30 µM
)
Figure 2. Structures and activities of the reported M1 PAMs 4–6. Mulitple productive replacements (tertiary hydroxyls, dioxalanes and spiro furans/pyrroles) were identified
for the isatin moiety of ML137 that maintained selective M1 PAM activity.
isatin replacements
O
(3o hydroxyls, spirooxetanes, spriopyrroles, etc.)
F3CO
O
N
modifications (alternate linkers, Ar, etc.)
alternative
substituents
O
1, ML 129
hM5 EC50 = 1.1 µM (91% AChmax
)
hM5 ACh fold shift (@30 µM) = 14-fold
hM1 - hM4 EC50 > 30 µM
Figure 3. Multi-dimenisional chemical optimization plan for ML129 (1).
O
HO
NO2
F3CO
F3CO
a
O
O
hM5 EC50 = 1.4 µM
75% ACh Max
N
N
O
O
1, ML 129
7
Scheme 1. Synthesis of 3° hydroxyl analog 7. Reagents and conditions: (a) DABCO, nitromethane (neat), 100%.
ML129, and while there was a slight elevation in the ACh max, all
of the analogs were >10 M as M5 PAMs, indicating a disconnect in
This led us to explore other 3° alcohols possessing heterocycles,
l
and most notably pyridines. Here, we employed two routes to sur-
vey pyridyl analogs 8 and pyridyl methyl homologs 11 (Scheme 2).
Again, utilizing ML129 as starting material, exposure to a function-
alized pyridyl boronic acid 9 under Cu(OTf)2 catalysis, affords ana-
logs 8 in 25–70% yields.22 We also employed direct lithiation/
addition in some instances.23 The homologated congeners 11 were
easily accessed following the Li protocol,24 employing picolines
under Bronsted acid catalysis. SAR again proved shallow, with all
the SAR between M1 and M5. We next explored novel 3° hydroxyl
analogs with diverse functionalities, which proved to be more
productive. Treatment of ML129 with DABCO in nitromethane
afforded the 3° alcohol 7 in quantitative yield (Scheme 1);21 impor-
tantly, 7 was of comparable potency and efficacy to ML129 (M5
EC50 = 1.4
lM, 75% ACh Max), but the nitro moiety was not attrac-
tive as an in vivo probe.

2998
P. R. Gentry et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2996–3000
O
F3CO
O
N
OMe
1, ML 129
R
R
(HO)2B
R
N
10
N
b
a
9
R
N
O
F
HO
HO
HO
N
F3CO
F3CO
N
O
F3CO
O
N
N
N
OMe
8a
OMe
OMe
µ
hM5 EC50 = 2.2
74% ACh Max
M
8
11
Scheme 2. Synthesis of pyridyl-based 3° hydroxyl analogs 8 and 11. Reagents and conditions: (a) Cu(OTf)2, 1,10-phenathroline, LiOH, DCE, reflux, 48 h, 5–45%; (b) TfOH,
dioxane, 180 °C, mw, 45 min, 22–74%.
OMe
O
O
F3CO
F3CO
b
a
O
N
N
OMe
OMe
1, ML 129
OH
12
OH
O
N
F3CO
F3CO
O
O
c
N
OMe
OMe
13
14
Scheme 3. Synthesis of a spiro-oxetane analog 14. Reagents and conditions: (a) methyl methoxyphosphonium bromide, LDA, THF, À78 °C to rt, 70%; (b) PTSA, aq
formaldehyde, Na2CO3, THF: H2O, rt, 82%; (c) Tf2O, lutidine, DCM, À78 °C to rt, 6%.
5- or 7-
O
O
R = F, Cl, Br
OMe, CF3
Me
O
O
O
O
N
N
Me
R
15, CID2145491
31% ACh Max@30 µM
16
23-60% ACh Max@30 µM
O
O
F3CO
F3CO
O
O
N
N
O
O
1, ML 129
17
Figure 4. M5 Triple-add HTS PAM screening hit 15 and related analogs 16. Could juxtaposition of ML129 into 17 afford an improved M5 PAM?

P. R. Gentry et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2996–3000
2999
O
O
F3CO
F3CO
ML326
a
O
O
O
hM5 EC50 = 409 nM (91% AChmax
hM5 ACh fold shift (@30 µM) = 20-fold
)
N
N
H
hM1 - hM4 EC50 > 30 µM
18
17
Scheme 4. Synthesis of phenethyl ether analog 17 (ML326). Reagents and conditions: (a) (2-bromoethoxy)benzene, K2CO3, KI, ACN, mw, 160 °C, 10 min, 82%.
Table 1
Structure and activities of analogs 19
O
F3CO
O
N
R
O
19
Compd
R
M5EC50
(l
m)
pEC 50 SEM
%ACh Max
ML326
19a
19b
19c
19d
H
4-OPh
4-Cl
4-OMe
3-Me
0.41
1.05
0.45
0.59
0.53
6.39 0.04
5.98 0.04
6.35 0.04
6.23 0.04
6.28 0.03
91 1.6
93 1.9
92 1.7
89 1.7
91 1.3
All values are the average of three independent experiments.
ML129 provided 12, which was smoothly converted into the diol
13 in 57% overall yield. Mono-triflation of 13 and nucelophilic dis-
placement proceeded in low yield (ꢀ6%) to the spioroxetane 14.25
Interestingly, 14 was inactive as an M5 PAM, and only afforded a
marginal base-line increase in the ACh Max.
Other modifications, such as spiro-pyrrolidines and dioxalanes,
which were well tolerated for M1 PAM activity in the ML137 ser-
ies,19,20 were uniformly inactive on the ML129 scaffold. Clearly,
SAR was steep and did not cross-over between M1 and M5. More-
over, despite the synthesis and screening of hundreds of analogs
of ML129, surveying multiple regions, we were unable to develop
a submicromolar M5 PAM.
At this point, we pursued a new high throughput screen (HTS)
for M5 run in triple-add mode performed on the MLPCN screening
deck (ꢀ360,000 compounds, PubChem AID 624103).
Early in the process, and prior to official ‘hit’ confirmation
(Fig. 4), we noticed a structurally related, weak single-point (31%
at 3 lM) hit, CID2145491 (15). This ‘hit’ possessed a phenethyl
ether linkage, a moiety we had not yet explored on the ML129 core,
and we were very surprised to find an isatin core without the 5-
OCF3 moiety, and a 7-methyl group)15 with activity at M5. We then
reviewed the preliminary HTS data, and found a number of related
compounds 16 with diverse functionality on the phenyl ring of the
phenethyl ether, as well as either a 5- or 7-methyl group on the
isatin core with weak M5 activity (23–60% ACh max at 30 lM).
Figure 5. In vitro pharmacological characterization of 17 (VU0467903, ML326). (A)
M5 PAM concentration-response-curve (CRC) afforded an EC50 of 409 nM, 91% ACh
Max; (B) rat M5 fold-shift assay with 1 (VU0238429, ML129) and 17, demonstrating
a robust 20-fold shift (human fold-shift data is the same). ACh EC50 = 4.58 nM,
pEC50 = 8.34 0.04, ACh + 429 EC50 = 263.4 pM, pEC50 = 9.58 0.06, ACh + 903
EC50 = 225.4 pM, pEC50 = 9.65 0.06; (C) rat mAChR selectivity data with 17,
Based on these data, we immediately questioned if the juxtaposi-
tion of ML129 with 15, leading to analog 17 would lead to an im-
proved M5 PAM.
To evaluate this possibility, we prepared 17 by alkylating the
5-OCF3 isatin 18 with commercial (2-bromoethyoxy) benzene
under microwave-assisted conditions in 82% yield (Scheme 4).26
Gratifyingly, this structural modification led to one of the most
potent M5 PAM we have ever identified. Compound 17
(VU0467903), later declared an MLPCN probe and given the desig-
nation ML326, was a potent M5 PAM (Fig. 5) on both human
(EC50 = 409 nM, 91% ACh Max, pEC 50 = 6.39 0.04) and rat M5
(EC50 = 500 nM, 59% ACh Max). In fold-shift assays, ML326 afforded
a robust 20-fold leftward shift of the ACh CRC, and was found to
indicating >30 lM versus M1–M4 (human data is the same).
analogs 11 showing no activity as M5 PAMs; however, one of the 3-
pyridyl-based 3° hydroxyls 8a proved to be a moderately active M5
PAM (M5 EC50 = 2.2 l
M, 74% ACh Max), but SAR was steep.3
We therefore elected to introduce a more subtle variant of the
istain carbonyl in the form of a spiro-oxetane, and evaluate this
carbonyl isostere for M5 activity (Scheme 3). A Wittig reaction with

3000
P. R. Gentry et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2996–3000
8. Steidl, S.; Miller, A. D.; Blaha, C. D.; Yeomans PLoS ONE 2011, 6(11), e27538.
highly selective versus M1–M4 (EC50s >30 lM). Very encouraged by
http://dx.doi.org/10.1371/journal.pone.0027538.
the initial in vitro potency and selectivity profile for ML326 we pre-
pared gram quantities and set about characterizing it through our
in-house tier 1 DMPK assays (PPB, rat/human microsomal stability
with predicted intrinsic clearance, P450 inhibition, rat brain homog-
enate binding, etc.) and single dose PK/CNS exposure in rat. Many
of these experiments were initiated in parallel, including the col-
lection of rat plasma/brain samples; however we encountered
insurmountable LCMS/MS analytical quantization issues due to
poor ionization of ML326 using ESI, APPI, and APCI ionization
probes, which prevented the determination of routine tier 1 DMPK
parameters and in vivo rat exposure. Alternative methods includ-
ing chemical derivatization and UV absorbance also failed to pro-
vide the requisite sensitivity for detection of ML326 thus
preventing the completion of numerous studies. However, we were
able to assess that ML326 had relatively clean CYP and ancillary
pharmacology profiles.
Based on this promising data, we prepared other analogs 19
(Table 1) with substituents in the phenyl ring of 17, following
the route depicted in Scheme 4. While this afforded a number of
potent and selective M5 PAMs, they all suffered from the same poor
ionization profiles which precluded extensive DMPK profiling.
Based on the ionization issue and relatively flat SAR, this series is
no longer the subject of chemical optimization.
9. Vilaro, M. T.; Palacios, J. M.; Mengod, G. Neurosci. Lett. 1990, 114, 154.
10. Weiner, D. M.; Levey, A. I.; Brann, M. R. Proc. Natl. Acad. Sci. U.S.A. 1990, 87,
7050.
11. Anney, R. J. L.; Lotfi-Miri, M.; Olsson, C. A.; Reid, S. C.; Hemphill, S. A.; Patton, G.
C. BMC Genet. 2007, 8. http://dx.doi.org/10.1186/1471-2156-8-46.
12. Yamada, M.; Lamping, K. G.; Duttaroy, A.; Zhang, W.; Cui, Y.; Bymaster, F. P.;
McKinzie, D. L.; Felder, C. C.; Deng, C.-X.; Faraci, F. M.; Wess, J. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 14096.
13. Thomsen, M.; Wörtwein, G.; Fink-Jessen, A.; Woldbye, D. P. D.; Wess, J.; Caine,
S. B. Psychopharmacology 2007, 192, 97.
14. Araya, R.; Noguchi, T.; Yuhki, M.; Kitamura, N.; Higuchi, M.; Saido, T. C.; Seki,
K.; Itohara, S.; Kawano, M.; Tanemura, K.; Takashima, A.; Yamada, K.; Kondoh,
Y.; Kanno, I.; Wess, J.; Yamada, M. Neurobiol. Dis. 2006, 24, 334.
15. Bridges, T. M.; Marlo, J. E.; Niswender, C. M.; Jones, C. K.; Jadhav, S. B.; Gentry, P.
R.; Plumley, H. C.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2009,
52, 3445.
16. Bridges, T. M.; Kennedy, J. P.; Cho, H. P.; Breininger, M. L.; Gentry, P. R.;
Hopkins, C. R.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2010, 20, 558.
17. Bridges, T. M.; Kennedy, J. P.; Hopkins, C. R.; Conn, P. J.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2010, 20, 5617.
18. Bridges, T. M.; Kennedy, J. P.; Cho, H. P.; Conn, P. J.; Lindsley, C. W. Bioorg. Med.
Chem. Lett. 2010, 20, 1972.
19. Melancon, B. J.; Poslunsey, M. S.; Gentry, P. R.; Tarr, J. C.; Mattmann, M. E.;
Bridges, T. M.; Utley, T. J.; Sheffler, D. J.; Daniels, J. S.; Niswender, C. M.; Conn, P.
J.; Lindsley, C. W.; Wood, M. R. Bioorg. Med. Chem. Lett. 2013, 23, 412.
20. Poslunsey, M. S.; Melancon, B. J.; Gentry, P. R.; Bridges, T. M.; Utley, T. J.;
Sheffler, D. J.; Daniels, J. S.; Niswender, C. M.; Conn, P. J.; Lindsley, C. W.; Wood,
M. R. Bioorg. Med. Chem. Lett. 2013. http://dx.doi.org/10.1016/
j.bmcl.2013.01.017.
21. Meshram, H. M.; Ramesh, P.; Kumar, S.; Swetha, A. Tetrahedron Lett. 2011, 52,
5862.
22. Zhang, J.; Chen, J.; Ding, J.; Liu, M.; Wu, H. Tetrahedron 2011, 67, 9347.
23. Scheme to access analogs 8 via lithiation/addition sequence.
In summary, further elaboration of the ML129 M5 PAM structure
has been explored, and SAR was particularly steep. Insight from a
weak M5 PAM HTS hit led to the hybridization with ML129 to afford
ML326 (VU0467903), the first highly selective (>30 lM versus
M1–M4) and sub-micromolar (human (EC50 = 409 nM, 91% ACh Max)
and rat M5 (EC50 = 500 nM, 59% ACh Max)) M5 PAM. Interestingly,
the preliminary HTS hits that inspired the structural modifications
to ML129, leading to ML326 did not confirm upon re-order and full
CRC. Finally, the poor ionization of ML326 precluded in-depth
DMPK profiling; however, ML326 is a valuable in vitro probe and
is serving as an important probe in electrophysiology studies,
which will be reported shortly.
R
R
I
O
HO
N
F3CO
F3CO
N
O
nBuLi
O
O
N
N
THF
-78oC
15 min
O
50-91%
1, ML129
8
Acknowledgments
The authors thank the NIH (U54MH084659) and William K.
Warren, Jr. who funded the William K. Warren, Jr. Chair in Medi-
cine (to C.W.L.). Vanderbilt University is a Specialized Chemistry
Center within the MLPCN and all the ML# probes are freely avail-
able upon request. Scripps was supported by the National Institute
of Health Molecular Library Probe Production Center grant U54
MH084512. We thank Lina DeLuca (Lead Identification Division,
Scripps Florida) for compound management.
24. Niu, R.; Xiao, J.; Liang, T.; Li, X. Org. Lett. 2012, 14, 676.
25. Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.; Schuler, F.;
Rogers-Evans, M.; Muller, K. J. Med. Chem. 2010, 53, 3227.
26. 1-(2-phenoxyethyl)-5-(trifluoromethoxy)indoline-2,3-dione, ML326: The title
compound was synthesized in one step from commercially available starting
materials according to the following procedure. Into
a 20 mL microwave
reaction vial, containing magnetic stir bar, were weighed 5-
a
(trifluoromethoxy)isatin (460 mg, 2.0 mmol), K2CO3 (550 mg, 4.0 mmol), KI
(33 mg, 0.20 mmol), followed by acetonitrile (20 mL, 0.1 M) and 2-bromoethyl
phenyl ether (480 mg, 2.4 mmol). After being sealed with a crimp cap, the
vessel was placed in a microwave reactor and heated to 160 °C for 10 min, with
magnetic stirring. After cooling to ambient temperature, the reaction was
diluted with CH2Cl2 (ꢀ20 mL) and washed with brine. The organic layer was
separated and dried over Na2SO4. Solvent was removed under reduced
pressure and the crude product was purified via flash column
chromatography (silica gel, hexane/ethyl acetate, 0–50% ethyl acetate
gradient). Product containing fractions were combined and the solvents
removed under reduced pressure to obtain 583 mg of ML326 (83% yield) as a
red-orange powder. TLC Rf = 0.79 (hexane/ethyl acetate 1:1); 1H NMR
(400 MHz, CDCl3 calibrated to 7.26) d 7.52–7.46 (m, 2H), 7.31–7.25 (m, 3H),
6.98 (t, J = 7.4 Hz, 1H), 6.82 (d, 2H), 4.28 (t, J = 5.0 Hz, 2H), 4.17 (t, J = 5.0 Hz,
2H); 13C NMR (125 MHz, CDCl3 calibrated to 77.16) d 182.25, 158.27, 157.93,
150.01, 145.44, 131.02, 129.78, 121.75, 118.32, 114.39, 112.89, 65.94, 40.62;
HRMS calcd for C17H13NO4F3[M+H+]; 352.0797 found: 352.0795.
References and notes
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