Gemfibrozil derivatives as activators of soluble guanylyl cyclase – A structure-activity study

Article abstract of DOI:10.1016/j.ejmech.2021.113729

Previous studies demonstrated that anti-hyperlipidemic drug gemfibrozil acts as NO- and heme-independent activator of NO receptor soluble guanylyl cyclase. A series of new gemfibrozil derivatives were synthesized and evaluated for sGC activation. The structure-activity relationship study identified the positions in gemfibrozil's scaffold that are detrimental for sGC activation and those that are amendable for optimizing modifications. Compared with gemfibrozil, compounds 7c and 15b were more potent activators of cGMP-forming activity of purified sGC and exhibited enhanced relaxation of preconstricted mouse thoracic aorta rings. These studies established the overall framework needed for futher improvement of sGC activators based on gemfibrozil scaffold.

Full text of DOI:10.1016/j.ejmech.2021.113729

European Journal of Medicinal Chemistry 224 (2021) 113729
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Gemfibrozil derivatives as activators of soluble guanylyl cyclase e A
structure-activity study
Kevin M. Gayler a, Jeremy M. Quintana a, Jordan Mattke a, Michael A. Plunk a,
Jessica H. Kostyo a, Johann W. Karunananthan a, Harold Nguyen a, Mina Shuda a,
Liam D. Ferreira a, Hannah Baker a, Alexandra L. Stinchcomb a, Iraida Sharina b,
,
, *
Robert R. Kane a **, Emil Martin b
a Department of Chemistry and Biochemistry, Institute of Biomedical Studies, Baylor University, Baylor University, Waco, TX, 76798, USA
b Department of Internal Medicine, Division of Cardiology, the University of Texas Health Science Center in Houston, McGoven Medical School, 1941 East
Road, Houston, TX, 77054, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies demonstrated that anti-hyperlipidemic drug gemfibrozil acts as NO- and heme-
independent activator of NO receptor soluble guanylyl cyclase. A series of new gemfibrozil derivatives
were synthesized and evaluated for sGC activation. The structure-activity relationship study identified
the positions in gemfibrozil's scaffold that are detrimental for sGC activation and those that are
amendable for optimizing modifications. Compared with gemfibrozil, compounds 7c and 15b were more
potent activators of cGMP-forming activity of purified sGC and exhibited enhanced relaxation of pre-
constricted mouse thoracic aorta rings. These studies established the overall framework needed for
futher improvement of sGC activators based on gemfibrozil scaffold.
Received 11 June 2021
Received in revised form
23 July 2021
Accepted 27 July 2021
Available online 3 August 2021
Keywords:
Gemfibrozil
Soluble guanylyl cyclase
Biochemistry
© 2021 Elsevier Masson SAS. All rights reserved.
Medicinal chemistry
1. Introduction
molecule to the ferrous sGC heme induces structural changes that
enhance several hundred fold the cGMP-forming activity of the
Nitric oxide (NO) signaling is one of the fundamental pathways
in mammalian physiology, which plays important roles in cardio-
vascular homeostasis, platelet function, gastrointestinal function,
neurotransmission and other processes. A key mediator of NO
signaling is soluble guanylyl cyclase (sGC), a heme protein with
high affinity for NO. NO-sensitive guanylyl cyclase is a hetero-
enzyme. The resulting increase of intracellular cGMP level triggers
cGMP-dependent processes that cause relaxation of smooth muscle
cells, affects vasodilatation and GI motility [1,2]; inhibits the
adhesion of leukocyte and platelet to vascular walls, diminishes
platelet aggregation; and decreases the proliferation and migration
of smooth muscles cells [3]. Proper sGC function is beneficial for
maintaining normal vascular plasticity, preventing atherosclerosis
[4], thrombosis [5] and stroke [6]. On the contrary, deficient sGC
function is associated with increased risk of myocardial infarction
[7], coronary artery disease [8e10], or vascular and GI dysfunctions
[11,12]. Not surprisingly, sGC is an important therapeutic target.
Pharmacological activation of sGC in cases of angina pectoris or
heart failure is achieved via the use of various NO releasing organic
nitrovasodilators, such as nitroglycerin, glyceryl trinitrate, iso-
sobide dinitrate and others. Although effective, the clinical appli-
cation of these NO-generating prodrugs is limited by the rapid
onset of tolerance and tachyphylaxis [13]. Moreover, under in-
flammatory conditions these agents may generate reactive nitrogen
species and cause damage to DNA, proteins and lipids [14].
dimeric protein composed of one
a and one b subunit. Both sub-
units are needed for a functional catalytic site, which uses GTP to
synthesize secondary cellular messenger cGMP. Humans and mice
have two functional isoforms of the
a subunit (a1 and a2) and one
functional b1 isoform. The heterodimer a1b1 (GC-1) is ubiquitously
expressed and has a higher level of expression than the a2b1 (GC-2)
counterpart, which has tissue-specific expression, and is more
prevalent in brain, kidney and placenta. The binding of a NO
* Corresponding author.
** Corresponding author.
E-mail addresses: Bob_kane@Baylor.edu (R.R. Kane), emil.martin@uth.tmc.edu
(E. Martin).
https://doi.org/10.1016/j.ejmech.2021.113729
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
Fortunately, sGC function may be stimulated NO-independently by
a number of allosteric regulators. Two types of such allosteric
regulators have been identified [15] - sGC stimulators and sGC ac-
tivators. Allosteric sGC stimulators potentiate NO signaling by
sensitizing sGC to lower doses of NO. Some of these stimulators are
approved as sGC-targeting therapeutics. For example, the sGC
stimulator riociguat has been approved for management of pul-
monary arterial hypertension and chronic thromboembolic pul-
monary hypertension [16,17], while vericiguat has been approved
for heart failure [18,19]. Although these NO-independent sGC
stimulators have found clinical use, they are effective only when
sGC heme is in reduced ferrous state. Many cardiovascular ailments
are associated with inflammatory conditions and increased burden
of oxidative stress. Under oxidative stress, sGC heme may be
oxidized to a ferric state [20e23], rendering sGC insensitive to NO
or allosteric stimulators. Therefore, NO-independent sGC activators
targeting sGC with ferric heme are of special interest. sGC activators
cinaciguat (also known as BAY58-2667) and ataciguat (also known
as HMR1766) have been proven effective in vitro and in vivo sGC
activators [24,25] and are considered as possible therapeutic agents
[26]. Although the clinical trial of cinaciguat for the treatment of
heart failure [27] showed promising results in unloading the heart,
it had to be stopped due to the development of hypotension. This
side effect is most likely related to the very high affinity of cinaci-
guat, which has an estimated EC50 in low nanomolar range [25].
Therefore, NO-independent sGC activators with lower affinity may
be useful. Previous studies demonstrated that the drug gemfibrozil,
which is used for treatment of type IV and V hyperlipidemias [28],
also acts as a heme-independent sGC activator with vasoactive and
anti-platelet properties [29]. This sGC-activating property may be
responsible for superior cardiovascular protection of gemfibrozil
over other fibrates [30]. The present study has been carried to
obtain preliminary structure-activity information on sGC activating
features of gemfibrozil, with the ultimate goal of obtaining a
gemfibrozil derivative with improved sGC affinity, activation and/or
vasodilating function.
Scheme 1. Synthesis of nor-methyl derivatives.
Scheme 2. Synthesis of aromatic variants.
The replacement of the gemfibrozil ether with an amine in 7a
allowed us to introduce branching at the heteroatom linker (a-to
the aryl group). Accordingly, amidation of amino ester 9 (an in-
termediate in the synthesis of 7a) with acid halides and acids,
2. Results and discussion
2.1. Design and synthesis
In order to explore the development of sGC activators based
upon the gemfibrozil scaffold we prepared a series of compounds.
Variations of the aryl terminus, the linking heteroatom, the side-
chain length and substitution, and the acid terminus were
explored, as depicted in Fig. 1.
We first focused our synthetic efforts on analogs with varied
alkyl chain lengths and substitution. Simple nor-methyl analogs
(4a-d) were readily accessible from the respective bromoacids (2a-
d) using a four-step sequence (Scheme 1). Using similar chemistry,
we were also able to examine the role of ether linkage, as well as
the substitution of the aryl ring. For this purpose,
d
ꢀchloroester 6
or -bromoacid 2c were employed to furnish dimethyl (7a-c) or
d
nor-methyl analogs (8a-b) with variations on the aromatic sub-
stituents, as well as ether, amine or sulfide linkages (Scheme 2).
Fig. 1. Gemfibrozil structure and Scaffold Modifications Sites.
Scheme 3. Synthesis of Aniline Analogs.
2

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
followed by saponification of the isobutyl ester, allowed us to
readily prepare several compounds to explore how branching by
chains of different nature affects sGC activation (Scheme 3). Several
simple derivatives containing hydrophobic side-chains (10a-c)
were initially prepared by this route. Similarly, substitution with
additional acidic residues (10d-f) was used to assess if increased
hydrogen bonding potential and/or hydrophilicity would signifi-
cantly impact sGC activation. Finally, an amine-linked dimeric
analog 11 was also synthesized.
We also investigated derivatives of the carboxyl terminus of
gemfibrozil, including simple amide derivatives 12a-e and dimeric
derivatives 13a-c (Scheme 4). Finally, ester 14 was used to prepare
mono-alkyl acids 15a-b after saponification (Scheme 5).
2.2. Biological results
Scheme 5. Synthesis of
a substituted derivatives.
Once the gemfibrozil derivatives described above were syn-
thesized, we subjected them to a battery of tests to evaluate how
they affect sGC activity.
only the 2-methyl group (although that compound also has a sul-
fide linkage rather than an ether, complicating any direct compar-
ison). Replacing the 2-methyl group with the bulkier 2-isopropyl, as
seen in compound 7c, is well tolerated for activation of both Fe2þ
-
2.2.1. sGC activation tests
sGC and Fe3þ-sGC. Although 8b, lacking both the aryl substituents
as well as the geminal dimethyl group, does not activate sGC, in-
clusion of the 2,5-dimethyl aryl substitution in 4d rescues the
ability to activate sGC and even results in lower EC50 value (Table 1).
Considering that previous work demonstrated that gemfibrozil
derivatives with 3,4,5-trimethyl and (to a lesser effect) 4-fluoro aryl
substituents also support sGC activation [29], these results
demonstrate that substitution of the aryl core is favorable and
tolerates different substitution patterns and substituents.
First, we compared the effect of 100 mM gemfibrozil or gemfi-
brozil derivatives on cGMP-forming activity of sGC. Previous
studies demonstrated that gemfibrozil activates sGC with ferrous
heme, but is more effective when sGC heme is oxidized into ferric
state [29]. Therefore, the initial screening of synthesized com-
pounds was performed using native sGC with ferrous heme (Fe2þ
-
sGC) and sGC with oxidized ferric heme (Fe3þ-sGC) (Fig. 2). Based
on the results of this initial screening, we selected 4c, 4d, 7a, 7c, 11
and 15b, which activated both Fe2þ-sGC and Fe3þ-sGC, for analysis
of dose-dependent activation of Fe3þ-sGC. The estimated EC50
values are reported in Table 1.
We next evaluated the importance of the gem-dimethyl sub-
stitution alpha to the pentanoic acid chain. As shown in Fig. 2,
removal of both methyl groups in compound 4d was tolerated by
Fe2þ-sGC, but diminished Fe3þ-sGC activation. Replacement of
gemfibrozil gem-dimethyl substitution by an ethyl group in com-
pound 15a blocked the activation of Fe2þ-sGC, and significantly
hampered the activation of Fe3þ-sGC. However, introduction of a
longer hexyl chain in 15b was very well tolerated and substantially
lowered the observed EC50 (Table 1).
While previous studies indicated that esterification of the car-
boxylic group is detrimental to sGC activating properties of gem-
fibrozil [29], we explored whether amide substitutions are
tolerated. Activating properties of 12a-12e compounds clearly
indicated that such substitution do not promote sGC activation.
Even the hexyl aliphatic chain, which improved sGC activating
properties when used in the nearby alpha position in 15a, had no
positive effect. Connecting two gemfibrozil cores via diamide links
of different length (13a-13c) also did not offer any additional
benefits.
Analysis of sGC-activating properties of the simple nor-methyl
derivatives 4a-4d revealed that the length of the carboxylic acid
chain is an important factor in determining the potency of activa-
tion (Fig. 1), but that the gem-dimethyl substitution
a to the car-
boxylic acid is less critical. Short chains in 4a and 4b were not
conducive to sGC activation, while longer pentanoic and hexanoic
chains of 4c and 4d, respectively, were more favorable for activa-
tion. The decreased activation by 4d vs 4c suggests that the five
carbon chain (as found in the parent gemfibrozil) is optimal for sGC
activation.
Analysis of these gemfibrozil derivatives also points to the
importance of the benzene ring substituents. The 2,5-dimethyl
substitution found in gemfibrozil provides more potent sGC acti-
vation than the mono-substituted compound 7b, which carries
We also evaluated the importance of the atom connecting the
carboxylic acid chain and the aryl moiety. Comparing gemfibrozil
and aniline 7a, which has a nitrogen connecting atom, demon-
strates that this modification is well tolerated, as 7a has a much
better affinity based on its lower EC50 (Table 1). On the other hand,
gemfibrozil provides more potent sGC activation than the sulfide-
linked 7b, although that derivative has only a 2-methyl aryl sub-
stitution, complicating any direct comparison.
Considering that the aniline moiety is well tolerated, we eval-
uated how substitution of aniline nitrogen affects sGC activation.
Acetylation, butyration, caproylation in 10a-10d were not condu-
cive to sGC activation. The introduction of an additional carboxylic
group afforded by modification with succinate (10d), glutarate
(10e) or adipiate (10f) resulted in weak, yet statistically significant
activation of Fe3þ-sGC, but not Fe2þ-sGC (Fig. 1). Therefore, we
Scheme 4. Synthesis of amide derivatives.
3

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
Fig. 2. Upregulation of sGC activity by gemfibrozil derivatives. Purified human sGC containing heme in ferrous (A) or ferric (B) state was pre-incubated at room temperature for
10 min with 100
M concentrations of gemfibrozil derivatives, gemfibrozil (Gem) or vehicle control (cnt) and then tested for cGMP-forming activity using the [32P]GTP assay, as
described in the Method Section. Data are shown as mean SD from three independent tests performed in triplicate. * indicates p < 0.05 vs control activity (t-test).
m
two different sites on sGC, where the first binding with a lower EC50
has an activating effect and binding to the second site (with higher
EC50) has an inhibitory effect. Previous computational modeling
predicted that gemfibrozil may have two binding sites within the
cavity of the heme-binding pocket of sGC [29]. The bi-phasing effect
of 7c and 15b is consistent with the two-binding site hypothesis.
Similarly, although compound 7a had the lowest EC50 value of
50 16 mM, it exhibited only a modest sGC activation at 100 mM and
flattening of the curve, which could be explained by the inhibitory
effect of the lower affinity binding site.
Table 1
sGC-stimulating gemfibrozil derivatives.
Compound
cGMP-forming activity test
EC50,
Ma fold stimulation at 100
Gemfibrozil 624 217
vasorelaxation test
IC50, mM
Ma MECb,
m
mM
m
4
310 115 30
812 453 100
4c
707 272 2.9
389 151 2.1
50 16
166 41
119 84
128 41
4d
7a
7c
11
15b
>1000
300
2.9
2.6
2.2
4.1
721 161 100
71 24
261 77
10
10
10
45
8
a
Presented as mean SD.
MEC e apparent minimal effective concentration, defined as lowest concen-
2.2.2. Vasodilator action of generated derivatives
b
Regulation of vascular tone via promotion of vasodilation is one
of the most important physiologic function of sGC. Previous studies
demonstrated that activation of sGC by gemfibrozil results in
vasodilation of isolated aortic rings [29]. Therefore, we tested the
compounds 4c, 4d, 7a, 7c, 11 and 15b for their ability to inhibit the
contraction of phenylephrine-treated isolated mouse aorta. Typical
traces of changes in aortic ring tension in response to phenyleph-
rine contraction and the vasoactive function of cumulative doses of
gemfibrozil, 7c, and 15b are shown in Fig. 4a. Using this approach
we evaluated the dose-dependent vasodilatory effect of these
compounds and estimated the minimally effective concentration
(MEC) and the half-maximal inhibitory concentration IC50 (Table 1).
Although the general trend that compounds with improved EC50, as
compared with gemfibrozil, also exhibited a better IC50 in vasodi-
lation tests was evident, there were some exceptions. For instance,
compounds 4d and 7a exhibited improved affinity in the tests with
purified sGC, but had weaker MEC and IC50 values in vasodilation
experiment. This difference is most likely due to affected mem-
brane permeability and/or bioavailability. The dose-dependent
vasoactivity curves for 7c and 15b, which exhibited best vaso-
dilatory IC50 values, are shown in Fig. 4b together with gemfibrozil
and 4c.
tration exhibiting statistically significant difference from untreated conditions.
prepared an amide-linked dimer 11, which exhibited sGC activation
and had a lower EC50 value, as compared to gemfibrozil.
Of interest, examination of the activation curves for the active
compounds revealed anomalous behavior for two of the de-
rivatives. As can be seen in Fig. 3, the activation curves for 4c, 4d, 7a,
11 followed a typical S-shaped pattern similar to gemfibrozil, while
compounds 7c and 15b exhibit a bell-shaped curve. The bell-
shaped curve suggests a bi-phasic binding of these compounds to
3. Conclusion
In summary, we described the synthesis of gemfibrozil de-
rivatives and the analysis of their sGC activating properties. The
structure-activity relationship study identified the positions in
gemfibrozil's scaffold that are detrimental for sGC activation and
those that are amendable for optimizing modifications. We deter-
mined that short carboxylic acid chains, modification of gemfi-
brozil's carboxyl group, and the lack of benzene ring modification
are detrimental for sGC activating properties. We also demon-
strated that some modification of the alpha position of the
Fig. 3. Dose-dependent activation of sGC by gemfibrozil derivatives. Purified human
sGC with ferric heme pre-incubated with different concentrations of indicated com-
pounds was tested for cGMP-forming activity. Data are mean SD (n ¼ 9).
4

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
Fig. 4. Vasoactivity of 7c and 15b. (A): Representative wire myograph traces of changes in isometric tension of isolated mouse aortic rings in response to gemfibrozil, 7c or 15b or
solvent (DMSO). Mouse aortic rings were isolated and prepared as described in Methods. After the contraction induced by 1 M phenylephrine stabilized, cumulative doses of
m
compounds were added to the organ bath and changes in isometric tension were recorded. Upticks in the traces mark when indicated amount of agonists was added. (B): Relaxation
of pre-constricted mouse aortic rings in response to different concentrations of gemfibrozil, 4c, 7c or 15b. Data are mean SD (n ¼ 5), * - p < 0.05 (one-way ANOVA followed by
Tukey's multi comparison test).
carboxylic acid, introduction of an additional carboxylic acid chain
or creation of dimeric gemfibrozil derivatives with preserved car-
boxylic acid chains might offer additional directions for optimiza-
tion of sGC activating properties in new gemfibrozil derivatives.
These studies also revealed a bi-phasic response of sGC to some of
gemfibrozil derivatives, providing additional support to the notion
that sGC possesses two gemfibrozil-binding sites. Several com-
pounds with improved EC50 values in sGC activity tests were
generated. Two sGC-activating compounds, 7c and 15b, exhibited
an enhanced vasodilatory effect, likely due to a combination of
improved sGC affinity and better bioavailability. Further studies are
needed to determine the effect of 7c and 15b on in vivo sGC activity
and function. These SAR studies established the overall framework
needed for future development of sGC activators based on gemfi-
brozil scaffold. Since gemfibrozil-based sGC activators are more
potent activation of sGC with ferric heme, the active compounds
reported here and future improved derivatives may be beneficial in
clinical setting for pharmacological upregulation of sGC cardio-
vascular function in conditions of increased burden of oxidative
stress and diminished NO-dependent sGC activation.
constants (J) are reported in hertz (Hz). NMR peak pattern abbre-
viations are as follows: s ¼ singlet, d ¼ doublet, dd ¼ doublet of
doublets,
t
¼
triplet, at
¼
apparent triplet,
q
¼
quartet,
m ¼ multiplet. NMR spectra were calibrated relative to their
respective residual NMR solvent peaks, chloroform-d ¼ 7.26 ppm
(1H NMR)/77.16 ppm (13C NMR), DMSO ¼ 2.50 ppm (1H NMR)/
39.52 ppm (13C NMR). HRMS was performed in the Baylor Uni-
versity Mass Spectrometry Center on a Thermo Scientific LTQ
Orbitrap Discovery spectrometer using þ ESI or eESI and reported
for the molecular ion ([MþH]þ
&
[MþNa]þ or [M
ꢀ
H]-
respectively).
4.2. General procedure for synthesis of 3a-d
To a 6 dram vial was added acid 2a-d, followed by SOCl2 (2 mL).
The reaction vessel was capped and heated to 75 ꢁC for 2 h and the
solvent was removed by rotary evaporation. To the crude acid
chloride was added isobutanol (10 mL), and the resulting mixture
was allowed to sit at 25 ꢁC for 12 h. The solvent was removed by
rotary evaporation to provide the crude product which was suffi-
ciently pure to carry forward.
4. Experimental section
4.1. Reagents and synthesis
4.2.1. Isobutyl 2-bromoacetate (3a) was synthesized according to
the above general procedure using 2a (1.0 g, 7.2 mmol,1 equiv.), 35%
yield
Unless otherwise stated all materials were used as received and
reactions were performed under anhydrous conditions under an
atmosphere of N2. Reactions in vials were performed in oven-dried
nitrogen-flushed vials that were either capped (most reactions) or
fitted with a septum to allow nitrogen atmosphere. THF and
anhydrous DMF were purchased from Sigma-Aldrich (St. Louis, MO,
USA); DCM and MeCN were purchased from Fisher Scientific
(Waltham, MA, USA); TEA was purchased from Fisher Scientific
(Waltham, MA, USA). TEA and DIPEA were dried over CaH2, distilled
and stored over 4 Å molecular sieves. THF and MeCN were dried
and stored over 4 Å molecular sieves. Chloroform-d and DMSO‑d6
were purchased from Cambridge Isotopes (Tewksbury, MA, USA).
Reactions were monitored using normal phase thin-layer chro-
matography (TLC) on Millipore glass-backed 60 Å plates (indicator
1H NMR (Chloroform-d, 600 MHz)
d
3.95 (dd, J ¼ 6.7, 1.1 Hz, 2H),
2.84 (d, J ¼ 1.1 Hz, 2H), 1.98 (hept, J ¼ 7.0 Hz, 1H), 0.95 (d, J ¼ 6.7 Hz,
6H)$13C NMR (Chloroform-d, 151 MHz)
d 167.4, 72.4, 27.8, 26.0,
19.1. þ ESI-HRMS m/z: calc'd for [MþH]þ C6H12BrO2þ ¼ 195.0015,
C6H12BrOþ2 found 195.0014.
4.2.2. Isobutyl 4-bromobutanoate (3b) was synthesized
according to the above general procedure using 2b (1.0 g,
5.99 mmol, 1 equiv.), 87% yield
1H NMR (600 MHz, Chloroform-d)
d
3.86 (d, J ¼ 6.7 Hz, 2H), 3.46
(t, J ¼ 6.5 Hz, 2H), 2.50 (t, J ¼ 7.2 Hz, 2H), 2.17 (p, J ¼ 6.8 Hz, 2H), 1.92
F-254, 250 mm). Column chromatography was perform using Sili-
cycle SiliaFlash® P60 (230e400 mesh) silica gel as the stationary
phase using eluents indicated in the experimental section. Chem-
(hept, J ¼ 6.7 Hz, 1H), 0.92 (d, J ¼ 6.7 Hz, 6H)$13C NMR (151 MHz,
Chloroform-d)
d
172.7, 70.8, 32.9, 32.6, 27.9, 27.8, 19.2. þ ESI-HRMS
m/z: calc'd for [MþNa]þ C8H15BrNaO2þ ¼ 245.0148, C8H15BrNaOþ2
ical shifts (
d
) are reported in parts per million (ppm) and coupling
found 245.0150.
5

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
4.2.3. Isobutyl 5-bromopentanoate (3c) was synthesized
according to the above general procedure, using 2c (1.0 g,
5.52 mmol, 1 equiv.), 88% yield
4.3.4. 6-(2,5-dimethylphenoxy)hexanoic acid (4d) was
synthesized according to the above general procedure, using K2CO3
(68 mg, 0.492 mmol, 2.5 equiv.), 2,5-dimethylphenol (24 mg,
0.196 mmol, 1 equiv.), MeCN (2 mL) followed by 3d (50 mg,
0.199 mmol, 1 equiv.), 42% yield
1H NMR (600 MHz, Chloroform-d)
d
3.85 (d, J ¼ 6.7 Hz, 2H), 3.41
(t, J ¼ 6.7 Hz, 2H), 2.35 (t, J ¼ 7.3 Hz, 2H), 1.93e1.88 (m, 3H),
1.81e1.75 (m, 2H), 0.92 (d, J ¼ 6.7 Hz, 6H)$13C NMR (151 MHz,
1H NMR (600 MHz, Chloroform-d)
d
7.00 (d, J ¼ 7.4 Hz, 1H), 6.66
Chloroform-d)
d
173.2, 70.6, 33.3, 33.0, 32.0, 27.7, 23.6, 19.1. þ ESI-
(d, J ¼ 7.5 Hz, 1H), 6.62 (s, 1H), 3.95 (t, J ¼ 6.3 Hz, 2H), 2.41 (t,
J ¼ 7.4 Hz, 2H), 2.31 (s, 3H), 2.17 (s, 3H), 1.85e1.80 (m, 2H), 1.73 (p,
J ¼ 7.5 Hz, 2H), 1.60e1.51 (m, 2H)$13C NMR (151 MHz, Chloroform-
HRMS m/z: calc'd for [MþNa]þ C9H17BrNaO2þ
¼
259.0304,
C9H17BrNaOþ2 found ¼ 259.0305.
d) d 178.9,157.1,136.6,130.4,123.8,120.8,112.1, 67.6, 34.0, 29.2, 25.9,
4.2.4. Isobutyl 6-bromohexanoate (3d) was synthesized
according to the above general procedure using 2d (1.0 g, 5.12 mmol,
1 equiv.), 92% yield
24.6, 21.6, 15.9.
þ
ESI-HRMS m/z: calc'd for [MþNa]þ
C14H20NaOþ3 ¼ 259.1305, C14H20NaOþ3 found ¼ 259.1302.
1H NMR (600 MHz, Chloroform-d)
d
3.86 (d, J ¼ 6.7 Hz, 2H), 3.41
4.4. Synthesis of isobutyl 5-chloro-2,2-dimethylpentanoate (6)
(t, J ¼ 6.8 Hz, 2H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.97e1.86 (m, 3H), 1.66 (dt,
J ¼ 15.3, 7.5 Hz, 2H), 1.48 (p, J ¼ 7.7 Hz, 2H), 0.93 (d, J ¼ 6.7 Hz,
To a round bottom flask under an atmosphere of N2 was added
diisopropylamine (2 mL, 14.5 mmol, 2.1 equiv), followed by THF
(5 mL), then cooled to ꢀ78 ꢁC for 20 min. To the above solution was
added 5 M n-BuLi in hexanes (2.7 mL, 13.9 mmol, 2 equiv.) then
allowed to warm to 25 ꢁC for 1 h before being cooled again
to ꢀ78 ꢁC. To a separate round bottom flask was added isobutyl
isobutyrate (1.0 g, 6.93 mmol, 1 equiv.) and THF (15 mL), then
cooled to ꢀ78 ꢁC for 30 min, after which the LDA solution was
added dropwise and the mixture was stirred for 1 h. The reaction
mixture was warmed to 0 ꢁC in an ice bath, followed by the drop-
6H)$13C NMR (151 MHz, Chloroform-d)
d 173.7, 70.7, 34.3, 33.6,
32.6, 27.9, 27.8, 24.3, 19.3. þ ESI-HRMS m/z: calc'd for [MþH]þ
C10H20BrOþ2 ¼ 251.0641, C9H11BrNaO2þfound ¼ 251.0638.
4.3. General procedure for synthesis of 4a-d
To a 6 dram vial was added esters 3a-d, followed by K2CO3 (2.5
eq) 2,5-dimethylphenol (1 eq), and MeCN (2 mL)and the reaction
heated to 82 ꢁC for 12 h. After cooling to 25 ꢁC,1 M NaOH (2 mL) was
added and the reaction heated to 100 ꢁC for 24 h. The resulting
mixture was then partitioned between EtOAc (5 mL) and brine
(10 mL) and the organic phase dried with MgSO4 and evaporated by
rotary evaporation. The resulting residue was purified by silica
chromatography (10e20% EtOAc in hexanes).
wise addition of 1-bromo-3-chloropropane (682 mL, 6.93 mmol).
The reaction was allowed to warm to 25 ꢁC over 2 h, followed by
removal of the solvent by rotary evaporation. The crude residue was
purified by silica chromatography (10e20% EtOAc in hexanes) to
provide the product as a clear oil in 82% yield.1H NMR (Chloroform-
d, 600 MHz)
d
3.85 (d, J ¼ 6.5 Hz, 2H), 3.51 (t, J ¼ 6.5 Hz, 2H), 1.93
4.3.1. 2-(2,5-dimethylphenoxy)acetic acid (4a) was synthesized
according to the above general procedure using K2CO3 (68 mg,
0.492 mmol, 2.51 equiv.), 2,5-dimethylphenol (24 mg, 0.196 mmol, 1
equiv.), MeCN (2 mL) followed by 3a (50 mg, 0.256 mmol, 1.3
equiv.), 32% yield
(hept, J ¼ 6.7 Hz, 1H), 1.75e1.70 (m, 2H), 1.68e1.63 (m, 2H), 1.19 (s,
6H), 0.94 (d, J ¼ 6.7 Hz, 6H)$13C NMR (Chloroform-d, 151 MHz)
d
177.5, 70.6, 45.3, 42.1, 37.9, 28.4, 27.8, 25.2, 19.1. þ ESI-HRMS m/z:
calc'd for [MþNa]þ C11H21ClNaO2þ
found ¼ 243.1124.
¼
243.1122, C11H21ClNaO2þ
1H NMR (Chloroform-d, 600 MHz)
d
7.04 (d, J ¼ 7.5 Hz, 1H), 6.75
(d, J ¼ 7.5 Hz, 1H), 6.56 (s, 1H), 4.68 (s, 2H), 2.31 (s, 3H), 2.25 (s,
3H)$13C NMR (Chloroform-d, 151 MHz)
173.8, 155.6, 136.9, 131.1,
4.5. Synthesis of 5-((2,5-dimethylphenyl)amino)-2,2-
dimethylpentanoic acid (7a)
d
124.2, 122.7, 112.5, 63.3, 21.5, 15.9. þ ESI-HRMS m/z: calc'd for
[MþNa]þ
C10H12NaOþ3
¼
203.0679,
To a 6 dram vial was added 9 (75 mg, 0.24 mmol,1 equiv.), MeOH
(1.0 mL), and 10 M NaOH (0.7 mL). The reaction mixture was
allowed to stir at 25 ꢁC for 22 h. To the reaction mixture was added
MeOH (0.5 mL) and was heated to 80 ꢁC for 3 d. The reaction
mixture cooled to 25 ꢁC, the volatile solvents were removed by
rotary evaporation and the remaining aqueous solution was acidi-
fied to pH 5 with 3 M HCl resulting in the formation of a white
precipitate. The white precipitate was then extracted with EtOAc
(5 mL), the organic layer dried with Na2SO4 and the solvent
removed by rotary evaporation. The product was partially purified
by trituration with hexanes, then the solid material was dissolved
and passed through a silica plug (60% EtOAc in hexanes) to provide
C9H11BrNaO2þfound ¼ 203.0677.
4.3.2. 4-(2,5-dimethylphenoxy)butanoic acid (4b) was
synthesized according to the above general procedure using K2CO3
(68 mg, 0.492 mmol, 2.5 equiv.), 2,5-dimethylphenol (24 mg,
0.196 mmol, 1 equiv.), MeCN (2 mL) followed by 3b (50 mg,
0.224 mmol, 1.1 equiv.), 32% yield
1H NMR (Chloroform-d, 600 MHz)
d
7.04 (d, J ¼ 7.5 Hz, 1H), 6.75
(d, J ¼ 7.5 Hz, 1H), 6.56 (s, 1H), 4.68 (s, 2H), 2.31 (s, 3H), 2.25 (s,
3H)$13C NMR (Chloroform-d, 151 MHz)
173.8, 155.6, 136.9, 131.1,
d
124.2, 122.7, 112.5, 63.3, 21.5, 15.9. þ ESI-HRMS m/z: calc'd for
[MþNa]þ C12H16NaOþ3 ¼ 231.0992, C12H16NaOþ3 found ¼ 231.0990.
the product in 70% yield.1H NMR (600 MHz, Chloroform-d)
d 6.93
(d, J ¼ 7.4 Hz, 1H), 6.47 (d, J ¼ 7.3 Hz, 1H), 6.42 (s, 1H), 3.14 (t,
4.3.3. 5-(2,5-dimethylphenoxy)pentanoic acid (4c) was
synthesized according to the above general procedure, using K2CO3
(68 mg, 0.492 mmol, 2.5 equiv.), 2,5-dimethylphenol (24 mg,
0.196 mmol, 1 equiv.), MeCN (2 mL) followed by 3c (50 mg,
0.211 mmol,1.1 equiv), 33% yield
J ¼ 6.5 Hz, 2H), 2.29 (s, 3H), 2.09 (s, 3H), 1.70e1.65 (m, 4H), 1.24 (s,
6H)$13C NMR (151 MHz, Chloroform-d)
d 184.0, 146.2, 136.9, 130.0,
119.0,117.6,110.7, 44.4, 42.2, 37.9, 25.5, 25.2, 21.7,17.1.-ESI-HRMS m/
z: calc'd for [M
found ¼ 248.1657.
ꢀ
H]- C15H22NOꢀ2
¼
248.1656, C15H22NO2ꢀ
1H NMR (600 MHz, Chloroform-d)
d
7.00 (d, J ¼ 7.4 Hz, 1H), 6.66
(d, J ¼ 7.5 Hz, 1H), 6.62 (s, 1H), 3.97 (t, J ¼ 5.7, 2H), 2.46 (t, J ¼ 7.0 Hz,
4.6. Synthesis of 2,2-dimethyl-5-(o-tolyloxy)pentanoic acid (7b)
2H), 2.31 (s, 3H), 2.17 (s, 3H), 1.90e1.78 (m, 4H)$13C NMR (151 MHz,
Chloroform-d)
d
178.9, 157.0, 136.6, 130.5, 123.8, 120.9, 112.0, 67.3,
To a 2 dram vial O-cresol (576.3 mg, 4.17 mmol, 3 equiv.) was
added to a solution of 6 (305.7 mg, 1.4 mmol, 1 equiv.), NaI
(675.0 mg, 4.17 mmol 3 equiv.), and K2CO3 (967.5 mg, 6.95 mmol, 5
33.6, 28.9, 21.7, 21.6, 16.0. þ ESI-HRMS m/z: calc'd for [MþNa]þ
C13H18NaOþ3 ¼ 245.1148, C13H18NaOþ3 found ¼ 245.1148.
6

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
equiv.) in DMSO (2 mL). The reaction mixture was stirred at 80 ꢁC
for 47 h. The mixture was then diluted with EtOAc (15 mL) and
washed with 1 M NaOH (15 mL), then brine. The organic layer was
dried with Na2SO4 and the solvent was removed by rotary evapo-
ration to provide a 299.6 mg of a brown oil that was used without
further purification. The above oil was dissolved in MeOH (2.5 mL)
and added to 6 dram vial. To this solution was added 10 M NaOH
(2.5 mL) and the reaction mixture was stirred at 80 ꢁC for 48 h. The
volatiles were removed from the reaction mixture by rotary evap-
oration. The remaining aqueous solution was washed with EtOAc
(5 mL), then acidified with 3 M HCl (10 mL) and extracted with
EtOAc (5 mL). Organic layer was dried with Na2SO4 and the solvent
was removed by rotary evaporation. The reaction mixture was
purified by silica chromatography (20% EtOAc in hexanes) to pro-
vide the product in 22% yield. 1H NMR (600 MHz, Chloroform-d)
the reaction mixture was stirred for 2 h. The solvent was then
removed by rotary evaporation and the resulting residue was pu-
rified by silica chromatography (10% EtOAc in Hexanes) to provide
isopropyl 6-bromohexanoate in 82% yield. 1H NMR (DMSO‑d6,
600 MHz)
d
5.00 (sept., J ¼ 6.3 Hz, 1H), 3.40 (t, J ¼ 6.8 Hz, 2H), 2.28
(t, J ¼ 7.4 Hz, 2H), 1.91e1.84 (m, 2H), 1.68e1.61 (m, 2H), 1.50e1.43
(m, 2H), 1.23 (d, J ¼ 6.3 Hz, 6H)$13C NMR (DMSO‑d6, 151 MHz)
d
173.1, 67.7, 34.6, 33.7, 32.6, 27.8, 24.3, 22.0. þ ESI-HRMS m/z: calc'd
for [MþNa]þ C9H17BrNaO2þ 259.0306, C9H17BrNaOþ2 found
259.0306.
To a 6 dram vial was added isopropyl 6-bromohexanoate
(500 mg, 2.11 mmol, 1 equiv.) dissolved in THF (1 mL). Thio-
phenol (216 mL, 2.11 mmol, 1 equiv.) was added as a solution in 1 M
NaOH (2 mL) and THF (1 mL), then the reaction mixture was stirred
at 25 ꢁC. The solution was subjected to rotary evaporation to
remove the THF, quenched with 3 M HCl (5 mL) and extracted with
DCM (5 mL). The organic phase was dried with MgSO4 and evap-
orated to provide the isopropyl ester of 8a in 87% yield.1H NMR
d
7.13 (t, J ¼ 7.1 Hz, 2H), 6.84 (t, J ¼ 7.4 Hz, 1H), 6.78 (d, J ¼ 8.3 Hz,
1H), 3.95 (t, J ¼ 6.1 Hz, 2H), 2.22 (s, 3H), 1.86e1.78 (m, 2H),
1.78e1.73 (m, 2H), 1.26 (s, 6H)$13C NMR (151 MHz, Chloroform-d)
d
183.2, 157.2, 130.7, 126.9, 126.8, 120.3, 111.0, 68.0, 42.0, 37.0, 25.3,
(DMSO‑d6, 600 MHz)
d
7.32 (dd, J ¼ 8.1, 1.4 Hz, 2H), 7.23 (t, J ¼ 7.5,
25.2,
16.3.
þ
ESI-HRMS
m/z:
calc'd
for
[MþH]þ
2H), 7.16 (tt, J ¼ 7.3, 0.8 Hz, 1H), 5.00 (sept., J ¼ 6.3 Hz, 1H), 2.91 (t,
J ¼ 7.3 Hz, 2H), 2.26 (t, J ¼ 7.4 Hz, 2H), 1.70e1.60 (m, 4H), 1.49e1.43
(m, 2H), 1.22 (d, J ¼ 6.3 Hz, 6H)$13C NMR (DMSO‑d6, 151 MHz)
C14H20NOþ3 ¼ 259.1310, C14H20NOþ3 found 259.1306.
4.7. Synthesis of 5-(2-isopropyl-5-methylphenoxy)-2,2-
dimethylpentanoic acid (7c)
d 173.3, 136.9, 129.1, 129.0, 125.9, 67.6, 34.6, 33.6, 28.9, 28.4, 24.7,
22.0. þ ESI-HRMS m/z: calc'd for [MþH]þ C15H23SO2þ 267.1413,
C
15H23SO2þ found 267.1413.
To a 2 dram vial was added DMSO (2 mL), 6 (305.7 mg,
1.39 mmol, 1 equiv.), NaI (625.0 mg, 4.17 mmol, 3 equiv.), K2CO3
(967.5 mg, 6.95 mmol, 5 equiv.), and thymol (626.4 mg, 4.17 mmol,
3 equiv.) The reaction mixture was heated to 80 ꢁC for 3 d. The
reaction was allowed to cool to 25 ꢁC and was quenched with brine
(50 mL), extracted with EtOAc (20 mL). The organic extract was
dried with Na2SO4 and the solvent removed by rotary evaporation.
The crude mixture was purified by silica chromatography (2.5%
EtOAc in hexanes) to provide the isobutyl ester of 7c in 40% yield. 1H
In a 6 dram vial the isopropyl ester of 8a (489 mg, 1.84 mmol, 1
equiv.) dissolved in THF (2 mL) and added to of 10 M NaOH (2 mL) in
a round bottom flask. The mixture was heated to 100 ꢁC for 16 h,
then allowed to cool to 25 ꢁC. The crude material was acidified with
3 M HCl (10 mL) and cooled to 4 ꢁC for 2 h, then filtered to afford the
product in 18% yield. 1H NMR (DMSO‑d6, 600 MHz)
d 7.31 (d,
J ¼ 8.1 Hz, 2H), 7.28 (t, J ¼ 7.5 Hz, 2H), 7.17 (t, J ¼ 7.3 Hz, 1H), 2.92 (t,
J ¼ 7.3 Hz, 2H), 2.36 (t, J ¼ 7.4 Hz, 2H), 1.71e1.63 (m, 4H), 1.52e1.46
(m, 2H)$13C NMR (DMSO‑d6, 151 MHz)
d 177.4, 136.8, 129.2, 129.0,
NMR (400 MHz, Chloroform-d)
d
7.13 (d, J ¼ 7.7 Hz, 1H), 6.77 (d,
126.0, 33.6, 33.6, 28.9, 28.3, 24.4. þ ESI-HRMS m/z: calc'd for
J ¼ 7.6 Hz, 1H), 6.68 (s, 1H), 3.98 (q, J ¼ 5.6, 5.1 Hz, 2H), 3.91 (d,
J ¼ 6.6 Hz, 2H), 3.33 (hept, J ¼ 7.0 Hz, 1H), 2.35 (s, 3H), 1.99 (hept,
J ¼ 13.3, 6.7 Hz, 1H), 1.85e1.75 (m, 4H), 1.28 (s, 6H), 1.25 (d,
J ¼ 7.0 Hz, 6H), 0.99 (d, J ¼ 6.7 Hz, 6H)$13C NMR (151 MHz, Chlo-
[MþNa]þ C12H16NaO2Sþ 247.0763, C12H16NaO2Sþ found 247.0762.
4.9. Synthesis of 6-phenoxyhexanoic acid (8b)
roform-d)
d
189.0, 177.8, 156.1, 136.3, 134.1, 125.9, 121.0, 112.2, 70.6,
In
0.211 mmol, 1 equiv.) was dissolved in a mixture of THF (2 mL) and
1 M NaOH (2 mL), and phenol (18.6 L, 0.211 mmol, 1 equiv.) was
a 6 dram vial isopropyl 6-bromohexanoate (50 mg,
68.1, 42.3, 37.4, 27.9, 26.7, 25.3, 25.3, 22.8, 21.4, 19.2. þ ESI-HRMS m/
z: calc'd for [MþNa]þ C21H34NaO3þ ¼ 357.2406, C21H34NaOþ3 found
357.2409.
m
added and the mixture vigorously stirred for 8 h at 80 ꢁC. 10 M
NaOH (2 mL) was then added and the mixture was heated to 100 ꢁC
for 48 h, and then 3 M HCl (10 mL) was used to quench the reaction.
The mixture was extracted with EtOAc (10 mL), and the organic
layer dried with MgSO4, and the solvent removed by rotary evap-
oration. The crude residue was then purified by silica chromatog-
raphy (10% EtOAc in Hexanes) to afford the product in 46% yield.1H
To a 6 dram vial was added the isobutyl ester of 7c (186.7 mg,
0.56 mmol, 1 equiv.), 10 M NaOH (2 mL), and MeOH (2 mL) and
heated to 80 ꢁC and stirred for 52 h. The volatiles were removed
with rotary evaporation, reaction was quenched by the addition of
12 M HCl (2 mL). The crude mixture was extracted with EtOAc
(5 mL) and the organic extract washed with brine (5 mL). The
organic layer was then dried with Na2SO4 and evaporated with
rotary evaporation. The crude product was purified by silica chro-
matography (20% EtOAc in hexanes) to provide the product in 77%
NMR (Chloroform-d, 600 MHz)
d
7.27 (t, J ¼ 8.3 Hz, 2H), 6.93 (t,
J ¼ 7.3 Hz,1H), 6.88 (d, J ¼ 8.0 Hz, 2H), 3.96 (t, J ¼ 6.4 Hz, 2H), 2.40 (t,
J ¼ 7.4 Hz, 2H), 1.85e1.78 (m, 2H), 1.76e1.70 (m, 2H), 1.58e1.50 (m,
yield.1H NMR (600 MHz, Chloroform-d)
d
7.08 (d, 1H), 6.73 (d,
2H)$13C NMR (Chloroform-d, 151 MHz)
d 178.3, 159.1, 129.6, 120.7,
J ¼ 7.1 Hz, 1H), 6.63 (s, 1H), 3.93 (t, 2H), 3.31e3.22 (sept, 1H), 2.31 (s,
114.6, 67.6, 33.8, 29.1, 25.8, 24.6.-ESI-HRMS m/z: calc'd for [M ꢀ H]-
3H), 1.84e1.78 (m, 2H), 1.78e1.72 (m, 2H), 1.26 (s, 6H), 1.20 (d, 6H).
C12H15Oꢀ3 207.1027, C12H15Oꢀ3 found 207.1026.
13C NMR (151 MHz, Chloroform-d)
d 156.0, 136.3, 134.0, 125.9, 121.0,
112.2, 67.9, 41.9, 37.0, 26.7, 25.2, 25.0, 22.8, 21.4.-ESI-HRMS m/z:
4.10. Synthesis of isobutyl 5-((2,5-dimethylphenyl)amino)-2,2-
dimethylpentanoate (9)
calc'd for [M ꢀ H]- C17H25Oꢀ3 277.1809, C17H25Oꢀ3 found 277.1809.
4.8. Synthesis of 6-(phenylthio)hexanoic acid (8a)
To a 6 dram vial was added DMF (9 mL), 6 (2.0 g, 9 mmol, 1
equiv.), NaI (4.1 g, 27 mmol, 3 equiv.), K2CO3 (3.4 g, 24.8 mmol, 2.75
equiv.), and 2,5-dimethylaniline (1.2 g, 9.9 mmol, 1.1 equiv.). The
reaction mixture was heated to 62 ꢁC and stirred for 23 h, then was
allowed to cool to 25 ꢁC, quenched with brine (50 mL), and
extracted with EtOAc (20 mL). The organic extract was dried with
In a 6 dram vial, 6-bromohexanoic acid (2e) (502 mg, 3.03 mmol,
1 equiv.) was added to thionyl chloride (1 mL, 13.7 mmol), which
was heated to 75 ꢁC for 1 h under nitrogen. The mixture was then
allowed to cool to 25 ꢁC before isopropanol (20 mL) was added and
7

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
Na2SO4 and the solvent removed by rotary evaporation. The crude
mixture was partially purified by silica chromatography (5e10%
EtOAc in hexanes). The partially purified product was dissolved in a
2:1 hexanes to 4 M HCl in dioxane mixture (15 mL), followed by
addition of 3 M HCl in H2O (5 mL) and vigorously shaken to form a
white precipitate, which was filtered and washed with EtOAc
(5 mL) to provide the product in 50% yield.1H NMR (600 MHz,
To a 6 dram vial was added the ester of the desired product
followed by 4:1 MeOH:10 M NaOH solution (2 mL) and was stirred
at 25 ꢁC for 72 h, then the temperature was increased to 40 ꢁC for
24 h, then to 60 ꢁC for 48 h. The organic solvent was removed by
rotary evaporation, 3 M HCl added until the reaction mixture pH
was 2, the aqueous solution extracted with EtOAc (5 mL), and the
organic extract dried with Na2SO4. The organic solvent was
removed by rotary evaporation and the product was purified as
specified.
DMSO‑d6)
d
6.49 (d, J ¼ 7.8 Hz, 1H), 6.44 (d, J ¼ 7.8 Hz, 1H), 6.37 (s,
1H), 3.03 (d, J ¼ 6.5 Hz, 2H), 2.55e2.51 (m, 4H), 1.59 (s, 3H), 1.57 (s,
3H), 1.08 (hept, J ¼ 13.4 Hz, 1H), 0.91 (ddt, J ¼ 10.6, 7.5, 3.4 Hz, 2H),
0.86e0.80 (m, 2H), 0.41 (s, 6H), 0.12 (d, J ¼ 6.7 Hz, 6H)$13C NMR
4.12.1. Isobutyl 5-(N-(2,5-dimethylphenyl)butyramido)-2,2-
dimethylpentanoate (10b isobutyl ester) was synthesized using
(151 MHz, DMSO‑d6)
d 176.5, 137.3, 132.3, 131.4, 129.4, 127.0, 122.1,
69.6, 50.4, 41.0, 35.7, 26.8, 23.3, 20.8, 18.6, 17.2, 14.3. þ ESI-HRMS m/
the above general procedure using butyric acid (16.2
1.1 equiv), HATU (68.4 mg, 0.18 mmol, 1.1 equiv.), TEA (67.5
0.48 mmol, 3 equiv.), and DMF (1 mL)
m
L, 0.18 mmol,
z: calc'd for [MþH]þ C19H32NO2þ
found ¼ 306.2433.
¼
306.2428, C19H32NO2þ
mL,
After addition of 9 (50 mg, 0.16 mmol, 1 equiv.) the reaction was
stirred for 47 h followed by addition of TEA (67.5 L, 0.48 mmol, 3
4.11. Synthesis of 5-(N-(2,5-dimethylphenyl)acetamido)-2,2-
dimethylpentanoic acid (10a)
m
equiv.), and stirred for an additional 26 h. Silica chromatography
(20-40-60% EtOAc in hexanes), provided the isobutyl ester of 10b in
To a 1 dram vial was added 9 (50.0 mg, 0.16 mmol, 1 equiv.), THF
34% yield.1H NMR (600 MHz, Chloroform-d)
d
7.15 (d, J ¼ 7.7 Hz,
(0.75 mL), and TEA (67.5
was then added to increase homogeneity of the reaction, followed
by the addition of AcCl (22.8 L, 0.32 mmol, 2 equiv.). The reaction
mL, 0.48 mmol, 3 equiv.). DCM (0.75 mL)
1H), 7.06 (d, J ¼ 7.8 Hz, 1H), 6.85 (s, 1H), 4.08e4.00 (m, 1H),
3.82e3.73 (m, 2H), 3.05e2.98 (m, 1H), 2.32 (s, 3H), 2.13 (s, 3H), 1.93
(dt, J ¼ 15.2, 7.4 Hz, 1H), 1.89e1.75 (m, 2H), 1.60e1.39 (m, 6H), 1.15
(d, J ¼ 2.8 Hz, 6H), 0.86 (dd, J ¼ 6.7, 2.5 Hz, 6H), 0.81 (t, J ¼ 7.4 Hz,
m
mixture was allowed to stir at 25 ꢁC for 24 h and diluted with EtOAc
(3 mL) and filtered. The solvent was removed with rotary evapo-
ration and the mixture was purified with by silica chromatography
(20% then 60% EtOAc in hexanes) to provide the isobutyl ester of
3H)$13C NMR (151 MHz, Chloroform-d)
d 177.9, 173.0, 141.2, 136.9,
132.4, 131.3, 129.9, 129.0, 70.5, 48.5, 42.4, 37.9, 36.0, 27.9, 25.5, 25.2,
23.5, 20.9, 19.2, 18.7, 17.2, 14.0. þ ESI-HRMS m/z: calc'd for [MþNa]þ
C17H25NaNOþ3 ¼ 398.2671, C17H25NaNOþ3 found ¼ 398.2671.
10a in 72% yield.1H NMR (600 MHz, Chloroform-d)
d 7.15 (d,
J ¼ 7.7 Hz, 1H), 7.06 (d, J ¼ 6.9 Hz, 1H), 6.87 (s, 1H), 4.00e3.99 (m,
1H), 3.82e3.73 (m, 2H), 3.10e3.00 (m, 1H), 2.32 (s, 3H), 2.15 (s, 3H),
1.84 (sept J ¼ 13.3 Hz, 1H), 1.72 (s, 3H), 1.58e1.38 (m, 4H), 1.15 (s,
3H), 1.15 (s, 3H), 0.86 (dd, J ¼ 6.7, 2.3 Hz, 6H)$13C NMR (151 MHz,
4.12.2. 5-(N-(2,5-dimethylphenyl)butyramido)-2,2-
dimethylpentanoic acid (10b) was synthesized using the above
general procedure, using the isobutyl ester of 10b (20.4 mg,
0.054 mmol, 1 equiv.)
Chloroform-d)
d 177.8, 170.6, 141.6, 137.0, 132.3, 131.4, 129.6, 129.1,
70.5, 48.4, 42.4, 37.9, 27.9, 25.4, 25.2, 23.5, 22.4, 20.9, 19.2,
The product was purified by dissolution in hexanes and was
decanted to from insoluble impurities. The hexanes solution was
evaporated to provide the product 79% yield.1H NMR (600 MHz,
17.2.
þ
ESI-HRMS
m/z:
calc'd
for
[MþNa]þ
C21H33NaNOþ3 ¼ 370.2358, C21H33NaNOþ3 found ¼ 370.2358.
To a 6 dram vial was added the isobutyl ester of 10a (36.7 mg,
0.12 mmol, 1 equiv.), followed by MeOH (1.75 mL) and 10 M NaOH
(1.25 mL), and the mixture stirred at 25 ꢁC for 24 h. The organic
solvent was removed by rotary evaporation and 3 M HCl was added
until reaction mixture pH was 2. The solution was then extracted
with EtOAc (5 mL), the organic extract dried with Na2SO4, and the
solvent removed by rotary evaporation. The product was purified
by trituration with hexanes to provide the product in 35% yield.1H
Chloroform-d)
d
7.16 (d, J ¼ 7.9 Hz, 1H), 7.06 (d, J ¼ 8.4 Hz, 1H), 6.88
(s, 1H), 4.14e4.07 (m, 1H), 3.05e2.97 (m, 1H), 2.31 (s, 3H), 2.13 (s,
3H), 1.99e1.91 (m, 1H), 1.85e1.77 (m, 1H), 1.66e1.43 (m, 7H), 1.18 (d,
J ¼ 9.3 Hz, 6H), 0.81 (t, J ¼ 7.4 Hz, 3H)$13C NMR (151 MHz, Chlo-
roform-d)
d 173.3, 141.1, 137.0, 132.4, 131.4, 129.9, 129.1, 48.4, 42.0,
37.6, 36.0, 25.4, 24.9, 23.3, 20.9, 18.7, 17.2, 14.0.-ESI-HRMS m/z:
calc'd for [M
H]- C19H28NOꢀ3 318.2075, C19H28NOꢀ3
found ¼ 318.2077.
ꢀ
¼
NMR (600 MHz, Chloroform-d)
d
7.15 (d, J ¼ 7.8 Hz, 1H), 7.06 (d,
J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 1.8 Hz,1H), 4.06 (ddd, J ¼ 13.2, 8.7, 6.7 Hz,
1H), 3.41e3.32 (m, 1H), 3.05 (ddd, J ¼ 13.1, 8.4, 4.6 Hz, 1H), 2.31 (s,
3H), 2.15 (s, 3H), 1.74 (s, 3H), 1.71e1.40 (m, 4H), 1.17 (d, J ¼ 10.2 Hz,
4.12.3. Isobutyl 5-(N-(2,5-dimethylphenyl)pentanamido)-2,2-
dimethylpentanoate (10c isobutyl ester) was synthesized using
the above general procedure using caproic acid (22.5
mL, 0.18 mmol,
6H)$13C NMR (151 MHz, Chloroform-d)
d
183.0, 171.1, 141.4, 137.1,
1.1 equiv), HATU (68.4 mg, 0.18 mmol, 1.1 equiv.), TEA (67.5
0.48 mmol, 3 equiv.), and DMF (1 mL)
mL,
132.2, 131.4, 129.6, 129.2, 48.3, 41.9, 37.6, 25.4, 24.9, 23.3, 22.3, 20.9,
17.1.-ESI-HRMS m/z: calc'd for [M ꢀ H]- C17H25NO3ꢀ ¼ 290.1762,
After addition of 9 (50 mg, 0.16 mmol, 1 equiv.) the reaction was
stirred for 47 h followed by addition of TEA (67.5 L, 0.48 mmol, 3
C
17H25NOꢀ3 found ¼ 290.1761.
m
equiv.) and the reaction was stirred for an additional 26 h. Silica
chromatography (20-40-60% EtOAc in hexanes), provided the iso-
butyl ester of 10c in 35% yield.1H NMR (600 MHz, Chloroform-d)
4.12. General procedure for synthesis of 10b-f
To a 1 dram vial was added the appropriate acid, HATU, TEA, and
DMF and the mixture stirred for 15 min at 25 ꢁC. To the reaction
mixture was added 9 (50 mg, 0.16 mmol, 1 equiv.) and the resulting
mixture stirred for the specified time at 25 ꢁC. The reaction was
quenched with 3 M HCl (2 mL) and extracted with EtOAc (2 mL).
The organic layer was further washed with 1 M NaOH (2 mL) and
the organic layer dried with Na2SO4. The solvent was removed with
rotary evaporation. The crude material was purified by silica
chromatography using the eluents indicated to provide the ester
precursor of the desired product.
d
7.15 (d, J ¼ 7.8 Hz, 1H), 7.06 (d, J ¼ 9.9 Hz, 1H), 6.85 (s, 1H),
4.07e3.99 (m, 1H), 3.82e3.73 (m, 2H), 3.05e2.98 (m, 1H), 2.32 (s,
3H), 2.13 (s, 3H),1.89e1.77 (m,1H),1.58e1.49 (m, 6H),1.52e1.36 (m,
1H) 1.25e1.16 (m, 3H), 1.18e1.16 (m, 1H), 1.15 (d, J ¼ 2.9 Hz, 6H),
1.14e1.12 (m, 1H), 0.86 (dd, J ¼ 6.7, 2.5 Hz, 6H), 0.82 (t, J ¼ 7.2 Hz,
3H)$13C NMR (151 MHz, Chloroform-d)
d 177.9, 173.2, 141.2, 136.9,
132.4, 131.3, 129.8, 129.0, 70.5, 48.5, 42.4, 37.9, 34.1, 31.7, 27.9, 25.5,
25.2, 25.1, 23.5, 22.6, 20.9, 19.2, 17.2, 14.0. þ ESI-HRMS m/z: calc'd
for [MþNa]þ C25H41NaNOþ3
found ¼ 426.2982.
¼
426.2984, C25H41NaNO3þ
8

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
4.12.4. 5-(N-(2,5-dimethylphenyl)pentanamido)-2,2-
dimethylpentanoic acid (10c) was synthesized using the above
general procedure, using the isobutyl ester of 10c (22.9 mg,
0.057 mmol, 1 equiv.)
The product was purified by dissolution in hexanes and was
decanted to from insoluble impurities. The hexanes solution was
evaporated to provide the product in 76% yield.1H NMR (600 MHz,
stirred for 48 h at 25 ꢁC. Silica chromatography (20e40% EtOAc in
hexanes) provided the diester of 10e in 45% yield.1H NMR
(600 MHz, Chloroform-d)
d
7.15 (d, J ¼ 7.8 Hz,1H), 7.06 (d, J ¼ 7.8 Hz,
1H), 6.84 (s, 1H), 4.07e3.97 (m, 1H), 3.84e3.72 (m, 2H), 3.59 (s, 3H),
3.02 (ddd, J ¼ 13.5, 8.8, 4.8 Hz, 1H), 2.31 (s, 3H), 2.27 (t, J ¼ 6.6 Hz,
2H), 2.12 (s, 3H),1.99e1.92 (m,1H),1.88e1.77 (m, 2H),1.58e1.53 (m,
2H), 1.52e1.46 (m, 4H), 1.43e1.36 (m, 1H), 1.15 (s, 6H), 0.86 (dd,
Chloroform-d)
d
7.16 (d, J ¼ 7.8 Hz, 1H), 7.06 (d, J ¼ 5.8 Hz, 1H), 6.88
J ¼ 6.7, 2.4 Hz, 6H)$13C NMR (151 MHz, Chloroform-d)
d 177.9, 173.8,
(s, 1H), 4.14e4.06 (m, 1H), 3.01 (dd, J ¼ 17.9, 8.3 Hz, 1H), 2.31 (s, 3H),
2.13 (s, 3H), 1.95 (dt, J ¼ 15.2, 7.5 Hz, 1H), 1.83 (dt, J ¼ 15.3, 7.5 Hz,
1H), 1.65e1.44 (m, 6H), 1.27e1.19 (m, 2H), 1.18 (d, J ¼ 9.5 Hz, 6H),
1.16e1.09 (m, 2H), 0.82 (t, J ¼ 7.2 Hz, 3H)$13C NMR (151 MHz,
172.1, 140.9, 137.0, 132.4, 131.4, 129.7, 129.2, 70.5, 51.5, 48.5, 42.3,
37.9, 33.4, 33.1, 27.9, 25.5, 25.1, 23.4, 20.9, 20.6, 19.2, 17.1. þ ESI-
HRMS m/z: calc'd for [MþNa]þ C25H39NaNO5þ
¼
456.2726,
C
25H39NaNOþ5 - found ¼ 456.2721.
Chloroform-d)
d 183.5, 173.5, 141.1, 137.0, 132.4, 131.4, 129.9, 129.1,
48.3, 42.0, 37.6, 34.0, 31.6, 25.4, 25.1, 24.9, 23.3, 22.5, 20.9, 14.0.-ESI-
HRMS m/z: calc'd for [M ꢀ H]- C21H32NOꢀ3 ¼ 346.2388, C21H32NOꢀ3
found ¼ 346.2390.
4.12.8. 5-(4-carboxy-N-(2,5-dimethylphenyl)butanamido)-2,2-
dimethylpentanoic acid (10e) was synthesized using the above
general procedure, using the diester of 10e (22.3 mg, 0.051 mmol, 1
equiv.)
4.12.5. Isobutyl 5-(N-(2,5-dimethylphenyl)-4-methoxy-4-
oxobutanamido)-2,2-dimethyl-pentanoate (10d diester) was
synthesized using the above general procedure using monomethyl
succinic acid (24.8 mg, 0.18 mmol, 1.1 equiv), HATU (68.4 mg,
The product was purified silica chromatography (80% EtOAc in
hexanes plus 2% AcOH). The product was purified was further pu-
rified by precipitation from DCM with hexanes to provide the
product in 45% yield.1H NMR (600 MHz, Chloroform-d)
d 7.16 (d,
0.18 mmol, 1.1 equiv.), TEA (67.5
(1 mL)
After addition of 9 (50 mg, 0.16 mmol, 1 equiv.), the reaction was
stirred for 26 h followed by addition of monomethyl succinic acid
(24.8 mg, 0.18 mmol, 1.1 equiv), HATU (68.4 mg, 0.18 mmol, 1.1
m
L, 0.48 mmol, 3 equiv.), and DMF
J ¼ 7.9 Hz, 1H), 7.07 (d, J ¼ 7.9 Hz, 1H), 6.91 (s, 1H), 4.16e4.09 (m,
1H), 3.00e2.93 (m, 1H), 2.32 (s, 3H), 2.27 (t, J ¼ 7.1 Hz, 1H), 2.13 (s,
3H), 2.05e1.97 (m, 2H), 1.90e1.82 (m, 1H), 1.67e1.56 (m, 3H),
1.56e1.45 (m, 4H), 1.17 (d, J ¼ 13.6 Hz, 6H)$13C NMR (151 MHz,
Chloroform-d)
d 183.7, 178.5, 172.8, 140.7, 137.2, 132.2, 131.5, 129.8,
equiv.), TEA (67.5
action was stirred for an addition 24 h. Silica chromatography
m
L, 0.48 mmol, 3 equiv.) was added and the re-
129.4, 48.4, 42.0, 37.6, 33.4, 32.8, 25.8, 24.7, 23.4, 20.9, 20.4, 17.1.-
ESI-HRMS m/z: calc'd for [M ꢀ H]- C20H28NO5ꢀ ¼ 362.1973,
(20e40% EtOAc in hexanes), to provide the diester of 10d in 26%
C
20H28NOꢀ5 found ¼ 362.1974.
yield.1H NMR (600 MHz, Chloroform-d)
d
7.19 (d, J ¼ 7.7 Hz, 1H),
7.09 (d, J ¼ 7.0 Hz, 1H), 6.92 (s, 1H), 4.06 (ddd, J ¼ 13.2, 9.4, 5.9 Hz,
1H), 3.84e3.75 (m, 2H), 3.67 (s, 3H), 3.09e3.01 (m, 1H), 2.66e2.58
(m, 1H), 2.58e2.50 (m, 1H), 2.34 (s, 3H), 2.32e2.24 (m, 1H), 2.20 (s,
3H), 2.17e2.07 (m, 1H), 1.87 (dt, J ¼ 13.3, 6.7 Hz, 1H), 1.61e1.38 (m,
4H), 1.17 (s, 6H), 0.88 (dd, J ¼ 6.7, 2.6 Hz, 6H)$13C NMR (151 MHz,
4.12.9. Methyl 6-((2,5-dimethylphenyl) (5-isobutoxy-4,4-
dimethyl-5-oxopentyl)amino)-6-oxohexanoate (10f diester) was
synthesized using the above general procedure using monomethyl
adipic acid (28.8 mg, 0.18 mmol, 1.1 equiv), HATU (68.4 mg,
0.18 mmol, 1.1 equiv.), TEA (67.5
(1 mL)
mL, 0.48 mmol, 3 equiv.), and DMF
Chloroform-d)
d 177.9, 173.7, 171.3, 140.6, 137.1, 132.6, 131.5, 129.8,
129.3, 70.5, 51.8, 48.6, 42.3, 37.8, 29.4, 29.2, 27.9, 25.5, 25.1, 23.4,
After addition of 9 (50 mg, 0.16 mmol, 1 equiv.), the reaction was
stirred for 26 h, followed by addition of monomethyl succinic acid
(24.8 mg, 0.18 mmol, 1.1 equiv), HATU (68.4 mg, 0.18 mmol, 1.1
20.9, 19.2, 17.2.
þ
ESI-HRMS m/z: calc'd for [MþNa]þ
C24H37NaNOþ5 ¼ 442.2569, C24H37NaNOþ5 found ¼ 442.2562.
equiv.), TEA (67.5 mL, 0.48 mmol, 3 equiv.). The reaction was stirred
4.12.6. 5-(3-carboxy-N-(2,5-dimethylphenyl)propanamido)-2,2-
dimethylpentanoic acid (10d) was synthesized using the above
general procedure, using the diester of 10d (17.3 mg, 0.041 mmol, 1
equiv.)
The product was partially purified with silica chromatography
(80% EtOAc in hexanes plus 2% AcOH). The product was purified
was further purified by precipitation from DCM with hexanes to
provide the product in 37% yield.1H NMR (600 MHz, Chloroform-d)
for an additional 24 h. After evaporation of the solvent silica
chromatography (20e40% EtOAc in hexanes), provided the diester
of 10f in 45% yield.1H NMR (600 MHz, Chloroform-d)
d 7.14 (d,
J ¼ 7.7 Hz, 1H), 7.05 (d, J ¼ 9.6 Hz, 1H), 6.83 (s, 1H), 4.00 (ddd,
J ¼ 13.8, 9.4, 5.5 Hz, 1H), 3.81e3.72 (m, 2H), 3.60 (s, 3H), 3.01 (ddd,
J ¼ 13.5, 8.7, 4.9 Hz, 1H), 2.31 (s, 3H), 2.21 (t, J ¼ 7.3 Hz, 2H), 2.11 (s,
3H), 1.96 (dt, J ¼ 15.1, 7.3 Hz, 1H), 1.88e1.77 (m, 3H), 1.58e1.52 (m,
2H), 1.52e1.46 (m, 4H), 1.40 (ddt, J ¼ 16.3, 9.0, 4.5 Hz, 1H), 1.14 (d,
J ¼ 2.8 Hz, 6H), 0.85 (dd, J ¼ 6.7, 2.5 Hz, 6H)$13C NMR (151 MHz,
d
7.18 (d, J ¼ 7.7 Hz, 1H), 7.09 (d, J ¼ 7.7 Hz, 1H), 6.99 (s, 1H), 4.15 (dt,
J ¼ 14.3, 7.7 Hz, 1H), 3.02 (ddd, J ¼ 13.1, 7.7, 5.2 Hz, 1H), 2.71 (ddd,
J ¼ 16.8, 9.1, 3.8 Hz, 1H), 2.53 (ddd, J ¼ 17.0, 7.3, 3.8 Hz, 1H),
2.41e2.31 (m, 1H), 2.30 (s, 3H), 2.17 (s, 3H), 2.04 (ddd, J ¼ 17.0, 7.2,
4.0 Hz, 1H), 1.69 (td, J ¼ 12.3, 3.9 Hz, 1H), 1.65e1.44 (m, 3H), 1.19 (s,
Chloroform-d) d 177.8, 174.0, 172.5, 141.0, 137.0, 132.3, 131.4, 129.7,
129.1, 70.5, 51.5, 48.5, 42.3, 37.9, 33.9, 33.7, 27.8, 25.5, 25.1, 24.8,
24.7, 23.4, 20.9, 19.1, 17.2. þ ESI-HRMS m/z: calc'd for [MþNa]þ
C26H41NaNOþ5 ¼ 470.2882, C26H41NaNOþ5 found ¼ 470.2869.
3H), 1.16 (s, 3H)$13C NMR (151 MHz, Chloroform-d)
d 183.1, 177.8,
172.3, 140.4, 137.3, 132.4, 131.6, 129.9, 129.5, 48.7, 42.0, 37.6, 29.8,
29.0, 24.6, 23.4, 20.9, 17.1.-ESI-HRMS m/z: calc'd for [M ꢀ H]-
C19H26NOꢀ5 ¼ 348.1816, C19H26NOꢀ5 found ¼ 348.1820.
4.12.10. 6-((4-carboxy-4-methylpentyl) (2,5-dimethylphenyl)
amino)-6-oxohexanoic acid (10f) was synthesized using the above
general procedure, using the diester of 10f (32.0 mg, 0.07 mmol, 1
equiv.) The product was purified by silica chromatography (80%
EtOAc in hexanes plus 2% AcOH)
4.12.7. Isobutyl 5-(N-(2,5-dimethylphenyl)-5-methoxy-5-
oxopentanamido)-2,2-dimethyl-pentanoate (10e diester) was
synthesized using the above general procedure using monomethyl
glutaric acid (52.6 mg, 0.36 mmol, 2.2 equiv.), HATU (136.9 mg,
The product was purified was further purified by precipitation
from DCM with hexanes to provide the product in a 59% yield.1H
NMR (600 MHz, Chloroform-d)
d
7.16 (d, J ¼ 7.9 Hz, 1H), 7.07 (d,
0.36 mmol, 2.2 equiv.), TEA (118
(1 mL)
m
L, 0.84 mmol, 6 equiv.), and DMF
J ¼ 7.9 Hz, 1H), 6.91 (s, 1H), 4.16e4.09 (m, 1H), 3.00e2.93 (m, 1H),
2.32 (s, 3H), 2.27 (t, J ¼ 7.1 Hz, 2H), 2.13 (s, 3H), 2.05e1.97 (m, 1H),
1.90e1.82 (m, 1H), 1.67e1.56 (m, 4H), 1.56e1.43 (m, 4H).1.17 (d,
After addition of 9 (50 mg, 0.16 mmol, 1 equiv.) the reaction was
9

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
J ¼ 13.6 Hz, 6H)$13C NMR (151 MHz, Chloroform-d)
d
183.4, 178.8,
4.14.1. 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanamide
(12a) was synthesized by the above general procedure using 7 N
ammonia in MeOH (150 mL, 1.05 mmol, 5.25 equiv.), Gemfibrozil (1)
(50 mg, 0.2 mmol, 1 equiv.), HATU (83.6 mg, 0.22 mmol, 1.1 equiv.),
and DMF (1 mL)
173.2, 141.0, 137.2, 132.3, 131.5, 129.8, 129.3, 48.7, 42.1, 37.7, 34.1,
33.5, 25.7, 24.6, 24.5, 23.5, 20.9, 17.2.-ESI-HRMS m/z: calc'd for
[M ꢀ H]- C21H30NO5ꢀ ¼ 376.2129, C21H30NOꢀ5 found ¼ 376.2124.
4.13. Synthesis of 5,5'-(adipoylbis((2,5-dimethylphenyl)azanediyl))
bis(2,2-dimethyl-pentanoic acid) (11)
After the addition of TEA (27.9
mixture was stirred for 2 h. Centrifugal evaporation provided the
product in 95% yield.1H NMR (Chloroform-d, 600 MHz)
7.00 (d,
mL, 0.2 mmol, 1 equiv.) the
d
To a 1 dram vial was added SOCl2 (0.5 mL) followed by adipic
acid (32.2 mg, 0.22 mmol, 1 equiv.) and the resulting mixture was
heated to 75 ꢁC for 24 h SOCl2 was removed by rotary evaporation
and the acid chloride was used without further purification. The
crude acid chloride was dissolved in DCM (1 mL) and added to a 6
dram vial containing 9 (135 mg, 0.44 mmol, 2 equiv.) and DIPEA
J ¼ 7.5 Hz, 1H), 6.66 (d, J ¼ 7.4 Hz, 1H), 6.61 (s, 1H), 5.68 (brs, 1H),
5.45 (brs, 1H), 3.93 (t, J ¼ 6.1 Hz, 2H), 2.30 (s, 3H), 2.17 (s, 3H),
1.83e1.78 (m, 2H), 1.73e1.70 (m, 2H), 1.24 (s, 6H)$13C NMR (Chlo-
roform-d, 151 MHz)
d 180.3, 157.0, 136.7, 130.5, 123.6, 120.9, 112.2,
68.0, 42.1, 37.7, 25.8, 25.3, 21.6, 16.0. þ ESI-HRMS m/z: calc'd for
[MþNa]þ C15H23NNaOþ2 273.1620, C15H23NNaOþ2 found 273.1620.
(307.4 mL, 1.76 mmol, 8 equiv.). The reaction mixture was allowed to
stir at 25 ꢁC for 3 days and the solvents were removed by rotary
evaporation. To the reaction mixture was added 5 mL 3 M HCl,
which was extracted with EtOAc (5 mL) and the organic extract
dried with Na2SO4. The organic solvent was removed by rotary
evaporation and the product was purified silica chromatography
(20%e40% EtOAc in hexanes) to provide the isobutyl ester of 11 in a
4.14.2. 5-(2,5-dimethylphenoxy)-N,2,2-trimethylpentanamide
(12b) was synthesized by the above general procedure using 2 M
methylamine in MeOH (500 mL, 1.0 mmol, 5 equiv.), Gemfibrozil (1)
(50 mg, 0.2 mmol, 1 equiv.), HATU (83.6 mg, 0.22 mmol, 1.1 equiv.)
and DMF (1 mL)
57% yield.1H NMR (600 MHz, Chloroform-d)
d
7.12 (d, J ¼ 7.7 Hz,
After the addition of TEA (27.9
mixture was stirred for 2 h. Centrifugal evaporation provided the
product in 94% yield.1H NMR (Chloroform-d, 600 MHz)
7.00 (d,
mL, 0.2 mmol, 1 equiv.) the
2H), 7.04 (d, J ¼ 7.8 Hz, 2H), 6.79 (s, 2H), 3.99e3.93 (m, 2H),
3.82e3.71 (m, 4H), 3.03e2.95 (m, 2H), 2.30 (s, 6H), 2.08 (d,
J ¼ 4.6 Hz, 6H), 1.85 (dtd, J ¼ 26.9, 13.4, 11.6, 6.5 Hz, 4H), 1.75e1.68
(m, 2H), 1.51e1.44 (m, 6H), 1.42e1.34 (m, 6H), 1.13 (s, 12H), 0.84 (dd,
d
J ¼ 7.4 Hz, 1H), 6.66 (d, J ¼ 7.4 Hz, 1H), 6.60 (s, 1H), 5.72 (brs, 1H),
3.91 (t, J ¼ 5.8 Hz, 2H), 2.80 (d, J ¼ 4.7 Hz, 3H), 2.30 (s, 3H), 2.17 (s,
3H), 1.76e1.67 (m, 4H), 1.21 (s, 6H)$13C NMR (Chloroform-d,
J ¼ 6.7, 2.6 Hz, 12H)$13C NMR (151 MHz, Chloroform-d)
d 177.9,
151 MHz)
d 178.2, 157.0, 136.7, 130.4, 123.6, 120.9, 112.2, 68.1, 42.0,
172.7,141.0, 136.9,131.3, 129.7,129.1, 70.5, 48.7, 42.3, 37.8, 33.9, 33.9,
27.8, 25.5, 25.1, 25.0, 25.0, 23.4, 20.9, 19.1, 17.2. þ ESI-HRMS m/z:
calc'd for [MþNa]þ C44H68N2NaOþ6 ¼ 743.4975, C44H68N2NaOþ6
found ¼ 743.4967.
37.6, 26.6, 25.7, 25.3, 21.6, 16.0. þ ESI-HRMS m/z: calc'd for
[MþNa]þ C16H25NNaOþ2 286.1778, C16H25NNaOþ2 found 286.1778.
To a 6 dram vial was added the isobutyl ester of 11 (91.1 mg,
0.13 mmol, 1 equiv.), 10 M NaOH (0.5 mL), and MeOH (1.5 mL) and
was heated to 80 ꢁC. The reaction was stirred for 2 days and organic
solvents were removed by rotary evaporation. To the reaction
mixture was added 3 M HCl until reaction mixture pH was 2,
extracted with EtOAc (5 mL), and dried with Na2SO4. The organic
solvent was removed by rotary evaporation and the product was
purified silica chromatography (60%e100% EtOAc in hexanes þ 2%
AcOH) to provide the product in a 76% yield. Rotamer A: 1H NMR
4.14.3. 5-(2,5-dimethylphenoxy)-N-ethyl-2,2-
dimethylpentanamide (12c) was synthesized by the above general
procedure using 2 M ethylamine in THF (500 mL, 1.0 mmol, 5 equiv.),
Gemfibrozil (1) (50 mg, 0.2 mmol, 1 equiv.), HATU (83.6 mg,
0.22 mmol, 1.1 equiv.), and DMF (1 mL)
After the addition of TEA (27.9
action mixture was stirred for 2 h. Centrifugal evaporation provided
the product in 90% yield.1H NMR (Chloroform-d, 600 MHz)
7.00
mL, 0.2 mmol, 1 equiv.) the re-
d
(d, J ¼ 7.5 Hz, 1H), 6.66 (d, J ¼ 7.5 Hz, 1H), 6.60 (s, 1H), 5.66 (brs, 1H),
3.91 (t, J ¼ 5.9 Hz, 2H), 3.31e3.26 (m, 2H), 2.30 (s, 3H), 2.17 (s, 3H),
1.76e1.73 (m, 2H), 1.70e1.67 (m, 2H), 1.20 (s, 6H), 1.13 (t, J ¼ 7.3 Hz,
(600 MHz, Chloroform-d)
d
7.13 (d, J ¼ 4.5 Hz, 2H), 7.07 (s, 2H), 6.95
(s, 2H), 4.22 (dt, J ¼ 13.4, 7.3 Hz, 2H), 2.83e2.76 (m, 2H), 2.35 (s, 6H),
2.09 (s, 6H), 1.99e1.87 (m, 4H), 1.65e1.54 (m, 8H), 1.53e1.45 (m,
4H), 1.20 (s, 6H), 1.16 (s, 6H)$13C NMR (151 MHz, Chloroform-d)
3H)$13C NMR (Chloroform-d, 151 MHz)
d 177.7, 157.4, 137.0, 130.8,
124.0, 121.2, 112.6, 68.5, 42.3, 38.1, 34.9, 26.1, 25.6, 21.9, 16.3,
15.5. þ ESI-HRMS m/z: calc'd for [MþNa]þ C17H27NNaO2þ 300.1934,
C17H27NNaOþ2 found 300.1934.
d
183.6, 173.0, 141.3, 137.2, 132.2, 131.3, 130.1, 129.1, 48.4, 42.2, 37.9,
33.7, 25.6, 25.1, 24.7, 23.8, 21.0, 17.1. Rotamer B:1H NMR (600 MHz,
Chloroform-d)
d
7.14 (d, J ¼ 4.5 Hz, 2H), 7.05 (s, 2H), 6.88 (s, 2H),
4.02 (dt, J ¼ 7.2, 6.4 Hz, 2H), 3.04e2.98 (m, 2H), 2.32 (s, 6H), 2.11 (s,
6H), 1.75 (dt, J ¼ 13.6, 6.6 Hz, 4H), 1.65e1.54 (m, 8H), 1.53e1.45 (m,
4H), 1.18 (s, 6H), 1.17 (s, 6H)$13C NMR (151 MHz, Chloroform-d)
4.14.4. N-butyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanamide (12d) was synthesized by the above general
procedure using Gemfibrozil (1) (50 mg, 0.2 mmol, 1 equiv.), HATU
d
183.3, 173.1, 141.4, 137.0, 132.4, 131.4, 129.8, 129.1, 48.8, 42.2, 37.9,
33.7, 25.4, 25.0, 24.7, 23.8, 20.9, 17.3.-ESI-HRMS m/z: calc'd for
(76 mg, 0.2 mmol, 1 equiv.), TEA (27.9
DMF (1 mL)
m
L, 0.2 mmol, 1 equiv.) and
[M ꢀ H]- C36H51N2O6ꢀ ¼ 607.3753, C36H51N2Oꢀ6 found ¼ 607.3750.
After the addition of n-butyl amine (98.6
mL, 1 mmol, 5 equiv.)
4.14. General procedure for synthesis of 12a-e, 13a-c
the reaction was stirred for 23 h. Silica chromatography using (40%
EtOAc in hexanes) provided the product in a 47% yield.1H NMR
To a 2 dram vial was added gemfibrozil (1), HATU, then DMF
followed by either the desired amine or TEA as indicated. The re-
action mixture was stirred for 15 min at 25 ꢁC, followed by addition
of desired amine or TEA as indicated. The reaction was stirred at
25 ꢁC for the specified amount of time. The reaction was diluted
with EtOAc (5 mL) and washed sequentially with water (5 mL), 1 M
HCl (5 mL), and brine (5 mL). The organic phase was then dried over
MgSO4 and the solvent removed by rotary evaporation. Product was
purified as specified.
(600 MHz, Chloroform-d)
d
7.00 (d, J ¼ 7.5 Hz, 1H), 6.67e6.65 (d,
J ¼ 7.5 Hz 1H), 6.61 (s, 1H), 5.65 (t, J ¼ 5.8 Hz, 1H), 3.92 (t, J ¼ 6.0 Hz,
2H), 3.25 (td, J ¼ 7.2, 5.6 Hz, 2H), 2.30 (s, 3H), 2.17 (s, 3H), 1.78e1.72
(m, 2H), 1.71e1.67 (m, 2H), 1.50e1.45 (m, 2H), 1.38e1.31 (m, 2H),
1.21 (s, 6H), 0.92 (t, J ¼ 7.4 Hz, 2H)$13C NMR (151 MHz, Chloroform-
d) d 177.3,156.9,136.5,130.3,123.5,120.7, 112.1, 68.0, 41.8, 39.3, 37.6,
31.9, 25.6, 25.1, 21.4, 20.1, 15.8, 13.8. þ ESI-HRMS m/z: calc'd for
[MþNa]þ C19H31NNaOþ2
Oþ2 ¼ 328.2247.
¼
328.2247, found C19H31NNa
10

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
4.14.5. 5-(2,5-dimethylphenoxy)-N-hexyl-2,2-
dimethylpentanamide (12e) was synthesized by the above general
procedure using Gemfibrozil (1) (50 mg, 0.2 mmol, 1 equiv.), HATU
130.4, 123.6, 120.9, 112.2, 68.1, 42.0, 39.2, 37.7, 29.7, 26.2, 25.7, 25.3,
21.5, 15.9.
þ
ESI-HRMS
m/z:
calc'd
for
[MþNa]þ
C36H56N2NaOþ4 ¼ 603.4132, found C36H56N2NaOþ4 ¼ 603.4131.
(83.6 mg, 0.22 mmol, 1.1 equiv.), TEA (27.9
and DMF (1 mL)
mL, 0.2 mmol, 1 equiv.),
After the addition of n-hexylamine (12.84
the reaction was stirred for 2 h. Centrifugal evaporation provided
the product in 92% yield.1H NMR (Chloroform-d, 600 MHz)
7.00
mL, 1.0 mmol, 5 equiv.)
4.15. Synthesis of isobutyl 5-(2,5-dimethylphenoxy)pentanoate (14)
d
In a 6 dram vial 3c (50 mg, 0.211 mmol,1.1 equiv.) was added to a
solution of K2CO3 (68 mg, 0.492 mmol, 2.5 equiv.) and 2,5-
dimethylphenol (24 mg, 0.196 mmol, 1 equiv.) in MeCN (2 mL).
The mixture was heated to 82 ꢁC for 14 h then extracted with EtOAc
(5 mL) and the organic phase was dried with MgSO4 and evapo-
rated by rotary evaporation. The resulting residue was purified by
silica chromatography (10e25% EtOAc in hexanes) to provide the
(d, J ¼ 7.4 Hz, 1H), 6.66 (d, J ¼ 7.4 Hz, 1H), 6.61 (s, 1H), 5.65 (brs, 1H),
3.91 (t, J ¼ 5.8 Hz, 2H), 3.24 (q, J ¼ 6.6 Hz, 2H), 2.30 (s, 3H), 3.17 (s,
3H), 1.78e1.73 (m, 2H), 1.70e1.67 (m, 2H), 1.51e1.46 (m, 2H),
1.33e1.28 (m, 6H), 1.21 (s, 6H), 0.88 (t, J ¼ 6.6 Hz, 3H)$13C NMR
(Chloroform-d, 151 MHz)
d 177.4, 157.0, 136.6, 130.4, 123.6, 120.9,
112.2, 68.1, 42.0, 39.7, 37.7, 31.6, 29.8, 26.7, 25.7, 25.3, 22.7, 21.5, 15.9,
14.1 þ ESI-HRMS m/z: calc'd for [MþNa]þ C15H20NaO3þ 273.1463,
product in 78% yield. 1H NMR (600 MHz, Chloroform-d)
d 7.02 (d,
C
15H20NaOþ3 found 273.1463.
J ¼ 7.5 Hz, 1H), 6.68 (d, J ¼ 7.5 Hz, 1H), 6.63 (s, 1H), 3.97 (t, J ¼ 5.7 Hz,
2H), 3.88 (d, J ¼ 6.7 Hz, 2H), 2.43 (t, J ¼ 7.1 Hz, 2H), 2.32 (s, 3H), 2.19
(s, 3H), 1.94 (hept, J ¼ 6.7 Hz, 1H), 1.87e1.84 (m, 4H), 0.95 (d,
4.14.6. N,N'-(ethane-1,2-diyl)bis(5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanamide) (13a) was synthesized by the above
general procedure using Gemfibrozil (1) (100 mg, 0.4 mmol, 2
J ¼ 6.7 Hz, 6H)$13C NMR (151 MHz, Chloroform-d)
d 173.8, 157.0,
136.6, 130.4, 123.7, 120.8, 112.0, 70.6, 67.3, 34.2, 29.0, 27.9, 22.0, 21.5,
equiv), HATU (152.1 mg, 0.4 mmol, 2 equiv.), TEA (126.7
0.8 mmol, 4 equiv.) and DMF (1 mL) and stirred for 15 min
After addition of ethylenediamine (13.3 L, 0.2 mmol, 1 equiv.)
mL,
19.2, 15.9.
þ
ESI-HRMS
m/z:
calc'd
for
[MþNa]þ
C17H26NaOþ3 ¼ 301.1774, C17H26NaOþ3 found ¼ 301.1775.
m
the reaction was stirred for 4.5 h. Silica chromatography using (40%
EtOAc in hexanes) provided the product in 54% yield.1H NMR
(600 MHz, Chloroform-d)
d
6.99 (d, J ¼ 7.4 Hz, 2H), 6.67 (s, 2H), 6.65
4.16. Synthesis of 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid
(15a)
(d, J ¼ 7.6 Hz, 2H), 6.59 (d, J ¼ 1.6 Hz, 2H), 3.89 (t, J ¼ 5.8 Hz, 4H),
3.41e3.35 (m, 4H), 2.30 (s, 6H), 2.17 (s, 6H), 1.75e1.66 (m,8H), 1.21
(s, 12H)$13C NMR (151 MHz, Chloroform-d)
d
179.3, 157.0, 136.6,
In a 6 dram vial 5 M n-BuLi in hexanes (0.9 mL, 4.5 mmol, 15
equiv.) was added to a dry solution of diisopropylamine (0.8 mL,
5.7 mmol, 19 equiv.) under nitrogen while stirring at ꢀ78 ꢁC for
20 min in THF (3.5 mL). The reaction mixture was then allowed to
warm to room temperature for 1 h before being cooled again
to ꢀ78 ꢁC. To the cold LDA solution was added ethyl bromide
(0.8 mL, 10.7 mmol, 35.7 equiv.). A solution of 14 (82 mg, 0.3 mmol,
1 equiv.) was prepared in THF (15 mL) and added dropwise to the
solution of LDA and ethyl bromide then stirred at ꢀ78 ꢁC for 30 min.
The reaction mixture was then allowed to warm to 0 ꢁC in an ice
bath for 1 h. The ice bath was then removed, and the reaction was
allowed to warm to 25 ꢁC over 2 h before the solvent was removed
by rotary evaporation and the crude residue purified by silica
chromatography (0e5% EtOAc in Hexanes) to provide the product
130.4, 123.6, 120.8, 112.1, 68.0, 41.9, 40.8, 37.5, 25.6, 25.2, 21.5,
15.9.
þ
ESI-HRMS
m/z:
calc'd
for
[MþNa]þ
C32H48N2NaOþ4 ¼ 547.3506, found C32H48N2NaO4 ¼ 547.3505.
4.14.7. N,N'-(butane-1,4-diyl)bis(5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanamide) (13b) was synthesized by the above
general procedure using Gemfibrozil (1) (100 mg, 0.4 mmol, 2
equiv.), HATU (152.1 mg, 0.4 mmol, 2 equiv.), TEA (126.7
0.8 mmol, 4 equiv.) and DMF (1 mL)
mL,
After addition of putrescine (20.1 mL, 0.2 mmol, 1 equiv.) the
reaction was allowed to stir at 25 ꢁC for 4.5 h. Silica chromatog-
raphy (40% EtOAc in hexanes), provided the product in 79% yield.1H
NMR (600 MHz, Chloroform-d)
d
6.99 (d, J ¼ 7.5 Hz, 2H), 6.65 (dd,
J ¼ 7.5, 1.5 Hz, 2H), 6.60 (d, J ¼ 1.6 Hz, 2H), 5.93 (t, J ¼ 5.7 Hz, 2H),
3.90 (t, J ¼ 5.9 Hz, 4H), 3.26 (q, J ¼ 6.4 Hz, 4H), 2.30 (s, 6H), 2.16 (s,
6H), 1.77e1.65 (m, 8H), 1.55e1.48 (m, 4H), 1.20 (s, 12H)$13C NMR
as a clear oil in 38% yield. 1H NMR (600 MHz, Chloroform-d)
d 7.00
(d, J ¼ 7.4 Hz, 1H), 6.66 (d, J ¼ 7.5 Hz, 1H), 6.61 (s, 1H), 3.94 (s, 2H),
3.88 (d, J ¼ 6.6 Hz, 2H), 2.41e2.36 (m, 1H), 2.31 (s, 3H), 2.17 (s, 3H),
1.94 (hept, J ¼ 6.3 Hz, 1H), 1.81e1.76 (m, 3H), 1.73e1.64 (m, 2H),
1.61e1.54 (m, 1H), 0.96e0.90 (m, 9H)$13C NMR (151 MHz, Chloro-
(151 MHz, Chloroform-d)
d 177.7, 157.0, 136.6, 130.4, 123.5, 120.9,
112.2, 68.1, 41.9, 39.0, 37.6, 27.1, 25.6, 25.2, 21.5, 15.9. þ ESI-HRMS
m/z: calc'd for [MþNa]þ C34H52N2NaO4þ
¼
575.3819, found
form-d) d 176.3, 157.1, 136.6, 130.4, 123.7, 120.8, 112.1, 70.50, 67.5,
C
34H52N2NaOþ4 ¼ 575.3818.
47.2, 28.7, 27.9, 27.5, 25.6, 21.6, 19.3, 15.9, 11.9. þ ESI-HRMS m/z:
calc'd for [MþNa]þ C19H30NaO3þ
found ¼ 329.2090.
¼
329.2087, C19H30NaO3þ
4.14.8. N,N'-(hexane-1,6-diyl)bis(5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanamide) (13c) was synthesized by the above general
procedure Gemfibrozil (1) (100 mg, 0.4 mmol, 2 equiv.), HATU
(152.1 mg, 0.4 mmol, 2 equiv.), TEA (126.7
and DMF (1 mL)
After addition of hexamethylenediamine (23.2 mg, 0.2 mmol, 1
equiv.) the reaction was allowed to stir for 4.5 h, when an additional
HATU (0.3 mmol, 0.08 equiv.) was added and allowed to stir for an
additional 16 h. Silica chromatography using (40% EtOAc in hex-
anes), to provide the product in 28% yield.1H NMR (600 MHz,
In a 6 dram vial the above compound, (34.9 mg, 0.114 mmol, 1
equiv.), was added to a solution of 10 M NaOH (2 mL) and toluene
(2 mL) which was 110 ꢁC for 48 h. The resulting mixture was then
extracted with EtOAc (5 mL) and the organic phase dried with
MgSO4 and evaporated by rotary evaporation. The resulting residue
was purified by silica chromatography (10e25% EtOAc in hexanes)
to provide the product in 75% yield.1H NMR (600 MHz, Chloroform-
mL, 0.8 mmol, 4 equiv.)
d)
d
7.00 (d, J ¼ 7.5 Hz, 1H), 6.65 (d, J ¼ 7.4 Hz, 1H), 6.62 (s, 1H),
3.98e3.92 (m, 2H), 2.44e2.40 (m, 1H), 2.30 (s, 3H), 2.17 (s, 3H),
1.88e1.80 (m, 2H), 1.76e1.68 (m, 2H), 1.64e1.57 (m, 2H), 0.97 (t,
Chloroform-d)
d
6.99 (d, J ¼ 7.5 Hz, 2H), 6.65 (d, J ¼ 8.0 Hz, 2H), 6.60
(d, J ¼ 1.6 Hz, 2H), 5.79 (t, J ¼ 5.8 Hz, 2H), 3.91 (t, J ¼ 6.0 Hz, 4H), 3.22
(q, J ¼ 6.7 Hz, 4H), 2.30 (s, 6H), 2.17 (s, 6H), 1.74 (td, J ¼ 6.3, 2.4 Hz,
2H), 1.71e1.66 (m, 4H), 1.48 (q, J ¼ 6.8 Hz, 6H), 1.34e1.31 (m, 4H),
J ¼ 7.4 Hz, 3H)$13C NMR (151 MHz, Chloroform-d)
d 179.5, 157.0,
136.6, 130.5, 123.8, 120.9, 112.1, 67.5, 46.4, 28.4, 27.3, 25.4, 21.6, 15.9,
11.8. þ ESI-HRMS m/z: calc'd for [MþNa]þ C15H20NaO3þ ¼ 273.1461,
1.21 (s, 12H)$13C NMR (151 MHz, Chloroform-d)
d
177.5, 157.0, 136.6,
C
15H20NaOþ3 found ¼ 273.1463.
11

K.M. Gayler, J.M. Quintana, J. Mattke et al.
European Journal of Medicinal Chemistry 224 (2021) 113729
4.17. Synthesis of 2-(3-(2,5-dimethylphenoxy)propyl)octanoic acid
(15b)
4.20. Aortic ring relaxation
All animal experiments were performed in accordance to Public
Health Service's Guide for the Care and Use of Laboratory Animals,
the Foundation for Biomedical Research's Biomedical Investigator's
Handbook for Researchers and the guidelines of the Animal Welfare
Committee of the University of Texas Health Science Center (pro-
tocol AWC 18e085). C57Bl6 mice (8e10 weeks old, Jackson labo-
ratories, Ann Harbor, MI) were euthanized by CO2. After
thoracotomy, descending thoracic aorta was dissected, cut into 3-5-
mm-long segments and mounted on a four-channel wire Myograph
610 (DMT, Copenhagen, Denmark) under 0.9 g of passive tension.
All force measurements were recorded using Powerlab 400™ data
acquisition system and LabChart software. The rings were equili-
brated for 80 min in Krebs-Henseleit solution pH 7.4, oxygenated
with carbogen (95% O2, 5% CO2) with at least three buffer changes
every 20 min. After equilibration the rings were contracted with a
mixture of 500 nM phenylephrine and 100 nM of 5-
In a round bottom flask a 2 M solution LDA in THF/n-heptane/
ethylbenzene (0.9 mL, 1.8 mmol, 5 equiv.) was added to a solution
of dry THF (5 mL) and 1-bromohexane (0.8 mL, 5.7 mmol, 15.8
equiv.) under nitrogen while stirring at ꢀ78 ꢁC. A solution of 14
(100 mg, 0.36 mmol,1 equiv.) was then prepared in THF (15 mL) and
added dropwise to the solution of LDA and bromohexane at ꢀ78 ꢁC
for 30 min, after which the reaction mixture was allowed to warm
to 0 ꢁC in an ice bath for 1 h. The ice bath was then removed, and the
reaction was allowed to warm to 25 ꢁC over 2 h before the solvent
was removed by rotary evaporation. The crude residue was purified
by silica chromatography (0e5% EtOAc in Hexanes) to provide the
partially purified product as a clear oil. This material was carried
forward without further purification.
In a 6 dram vial the above material was added to a solution of
10 M NaOH (2 mL) and toluene (2 mL) which was 110 ꢁC for 48 h.
The resulting mixture was then extracted with EtOAc (5 mL) and
water (5 mL) and the organic phase was dried with MgSO4 and
evaporated by rotary evaporation. The resulting residue was puri-
fied by silica chromatography (10e25% EtOAc in hexanes) to pro-
vide the product in 32% yield. 1H NMR (Chloroform-d, 600 MHz)
hydroxytryptamine
to
achieve
submaximal
contraction
(~1.8e2.5 g of tension). After stabilization, gemfibrozil or gemfi-
brozil analogs were added cumulatively and changes in isometric
tension were recorded.
d
7.00 (d, J ¼ 7.5 Hz, 1H), 6.66 (d, J ¼ 7.5 Hz, 1H), 6.62 (s, 1H),
4.21. Statistical analysis
3.98e3.92 (m, 2H), 2.49e2.44 (m, 1H), 2.31 (s, 3H), 2.17 (s, 3H),
1.87e1.79 (m, 3H), 1.76e1.66 (m, 2H), 1.55e1.50 (m, 1H), 1.35e1.23
(m, 8H), 0.88 (t, J ¼ 6.9 Hz, 3H)$13C NMR (151 MHz, Chloroform-d)
Statistical analysis, nonlinear regression, calculation of IC50, EC50
and was performed using GraphPad Prism 5.1 (GraphPad, La Jolla,
USA). All results are expressed as mean SD of at least three in-
dependent experiments. Comparisons between two data groups
were performed using the two tailed student t-test. Two-way
ANOVA followed by Bonferroni's multi comparison test was used
for statistical evaluation of the dose-response curves. The p
value < 0.05 was considered statistically significant.
d
180.2, 157.0, 136.6, 130.5, 123.8, 120.9, 112.1, 67.5, 44.9, 32.4, 31.8,
29.4, 28.8, 27.4, 27.3, 22.7, 21.6, 15.9, 14.2. þ ESI-HRMS m/z: calc'd
for
[MþNa]þ
C19H30NaOþ3
¼
329.2087,
C19H30NaOþ3
found ¼ 329.2088.
4.18. Preparation of recombinant human sGC enzyme
Full-length sGC was purified from Sf9 cells as described previ-
ously [31]. Briefly, 6 L of Sf9 cells were harvested 72 h after being
infected with viruses expressing a1 and b1 sGC subunits. The cells
disrupted by sonication and centrifuged at 100,000ꢂg. The super-
natant was applied onto a 50 mL DEAE-FF Sepharose column and,
after extensive washes, sGC was eluted with 350 mM NaCl and
loaded onto a Ni-agarose column (30 mL bed volume). After washes
with 40 mM imidazole, sGC was eluted with 200 mM imidazole.
The fractions containing sGC were pooled, supplemented with
5 mM DTT and concentrated on a 10 kDa centrifugal filter
concentrator (Millipore, Bedford, MA). Concentrated sGC sample
was subjected to gel-filtration on Superdex 200 equilibrated with
50 mM triethanolamine pH 7.4, 250 mM NaCl, 1 mM MgCl2. For
long-term storage at ꢀ80 ꢁC, the sample was supplemented with
25% glycerol.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by National Institutes of Health (grant
HL139838 to E.M.).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113729.
4.19. Assay of sGC activity in vitro
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