Identification of new γ-hydroxybutenolides that preferentially inhibit the activity of mPGES-1

Article abstract of DOI:10.1016/j.bmc.2012.06.032

Microsomal prostaglandin E₂ synthase-1 (mPGES-1) has been recognized as novel, promising drug target for anti-inflammatory and anticancer drugs. mPGES-1 catalyzes the synthesis of the inducible prostaglandin E ₂ in response to pro-inflammatory stimuli, rendering this enzyme extremely interesting in drug discovery process owing to the drastic reduction of the severe side effects typical for traditional non-steroidal anti-inflammatory drugs. In the course of our investigations focused on this topic, we identified two interesting molecules bearing the γ- hydroxybutenolide scaffold which potently inhibit the activity of mPGES-1. Notably, the lead compound 2c that inhibited mPGES-1 with IC₅₀ = 0.9 μM, did not affect other related enzymes within the arachidonic acid cascade.

Full text of DOI:10.1016/j.bmc.2012.06.032

Bioorganic & Medicinal Chemistry 20 (2012) 5012–5016
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of new c-hydroxybutenolides that preferentially inhibit
the activity of mPGES-1
Rosa De Simone a, Ines Bruno a, , Raffaele Riccio a, Katharina Stadler b, Julia Bauer b, Anja M. Schaible c,
⇑
Stefan Laufer d, Oliver Werz c
a Department of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Department of Pharmaceutical Analytics, Pharmaceutical Institute, University Tuebingen, 72076 Tuebingen, Germany
c Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, D-07743 Jena, Germany
d Chair of Pharmaceutical/Medicinal Chemistry, Pharmaceutical Institute, University Tuebingen, 72076 Tuebingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Microsomal prostaglandin E2 synthase-1 (mPGES-1) has been recognized as novel, promising drug target
for anti-inflammatory and anticancer drugs. mPGES-1 catalyzes the synthesis of the inducible prostaglan-
din E2 in response to pro-inflammatory stimuli, rendering this enzyme extremely interesting in drug
discovery process owing to the drastic reduction of the severe side effects typical for traditional non-ste-
roidal anti-inflammatory drugs. In the course of our investigations focused on this topic, we identified two
Received 26 January 2012
Revised 11 June 2012
Accepted 13 June 2012
Available online 23 June 2012
interesting molecules bearing the
mPGES-1. Notably, the lead compound 2c that inhibited mPGES-1 with IC50 = 0.9 lM, did not affect other
related enzymes within the arachidonic acid cascade.
c-hydroxybutenolide scaffold which potently inhibit the activity of
Keywords:
c
-Hydroxybutenolide
Microsomal prostaglandin E2 synthase-1
Anti-inflammatory
Ó 2012 Elsevier Ltd. All rights reserved.
Suzuki coupling
1. Introduction
opment of safer drugs in inflammation disorders, because mPGES-1
seems not to affect the constitutive prostaglandins involved in gas-
tro-protection and in several important physiological functions.10
Moreover, inhibition of mPGES-1 activity could also represent a va-
lid strategy in the chemotherapy field.11 For example, it was shown
that cell proliferation and invasive activity in vitro as well as xeno-
graft formation in vivo were reduced by mPGES-1 knockdown and
conversely enhanced by mPGES-1 overexpression in lung and pros-
tate cancer cells.12,13 Several mPGES-1 inhibitors of natural or syn-
thetic origin have been recently identified, for review see,8,14 for
example also through structure-based virtual screening.15
Continuing our investigations in medicinal chemistry area, in the
recent years our studies16–19 have been directed towards the discov-
ery of new molecules targeting this interesting enzyme as promis-
ing candidates for the development of innovative therapeutics.8,14,20
In our approaches two main strategies were followed to affect
mPGES-1 levels: on one hand the direct inhibition of its catalytic
activity and, on the other hand, the modulation of the enzyme
expression. In the first case, recently, on the basis of an accurate
drug-receptor analysis, we rationally designed and synthesized a
small collection of fast synthetically accessible triazole-based
compounds as potential mPGES-1 inhibitors, some of which
showed an interesting pharmacological profile that encouraged us
to continue our investigations on this topic.19 As alternative ap-
proach, in the course of another previous project we were able to
identify compound 1 (Chart 1) as a potent modulator of mPGES-1
Several recent epidemiological and clinical studies confirmed
that the most widespread pathologies among population, in partic-
ular those afflicting aged people, have their base in inflammation.1–7
Hence, there is a growing need for effective and safe anti-inflamma-
tory drugs that in most cases, as in chronic affections, require a long-
term use. Accordingly, the identification of novel strategic targets to
address therapeutic intervention is considered extremely urgent. In
this perspective in the last years microsomal prostaglandin E2 syn-
thase-1 (mPGES-1) has been considered of great interest for new
efficient anti-inflammatory and anticancer drug discovery and
development.8 This enzyme is responsible, along the arachidonic
acid cascade, for the conversion of the cyclooxygenase (COX)-de-
rived unstable endoperoxide prostaglandin H2 (PGH2) into prosta-
glandin E2 (PGE2), and it is over-expressed in several
inflammatory disorders as well as in many human tumors.9
mPGES-1 is a glutathione dependent transmembrane enzyme
belonging to membrane-associated proteins involved in eicosanoid
and glutathione metabolism (MAPEG) family and it represents the
COX-2-coupled isoform that is more strictly involved in patholo-
gies.10 Pharmacological intervention with the enzyme levels
or activity has been indicated as a promising approach for the devel-
⇑
Corresponding author.
E-mail address: brunoin@unisa.it (I. Bruno).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.032

R. De Simone et al. / Bioorg. Med. Chem. 20 (2012) 5012–5016
5013
expression.16,17 Note that compound 1 selectively reduced the
LPS-induced expression of mPGES-1 protein in intact cells leading
to reduced PGE2 generation but not by affecting COX-2 or mPGES-
1 enzymatic activity.16,17
This finding is of great relevance in consideration that this com-
pound represents one of the few molecules able to produce a selec-
tive down-regulation of the expression of mPGES-1 enzyme so far
identified.
On the basis of this premises, we decided to explore more
chemical space generating a new collection of butenolides bearing,
as side chain, structurally different appendages (Chart 2) with the
aim of increasing the biological activity. The collection of desired
compounds has been successfully synthesized and subjected to
pharmacological screening. Even though none of the compounds
reduced the expression of mPGES-1 protein, we identified two very
interesting compounds able to efficiently inhibit the catalytic
activity of mPGES-1. Our data revealed that molecules belonging
to a chemical platform never experienced against this target can
fit the structural demand of enzyme catalytic pocket, hence paving
the way for the development of an innovative class of drug
candidates.
9a–f and 10a–e, were linked to the mucobromic acid 13 through a
Suzuki reaction.
In more detail, in order to reduce the polarity of the amido-aro-
matic intermediates and make easier the purification step on silica
columns, we first converted the boronic acids 4 and 5 into the cor-
responding pinacol esters 6 and 7. These last were subjected to
amidation reaction with the appropriate amines a–f, using the
same protocol applied in peptide synthesis. In our specific case,
we used triethylamine (TEA) as base, N-hydroxybenzotriazole
(HOBt) and N,N-dicyclohexylcarbodiimide (DIC) as carboxylic acid
activators and N,N-dimethylformamide (DMF) as solvent.
After purification on silica gel, the Suzuki coupling between
these advanced intermediates and the methoxy-ethoxy-methyl-
ether (MEM)-protected 3-bromo-c-hydroxybutenolide scaffold 8
afforded the protected adducts 11a–f and 12a–e (Scheme 2).
Indeed, because of the instability of the butenolide ring under basic
conditions required from the Suzuki coupling reaction, it was nec-
essary to protect the mucobromic acid 13 as MEM-derivative. For
the Suzuki coupling, we followed the experimental conditions pre-
viously optimized in our laboratory21 providing [1,10-bis-(diphen-
ylphosphino)ferrocene]-dichloropalladium (II) (Pd(dppf)-Cl2) for
the use as a catalyst and CsF as a base in a mixture of tetrahydro-
furan/water (THF/H2O) 1:1, under microwave irradiation.
2. Results and discussion
The last step, consisting in the removal of the protecting MEM
group, using a solution of trifluoroacetic acid/triisopropylsilane
and water (TFA/TIS/H2O) 95:2.5:2.5, afforded our products 2a–f
and 3a–e in good yields together with lower amounts of bis-substi-
tuted adducts as major by-products.
Before submitting the synthesized compounds 2a–f and 3a–e
to the biological screening, their purities (>95%) were verified by
Agilent Technologies 1200 series high-performance liquid chroma-
tography (HPLC) with ultraviolet (UV) detection at 280 nm (meth-
2.1. Chemistry
The synthesis of compounds 2a–f and 3a–e, characterized by
complex amido–aromatic fragments linked to the c-hydroxybute-
nolide scaffold, required the synthetic procedure outlined in
Scheme 2.
Specifically, the retro-synthetic analysis (Scheme 1) suggested
us to construct the amido-aromatic appendages through an amida-
tion reaction, starting from the appropriate amines a–f and carbox-
ylic acids 4–5. As next step, the advanced intermediates obtained
od: Jupiter C-18 column, 250–4.60 mm, 5 lm, 300 Å; 1.0 mL/min
flow rate; 5–100% in 35 min of 0.1% TFA/CH3CN-0.1% TFA/H2O).
2.2. Biological evaluation
In order to investigate whether or not the test compounds 2a–f
and 3a–e modulate the expression of mPGES-1, A549 cells were
S
Br
pretreated with the test compounds (10
lM, each) or with dexa-
methasone (1 M, positive control) and then stimulated for 48
l
HO
O
O
hours with IL-1b (or DMSO as vehicle) to induce the expression
of mPGES-1. Compared to vehicle treatment, IL-1b up-regulated
mPGES-1 protein as determined by Western blot and this was
1
Chart 1. Chemical structure of compound 1.
H
N
O
N
N
N
HN
N
NH
O
O
O
O
O
O
Br
Br
Br
Br
Br
Br
O
O
O
O
O
O
HO
HO
N
HO
HO
HO
HO
O
2e
O
O
O
O
O
2d
2f
2c
2a
2b
O
O
O
O
O
O
NH
NH
N
N
Br
Br
Br
Br
Br
O
O
HO
O
O
O
HO
O
HO
HO
HO
O
O
O
O
3e
3d
3a
3b
3c
Chart 2. Collection of compound 1 derivatives.

5014
R. De Simone et al. / Bioorg. Med. Chem. 20 (2012) 5012–5016
R1
N
activity relationships for these c-hydroxybutenolides seem evident,
R1
N
O
OH
O
O
and comparison of the overall structures of 2c and 2d reveal no
obvious similarity to other known mPGES-1 inhibitors identified
thus far.
Because many structural diverse inhibitors of mPGES-1 also
inhibited other enzymes within eicosanoid biosynthesis, in partic-
ular 5-LO and COX enzymes, we analyzed if the lead compound 2c
could inhibit the activities of 5-, 12-, and 15-LOs, COX-1/2, and
R2
R2
R2
R1
Br
Br
HO
N
+
H
O
Br
O
B
B
OH
HO
OH
HO
O
HO
O
Scheme 1. Retro-synthetic approach for the synthesis of 2a–f and 3a–e.
cPLA2. At a concentration of 10 lM, compound 2c failed to affect
the activity of COX-1 and COX-2 (isolated enzymes), of 12-LO
and COX-1 in human platelets and of 15-LO in eosinophils (Supple-
mentary data Fig. S1). Similarly, 5-LO activity in neutrophils was
reduced by only 22%, and the activity of human recombinant
prevented by 1
l
M dexamethasone. However, as shown in Figure
1, none of the test compounds 2a–f and 3a–e markedly reduced
the expression of mPGES-1 protein.
Next, we evaluated the ability of the test compounds to inter-
fere with the activity of mPGES-1. Microsomes of IL-1b-stimulated
A549 cells expressing mPGES-1 were pre-incubated with the test
compounds (10
(compound 14, Chart 3, 10
conversion of PGH2 (20
by RP-HPLC.
cPLA2
a in cell-free assays was not suppressed by 10 lM of com-
pound 2c (Supplementary data Fig. S1). Nevertheless, 2c might
act as non-specific mPGES-1 binder, as reported for other
mPGES-1 inhibitors,22 and more detailed analysis of the molecular
interactions (e.g. by surface plasmon resonance spectroscopy) are
l
M) or with the reference inhibitor MK-886
M) and PGE2 formed by enzymatic
M as exogenous substrate) was analyzed
l
l
currently under investigation. Finally, analysis of 2c (10 lM) in a
cell viability assay using either RAW267.4 cells or primary human
monocytes treated for 24 h revealed no significant cytotoxic effects
(see Supplementary data). Taken together, compound 2c is a po-
tent and rather selective inhibitor of mPGES-1, and constitutes a
promising candidate as anti-inflammatory drug candidate, suitable
for further preclinical analysis. Ongoing distinct biological cell-
based experiments cellular studies and animal experiments will
further reveal the pharmacological potential of compound 2c.
The reference mPGES-1 inhibitor 14 (10 lM) suppressed
mPGES-1 activity by 82% (Fig. 2A). The structurally related com-
pounds 2c and 2d, carrying a phenyl- or benzylcarboxamide in
p-position of the linking benzene, significantly inhibited mPGES-1
activity, while the other compounds failed in this respect (Fig. 2).
For compound 1, only moderate mPGES-1 inhibition was evident
(IC50 >10
l
M, Fig. 2A). More detailed concentration-response anal-
M for 2d and 2c,
ysis revealed IC50 values of 5.6 0.4 and 0.9 0.2
l
respectively (Fig. 2). While 2c as N-phenyl-substituted amide is
quite potent, the related 2d differs only by its N-benzyl moiety in
structure which is seemingly detrimental. Also, relocation of the
3. Experimental Section
c-hydroxybutenolide scaffold at the benzamide of 2c and 2d from
3.1. Compounds and chemistry
4- to 3-position (yielding 3c and 3d, respectively) led to inactive
derivatives. Also, compounds where the benzamide nitrogen was
incorporated into a pyrolidine, morpholine, piperidine or pipera-
zine moiety failed to inhibit mPGES-1. Together, concrete structure
3.1.1. Methods and materials
All water and air sensitive reactions were carried out under an
inert atmosphere (N2) in oven- or flame-dried glassware. CH2Cl2
O
HO
OH
B
4-5
OH
Pinacol
EtOAc
O
NH
Br
Br
HO
N
N
O
B
O
HO
O
a
O
b
O
13
6-7
c
R
RH
HOBt
DIC
HN
MEM-Cl
DIPEA
CH2Cl2
N
N
e
DMF
N
H
f
Br
Br
d
O
O
O
O
O
O
O
B
R
8
O
9a-f
or
10a-e
Pd(dppf)Cl2
TBAB
CsF
H2O/THF
O
R
O
R
Br
O
Br
O
O
O
TFA (95%)
TIS (2.5%)
H2O (2.5%)
O
HO
2a-f
O
O
11a-f
or
12a-e
or
3a-e
Scheme 2. Synthetic protocol to generate derivatives 2a–f and 3a–e.

R. De Simone et al. / Bioorg. Med. Chem. 20 (2012) 5012–5016
5015
and THF were distilled from CaH2 immediately prior to use. Water
was degassed under vacuum (10 mbar). All reagents were used
from commercial source (Sigma–Aldrich) without any further
purification.
Microwave reactions were performed on a CEM DiscoverÒ
single mode platform using 10 mL pressurized vials.
Reactions were monitored on silica gel 60 F254 (Merck) plates
and visualized with potassium permanganate or ninhydrine and
under UV (k = 254 nm, 365 nm).
Flash column chromatography was performed using Merck 60/
230–400 mesh silica gel. Analytical and semi-preparative reverse-
phase HPLC purifications were performed on an Agilent Technolo-
Figure 1. Effects of the test compounds on mPGES-1 expression. A549 cells were
pre-incubated with the test compounds 2a–f and 3a–e (10 M, each), dexameth-
asone (1 M), or vehicle (DMSO, 0.3%) for 30 min and then IL-1b (2 ng/mL) was
added. After 48 h, cells were harvested, lysed and mPGES-1 protein (and b-actin as
control) was analyzed by Western blot. Data shown are representative for at least 3
experiments. Semi-quantification by densitometry of the mPGES-1 protein bands
(in%) is given as mean SD, n = 3; the 100% value corresponds to the uninhibited
vehicle (DMSO) control.
l
l
gies 1200 series using Jupiter C-18 column (250 4.60 mm, 5
300 Å; 250 10.00 mm, 10 m, 300 Å, respectively).
lm,
l
3.1.2. Synthesis of 3,4-dibromo-5-(2-methoxy-ethoxymethoxy)-
5H-furan-2-one 8
Mucobromic acid 13 (100 mg, 0.387 mmol) was dissolved in
10 mL of dry CH2Cl2 and MEM-Cl (66
lL, 0.581 mmol) was added to
the solution. N,N-diisopropylethylamine
(DIPEA, 101 L,
l
S
0.581 mmol) was added dropwise over a period of 15 min. After
4 h, the reaction mixture was quenched with 20 mL of HCl 1 M. The
aqueous layer was extracted with CH2Cl2 (3 Â 30 mL) and the organ-
ics were dried with Na2SO4, filtered and concentrated in vacuo. The
crude dark oil obtained was purified by flash chromatography (5%
diethyl ether/n-hexane to 20% diethyl ether/n-hexane) to give com-
pound 8 (115 mg, 85% yield).
N
COOH
Cl
14
Chart 3. Chemical structures of MK886 (14).
3.1.3. Esterification of boronic acids 4 and 5
The boronic acids 4 and 5 (0.667 mmol) were dissolved in 6 mL
of ethyl acetate and, stirring the solution, pinacol (0.667 mmol)
was added. After 4 h the reaction was stopped by adding anhy-
drous Na2SO4 (1 g) and CaCl2 (1 g). The mixture was filtered and
concentrated in vacuo (Yield: 91% of 6 and 90% of 7).
3.1.4. Synthesis of amides 9a–f and 10a–e
The pinacol ester 6 or 7 (1 equiv) and the appropriate amine a–f
(2 equiv) were dissolved in N,N-dimethylformamide (DMF). Trieth-
ylamine (TEA), N-hydroxybenzotriazole (HOBt) and N,N-dicyclo-
hexylcarbodiimide (DIC) (2 equiv of each) were added. The
mixture was left at room temperature for 48 h under stirring.
When TLC showed the consumption of the pinacol ester 6 or 7,
the reaction was stopped by adding HCl 1 N (10 mL). The aqueous
phase was extracted with ethyl acetate (3 Â 10 mL) and the organ-
ic phase was washed firstly with a saturated solution of NaHCO3
and then with brine. Afterward, the organics were dried over
Na2SO4, filtered and concentrated in vacuo. The crude was purified
by flash chromatography (10% diethyl ether/n-hexane to 70%
diethyl ether/n-hexane).
3.1.5. Microwave-assisted Suzuki coupling
In a CEM Discover vial, intermediate 8 (1 equiv), the appropriate
boronic esters 9a–f or 10a–e (1.5 equiv), Pd(dppf)Cl2 (0.03 equiv),
TBAB (0.5 equiv) and CsF (4 equiv) were placed. Under argon,
water (500 lL) and THF (500 lL) were added. The mixture was
irradiated for 3–6 min, setting the power at 200 W, the tempera-
ture at 120 °C, the pressure at 250 psi and the Power Max ON. At
the end of the reaction, the vial was cooled to 50 °C by gas jet cool-
ing before it was opened. After diluting (10 mL) with CH2Cl2, 10 mL
of an aqueous solution of HCl 1 N was added. The aqueous layer
was extracted with CH2Cl2 (3 Â 10 mL). The organics were then
dried over Na2SO4, filtered and concentrated in vacuo. The crude
products were purified by flash chromatography (10% diethyl
ether/n-hexane to 40% diethyl ether/n-hexane) to furnish com-
pounds 11a–f or 12a–e.
Figure 2. Inhibition of mPGES-1 activity by the test compounds. Microsomal
preparations of IL-1b-stimulated A549 cells were pre-incubated with (A) the test
compounds or the reference 14 at a concentration of 10
indicated concentrations of compound 2d (left panel) or 2c (right panel). After
10 min on ice, 20 M PGH2 was added to start mPGES-1 mediated PGE2 formation.
lM, each, or (B) with the
l
Data are given as mean S.E., n = 3–4. The 100% value corresponds to the
uninhibited vehicle (DMSO) control with an average of 0.8 nmol PGE2.

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R. De Simone et al. / Bioorg. Med. Chem. 20 (2012) 5012–5016
3.1.6. MEM-cleavage to generate desired products 2a–f and
3a–e
Acknowledgments
The MEM-protected intermediates 11a–f and 12a–e were
dissolved in a solution of TFA/TIS/H2O 95:2.5:2.5. The mixture
was stirred at room temperature for 1.5 h and concentrated in va-
cuo to leave a dark oil purified by flash chromatography (100% n-
hexane to 30% diethyl ether/n-hexane). All the products 2a–f and
3a–e were obtained as white solids.
Financial support from the MIUR (PRIN-06) and Università degli
Studi di Salerno. Both the institutions are gratefully acknowledged.
Supplementary data
Supplementary data (HPLC conditions, mass spectrometry (MS)
and nuclear magnetic resonance (NMR) data for the tested com-
pounds 2a–f and 3a–e and their precursors; protocol of the addi-
tional bioactivity assays) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2012.06.032. These data include MOL files and InChiKeys of
the most important compounds described in this article.
3.2. Biological assays
3.2.1. Materials and cells
The antibody against human mPGES-1 was from Cayman Chem-
ical (Ann Arbor, MI), the antibody against COX-2 was obtained
from Enzo Life Sciences (Loerrach, Germany).
Materials used: DMEM/High Glucose (4.5 g/l) medium, penicil-
lin, streptomycin, trypsin/EDTA solution, PAA Laboratories (Linz,
Austria); PGH2, Larodan (Malmö, Sweden); 11b-PGE2, PGB1,
MK-886 (compound 14, 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-
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G. J. Med. Chem. 2011, 54, 1565.
3.2.2. Preparation of crude mPGES-1 in microsomes of A549
cells and determination of PGE2 synthase activity
Preparation of A549 cells and determination of mPGES-1 activ-
ity was performed as described previously.23 In brief, cells were
treated with 1 ng/mL IL-1b for 48 h at 37 °C and 5% CO2. After
sonication, the homogenate was subjected to differential centrifu-
gation at 10,000Âg for 10 min and 174,000Âg for 1 h at 4 °C. The
pellet (microsomal fraction) was resuspended in 1 mL homogeni-
zation buffer (0.1 M potassium phosphate buffer pH 7.4, 1 mM
phenylmethanesulphonyl fluoride, 60
lg/mL soybean trypsin
inhibitor, 1 g/mL leupeptin, 2.5 mM glutathione, and 250 mM
l
sucrose), and the total protein concentration was determined.
Microsomal membranes were diluted in potassium phosphate
buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione. Test com-
pounds or vehicle were added, and after 15 min at 4 °C, the reac-
20. Radmark, O.; Samuelsson, B. J. Intern. Med. 2010, 268, 5–14.
21. Aquino, M.; Guerrero, M. D.; Bruno, I.; Terencio, M. C.; Paya, M.; Riccio, R.
Bioorg. Med. Chem. 2008, 16, 9056.
22. Wiegard, A.; Hanekamp, W.; Griessbach, K.; Fabian, J.; Lehr, M. Eur. J. Med.
Chem. 2012, 48, 153.
tion (100
(20 M). After 1 min at 4 °C, the reaction was terminated using
stop solution (100 L; 40 mM FeCl2, 80 mM citric acid, and
10 M of 11b-PGE2 as internal standard). PGE2 was separated
lL total volume) was initiated by addition of PGH2
l
l
l
23. Koeberle, A.; Zettl, H.; Greiner, C.; Wurglics, M.; Schubert-Zsilavecz, M.; Werz,
O. J. Med. Chem. 2008, 51, 8068.
by solid phase extraction and analyzed by RP-HPLC as
described.23