Modulating the Serotonin Receptor Spectrum of Pulicatin Natural Products

Article abstract of DOI:10.1021/acs.jnatprod.7b00317

Serotonin (5-HT) receptors are important in health and disease, but the existence of 14 subtypes necessitates selective ligands. Previously, the pulicatins were identified as ligands that specifically bound to the subtype 5-HT_2B in the 500 nM to 10 μM range and that exhibited in vitro effects on cultured mouse neurons. Here, we examined the structure-activity relationship of 30 synthetic and natural pulicatin derivatives using binding, receptor functionality, and in vivo assays. The results reveal the 2-arylthiazoline scaffold as a tunable serotonin receptor-targeting pharmacophore. Tests in mice show potential antiseizure and antinociceptive activities at high doses without motor impairment.

Full text of DOI:10.1021/acs.jnatprod.7b00317

Article
pubs.acs.org/jnp
Modulating the Serotonin Receptor Spectrum of Pulicatin Natural
Products
Zhenjian Lin,† Misty D. Smith,‡,§ Gisela P. Concepcion,⊥ Margo G. Haygood,† Baldomero M. Olivera,∥
Alan Light,# and Eric W. Schmidt*,†,∥
†Department of Medicinal Chemistry, ‡Department of Pharmacology and Toxicology, §School of Dentistry, ∥Department of Biology,
#
and Department of Anesthesiology, University of Utah, Salt Lake City, Utah 84112, United States
⊥Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines
S
* Supporting Information
ABSTRACT: Serotonin (5-HT) receptors are important in health and
disease, but the existence of 14 subtypes necessitates selective ligands.
Previously, the pulicatins were identified as ligands that specifically bound to
the subtype 5-HT2B in the 500 nM to 10 μM range and that exhibited in vitro
effects on cultured mouse neurons. Here, we examined the structure−activity
relationship of 30 synthetic and natural pulicatin derivatives using binding,
receptor functionality, and in vivo assays. The results reveal the 2-arylthiazoline
scaffold as a tunable serotonin receptor-targeting pharmacophore. Tests in
mice show potential antiseizure and antinociceptive activities at high doses
without motor impairment.
iderophores in the yersiniabactin family are widely
distributed among diverse bacterial phyla, where they are
known to be responsible for iron import and for interspecies
competition for iron.1 Among the probable breakdown
products of yersiniabactin-like siderophores is a family of
small heterocycles, such as aerugine, pulicatins, and relatives
(Figure 1), which are sometimes found in great abundance as
serotonin itself, bacteria inhabiting the gut produce secondary
metabolites with the potential to affect the serotonin system.7
Beyond the gut−brain axis, the 14 serotonin receptor
subtypes play central roles in human physiology.8 For example,
the 5-HT2B subtype is found in many tissues, including the
brain, gastrointestinal tract, cardiovascular system, and dorsal
root ganglion (DRG) neurons.9 In the brain, the 5-HT2B
receptor is the downstream target of selective serotonin
reuptake inhibitors (SSRIs), since it is the major serotonin-
responsive modulator of depression.10 In the gut and in the
DRG, 5-HT2B signaling promotes hyperalgesia, at least in part
by regulating the capsaicin receptor, TRPV1.11
S
5-HT2B receptors regulate aspects of cardiovascular function,
and agonists can cause the negative side effect of damage to the
heart.12 Cardiovascular damage is associated with specific
kinetics of ligand−protein interaction.12 In addition to 5-HT2B,
14 other receptor subtypes also contribute to many different
physiological functions.13,14 For example, 5-HT3 antagonists
control nausea and vomiting.15 5-HT1B agonists are useful in
treating migraine.16 Because of the many different functions of
serotonin receptors, it is important to develop agents with
different spectra of selectivity, different kinetics, and different
tissue distribution.
Figure 1. Structures of key metabolites pulicatin B (1), its probable
precursor siderophore (10), and aerugine (7).
fermentation products.2 The proposal that these are breakdown
products of larger siderophores recently gained support from
biosynthetic work on a methyltransferase that installs the
thiazoline 5-methyl group.3
Previously, we reported that pulicatins and aerugine bind to
serotonin (5-HT) receptors, with micromolar to nanomolar
affinity.4 In addition, aerugine relatives have other demon-
strated activities on neurons.5 Interestingly, 5-HT is also a
critical component of the gut−brain axis and is regulated in part
by the human microbiome.6 While research has largely focused
on bacteria that directly or indirectly regulate the metabolism of
Here, we further investigate pulicatin activity in vivo and in
vitro. Previously, ligand displacement assays with natural
materials showed that natural pulicatin derivatives 2 and 5−
Received: May 15, 2017
Published: July 26, 2017
© 2017 American Chemical Society and
American Society of Pharmacognosy
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Table 1. Thiazoline Ring Substituent Pulicatin Derivative Binding Affinity in Ligand Displacement Assays Using a Series of
a
Serotonin Receptors, with Ki Shown in nM
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Table 1. continued
a
Data for 2 and 5−10 were previously reported18 and are provided here for comparison. Bold: most potent binding target. In this and ensuing
b
figures, affinity is rounded to two significant figures. Note: “>” means >10 μM; “-” indicates not tested; rac, racemic. R is positioned on the
following scaffold:
10 exhibited selective binding to the 5-HT2B receptor, with Ki
from 510 to 4700 nM (Table 1).4 The assays were performed at
the Psychoactive Drug Screening Program (PDSP). Test
compounds compete with radiolabeled lysergic acid dieth-
ylamide (LSD) or with other ligands for other serotonin
receptor subtypes, and therefore constants are reported as
inhibition of LSD binding. Subsequently, additional functional
tests were performed for some compounds. Comparative
binding constants for serotonin and a large number of
serotonin receptor-binding compounds are provided at the
PDSP Web site (https://kidbdev.med.unc.edu/databases/pdsp.
php). In the pulicatin series, because binding affinity and other
bioassay outcomes sometimes gave different answers, here we
sought in part to use synthesis to generate sufficient
compounds for further biological evaluation.
We also sought to create analogues exhibiting greater
potency and selectivity. While the initial analogues were not
exceptionally potent, they are very small molecules, exhibit
excellent solubility in organic and aqueous solvents, and were
quite selective in initial assays; in short, they are good initial hit
compounds for a medicinal chemistry campaign. Based upon
our initial structure−activity relationship (SAR) findings from
naturally occurring compounds, we expected that subtle
changes in pulicatin structure could yield potent and selective
serotonin receptor modulating ligands.4 Most importantly, we
noted that addition of a methyl group to the thiazoline ring
(e.g., compounds 1−4 in comparison to 7) improved binding
affinity by >3-fold. Moreover, greater sp2 substitution did not
substantially affect potency. Finally, configuration led to subtle
differences in binding affinity. However, none of these changes
modified potency or selectivity by 10-fold or more. Therefore,
we aimed to explore more significant changes to either the
thiazoline ring or the aromatic moiety to determine which
factors were most important in determining selectivity. We
systematically explored changes to each ring using synthetic
methods.
show that simple thiazoline natural products are tunable and
selective ligands for serotonin receptors.
RESULTS AND DISCUSSION
■
Synthesis of Pulicatin B. A simple synthetic strategy was
generated based upon the previously published method17 that
efficiently provides pulicatin derivatives with high enantiomeric
excess (ee), although in our hands ee was modest (Scheme 1).
We synthesized pulicatin B (1) in three steps from salicylic acid
and L-threonine methyl ester in 35% yield (ee 50%), enabling
us to provide 1 in gram scale for animal testing. D-Threonine
methyl esters were used in the synthesis of the enantiomer of
pulicatin B (3) (racemic). In the formation of 4,5-substituted
Δ2-thiazolines by Lawesson’s reagent, the trans-Δ2-thiazoline
dominated. Very little cis form (∼2%) was produced. Moreover,
the subsequent reduction reaction of methyl ester using NaBH4
further enriched the trans-Δ2-thiazoline isomer. Hence, the
racemic cis isomer, pulicatin A (4), could be synthesized in very
low yield (<1%), and most synthetic work focused on
generating trans isomers. This racemization can be rationalized
by a proposed reaction mechanism for Lawesson’s reagent in
related transformations.17
Effect of Thiazoline Ring Substituent Modifications
on Receptor Binding Affinity. Because of the apparent
sensitivity of the binding affinity to slight changes in thiazoline
ring configuration and substituents, we synthesized analogues
11−16 for further SAR studies (Table 1). The idea was based
on preliminary molecular modeling suggesting that the primary
OH group might bind to the 5-HT2B receptor in the same
location as the amine in serotonin (data not shown).18 All
derivatives lost the selective 5-HT2B binding observed for the
natural compounds, and instead binding to a variety of different
5-HT receptors was observed. Intriguingly, a compound linked
to histidine (11) showed a change in selectivity to the 5-HT2A
receptor at 310 nM. The most striking shift was observed for
imidazole-linked pulicatin B derivatives (13−15). For example,
13 exhibited ligand displacement at the 5-HT2B receptor at 47
nM, while the best other analogue, pulicatin B (1), exhibited
binding affinity at 130 nM. However, this increased affinity
Here, we synthesized enantiomerically enriched 1, 3, and
racemic 4 and generated an additional set of synthetic
analogues 11−30 for SAR studies (Tables 1−3). The results
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Table 2. Aromatic Ring Substituent Effects on Ligand Displacement Assays Using a Series of Serotonin Receptors, with Ki
Shown in nM
a
b
Bold: most potent binding target. Note: “>” means >10 μM. R is positioned on the following scaffold, with configurations at 1 and 2 positions as
shown.
came at the expense of selectivity, since nanomolar to low
micromolar affinity was observed for 13 at the 1B, 2A, 2B, 2C,
3, 6, and 7 receptors. By contrast, the loss of hydrophilic
functional groups at the thiazoline ring substituent (16) totally
abolished affinity. These results indicated that addition of basic
nitrogen to the pulicatin skeleton tended to decrease selectivity
in binding assays.
Effect of Aromatic Ring Substitution Modifications.
Because the natural pulicatin derivatives exhibited the greatest
selectivity, using methods optimized for synthesis of 1, we
modulated the aromatic ring substitution of 1 to generate
different aryl derivatives 17−26 (Table 2). Although all aryl
derivatives had less affinity than the natural products at the 5-
HT2B receptor, all derivatives retained binding affinity at 2B at
<10 μM except for 21 and 26. In addition, all compounds
showed affinity for at least one serotonin receptor except for
furan 21, which showed no affinity for any receptors tested at
concentrations of <10 μM. All compounds except for 24−26
retained the greatest affinity for the 5-HT2B receptor; 24−26
had the highest affinity for 5-HT2C, while 24 had preferential
binding at the 5-HT2A receptor. These results showed that the
natural aromatic ring substitutions provided the greatest
selectivity for the 5-HT2B receptor.
SAR of Imidazole Group. Because of the potent binding
affinity of imidazole derivatives 13−15, we synthesized
additional analogues 27−30 with differing aryl groups (Table
3). In this series, we observed a greater amount of the cis isomer
than in other syntheses, enabling the purification and testing of
both cis and trans racemates. The resulting derivatives had
reduced affinity for the 5-HT2B receptor compared to 13;
however, they exhibited a shift in spectrum of binding affinity.
Compounds 27 and 28, containing benzoate in place of
salicylate, had greater affinity at the 5-HT3 receptor.
Compounds 29 and 30, containing the biphenyl group,
bound preferentially at the 5-HT2A reeptor. This latter result
is reminiscent of the shift to 2A binding observed with 24−26.
In sum, modest changes to the pulicatin structure did not
improve selective binding to the 5-HT2B receptor, but instead
reduced the selectivity or altered the preferred binding partner.
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Table 3. Imidazole Derivative Binding Affinity in Ligand Displacement Assays Using a Series of Serotonin Receptors, with Ki
a
Shown in nM
a
b
Bold: most potent binding target. Note: “>” means >10 μM; “-” indicates not tested. R is positioned on the following scaffold, with configurations
at 1 and 2 positions as shown.
Scheme 1. Synthetic Strategy
By modulating pulicatins, a library of 5-HT receptor-binding
ligands with different selectivities was generated.
displacement assay binding affinities. Imidazoles 13 and 15
were more active, with 13 showing 0.8 μM antagonism in Ca-
release assays, but no activity in Tango assays. Strikingly, 24
and 25 were Tango partial agonists (50%) of the 5-HT1B
receptor at 29 and 180 nM, respectively, and antagonists at 2
μM (100%), despite their lack of ligand displacement affinity at
the same receptor at 10 μM.
Mouse in Vivo Assays. We examined the pulicatins in a
series of assays aimed to examine their potential 5-HT receptor
activity in vivo based upon some of the known activities.19,20
Functional Assays with Pulicatin Derivatives. Selected
compounds were used in functional assays against various
serotonin receptors. The experiments revealed that ligand
displacement assays with pulicatin derivatives provided results
that were grossly different than those from functional assays
(Table 4). Compounds 1 and 2 did not exhibit agonist activity
at 5-HT2B, but were antagonists at 18 and 30 μM, respectively.
These values differ by about 50−140× from the ligand
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Table 4. Summary of Receptor Antagonist Assays with Pulicatin Derivatives in Specific 5-HT Receptor Subtypes
antagonist IC50 (μM)
Tango antagonist IC50 (μM)
compound
2A
2B
2C
7A
1A
1B
4
1D
1
18
30
2
11
12
13
15
24
25
26
27
28
10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.8
6.8
>10
13
3.8
>10
>10
>10
>10
>10
>10
2.1
1.9
>10
>10
10
>10
>10
>10
The antiseizure activity of pulicatin B (1) was tested in mice at
the NIH/NINDS Epilepsy Therapy Screening program
contract site at the University of Utah. Four mice each were
used for initial screening in the maximal electroshock seizure
(MES)21,22 and 6 Hz psychomotor seizure tests23 (Table 5).
32 mA, at 44 mA the ED50s increase to >600, 1089, and 310
mg/kg, respectively (generally, well above toxic dose levels).
Thus, 1 has a somewhat favorable profile in this initial assay.
Because of the association of 5-HT2B and pain,20 we further
evaluated 1 in the tail flick and hot plate assays of acute thermal
nociception (Figure 2).25−27 By one-way ANOVA, there was
Table 5. Summary of in Vivo Epilepsy and Rotarod Minimal
a
Motor Impairment Studies
time (h)
0.25
0.5
1.0
2.0
4.0
b
test
dose (mg/kg) N/F
N/F N/F N/F N/F
6 Hz (32 mA)
6 Hz (44 mA)
MES
100
100
100
100
NT
2/4
0/4
0/4
3/4
1/4
0/4
0/4
NT
1/4
0/4
0/4
1/4
0/4
NT
0/4
motor impairment
0/4
0/4
a
Doses of 10, 30, and 100 mg/kg were used; only the highest dose
b
evaluated is shown. N/F = number of animals active or impaired over
the number tested.
Using intraperitoneal dosing using four mice in each condition,
1 was found to be inactive at doses up to 100 mg/kg in the
MES test; in the 6 Hz test at 32 mA, at the highest dose of 100
mg/kg, three of four mice were protected from seizure for 30
min, while one out of four mice was still protected after 2 h. No
acute motor impairment was observed in any of the 24 mice
used in the initial testing, as defined using the rotorod test as
previously described.24 The effect on the 6 Hz test was
evaluated by repeating the test with four mice at the 100 mg/kg
dose at 44 mA. After 0.25 h, two out of four mice were
protected, with one out of four protected at 0.5 and 1 h; no
mice were protected at the 2 or 4 h time point. No overt
impairment of neurological or motor function was observed in
the four mice on the rotorod, on which a mouse can normally
maintain its equilibrium for extended periods as the rod rotates
at a fixed speed of 6 rpm. If the animal falls off the rotorod
three times in a 1 min observation period, it is considered to
cause minimal motor impairment. These experiments indicate a
potentially modest protection in the 6 Hz test at a relatively
high, but nonimpairing, dose. In the 6 Hz test, the compound
was active at both the 32 and 44 mA current stimulation
intensities. This is significant because seizures stimulated at 44
mA are more recalcitrant to many established antiseizure drugs,
such as phenytoin, lamotrigine, and ethosuximide.23 For
example, in a previous report from the same lab that tested
our sample,23 while ethosuximide, levetiracetam, and valproic
acid exhibit ED50s of 167, 19.4, and 126 mg/kg respectively at
Figure 2. Results of compound 1 in the tail flick and hot plate assays.
not a significant effect on the latency of response at 100 mg/kg
in the tail flick assay. By contrast, a statistically significant result
was detected in the hot plate assay. These results should be
considered as an initial indication that synthetic 1 might have
antinociceptive efficacy in mice. In identical assays performed in
the same lab, antinociceptive compounds generally act at doses
in the range of ∼1 (i.e., morphine) to ∼300 (i.e., aspirin) mg/
kg.28
CONCLUSIONS
Here, we describe the synthesis and pharmacological properties
of 30 natural and synthetic pulicatin derivatives. Despite
■
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substantial attempts at synthetic optimization, no derivative
exhibited a more favorable potency/selectivity profile for the 5-
HT2B receptor than the initially identified pulicatins A (2) and
B (1), which were highly selective for the 5-HT2B receptor in
ligand binding assays. However, several ligands displayed
greater potency with a decrease in selectivity, and in several
cases we changed the selectivity from 5-HT2B to other
receptors, such as 5-HT3 or 5-HT1B. These studies define
functional groups of importance to selective binding of
serotonin receptors, which are readily apparent in Tables 1−4.
Binding values did not correlate well with results from
functional agonist/antagonist assays. Although radiolabeled
lysergic acid diethylamide was readily displaced from the
receptors, a much higher concentration of ligand was required
to antagonize the action of serotonin. Some ligands that did not
score in binding assays were active in functional assays. This
could have to do with the different kinetics or conditions in the
assays or with the known differences in binding between
serotonin and LSD.29
We sought to determine whether binding data would
translate to in vivo effects. Pulicatin B (1) was selected for
initial testing because of its selectivity and ease of synthesis and
because it was previously shown to be active in mouse
neurons.4 As a caveat for all of the work described, it is
important to note that we do not directly examine the effects of
1 on 5-HT2B in animals. We considered this of lesser
importance in this initial work because of the complex
pharmacology surrounding the aminergic receptors in vivo,
where many useful compounds are more complex than thought
from initial assays. Instead, here we sought in vivo data that
might suggest further use or abandonment of this compound
series. Surprisingly, we found that the compound 1 exhibited no
motor impairment, but was modestly effective at reducing
limbic seizures induced by 6 Hz at 32 and 44 mA. This is
potentially significant because 44 mA seizures are considered to
be more resistant to therapeutics. Moreover, initial results
suggested that 1 may be effective in reducing acute thermal
pain. By contrast, we did not achieve statistical significance for
an effect on pain in the tail flick test, and the compound was
inactive at 100 mg/kg in the MES test.
Finally, the pulicatins were initially isolated from bacteria
associated with an animal (cone snail). It would be interesting
to determine whether pulicatins or their relatives ever reach
sufficient local concentration to impact seritonergic signaling in
humans or other animals. The ready tunability of these ligands
to different 5-HT receptor subtypes would then indicate the
potential for complex interactions between microbe and host.
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV spectra were obtained
using a PerkinElmer Lambda2 UV/vis spectrometer. NMR data were
collected using either a Varian INOVA 500 (1H 500 MHz, 13C 126
MHz) NMR spectrometer with a 3 mm Nalorac MDBG probe or a
Varian INOVA 600 (1H 600 MHz, 13C 150 MHz) NMR spectrometer
equipped with a 5 mm 1H[13C,15N] triple resonance cold probe with a
z-axis gradient, utilizing residual solvent signals for referencing
(CDCl3: δH 7.26, δC 77.36; acetone-d6: δH 2.09, δC 30.60; CD3CN:
δH 1.96, δC 1.79; CD3OD: δH 3.34, δC 49.86; DMSO-d6: δH 2.54, δC
40.45). High-resolution mass spectra (HRMS) were obtained using a
Bruker APEXII FTICR mass spectrometer equipped with an actively
shielded 9.4 T superconducting magnet (Magnex Scientific Ltd.), an
external Bruker APOLLO ESI source, and a Synrad 50W CO2 CW
laser. Supelco Discover HS (4.6 × 150 mm) and semipreparative (10
× 150 mm) C18 (5 μm) columns were used for analytical and
semipreparative HPLC, respectively, as conducted on a Hitachi Elite
Lachrom System equipped with a diode array L-2455 detector. The
enantiomeric excess was calculated from the area of the HPLC peak
using a silica-based protein phase enantiomer separation column,
RESOLVOSIL BSA-7, eluting with 0.1 M phosphate buffer pH 7.5 and
2% 1-propanol. Unless stated otherwise, all reagents and solvents were
purchased from commercial suppliers, and all reactions were carried
out under anhydrous conditions with an argon atmosphere. Yields
1
were calculated by HPLC or H NMR spectroscopy.
Representative Procedures. Methyl N-(2-hydroxybenzoyl)-L-
threoninate (1A). Salicylic acid (138 mg, 1 mmol) was dissolved in
CH2Cl2 (15 mL). To the solution were added in order ethylene
dichloride (EDC) (178 μL), Et3N (140 μL), and L-threonine methyl
ester hydrochloride (169 mg, 1 mmol). The mixture was stirred for 18
h at room temperature (rt). The mixture was concentrated by rotary
evaporation at 25 °C. To the residue were added H2O (15 mL) and
EtOAc (20 mL). The EtOAc layer was then washed with brine, dried
over CaCl2, and evaporated to dryness to afford the desired amide
(1A, 200 mg, 79%). Spectroscopic data agreed with published
results.34
Heterocyclization of 1A. Compound 1A (200 mg) and 2,4-bis(p-
methoxyphenyl)-1,3,2,4-dithiaphosphetane 2,4-disulfide (320 mg, 1
equiv) were dissolved in anhydrous toluene (150 mL). The solution
was heated to reflux under an argon atmosphere for 18 h. After
removal of the solvent, the residue was purified by silica gel column
chromatography with hexane−EtOAc (25:1) to yield 1B (100 mg,
50%): 1H NMR (500 MHz, CDCl3) δH 7.42 (1H, d, J = 7.9 Hz), 7.38
(1H, dd, J = 7.9, 7.9 Hz), 7.04 (1H, d, J = 7.9 Hz), 6.88 (1H, dd, J =
7.9, 7.9 Hz), 5.21 (1H, d, J = 7.7 Hz), 4.22 (1H, dq, J = 7.7, 6.9 Hz),
3.84 (3H, s), 1.31 (3H, d, J = 6.9 Hz); 13C NMR (125 MHz, CDCl3)
δC 174.5, 159.6, 134.0, 130.9, 119.2, 117.7, 79.8, 52.7, 45.9, 18.3;
ESIMS m/z 252 [M + H]+.
Methyl Ester Reduction of 1B. Compound 1B (100 mg) was
dissolved in methanol (70 mL) with stirring at rt, and NaBH4 (150
mg) was added to the solution. After 1 min, the reaction was quenched
with trifluoroacetic acid (TFA), and the solvent was removed by rotary
evaporation. The residue was extracted with H2O (20 mL) and CHCl3
(40 mL). The organic layer was dried to yield compound 1 (80 mg,
90%). Spectroscopic data agreed with natural pulicatin B.4
5-HT2B is especially important in hyperalgesia and
neuralgia.30 These effects are mediated largely through pain
sensory neurons in the GI and in the dorsal horn. Therefore,
we performed preliminary testing to demonstrate that
pulicatins may be effective at reducing acute thermal pain.
Both the tail flick and the hot-plate tests evaluate in vivo
sensitivity to acute heat-induced pain stimuli.31 The tail flick is a
spinally mediated flexor withdrawal reflex, whereas the hot plate
test is supraspinally mediated. This may explain why the
compound is more effective in one model compared to the
other.32
The major application of 5-HT2B antagonists is in chronic
pain conditions, where the 5-HT2B receptor is greatly
upregulated.33 It may be that pulicatin B (1) is more effective
at a lower dose under this condition, where 5-HT2B is one of
the dominant determinants of pain. The lack of observed
impairment caused by 1 and the simplicity of synthesis are
promising in this regard. Because of the in vivo activity observed
in this initial study, in future work, we plan to test these in
spinal nerve ligation models, where the upregulation of 5-HT2B
may be more important in mediating pain, and a significant
effect might be clearer.
For the synthesis of pulicatin A, the cis isomer of compound 1B (6
mg) was dissolved in precooled MeOH (−10 °C, 2 mL), and NaBH4
(15 mg) was added to the solution. After 10 s, the reaction was
quenched with TFA, and the solvent was removed by rotary
evaporation to yield compound 2 (2.1 mg, 39%). Spectroscopic data
agreed with natural pulicatin A.4
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1
Preparation of Compounds 11 and 12. Compound 1B (trans:cis
2:1, 5 mg) was dissolved in 10% LiOH solution in H2O−MeOH (1:1,
2 mL). The mixture was stirred for 4 h at rt. The MeOH was removed
under vacuum, EtOAc (4 mL) was added, and the organic layer was
concentrated. After drying overnight on a lyophylizer, the residue
containing 11D was dissolved in anhydrous CH2Cl2 (4 mL). After
adding L-histamine methyl ester (3.5 mg), EDC (8 μL), and Et3N (3
μL), the mixture was stirred for 18 h at rt. The solution was
concentrated by rotary evaporation at 25 °C. To the residue were
added H2O (5 mL) and EtOAc (10 mL), and the resulting suspension
was mixed well. The EtOAc layer was then washed with brine, dried
over CaCl2, and then evaporated to dryness to afford the desired
amide mixture (11 and 12), which was further purified by C18 HPLC
with 40% MeCN in H2O to obtain the pure isomers 11 (trans, 2 mg,
40%) and 12 (cis, 1.0 mg, 40%).
organic layer was dried to yield compound 13I (143.5 mg, 70%): H
NMR (600 MHz, CD3OD) δH 7.84 (1H, d, J = 8.1 Hz), 7.32 (1H, m),
6.85 (1H, d, J = 8.1 Hz), 6.78 (1H, dd, J = 8.1, 8.0 Hz), 4.49 (1H, m),
4.01 (1H, m), 3.33 (1H, m), 3.15 (1H, m), 1.26 (3H, d, J = 6.5 Hz);
key HSQC (600 MHz, CD3OD) δC 136.7 (CH), 130.8 (CH), 121.5
(CH), 120.4 (CH), 71.9 (CH), 57.7 (CH), 27.6 (CH2), 22.5 (CH3);
ESIMS m/z 276 [M + H]+.
Preparation of Compound 13. Compound 13I (143 mg) and 2,4-
bis(p-methoxyphenyl)-1,3,2,4-dithiaphosphetane 2,4-disulfide (210
mg, 1 equiv) were dissolved in anhydrous toluene (150 mL). The
solution was heated to reflux under an argon atmosphere for 18 h.
After removal of the solvent, the residue was purified by a silica gel
column with hexane−EtOAc (20:1) followed by C18 HPLC (35%
1
MeCN in H2O with 1% TFA) to yield 13 (5 mg, 3.5%): H NMR
(600 MHz, DMSO-d6) δH 8.90 (1H, brs), 7.83 (1H, brs), 7.73−7.76
(2H, m), 7.25−7.28 (2H, m), 5.21 (1H, brs), 4.28 (1H, brs), 3.62
(1H, dd, J = 11.0, 3.4 Hz), 3.52 (1H, dd, J = 11.0, 6.4 Hz), 1.79 (3H, d,
J = 6.7 Hz); 13C NMR (126 MHz, acetone-d6) δC 171.3, 159.1, 133.9,
133.2, 130.9, 130.6, 118.9, 117.0, 116.8, 116.2, 81.2, 48.0, 28.6, 21.2;
HRESIMS m/z 274.1004 [M + H]+ (calcd for C14H15N3OS,
274.1014).
Preparation of Compound 14. L-Histidinol dihydrochloride (214
mg) and aspirin acyl chloride (198 mg) were suspended in anhydrous
pyridine (100 mL). The mixture was stirred for 10 h at rt and dried by
rotary evaporation followed by lyophilizing overnight. The residue was
directly subjected to the heterocyclization described in the preparation
1
11D: H NMR (500 MHz, CDCl3) δH 7.47 (brs 1H), 7.44 (d, J =
7.8 HZ, 1H), 7.16 (brs, 1H), 6.89 (dd, J = 7.8, 7.5 Hz), 5.05 (brs 1H),
4.28 (brs, 1H), 1.61 (d, J = 6.0 Hz); ESIMS m/z 238 [M + H]+.
1
11: H NMR (600 MHz, acetone-d6) δH 8.91 (1H, brs), 8.36 (1H,
d, J = 8.3 Hz), 7.53 (1H, brs), 7.46−7.48 (2H, m), 6.96−7.01 (2H, m),
5.03 (1H, d, J = 5.7 Hz), 4.92 (1H, m), 4.30 (1H, dq, J = 7.0, 5.6 Hz),
3.77 (3H, s), 3.50 (1H, dd, J = 16.0, 4.3 Hz), 3.29 (1H, dd, J = 16.0,
9.5 Hz), 1.55 (3H, d, J = 6.8 Hz); HSQC (600 MHz, acetone-d6) δC
135.1 (CH), 132.5 (CH), 120.7 (CH), 118.6 (CH), 85.6 (CH), 53.7
(CH3), 53.4 (CH3), 48.5 (CH), 27.7 (CH2), 22.4 (CH3); HRESIMS
m/z 389.1268 [M + H]+ (calcd for C18H20N4O4S, 389.1278).
1
12: H NMR (600 MHz, acetone-d6) δH 8.89 (1H, brs), 8.30 (1H,
1
of 1B to yield compound 14 (4.6 mg 1.7%): H NMR (600 MHz,
d, J = 8.3 Hz), 7.57 (1H, brs), 7.45−7.48 (2H, m), 6.96−7.00 (2H, m),
5.05 (1H, d, J = 5.9 Hz), 4.90 (1H, m), 4.33 (1H, dq, J = 7.0, 5.9 Hz),
3.73 (3H, s), 3.49 (1H, dd, J = 16.0, 4.3 Hz), 3.35 (1H, dd, J = 16.0,
9.5 Hz), 1.53 (3H, d, J = 6.8 Hz); HSQC (600 MHz, acetone-d6) δC
135.4 (CH), 132.6 (CH), 120.9 (CH), 118.9 (CH), 85.6 (CH), 53.7
(CH3), 53.6 (CH3), 48.8 (CH), 28.0 (CH2), 22.9 (CH3); HRESIMS
m/z 389.1268 [M + H]+ (calcd for C18H20N4O4S, 389.1278).
Preparation of Compound 13F. L-Histidine (15.5 g) was dissolved
in anhydrous pyridine (100 mL), and Ac2O (15 mL) was added (note:
in the preparation of compound 15, benzoyl chloride was used instead
of Ac2O) into the solution under stirring in an ice bath. The mixture
was heated to reflux for 2 h. The reaction was quenched with 20%
Na2CO3 (100 mL), and pyridine was removed by rotary evaporation.
The residue was partitioned with EtOAc (200 mL) to yield 13F (19.0
g, 80%): 1H NMR (500 MHz, CD3OD) δH 7.74 (1H, s), 6.93 (1H, s),
4.65 (1H, dd, J = 8.5, 5.2 Hz), 3.12 (1H, dd, J = 15.1, 5.2 Hz), 2.92
(1H, dd, J = 15.1, 8.5 Hz), 2.16 (3H, s), 1.97 (6H, s); 13C NMR (126
MHz, CD3OD) δC 207.1, 171.7, 134.9, 133.1, 116.7, 59.0, 27.2, 26.0,
21.0; ESIMS m/z 196 [M + H]+.
DMSO-d6) δH 9.03 (1H, brs), 7.53 (1H, brs), 7.42 (2H, m), 6.95 (2H,
m), 5.05 (1H, m), 3.26 (1H, dd, J = 11.5. 7.2 Hz), 3.16 (1H, m), 3.10
(2H, m); 13C NMR (126 MHz, acetone-d6) δC 171.9, 158.9, 133.9,
133.3, 130.9, 130.6, 119.0, 117.1, 116.8, 116.1, 110.0, 74.9, 35.3;
HRESIMS m/z 260.0846 [M + H]+ (calcd for C13H13N3OS,
260.0858).
Compound 15: 1H NMR (500 MHz, acetone-d6) δH (cis) 8.88 (1H,
s), 7.31−7.55 (8H, m), 7.00 (2H, m), 5.40 (1H, brs), 5.39 (1H, brs),
3.01 (1H, m), 2.93 (1H, m); key HSQC (500 MHz, acetone-d6) δC
1
78.1 (CH), 54.7 (CH), 26.8 (CH2); (trans) H NMR (500 MHz,
acetone-d6) δH 8.88 (1H, s), 7.31−7.55 (8H, m), 7.00 (2H, m), 5.29
(1H, brs), 5.13 (1H, brs), 3.43 (2H, m); key HSQC (500 MHz,
acetone-d6) δC 82.6 (CH), 56.3 (CH), 38.6 (CH2); 13C NMR (125
MHz, acetone-d6) δC 171.7, 158.9, 140.8, 137.4, 133.5, 130.6, 128.9,
128.6, 128.5, 128.3, 128.0, 127.4, 119.1, 119.1, 116.9, 116.9, 115.9,
115.9, 82.6, 78.1, 56.3, 54.7, 28.6, 26.8, 18.0, 16.2; HRESIMS m/z
336.1160 [M + H]+ (calcd for C19H17N3OS, 336.1165).
The same procedure was used to synthesize compound 16: 1H
NMR (500 MHz, CDCl3) δH 7.45 (1H, dd, J = 7.9, 7.9 Hz), 7.40 (1H,
d, J = 7.9 Hz), 7.16 (1H, d, J = 7.9 Hz), 6.90 (1H, dd, J = 7.9, 7.9 Hz),
5.31 (1H, ddd, J = 8.6, 8.6, 2.8 Hz), 4.34 (1H, ddd, J = 8.3, 8.3, 3.3
Hz), 2.14 (3H, m), 1.96 (1H, m), 1.78 (2H. m); HSQC (500 MHz,
CDCl3) δC 135.6 (CH), 131.2 (CH), 117.9 (CH), 119.6 (CH), 78.7
(CH), 49.4 (CH), 34.2 (CH2), 36.7 (CH2), 23.4 (CH2); HRESIMS
m/z 220.0784 [M + H]+ (calcd for C12H13NOS, 220.0796).
Preparation of Compound 13G. Compound 13F (19.0 g) was
dissolved in 6 M HCl (100 mL), and the solution was heated to reflux
for 12 h. The mixture was dried under blowing air to yield compound
1
13G (12.0 g, 99%): H NMR (500 MHz, CD3OD) δH 9.04 (1H, s),
7.67 (1H, s), 4.71 (1H, m), 3.72 (1H, dd, J = 15.1, 5.2 Hz), 3.39 (1H,
dd, J = 15.1, 8.5 Hz), 2.47 (3H, s); 13C NMR (126 MHz, CD3OD) δC
202.1, 134.8, 126.8, 118.8, 58.1, 26.3, 24.3; ESIMS m/z 154 [M + H]+.
Preparation of Compound 13H. Compound 13G (153 mg) was
dissolved in anhydrous pyridine (50 mL). The solution was cooled in
an ice bath. Aspirin acyl chloride (198 mg) was added to the cooled
solution, and the mixture was stirred for 2 h in an ice bath. The
reaction was quenched by adding H2O (10 mL). The pyridine was
removed by rotary evaporation. The residue was purified by a silica gel
column with hexane−EtOAc (20:1) to yield compound 13H (204 mg,
75%): 1H NMR (500 MHz, DMSO-d6) δH 7.90 (1H, s), 7.86 (1H, d, J
= 8.6 Hz), 7.41 (1H, dd, J = 8.6, 8.6 Hz), 7.11 (1H, s), 6.90 (2H, m),
4.86 (1H, m), 3.24 (2H, brs), 2.17 (3H, s); 13C NMR (125 MHz,
DMSO-d6) δC 205.7, 171.2, 161.3, 136.6, 135.2, 134.8, 134.2, 127.3,
119.1, 118.7, 115.9, 60.7, 28.1, 27.4; ESIMS m/z 274 [M + H]+.
Preparation of Compound 13I. Compound 13H (204 mg) was
dissolved in MeOH (10 mL), NaBH4 (85 mg) was added to the
solution, and the mixture was stirred for 1 min and then quenched by
1% TFA in H2O (10 mL). The MeOH was removed by rotary
evaporation. The residue was washed with EtOAc (40 mL), and the
Compound 17: 1H NMR (500 MHz, acetone-d6) δH 7.89 (2H, d, J
= 7.5), 7.58 (1H, dd, J = 7.7, 6.7), 7.49 (2H, dd, J = 8.0, 7.7), 4.48 (1H,
m), 4.17 (1H, m), 3.81 (1H, dd, J = 11.0, 4.9 Hz), 3.69 (1H, dd, J =
11.0, 6.7 Hz), 1.48 (3H, d, J = 6.7 Hz); 13C NMR (126 MHz, acetone-
d6) δC 169.7, 132.5, 131.3, 128.7, 128.5, 84.9, 61.7, 46.8, 21.7;
HRESIMS m/z 208.0786 [M + H]+ (calcd for C13H14N2OS,
208.0791).
Compound 18: (mixture of cis and trans isomers, inseparable by C18
HPLC) 1H NMR (500 MHz, acetone-d6) δH (trans-18) 7.42 (1H, dd,
J = 7.8, 1.4 Hz), 7.32 (1H, dd, J = 7.7, 7.4 Hz), 6.72 (1H, d, J = 8.4),
6.61 (H, m), 4.47 (1H, m), 3.74 (1H, dd, J = 11.0, 4.8 Hz), 3.57 (1H,
dd, J = 11.0, 7.0 Hz), 3.91 (1H, m), 1.42 (3H, d, J = 6.7 Hz); (cis-18)
δH 7.48 (1H, dd, J = 7.8, 1.4 Hz), 7.32 (1H, dd, J = 7.7, 7.4 Hz), 6.72
(1H, d, J = 8.4), 6.61 (H, m), 4.47 (1H, m), 4.20 (1H, dd, J = 11.0, 6.5
Hz), 3.93 (1H, m), 3.96 (1H, m), 1.27 (3H, d, J = 6.7 Hz); 13C NMR
(126 MHz, acetone-d6) δC (trans-18) 168.2, 149.3, 132.1, 114.1, 110.0,
86.5, 62.3, 44.3, 21.7; (cis-18) 168.7, 149.3, 132.4, 114.1, 110.0, 81.5,
2367
DOI: 10.1021/acs.jnatprod.7b00317
J. Nat. Prod. 2017, 80, 2360−2370

Journal of Natural Products
Article
60.8, 44.8, 16.6; HRESIMS m/z 237.1050 [M + H]+ (calcd for
C12H16N2OS, 237.1062).
46.8, 31.2, 21.4, 19.2, 13.8; HRESIMS m/z 280.1360 [M + H]+ (calcd
for C15H21NO2S, 280.1366).
1
Compound 19: 1H NMR (500 MHz, CDCl3) δH 7.95 (1H, s), 7.74
(1H, dd, J = 7.7, 6.7 Hz), 7.68 (1H, d, J = 8.0 Hz), 7.47 (1H, dd, J =
7.7, 6.7 Hz), 7.31 (1H, s), 7.29 (1H, d, J = 8.0 Hz), 4.50 (1H, m), 4.05
(1H, m), 3.92 (1H, dd, J = 12.0, 5.2 Hz), 3.84 (1H, dd, J = 2.0, 4.4
Hz), 1.54 (3H, d, J = 6.7 Hz); 13C NMR (126 MHz, CDCl3) δ 175.3,
158.0, 114.1, 138.9, 128.8, 131.0, 126.1, 131.3, 130.0, 135.5, 121.1,
87.5, 65.7, 48.3, 24.4; HRESIMS m/z 274.1004 [M + H]+ (calcd for
C15H15NO2S, 274.1014).
Compound 25: H NMR (500 MHz, CDCl3) δH 7.68 (2H, d, J =
8.6 Hz), 6.84 (2H, d, J = 8.6 Hz), 4.29 (1H, m), 3.95 (2H, t, J = 6.9
Hz), 3.88 (1H, m), 3.79 (1H, m), 3.67 (1H, m), 1.76 (2H, m), 1.41
(3H, d, J = 6.7 Hz), 1.45−1.25 (6H, m), 0.89 (3H, t, J = 7.5 Hz);
HSQC (500 MHz, CDCl3) δC 129.9 (CH), 113.9 (CH), 86.1 (CH),
68.7 (CH2), 65.1 (CH2), 46.4 (CH), 28.6 (CH2), 27.5 (CH2), 26.7
(CH2), 21.2 (CH3), 14.9, (CH3); 13C NMR (126 MHz, CDCl3) δC
169.2, 162.6, 130.8, 126.1, 115.1, 86.6, 69.1, 64.2, 47.6, 32.4, 30.0, 26.5,
23.5, 22.2, 14.9; HRESIMS m/z 308.1671 [M + H]+ (calcd for
C17H25NO2S, 308.1679).
1
Compound 20: H NMR (500 MHz, CD3CN) δH 11.04 (1H, brs,
NH), 8.61 (1H, brs), 8.00 (1H, m), 7.66 (1H, m), 7.39 (2H, m), 4.35
(1H, dd, J = 10.4, 5.2 Hz), 4.23 (1H, dq, J = 7.4, 6.6 Hz), 3.94 (1H, dd,
J = 12.6, 3.9 Hz), 3.79 (1H, dd, J = 12.6, 5.0 Hz), 1.54 (3H, d, J = 6.7
Hz); 1H NMR (500 MHz, CDCl3) δH 9.00 (1H, brs, NH), 8.26 (1H,
d, J = 7.4 Hz), 7.68 (1H, s), 7.40 (1H, d, J = 7.4 Hz), 7.27 (2H, m),
4.40 (1H, dd, J = 11.2, 5.8 Hz), 3.90 (2H, m), 3.76 (1H, dd, J = 11.2,
5.7 Hz), 1.50 (3H, d, J = 6.6 Hz); HSQC (500 MHz, CDCl) δ 128.9
(CH), 123.3 (CH), 121.8 (CH), 121.6 (CH), 111.4 (CH), 85.2 (CH),
63.7 (CH2), 46.4 (CH), 21.2 (CH3); HRESIMS m/z 247.0895 [M +
H]+ (calcd for C13H14N2OS, 247.0900).
1
Compound 26: H NMR (500 MHz, CDCl3) δH 7.70 (2H, d, J =
8.6 Hz), 6.85 (2H, d, J = 8.6 Hz), 4.31 (1H, m), 3.97 (2H, t, J = 6.9
Hz), 3.84 (1H, m), 3.69 (1H, m), 3.67 (1H, m), 1.78 (2H, m), 1.46
(2H, m), 1.44 (3H, d, J = 6.7 Hz), 1.38−1.23 (p8H, m), 0.89 (3H, t, J
= 7.5 Hz); HSQC (500 MHz, CDCl3) δC 130.0 (CH), 114.1 (CH),
86.3 (CH), 68.3 (CH2); 63.8 (CH2), 46.6 (CH), 28.1 (CH2), 29.7
(CH2), 26.7 (CH2), 20.1 (CH3), 15.9 (CH3); 13C NMR (125 MHz,
CDCl3) δC 168.2, 161.7, 129.9, 125.8, 114.3, 85.9, 76.8, 68.2, 63.5,
46.8, 31.8, 29.3, 29.2, 29.1, 26.0, 22.6, 21.4, 14.0; HRESIMS m/z
336.1983 [M + H]+ (calcd for C19H29NO2S, 336.1992).
1
Compound 21: H NMR (500 MHz, CD3CN) δH 7.88 (1H, s),
1
Compound 27: H NMR (600 MHz, acetone-d6) δH 9.00 (1H,
7.54 (1H, s), 6.77 (1H, brs), 4.50 (1H, brs), 4.23 (1H, brs), 3.97 (2H,
brs), 1.55 (3H, d, J = 6.9 Hz); HRESIMS m/z 198.0579 [M + H]+
(calcd for C9H11NO2S, 198.0583).
brs), 7.89 (2H, m), 7.67 (1H brs), 7.60 (1H, m), 7.52 (2H, m), 4.75
(1H, brs), 4.04 (1H, brs), 3.35 (1H, m), 3.21 (1H, m), 1.52 (3H, brs);
key HSQC (600 MHz, acetone-d6) δC 83.1 (CH), 51.3 (CH), 29.7,
(CH2), 22.3 (CH3); HRESIMS m/z 258.1052 [M + H]+ (calcd for
C14H15N3OS, 258.1059).
In the structure identification of compound 21, we observed that
1
the H NMR spectrum of 21 gave very broad proton signals, even
though we tried four different NMR solvents (CDCl3, CD3OD,
DMSO-d6, and CD3CN). This suggested that the 21 presented as
slowly rotating isomers through the single bond between the furan ring
and the thiazoline ring. In order to stabilize the rotamers of 21, we
synthesized the p-bromobenzoyl ester of compound 21. Indeed, the
1H NMR spectrum of the p-bromobenzoyl ester of compound 21 gave
a single well-resolved set of peaks. Compound 21 (2 mg) was
dissolved in anhydrous pyridine (1 mL), one equivalent of p-
bromobenzoyl chloride was add to the solution, and reaction was
quenched by adding H2O (1 mL) after 4 h. The mixture was extracted
with EtOAc (2 mL) and purified by C18 HPLC (55% MeCN in water)
to yield the p-bromobenzoyl ester of compound 21 (1.8 mg).
1
Compound 28: H NMR (600 MHz, acetone-d6) δH 8.97 (1H,
brs), 7.89 (2H, m), 7.69 (1H brs), 7.57 (1H, m), 7.51 (2H, m), 4.75
(1H, brs), 4.04 (1H, brs), 3.35 (1H, m), 3.21 (1H, m), 1.52 (3H, brs);
key HSQC (600 MHz, acetone-d6) δC 79.1 (CH), 49.8 (CH), 26.9
(CH2), 17.9 (CH3); HRESIMS m/z 258.1052 [M + H]+ (calcd for
C14H15N3OS, 258.1059).
Compound 29: 1H NMR (600 MHz, DMSO-d6) δH 9.05 (1H, brs),
7.88 (2H, d, J = 8.7 Hz), 7.82 (2H, d, J = 8.7 Hz), 7.76 (2H, d, J = 7.7
Hz), 7.59 (1H, brs), 7.54 (2H, t, J = 7.7 Hz), 7.46 (1H, t, J = 7.7 Hz),
4.65 (1H, m), 3.92 (1H, m), 3.07 (1H, dd, J = 15.0, 6.0 Hz), 2.98 (1H,
dd, J = 15.0, 8.3 Hz), 1.41 (3H, d, J = 6.8 Hz); 13C NMR (126 MHz,
acetone-d6) δC 166.5, 143.9, 139.8, 133.6, 132.2, 131.2, 129.0 (2 × C),
128.9 (2 × C), 128.0 (2 × C), 127.0 (4 × C), 117.1, 82.6, 49.9, 28.6,
21.0; HRESIMS m/z 334.1363 [M + H]+ (calcd for C20H19N3S,
334.1378).
1
p-Bromobenzoyl ester of compound 21: H NMR (500 MHz,
CD3CN) δH 7.91 (2H, d, J = 8.1), 7.59 (2H, d, J = 8.1), 7.56 (1H, s),
6.94 (1H, s), 6.52 (1H, brs), 4.66 (1H, m), 4.58 (1H, m), 4.43 (1H,
1
m), 3.98 (1H, m), 1.51 (3H, d, J = 6.8 Hz); H NMR (500 MHz,
1
Compound 30: H NMR (500 MHz, acetone-d6) δH 8.97 (1H,
CDCl3) δH 7.93 (2H, d, J = 7.8), 7.60 (2H, d, J = 7.8), 7.57 (1H, s),
6.94 (1H, d, J = 3.5 Hz), 6.53 (1H, brs), 4.68 (1H, m), 4.59 (1H, dd, J
= 11.7, 4.7 Hz), 4.44 (1H, dd, J = 11.7, 6.5 Hz), 3.99 (1H, m), 1.52
(3H, d, J = 6.8 Hz); HSQC (500 MHz, CDCl3) δC 145.1 (CH), 131.9
× 2 (CH), 131.2 × 2 (CH), 114.5 (CH), 111.9 (CH), 82.3 (CH), 65.2
(CH2), 47.8 (CH), 22.4 (CH3); ESIMS m/z 380 [M + H]+.
brs), 7.99 (2H, d, J = 8.7 Hz), 7.78 (2H, d, J = 8.7 Hz), 7.74 (2H, d, J
= 7.7 Hz), 7.66 (1H, brs), 7.52 (2H, t, J = 7.7 Hz), 7.44 (1H, t, J = 7.7
Hz), 4.72 (1H, m), 4.25 (1H, m), 3.44 (1H, m), 3.38 (1H, m), 1.37
(3H, d, J = 6.8 Hz); 13C NMR (126 MHz, acetone-d6) δC 166.9, 143.9,
139.8, 133.6, 132.4, 129.0, 128.8, 128.0, 127.0, 116.8, 77.8, 48.4, 25.3,
16.2; HRESIMS m/z 334.1363 [M + H]+ (calcd for C20H19N3S,
334.1378).
1
Compound 22: H NMR (500 MHz, CDCl3) δH 7.76 (1H, d, J =
2.8 Hz), 7.43 (1H, d, J = 5.3 Hz), 7.28 (1H, dd, J = 5.3, 2.8 Hz), 4.28
(1H, m), 3.98 (1H, m), 3.93 (1H, dd, J = 11.5, 4.8 Hz), 3.69 (1H, dd, J
= 11.5, 5.1 Hz), 1.44 (3H, d, J = 7.1 Hz); 13C NMR (126 MHz,
CDCl3) δC 162.5, 136.6, 131.1, 130.1, 127.6, 85.2, 62.7, 47.7, 21.3;
HRESIMS m/z 214.0350 [M + H]+ (calcd for C9H11NOS2, 214.0355).
PDSP Screening. Percentage inhibition studies, Ki determinations,
and agonist and/or antagonist functional data were performed by the
National Institute of Mental Health’s Psychoactive Drug Screening
Program (PDSP). For experimental details refer to the PDSP Web
site: http://pdsp.med.unc.edu/.
In Vivo Assays. Animal Protocol. This work was performed under
IACUC protocol number 15-10007. NIH guidelines for animal
research were followed.
1
Compound 23: H NMR (500 MHz, CDCl3) δH 7.76 (1H, d, J =
2.8 Hz), 7.43 (1H, d, J = 5.3 Hz), 7.28 (1H, dd, J = 5.3, 2.8 Hz), 4.28
(1H, m), 3.98 (1H, m), 3.93 (1H, dd, J = 11.5, 4.8 Hz), 3.69 (1H, dd, J
= 11.5, 5.1 Hz), 1.44 (3H, d, J = 7.1 Hz); 13C NMR (126 MHz,
CDCl3) δC 164.8, 135.1, 129.3, 126.8, 126.4, 84.8, 62.7, 46.9, 21.2;
HRESIMS m/z 214.0350 [M + H]+ (calcd for C9H11NOS2, 214.0355).
Synthetic 1 was suspended in 30% polyethylene glycol 400 with a
few drops of DMSO. Compound 1 was administered to mice in a
volume of 0.01 mL/g of body weight via the i.p. route.
1
Compound 24: H NMR (500 MHz, CDCl3) δH 7.69 (2H, d, J =
Maximal Electroshock Test. For the MES, a drop of anesthetic and
electrolyte solution (0.5% tetracaine hydrochloride in 0.9% saline) was
applied to the eyes of each animal prior to corneal stimulation. The
electrical stimulus in the MES test (50 mA, 60 Hz, 0.2 s) was delivered
via corneal electrodes by an apparatus similar to that originally
described by Woodbury and Davenport.19 Abolition of the hind leg
tonic extensor component is taken as the end point for this test.
Efficacy on four mice was compared by evaluating the following i.p.
8.6 Hz), 6.84 (2H, d, J = 8.6 Hz), 4.30 (1H, ddd, J = 5.9, 5.8, 5.2 Hz),
3.97 (2H, t, J = 6.9 Hz), 3.89 (1H, dq, J = 6.6, 5.6 Hz), 3.83 (1H, m),
3.68 (1H, m), 1.76 (2H, m), 1.48 (2H, m), 1.45 (3H, d, J = 6.7 Hz),
0.96 (3H, t, J = 7.5 Hz); HSQC (500 MHz, CDCl3) δC 129.9 (CH),
114.2 (CH), 85.9 (CH), 67.9 (CH2), 63.5 (CH2), 46.8 (CH), 31.1
(CH2), 21.4 (CH3), 19.3, (CH2), 13.8 (CH3); 13C NMR (126 MHz,
CDCl3) δC 168.3, 161.7, 129.9 × 2, 125.7, 114.3 × 2, 85.8, 67.9, 63.5,
2368
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doses of 1: 100, 30, and 10 mg/kg at various time points (0.25, 0.5, 1,
2, and 4 h).
Officer J. Driscoll at NIMH, Bethesda, MD, USA. We thank the
NIH/NINDS Epilepsy Therapy Screening Program (ETSP) for
the epilepsy model testing performed.
6 Hz “Psychomotor” Seizure Test. The drug administration
approach employed in the 6 Hz test was identical to that described
above for the MES test. Mice were pretreated with 1 at the doses listed
above. At varying times (1/4, 1/2, 1, 2, and 4 h after treatment)
individual mice (four at each time point) were challenged with
sufficient current, 32 and 44 mA for 3 s, delivered through corneal
electrodes to elicit a partial psychomotor seizure.35 Data were
expressed as the number of mice protected out of the number of
animals tested over time.
Tail Flick Assay. Thermal pain sensitivity was measured using the
IITC Series 8 (model 336TG) automated plantar/tail analgesic meter
(IITC Life Science). Mice were placed in a restraint tube, and a light
beam (peak intensity 75 mJ, 50% maximal intensity) was focused on
the distal tail, 2−5 mm from the tip. Control mice received an
equivalent volume of vehicle at the same time of peak effect (TPE) for
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ACKNOWLEDGMENTS
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This research was supported by NIH R01GM107557 and
Fogarty International Center of the National Institutes of
Health under Award No. U19TW008163. The content is solely
the responsibility of the authors and does not necessarily
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