Discovery of sulfonamidebenzamides as selective apoptotic CHOP pathway activators of the unfolded protein response

Article abstract of DOI:10.1021/ml5003234

Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded p

Full text of DOI:10.1021/ml5003234

Letter
pubs.acs.org/acsmedchemlett
Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP
Pathway Activators of the Unfolded Protein Response
Daniel P. Flaherty,† Justin R. Miller,‡ Danielle M. Garshott,‡ Michael Hedrick,§ Palak Gosalia,§ Yujie Li,§
Monika Milewski,§ Eliot Sugarman,∥ Stefan Vasile,∥ Sumeet Salaniwal,§ Ying Su,§ Layton H. Smith,∥
Thomas D. Y. Chung,§ Anthony B. Pinkerton,§ Jeffrey Aube,† Michael U. Callaghan,‡
́
Jennifer E. Golden,*,† Andrew M. Fribley,*,‡ and Randal J. Kaufman*,⊥
†Delbert M. Shankel Structural Biology Center, University of Kansas Specialized Chemistry Center, 2034 Becker Drive, Lawrence,
Kansas 66047, United States
‡Carmen and Ann Adams Department of Pediatrics, Division of Hematology and Oncology, and the Karmanos Cancer Institute
Molecular Therapeutics Group, Wayne State University, 2228 Elliman Building, 421 East Canfield, Detroit, Michigan 48201, United
States
§Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, United
States
∥Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, Florida 32827,
United States
⊥Program in Degenerative Disease Research, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla,
California 92037, United States
S
* Supporting Information
ABSTRACT: Cellular proteins that fail to fold properly result
in inactive or disfunctional proteins that can have toxic
functions. The unfolded protein response (UPR) is a two-
tiered cellular mechanism initiated by eukaryotic cells that have
accumulated misfolded proteins within the endoplasmic
reticulum (ER). An adaptive pathway facilitates the clearance
of the undesired proteins; however, if overwhelmed, cells
trigger apoptosis by upregulating transcription factors such as
C/EBP-homologous protein (CHOP). A high throughput
screen was performed directed at identifying compounds that
selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a
sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50
= 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell
cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel.
KEYWORDS: CHOP activator, UPR modulator, UPR apoptotic pathway activator, anticancer
misfolded proteins immediately triggers an adaptive response
whereby the ER transmembrane protein inositol-requiring
enzyme, 1α (IRE1α), splices X-box binding protein 1 (XPBP1)
mRNA, unleashing its potential as a transcription factor, in an
effort to enhance protein folding and degradation of misfolded
proteins.5,6 Simultaneously, another ER transmembrane kinase,
RNA-like endoplasmic reticulum kinase (PERK), is activated to
cause an immediate transient attenutation of general mRNA
translation. If productive folding cannot be restored and the
burden of misfolded proteins persists, accumulation of
activating transcription factor 4 (ATF4) and C/EBP-homolo-
t has been estimated that nearly one-third of the human
Igenome encodes proteins that enter the secretory pathway
en route to their final destination. Proteins that are secreted,
cell membrane bound (including receptors), or function in a
lysosome are folded and post-translationally modified in the
endoplasmic reticulum (ER).1−3 Homeostatic maintenance
within the ER is imperative for proper manufacture and
modification of essential proteins. Disruption of this process by
pharmacological insults that perturb N-linked glycosylation,
disulfide bond formation or calcium or redox status can lead to
an accumulation of misfolded and nonfunctional proteins
within the organelle, a condition known as ER stress.2,4 The
unfolded protein response (UPR) is a coordinated attempt by
the cell to either restore protein folding or induce apoptosis if
homeostasis cannot be restored.2 The accumulation of
Received: August 1, 2014
Accepted: October 29, 2014
Published: October 29, 2014
© 2014 American Chemical Society
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gous protein (CHOP) leads to programmed cell death by
upregulating apoptotic genes and modulating the reinitiation of
protein synthesis.7−11 Indeed, recent studies suggest that
CHOP acts as a tumor suppressor gene.12 It has been shown
that ER stress and the UPR contribute to neurodegenerative13
and inflammatory14 diseases, metabolic disorders,15 diabetes,16
and cancer;17 modulating the UPR with chemical probes as a
means of addressing these conditions, especially cancer, has
been studied by our group and others.18−23
Previously reported UPR modulators include promoters of
the UPR adaptation mechanisms as well as those that inhibit
UPR pro-survival signaling.24 Compounds of the latter group
operate by modulating one or more components within the
apoptotic pathway, and many of these agents have been
pursued as cancer therapeutics. Sorafenib 1 is an FDA-approved
drug for certain kidney and liver cancers that is known to
inhibit multiple receptor tyrosine kinases and serine/threonine
kinases (Figure 1).25,26 More recently, the compound has been
(Tm)-treated positive controls. After retesting these at the
same concentration, compounds displaying ≥32% of Tm-
induced luciferase were subjected separately to dose−response
assays with CHO-CHOP-luc cells or CHO-XPB1-luc cells to
remove compounds that nonselectively activated the UPR.40
Several scaffolds were identified from this effort; however,
sulfonamidebenzamide 3 was one of the most promising,
exhibiting a CHOP AC50 = 1.9 μM and an XBP1 AC50 > 80 μM
(Figure 1). Analogues of 3 were generated to establish
structure−activity relationships (SAR) around the scaffold,
optimize the activity, and further characterize this structural
series in relevant cancer cell lines (Table 1).
The majority of analogues was synthesized by coupling 4-
nitrobenzenesulfonyl chloride 4 with various cyclic amines to
afford para-nitrophenylsulfonamide intermediates (Scheme 1).
The nitro group was subsequently reduced with Raney nickel
Table 1. CHOP and XBP1 Activity for Structural Changes in
3
UPR CHOP
UPR XBP1
AC50 SEM
AC50
a
a
compd
R1
R2
(μM)
(μM)
3
7
N-morpholine
5-NO2-furan
5-NO2-furan
1.9 0.4
1.4 0.4
>80
>80
3,5-(CH3)2-
N-morpholine
Figure 1. UPR activators sorafenib 1, bortezomib 2, and
sulfonamidebenzamide hit 3 with colored regions of SAR focus.
8
9
N-piperazine
5-NO2-furan
5-NO2-furan
26.4 1.5
2.0 0.4
>80
>80
4-hydroxy-N-
piperidine
10
11
N-piperidine
5-NO2-furan
5-NO2-furan
1.2 0.2
0.6 0.1
>80
>80
identified as an inducer of the UPR through inhibition of the
p97 ATPase, a cellular enzyme that regulates protein
degradation in the ER.27 Other p97 ATPase inhibitors have
also been reported,28,29 along with inhibitors of the molecular
chaperone, Hsp90.30 Lastly, bortezomib (Velcade) 2 is a
dipeptide boronic acid derivative that is FDA-approved for
multiple myeloma. While bortezomib 2 was found to effectively
inhibit the proteosome, thereby activating UPR-dependent
apoptotic cellular mechanisms,31−34 the compound has been
shown to also attenuate adaptive response cascades in human
multiple myeloma cells and select for cells that have reduced
expression of secretory proteins.35,36 While none of these
agents were derived from efforts to identify UPR-focused
targets, they were all found to directly or indirectly modulate
UPR activity. We hypothesized that a high throughput screen
(HTS) aimed at identifying compounds that broadly function
as pro-apoptotic UPR stimulants might provide novel starting
scaffolds for further development.23,37 Conceptually, small
molecules that selectively induced the apoptotic PERK-
CHOP pathway independently of the IRE1α-XBP1 pathway
were sought to properly interrogate the utility of a selective
CHOP activator.
This pursuit was initiated by performing an HTS of 331,676
Molecular Libraries Small Molecule Repository (MLSMR)
compounds as part of the NIH Molecular Libraries Probe
Production Centers Network (MLPCN).38 The HTS assay
assessed compounds at 10 μM for 6−8 h in Chinese hamster
ovary (CHO) K1 cells containing luciferase constructs that
individually reported on Chop activation or Xbp1 splicing.39 Hit
compounds were defined as those that generated ≥40%
luciferase in the CHOP-luc cells compared to tunicamycin
4-F-N-
piperidine
12
13
14
15
16
4-Cl-N-
piperidine
5-NO2-furan
5-NO2-furan
5-NO2-furan
5-NO2-furan
5-NO2-furan
0.8 0.04
0.7 0.1
0.6 0.1
0.7 0.1
0.7 0.1
>80
>80
>80
>80
>80
4-CH3-N-
piperidine
4-(CH3)2-N-
piperidine
4-tert-butyl-N-
piperidine
N-3-azaspiro
[5.5]undecane
17
18
19
20
21
N-pyrrolidine
phenyl
5-NO2-furan
5-NO2-furan
5-NO2-furan
5-NO2-furan
0.8 0.03
1.1 0.2
1.2 0.2
0.7 0.04
13.5 0.5
>80
>80
>80
>80
>80
4-pyran
cyclohexyl
N-morpholine
5-NO2-2-
thiophene
22
23
24
N-morpholine
N-morpholine
N-morpholine
2-thiophene
phenyl
>80
>80
>80
>80
>80
>80
4-NO2-
phenyl
25
26
4-(CH3)2-N-
piperidine
3-NO2-
phenyl
>80
>80
>80
>80
4-(CH3)2-N-
piperidine
1-CH3-5-
NO2-2-
imidazole
27
28
29
30
N-morpholine
N-morpholine
N-morpholine
2-furan
>80
>80
>80
>80
>80
>80
>80
>80
5-CH3-furan
5-Br-furan
5-CF3-furan
4-(CH3)2-N-
piperidine
a
Data were averaged from n ≥ 4 experiments.
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Scheme 1. General Synthesis of the Sulfonamidebenzamide
Scaffold
Table 2. MEF Proliferation Assay Data
a
CHOP AC
CHOP WT EC
CHOP KO EC
b5c0
,
50a
b5c0
d
,
compd
SEM (μM)
SEM (μM)
SEM (μM)
cLogP
10
11
12
13
14
15
1.2 0.2
0.6 0.1
0.8 0.04
0.7 0.1
0.6 0.1
0.7 0.1
7.8 1.2
17.5 2.7
4.8 1.1
4.6 1.2
3.0 0.6
>20
2.6
2.3
2.7
3.1
3.6
4.4
>20
>20
a
16.3 1.0
9.2 3.3
2.1 0.3
Reagents and conditions: (a) amine, pyridine, THF, 60 °C, 20 min,
38−98%; (b) Raney Ni, NaBH4, CH3OH/CH2Cl2, 0 °C, 30 min, 54−
98%; (c) acyl chloride, acetonitrile, 150 °C, μW, 20 min, 38−86%.
1.4 0.5
a
b
CHO-CHOP-luciferase cells. Data were averaged from n = 4
experiments performed with triplicate samples. MEF cells. Calcu-
c
d
and NaBH4 to unmask anilines 5, which were then treated with
acyl chlorides to deliver desired analogues 6. For several
derivatives, the requisite aniline 5 was commercially available.
Analogue 19 was prepared from an SN2 displacement of 4-
bromotetrahydropyran with 4-nitrothiophenol, followed by
subsequent oxidation of the thioether. Analogue 20 required
an adapted SNAr route,41 followed by oxidation of the resulting
thioether to the sulfone, thus affording a 4-nitrophenylsulfone
intermediate. These 4-nitrophenyl intermediates were trans-
formed uneventfully to their corresponding products as shown
in Scheme 1.
lated using CambridgeSoft ChemBioDraw Ultra 12.0.
diminished and appeared to track with changes in lipophilicity,
a result that could be due to permeability or transport
differences between cell types. Compound 11 was the least
lipophilic (cLogP 2.3) and required higher concentrations to
reduce proliferation. Compounds 10 and 12 were slightly more
lipophilic than 11 and selectively reduced proliferation of the
WT MEF cells. In four independent experiments using
triplicate samples Chop-null MEF cells were unaffected by
concentrations up to 20 μM after 24 h (Figure S1, Supporting
Information). Compounds 13−15 were comparatively more
lipophilic (cLogP 3.1−4.4) and were among the most potent
CHOP-luc cell activators. The fact that 13−15 reduced
proliferation in both MEF cell lines suggested that this subset
of analogues might utilize a Chop-independent mechanism of
growth inhibition.
Compound 12 appeared to have a well-balanced profile in
terms of cLogP, selectivity for activating the CHO-CHOP-
luciferase reporter versus the adaptive XBP1 reporter, and
reduced proliferation in a Chop-dependent fashion; therefore,
this compound was selected for further studies assessing affects
on UPR gene expression in human cells. Since our previous
work has demonstrated that oral squamous cell carcinoma
(OSCC) cell lines are sensitive to UPR-inducing compounds
compared to normal, nonmalignant cells, we performed dose−
response proliferation assays in a panel of OSCC cells. Cells
were treated with increasing concentrations of compound 12
for 24 h. Moderate single-digit micromolar potency was
Structural modifications initially focused on the morpholine
ring (red region of 3, Figure 1). Compounds bearing
morpholine ether oxygen alternatives were prepared to survey
the impact of hydrogen bonding, basicity, and lipophilicty at
that structural position on CHOP activation (Table 1). The
incorporation of the more basic, NH-donor of piperazine 8 was
not tolerated, resulting in a nearly 14-fold loss of activity. The
nonbasic phenolic analogue 9 activated CHOP on par with
activity observed with hit 3. However, replacement of polar
functionality at this 4-position with lipophilic components
resulted in the greatest improvement in CHOP activation.
Better activity was observed with 4-halogenated analogues 11
and 12 (0.6 and 0.8 μM, respectively) and 4-methylated
variants 13 and 14 (0.7 and 0.6 μM, respectively). In fact, a
range of lipophilic substitution was tolerated, including a tert-
butyl group and a spirocyclohexyl appendage (analogues 15
and 16). Replacement of the sulfonamide linkage with a
sulphone also produced respectable CHOP activation com-
pared to 3 (18−20). Compound lipophilicity ranged from 0.4−
4.6, with no apparent trend in the CHO−CHOP assay, and
these analogues did not induce the XBP1 pathway (AC50 > 80
μM). Several furyl group replacements were explored (blue
region of 3, Figure 1); however, none proved superior to the
parent 5-nitrofuryl moiety (21−26). Moreover, exchange of the
nitro group with a hydrogen atom (27), methyl group (28),
bromine atom (29), or a trifluoromethyl group (30) all resulted
in loss of activity. For this select group of analogues, the 5-
nitrofuryl functionality was necessary to retain CHOP activity.
Preliminary data indicated low micromolar (<10 μM) growth
inhibition in several human cancer cell lines exposed to
compounds that selectively activated the CHOP reporter (data
not shown). A panel of analogues (10−15) was subsequently
used for proliferation assays with wildtype or Chop knockout
murine embryonic fibroblast (MEF) cell lines. Although MEFs
were not expected to exactly recapitulate any human disease, we
hypothesized that wildtype (WT) cells would be more sensitive
to CHOP activators, while CHOP null cells would be
protected. MEF cell lines were treated in a dose response
fashion for 16 h, and proliferation was measured using an ATP-
based luminescent assay (Table 2). The ability of compounds
10−15 to inhibit proliferation in Chop-null MEF cells was
observed in UMSCC-23 (7.3
1.7 μM), HN12 (7.2
0.7
μM), and HN30 (9.0 1.1 μM); whereas A253, H460/T800,
and HN6 were more resistant (Figure S2, Supporting
Information). There is no obvious common thread we are
aware of to explain the differences in sensitivity (e.g., p53 status
or TGFβ-SMAD signaling defects). To assess the ability of
compound 12 to reduce proliferation in a broad range of
human cancers, it was screened using the National Cancer
Institute’s Developmental Therapeutics Program human tumor
cell line panel (NCI-60). Very promising low micromolar
potency against colon, melanoma, and renal cancer cell lines
was observed (Figure S3 and Table S1, Supporting
Information). Considered together, the OSCC and NCI-60
data indicate that compound 12 is not generally cytotoxic
across malignancies. Ongoing studies to identify specific
molecular signatures that regulate sensitivity will help to
identify which cancers/patients might benefit most from
compound 12 or its analogues.
Since compound 12 demonstrated promising antitumor
activity, we examined the expression of UPR and CHOP target
genes in OSCC. UMSCC23 cells were treated with compound
12 in a dose−response fashion for 6 h. Cells were then lysed,
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and cDNA pools were generated using random hexamers.
Quantitative real-time reverse transcription PCR (qRT-PCR)
analysis revealed increased transcripts for CHOP, spliced XBP1,
and GADD34 (Figure 2). Notably, CHOP transcripts were 5-
plasma, 12 was moderately stable (∼86% remaining), though
reduced stability in mouse plasma (52% remaining) was
observed. The compound was almost completely metabolized
in both human and mouse liver homogenates within 1 h, and
some toxicity was also noted toward human hepatocytes (∼11
μM). The promiscuity of compound 12 was also investigated
by profiling the analogue at a 10 μM concentration against a
Panlabs LeadProfiling Screen that included a panel of 67
GPCRs, nuclear receptors, transporters, and ion channels.44
Significant inhibition, defined as >50% of any single target, was
found to be 68% for the human dopamine receptor. Thus, the
off-target profile for 12 was determined to be relatively clean,
and the significance of dopamine transporter inhibition, though
noted, is measured given that modifications to the protoype
scaffold will be necessary to advance the compound series.
To further characterize the profile of compound 12, its
bioavailability was assessed in male CD-1 mice by IV (2 mg/kg
single dose) and IP (30 mg/kg single dose) administration.45
Plasma concentrations were determined at 7 intervals over 8 h
for each cohort (3 mice in each group). The IV dose of 12 was
readily cleared from circulation within 30 min of admin-
istration. Plasma concentrations of 12 following IP admin-
istration declined more gradually, although suboptimal
exposure was determined throughout the 8 h testing window
(Cmax = 0.14 μM; Tmax = 0.17 h). While these initial in vivo
results clearly suggest that further structural refinement will
likely be necessary in order to obtain sufficient exposure in
future in vivo studies, multiple variables such as metabolic
liability, biodistribution, and target identification should be
determined to better guide the optimization process.
In conclusion, a screening campaign focused on finding
selective inducers of the UPR apoptotic CHOP pathway
revealed a sulfonamidebenzamide scaffold with single-digit
micromolar cellular potency and no liability toward the
adaptive, XBP1 pathway of the UPR. An optimization effort
delivered analogues with improved, submicromolar potency
that were further characterized in terms of their ability to
selectively induce CHOP genes at the mRNA level and
demonstrated antiproliferative activity in multiple cancer cell
lines. Importantly, these outcomes lay the groundwork for
more advanced studies that will (1) examine the effects of these
compounds in an OSCC xenograft mouse model, (2) attempt
to discern the specific target within the CHOP pathway with
which these compounds interact, and (3) provide more
extensive SAR data directed at improving the overall
pharmacokinetic profile for downstream in vivo efficacy studies.
Figure 2. qRT-PCR studies with 12. Tunicamycin (Tm) was used as a
positive control for the induction of ER stress and the UPR.
fold higher than spliced XBP1 at 10 μM, indicating selectivity
similar to that observed using CHO-UPR-luciferase reporter
cells (Table 1). Furthermore, the DNA repair gene ERCC1 was
not increased suggesting compound 12-induced CHOP
expression is not a result of DNA-adduct formation and strand
breaks. Decreased gene expression for each transcript at 20 μM
coincided with cell death as evidenced by cells lifting off the
dish before the end of the experiment. Selective activation of
the apoptotic (CHOP) arm of the UPR and submicromolar
growth inhibition potency in a broad range of human cancer
cell lines support the notion that 12 is an appropriate chemical
probe for proof-of-concept in vivo xenograft studies.
While certain attributes such as aqueous solubility and
stability were assessed at an early stage of the project to guide
the hit selection process, more advanced in vitro pharmacology
was examined only for compound 12 in order to establish
baseline parameters for future optimization efforts. Chemical
stability of 12 was determined by treating 12 (10 μM in PBS at
pH 7.4 with 1% DMSO) separately with a 5-fold excess of thiol
nucleophiles, glutathione (GSH) or dithiothreitol (DTT), for 8
h at room temperature. In each case, 100% of 12 remained with
no detectable GSH or DTT adducts formed. These results
support that the acyl 5-nitro-2-furan was not prone to act as a
Michael acceptor.42
A number of ADME parameters were also examined for
compound 12 (Table S2, Supporting Information).43 Aqueous
stability in PBS and pION buffer through a biologically relevant
pH range was determined to be 8.7−9.7 μM. The solubility was
highest at physiological pH 7.4 and registered approximately
13−15-fold over its EC50 (0.8 μM), revealing that its potency
was not severely limited by its solubility. A PAMPA assay was
used as an in vitro model of passive, transcellular permeability.
Using UV spectroscopy to evaluate compound concentration
between different compartments, compound 12 was deter-
mined to have good permeability at pH values of 5.0, 6.2, and
7.4 in the donor compartment, with the highest permeability at
pH 6.2. The compound was highly plasma protein bound to
human plasma proteins (>99%), though it was somewhat less
tightly bound to mouse plasma proteins (∼84%). In human
ASSOCIATED CONTENT
■
S
* Supporting Information
General chemical methods, experimental and analytical
characterization for new intermediates and compounds 3 and
7−30, cell assay protocols, in vitro pharmacology assay
protocols, full NCI-60 panel results, and off-target PanLabs
profiling results and methods. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*(J.E.G.) E-mail: jengolden@ku.edu.
*(A.M.F.) E-mail: afribley@med.wayne.edu.
*(R.J.K.) E-mail: rkaufman@sanforburnham.org.
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Letter
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(18) Cross, B. C.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.;
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Funding
The authors gratefully acknowledge funding from the following
sources. Chemistry efforts at the University of Kansas
Specialized Chemistry Center were supported by NIH
U54HG005031 to J.A. KU NMR instrumentation was
supported by NIH S10RR024664 and NSF 0320648. High
throughput screening performed at the Conrad Prebys Center
for Chemical Genomics at the Sanford Burnham Medical
Research Institute was supported by NIH U54 HG00503.
R.J.K. acknowledges support from R03MH089782-01,
DK042394, DK088227, and HL052173. A.F. was supported
by DE019678 and the Wayne State University Fund for
Medical Research. Portions of this work were supported by
shared resources from the NCI Cancer Center grant
5P30CA030199.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ATF4, activating transcription factor 4; CHO, Chinese hamster
ovary; CHOP, C/EBP-homologous protein; DTT, dithiothrei-
tol; ER, endoplasmic reticulum; ERCC1, excision repair cross-
complementation group 1; GADD34, growth and DNA
damage 34; GSH, glutathione; HTS, high throughput screen;
IP, intraperitoneal; IV, intravenous; KO, knockout; MEF,
murine embryonic fibroblast; OSCC, oral squamous cell
carcinoma; PAMPA, parallel artificial membrane permeability
assay; PCR, polymerase chain reaction; SAR, structure−activity
relationship; UPR, unfolded protein response; UV, ultraviolet;
WT, wildtype; XBP1, X-box binding protein 1
(20) Axten, J. M.; Medina, J. s. R.; Feng, Y.; Shu, A.; Romeril, S. P.;
Grant, S. W.; Li, W. H. H.; Heerding, D. A.; Minthorn, E.; Mencken,
T. Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl) phenyl] acetyl}-
2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(GSK2606414), a potent and selective first-in-class inhibitor of protein
kinase R (PKR)-like endoplasmic reticulum kinase (PERK). J. Med.
Chem. 2012, 55, 7193−7207.
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(44) Formerly Ricerca Biosciences; see Table S3, Supporting
Information.
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