Identification of 5-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve Memory and Cognition

Article abstract of DOI:10.1021/acschemneuro.7b00282

Initial work in Drosophila and mice demonstrated that the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a master control gene for memory formation. The relationship between CREB and memory has also been found to be true in other species, including aplysia and rats. It is thus well-established that CREB activation plays a central role in memory enhancement and that CREB is activated during memory formation. On the basis of these findings, a phenotypic high-throughput screening campaign utilizing a CRE-luciferase (CRE-Luci) SK-N-MC cell line was performed to identify compounds that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin. A number of small-molecule hits of unknown mechanisms of action were identified in the screening campaign, including HT-0411. Follow-up studies suggested that the CREB activation by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-B). Further, HT-0411 was shown to improve 24 h memory in rodents in a contextual fear conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel, potent, and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of this and other related analogue series identified a number of potential clinical development candidates; ultimately, compound 8f was identified as a candidate molecule with high selectivity toward MAO-B (29-56 nM) over MAO-A (19% inhibition at a screening concentration of 50 μM), an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic properties in rodents and dogs, and efficacy in multiple rodent memory models.

Full text of DOI:10.1021/acschemneuro.7b00282

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Article
Identification of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-
yl)thiophene-2-Carboxamides as Novel and Selective Monoamine
Oxidase B Inhibitors Used to Improve Memory and Cognition
Alan Kaplan, Terence Keenan, Roderick Scott, Xianbo Zhou, Rusiko Bourchouladze,
Andrew J. McRiner, Mark E. Wilson, Darlene Romashko, Regina Miller, Matthew
Bletsch, Gary Anderson, Jennifer Stanley, Adia Zhang, Dong Lee, and John Nikpur
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00282 • Publication Date (Web): 31 Aug 2017
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Identification of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-
Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve
Memory and Cognition
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Authors:
Alan P. Kaplan,*1 Terence Keenan,1 Roderick Scott,1 Xianbo Zhou,2 Rusiko Bourchouladze,†
Andrew J. McRiner,3 Mark E. Wilson,1 Darlene Romashko,4 Regina Miller,5 Matthew Bletsch,6
Gary Anderson,1 Jennifer Stanley,1 Adia Zhang,1 Dong Lee,1 John Nikpur1
Author affiliation:
1 Dart NeuroScience, LLC, 12278 Scripps Summit Drive, San Diego, CA 92131, U.S.A.
2 SJN Biomed LTD, 398 West 2nd Ring Road, Kunming, 650118, China
3 X-Chem Pharmaceuticals, Inc., 100 Beaver Street, Suite 101, Waltham, MA 02453, U.S.A.
4 Aset Therapeutics, 25 Health Sciences Drive, Stony Brook, New York 11790, U.S.A.
5 Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492, U.S.A.
6 The Hain Celestial Group, 1111 Marcus Ave, New Hyde Park, NY 11042, U.S.A.
†
Deceased, August 6, 2011
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ABSTRACT
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Initial work in Drosophila and mice demonstrated that the transcription factor cAMP (cyclic
adenosine monophosphate) response element binding protein (CREB) is a master control gene
for memory formation. The relationship between CREB and memory has also been found to be
true in other species, including aplysia and rats. It is thus well established that CREB activation
plays a central role in memory enhancement and that CREB is activated during memory
formation. Based on these findings, a phenotypic high throughput screening campaign, which
utilized a CRE-luciferase (CRE-Luci) SK-N-MC cell line, was performed to identify compounds
that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin.
A number of small-molecule hits of unknown mechanisms of action were identified in the
screening campaign, including HT-0411. Follow-on studies suggested that the CREB activation
by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-
B). Further, HT-0411 was shown to improve 24-hour memory in rodents in a contextual fear
conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-
(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel,
potent and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral
evaluations of this and other related analog series identified a number of potential clinical
development candidates; ultimately, compound 8f was identified as a candidate molecule with
high selectivity towards MAO-B (29-56 nM) over MAO-A (no inhibition at 10 μM), an excellent
profile against a panel of other enzymes and receptors, good pharmacokinetic properties in
rodents and dogs, and efficacy in multiple rodent memory models.
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Keywords: MAO-B-inhibitors, CREB activation, long-term memory, synaptic plasticity,
phenotypic screening,
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INTRODUCTION
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The cAMP pathway (Figure 1), is a cascade of enzymatic reactions that transduce extracellular
neurotransmitter release into intracellular changes in the functional states of many proteins.
Neurotransmitters, their corresponding receptors and cytoplasmic protein kinase A (PKA) appear
to regulate short-term synaptic plasticity, while activation of the transcription factor cAMP
response element binding protein (CREB) is required to produce long-term synaptic plasticity.1, 2
These cellular effects are thought to be the physical representations of short-term and long-term
memories.
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In all species studied to date, activator and repressor forms of CREB that mediate “downstream”
gene transcription have been identified. Key behavioral experiments in Drosophila
melanogaster3,4,5 and mice6 originally established a critical role for CREB in the formation of
long-term memory. Drosophila are capable of learning the association between a particular smell
and the punishment of foot shock. After one training session, a majority of flies will avoid the
previously shock-paired odor when given a choice between that odor and a neutral odor in a
T-maze. Though such learning is initially quite robust, memory thereafter is transient, decaying
away within a day. In flies genetically engineered to produce high levels of CREB repressor,
long-term memory (LTM) specifically is blocked with no effects on learning or early memory.
Similarly, mice with a targeted disruption of the alpha and delta isoforms of CREB are shown to
be profoundly deficient in LTM.6 In flies genetically engineered to produce high levels of a
CREB activator, LTM is induced after just one training session. Over-expression of CREB
activator enhances memory by reducing the number of training sessions required to induce the
formation of LTM – the functional equivalent of a photographic memory.2
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Figure 1. The biochemistry of memory. The transcription factor cAMP response element-binding protein (CREB)
binds to cAMP response elements (CREs) in the promoter of many genes related to neuronal plasticity, thereby
driving their transcription and increasing the levels of their protein products. Elevations in cAMP, such as those
achieved by dopamine receptors (D1) on the surface of neurons lead to the activation of protein kinase A (PKA).
Traditionally, PKA was thought to lead to increased CREB-mediated gene expression via direct phosphorylation of
CREB. More recent insights have revealed alternate control mechanisms. PKA can phosphorylate the dopamine-
and cAMP-regulated neuronal phosphoprotein (DARPP-32) leading to the inactivation of protein phosphatase 1
(PP1), thereby preventing PP1-mediated de-phosphorylation of CREB. Further, PKA activity inactivates kinases that
normally phosphorylate the CREB-regulated transcription coactivator (CRTC). The de-phosphorylated form of
CRTC translocates to the nucleus and directly interacts with CREB to increase the transcription rate of CRE-
containing genes. CREB-mediated transcription can be monitored with artificial DNA constructs with CRE-
containing promoters driving the expression of reporter genes such as luciferase
Since these seminal experiments in Drosophila and mice, molecular studies in other animal
models, including aplysia,7 and rat,8 have begun to further elucidate the biochemistry of plasticity
and to link various aspects of synaptic plasticity with memory formation, leading to the following
general view of brain plasticity (Figure 2). A new experience is registered in a specific neural
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circuit as a change in neural activity. This neural activity induces a complex inter- and intra-
cellular biochemical response that is still being discovered. To-date, however, a few key aspects
of the process appear evolutionarily conserved. Stimulation of the NMDA receptor appears central
to induction of both the local, transient biochemical response at the synapse and the long-lasting
biochemical response in the cell nucleus (phosphorylation of CREB). Several intracellular
signaling pathways appear to be induced initially by neural activity, but CREB nonetheless appears
to be a dominant “downstream” target, regulating the conversion of early memory to lasting
memory. Down-regulation of CREB suppresses, while up-regulation of CREB enhances, a gene
transcriptional response underlying a synaptic growth process that yields lasting functional and
structural changes at synapses. In essence, CREB appears to act as a master switch that determines
when neural activity will give rise to lasting, structural changes in a circuit. A key factor
influencing this switch is repetition; when the CREB pathway is up-regulated, the number of
training sessions required for the formation of long-term memory is decreased.1, 2
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Figure 2. Brain plasticity and long-term memory. Developmental processes initially give rise to complex neural
circuitry, which then registers new experiences by an increase in neural activity. Neural activity induces a
biochemical response that includes the CREB pathway. When the CREB pathway is sufficiently stimulated it
regulates the expression of genes involved in a synaptic growth process, which ultimately changes the connections
among neurons in the circuit. These changes in synaptic connections, distributed throughout the circuit, represent
the physical basis of long-term memory
Based on this biology of memory consolidation, compounds that enhance the levels of cAMP in
the hippocampus, in conjunction with specific training, can potentially facilitate the process of
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long term memory formation. To this end, a program was initiated to screen a library of
compounds in a phenotypic cellular assay to identify compounds that upregulate CREB-
dependent gene expression as a starting point for drug discovery effort focused on the
identification of compounds that enhance long term memory. A primary hit was identified in the
screen, HT-0411, and this hit was shown to augment cognitive function in rodents. Efforts to
identify the mechanism of action of this hit suggested that its biological function was modulated
by inhibition of MAO-B. Based on these results, a medicinal chemistry effort was initiated,
culminating the identification of an optimized lead that was suitable for IND-enabling studies.
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RESULTS AND DISCUSSION
CRE-Luciferase Phenotypic Screen. A library of approximately 155,000 small molecules was
screened against an SK-N-MC cell line stably transfected with a CRE-luciferase (CRE-Luci)
reporter construct provided by Roche Pharmaceuticals (Figure 1), where the cell-line is
engineered to overexpress luciferase; i.e., Plasticity Genes = Luciferase). This screen was
designed to identify compounds that augment CREB activation in the presence of a suboptimal
dose of forskolin to elevate the intercellular levels of cAMP and facilitate the activation of
CREB-dependent transcription of luciferase. Due to the phenotypic nature of the screen, a
number of biological targets could be modulated to ultimately cause an increase in luciferase
activity; this screen was not intended to discriminate between targets or identify a specific target
that was responsible for increasing CREB-dependent transcription.
Compounds that provided a transcription induction ratio (TIR) in the screen > 2 stimulation
(relative to induction by 2µM forskolin as the control) were re-screened at four concentrations;
those hits that were confirmed with TIR > 1.5 were considered for further study. This screening
cascade yielded 178 confirmed active compounds, which were then re-tested in a 6-point dose
response assay to obtain EC50 values. The outcome of this phenotypic screen was the
identification of 84 confirmed active compounds (TIR > 2). Of these hits, 64 compounds were
deprioritized due to structural similarities to drugs that target known proteins (e.g.,
phosphodiesterase inhibitors, digitoxin derivatives, α- and β- adrenergic antagonists), while
additional compounds were removed from consideration due to stimulation of luciferase activity
in the absence of forskolin. A screen of the remaining 10 hits against a limited panel of
enzymes and receptors identified eight compounds that were inhibitors of phosphodiesterases, a
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protein family that was of lower priority for the program, and a ninth compound that was active
on a multiple of GPCRs (i.e. complex pharmacology). The screening results for the remaining
compound, HT-0411 (Figure 3), showed modest activity towards the GABA(B) and melatonin
(MT-2) receptors.
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Follow up experiments were pursued in an attempt to determine if the mechanism of action for
CREB activation by HT-0411 could be via either GABA(B) or MT-2. Baclofen, a known agonist
of GABA did not activate luciferase in the presence of forkskolin, nor did the GABA(B)
antagonist saclofen9 inhibit activation of luciferase in the presence of forskolin and HT-0411
(data not shown), suggesting that activation of GABA(B) by HT-0411 is not responsible for
CREB-dependent gene expression. In a similar experiment, the MT-2 receptor antagonist
prazosin10,11 was shown to have no effect on the ability of HT-0411 to activate CREB, implying
MT-2 was also not the mechanism of action for HT-0411.
Figure 3. Structure of HT-0411, a hit in the CRE-luci screen
In an effort to validate the utility of the CRE-Luci phenotypic screen, HT-0411 was evaluated in
a rodent behavior models to determine if the ability of the compound ability to activate CREB-
dependent gene expression in vitro translated to facilitation of 24-hour memory (i.e., long term
memory) in the rodent. Evaluation of mice in a contextual fear conditioning behavioral model
(cFC) upon intraperitoneal (i.p.) and oral (p.o) administration of HT-0411 demonstrated that
mice treated with HT-0411 prior to training show improvement in memory scores relative to
those mice treated with vehicle (Figure 4), suggesting that the HT-0411 upregulates CREB
function in vivo, though these results do not shed light on how such upregulation occurs at a
molecular level.
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Figure 4. HT-0411 Increases Freezing in Contextual Fear Conditioning In Mice. Mice were 0 (vehicle only),
1, 5, or 10 mg/kg p.o. HT-0411. Training was 30 minutes after oral administration. An unconditioned stimulus,
shock, was administered in a training chamber. For weak training, the unconditioned stimulus was delivered 2 times
with a 1-minute interval between shocks. After 24 hours, the mouse is returned to the training chamber and freezing
behavior, as a measure of memory, was evaluated for 3 minutes.
Chemistry. The modular structure of HT-0411 suggested a wide variety of analogs could be
obtained from the corresponding carboxylic acid regioisomers 6 or 6' and a diverse set of amines
(Figure 5), the full scope of which will be reported elsewhere. The focus herein is on of HT-0411
analogs derived from carboxylic acid 6 (Scheme 1). Although 6 was commercially available, a
synthetic route was developed in order to enable SAR studies and to unambiguously establish the
regiochemistry of the pyrazole ring. Accordingly, two complimentary approaches were taken. In
route A, commercially available malonate 1 was converted to enamine 2 by treatment with
ammonia gas,12 and the resulting enamine 2 was cyclized to hydroxypyrazole 3 via condensation
with methyl hydrazine in modest yield.13 Conversion of 3 to triflate 4 was followed by a Suzuki-
Miyaura coupling with thiophene-2-yl boronic acid under standard conditions to afford
thiophene 5 in excellent overall yield. Lithiation of 5 using n-butyl lithium/TMP base was
followed by the addition of carbon dioxide to form carboxylic acid 6. In route B, commercially
available 5-acetyl-thiophene-2-carboxylic acid ethyl ester (9) was transformed to 10 via Claisen
condensation with ethyl trifluoroacetate. Condensation of methylhydrazine with 10 afforded a
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mixture of regioisomers 11a and 11b, which were separated. The minor regioisomer 11b yielded
crystals, which were analyzed by X-ray (see Figure 1B in Supplemental Materials). Each
regioisomer was then saponified to give the corresponding acid, e.g., 11a afforded acid 6 and
11b afforded acid 6'. Coupling of 6 to an array of primary and secondary amines was achieved
through activation of the acid, followed by addition of the amine. Using this methodology, over
200 HT-0411 analogs were prepared. The regiochemistry of 6 was confirmed by preparation of
HT-0411 followed by X-ray analysis (see Figure 1A in Supplemental Materials).
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Figure 5. Points of Diversity for HT-0411 Analogs.
Scheme 1. Reagents and Conditions: (a) NH3, 85◦ C; (b) MeNHNH2, 85◦ C; (c) Tf2O, DCM, -10◦ C; (d) thiophen-
2-ylboronic acid, Pd(dppf)Cl2, K3PO4, dioxane, 100◦ C ; (e) nBuLi/TMP, -78◦ C , CO2; (f) (COCl)2, cat. DMF; (g)
R1R2NH, TEA, DCM, 0◦ C; (h) Ethyl trifluoroacetate, NaOEt, 40◦ C; (i) MeNHNH2, 75◦ C; (j) NaOH, dioxane, rt.
Using the CRE-Luci assay, the HT-0411 analogs were screened, using suboptimal doses of
forskolin; the ability of a compound to induce CREB-dependent synthesis of luciferase was
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expressed as a fold induction over forskolin (TIR, see above) and the percent of activity relative
to induction seen with HT-0411 + forskolin (PRS = percent relative to standard). While the CRE
CRE-Luci assay was sufficient to identify CREB activators (see Table 1 for representative
examples), the dynamic range of the CRE-Luci assay and inadequate measured dose response
were insufficiently reliably to differentiate the potency of the individual compounds and allow
prioritization of compounds for downstream studies such as in vitro/in vivo ADME studies or
subsequent evaluations for efficacy. For this reason, as a requirement to move the program
forward, resources shifted to the identification of the biological target of these CREB activators
in an effort to have an assay, or series of assays, that would provide greater differentiation of the
primary activity of the compounds and be more predictive of in vivo efficacy of the more
favorable compounds.
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Table 1. High Throughput Screening results of HT-0411 analogs in the SK-N-MC CRE-
Luciferase Assay
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Screening
Concentration
Compound
8a
R1
R2
Et
TIR*
1.68
PRS*
109
Me
5 μM
8b
8c
8d
3 μM
3 μM
3 μM
1.47
1.66
1.71
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102
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8f
100 nM
100 nM
1.54
1.65
95
89
*TIR = Transcriptional induction ratio, relative to forskolin alone; PRS = Percent relative to standard (HT-0411).
Assays were run in the presence of 5 μM forskolin and the PRS is measured relative to a 5 μM concentration of
HT-0411. The reported value is the average of 8 replicated measurements run in a single experiment.
Identification of MAO-B as Mechanism of Action. To explore the potential for HT-0411 to
interact with biological targets beyond those of the original panel screen (vide supra) a limited
selection of compounds, including HT-0411 and several related analogs were evaluated against
an additional panel of kinases (Invitrogen) at a screening concentration of 10 µM, which resulted
in no meaningful enzymatic inhibition.
As an extension to the original screen of HT-0411 against a panel of enzymes and receptors,
another multi- target, single point screen, with approximately 100 enzyme and receptor binding
assays, was performed with HT-0411. Results from the screen identified only three targets for
which there was either ≥ 50% inhibition of protein function by HT-0411, MAO-B (99%), or
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receptor binding, melatonin-1 receptor (MT-1) (57%) and melatonin-2 receptor (MT-2) (77%).
As a follow-up study, a full dose response was evaluated with these targets and the IC50 of
HT-0411 for MAO-B was measured to be 1.4 µM, while the IC50 against MT-1 and MT-2 were
5.3 and 3.2 µM, respectively.
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To confirm these results, evaluation of MT-1, MT-2 and MAO-B studies with HT-0411 were
repeated in a second lab. Upon retest, no significant inhibition of binding to either melatonin
receptor MT1 or MT-2 with 10 µM HT-0411 was detected, but the IC50 measured against
MAO-B was 192 nM for HT-0411, confirming the initial MAO-B inhibition result. The 10-fold
difference between the measured IC50 at the two labs, 192 nM versus 1.4 µM, may be attributed
to the differences in the MAO-B assay performed by the two vendors; one lab used human
MAO-B overexpressed in insect while the other lab used MAO-B isolated from a rat brain
membrane preparation. The affinity of HT-0411 for the closely related MAO-A was determined
and the compound was found to be a weak MAO-A inhibitor (22% inhibition of MAO-A at a
screening concentration of 10 μM) demonstrating that HT-0411 is a selective and specific MAO-
B inhibitor.
To provide further evidence to support the hypothesis that HT-0411 mediates activity in the
CRE-Luci assay through MAO-B inhibition, the MAO-B specific inhibitor Ro 16-6491 was
tested in the CRE-Luci assay using the SK-N-MC cell line and was found to be active (Table 2).
Taken together these results suggest that inhibition of MAO-B is a likely mechanism for HT-
0411 action. This conclusion is consistent with reports in the literature that L-deprenyl enhances
memory in animal models and in Parkinson and Alzheimer disease patients.14, 15, 16, 17
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Table 2. An MAO-B Inhibitor Potentiates CRE-Luciferase Activity
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Compound
RO16-6491
TIR
1.54
2.13
Screen Concentration (M)
PRS (HT-0411, 5 M)
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The results of the panel screens and the CRE-Luci assay results with additional MAO-B
inhibitors provided strong evidence that the mechanism of action of HT-0411 could be attributed,
at least in part, to the selective inhibition of MAO-B; a link between dopamine levels, cAMP and
downstream signaling via CREB has been previously established.18, 19
Mammalian dopamine receptors are divided into two general groups, D1/D5 and D2/D3/D4, based
on their ability to activate or inhibit adenylate cyclase. The D1 and D5 receptors are activators of
adenylate cyclase and are highly expressed in the striatum (predominantly D1) and the
hippocampus (predominantly D5). These two areas of the brain are recognized as important in
the control of cognitive functions and memory.20,21,22,23,24 The activation of adenylate cyclase
leads to the synthesis of cAMP and, ultimately, to CREB gene expression. It has been shown that
activation of the D1 and D5 receptors by either endogenous or exogenous dopamine agonists is
required for the biochemical strengthening of synaptic transmission (known as long-term
potentiation) in the corticostriatal pathways.25 Long-term potentiation is a process thought to be
central to memory formation;22 therefore, any conditions affecting the D1/D5 receptors (e.g.,
dopamine deficiencies associated with Parkinson’s disease) are likely to affect the ability to form
memories. Neurotransmitters such as dopamine, through their corresponding receptors, could
activate CREB transcription factor, which is required to produce long-term synaptic plasticity
and enhanced LTM formation. 1, 26
A number of MAO-B inhibitors have successfully been approved, primarily for the treatment of
Parkinson’s disease (PD). The first generation of nonselective, irreversible inhibitors of MAO-B
and MAO-A (e.g. tranylcypromine) induced serious side effects, including hepatotoxicity,
orthostatic hypotension, and most importantly the “cheese effect” characterized by an acute
hypertensive crisis when given in combination with foods containing tyramine. This potentially
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life-threatening side effect has largely limited the clinical use of drugs that irreversibly inhibit
MAO-A.27, 28 More selective inhibitors of MAO-B have been developed (e.g. L-deprenyl and
rasagiline), yet, despite their selectivity, these drugs still inhibit MAO-A at supra-therapeutic
doses, which has led to cautionary labeling regarding tyramine ingestion when doses are used
beyond the recommended therapeutic range.29, 30 These first generation, non-selective inhibitors
of MAO-A and MAO-B and the newer, more selective inhibitors of MAO-B are both irreversible
inhibitors of MAO enzyme activity and result in prolonged enzyme inhibition lasting many days
after discontinuation of the drug.31, 32 Both L-deprenyl and rasagiline are primarily used for the
treatment of PD as an adjunctive therapy with levodopa to raise central dopamine levels in
deficient patients and are known to improve motor functions and to delay or decrease the need
for levodopa. In addition to reducing the motor symptoms of PD, the possibility of a
neuroprotective, survival enhancing effect from L-deprenyl was first studied in 1985,33
suggesting that the use of a selective MAO-B inhibition is a viable approach to aid cognitive
function.
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As alluded to previously, potential drawbacks for the irreversible inhibitors of MAO-B include
the slow return of enzymatic activity after discontinuation of the drug, which could contribute to
adverse drug-drug interactions when used in combination with other drugs or could delay a new
drug therapy for which MAO-B inhibition is contraindicated (e.g. treatment with SSRIs). For
this reason, efforts in the drug discovery field to identify reversible inhibitors of MAO-B have
continued. Safinamide, a reversible MAO-B and glutamate release inhibitor, was approved in
Europe in 2015 and, more recently, in the United States. Safinamide has been shown to improve
learning of spatial memory tasks, enhance performance in passive and active avoidance tasks in
rodents, and has improved the performance of humans in word retrieval tasks, likely due to
glycine agonism.34
Because HT-0411 and it analogs are reversible inhibitors of MAO-B with good selectivity over
MAO-A, HT-0411 analogs were viewed as a reasonable and tractable alternative to current
MAO-B treatments.
Monamine Oxidase Inhibition: Lead Optimization. With MAO-B identified as the putative
biological target of interest, the entire set of HT-0411 analogs were screened against the enzyme
to gain structure-activity relationships (SAR). Typically, screening was performed with a limited
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number of inhibitor concentrations (e.g., 200 nM and/or 10 M), with the more active hits
further evaluated with a full dose response to determine IC50 values. Representative results from
the evaluation are presented in Table 3 and Table 4. These SAR suggest that the MAO-B
catalytic site accommodates a wide range of amide N-substituents, though smaller alkyl groups
and more extended alkyl groups are somewhat less preferred. Cyclic substituents are very well
tolerated and provide some of the most potent compounds identified (e.g., 8k). While some
substitution of the N-linked heterocycle is permissible (e.g., 8r) there are limits to the size of the
substituent (e.g., 8l).The preferred position for the N-alkyl substituent on the pyrazole ring is N-1
(Table 4).
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Table 3. Potency of HT-0411 Analogs Screened Against MAO-B
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Conc.
(μM)
Compound
R1
R2
% Inhib IC50 (μM)
0.1861
ND
HT-0411
8a
Me
Me
Me
Et
-
-
0.3122
ND
0.462
0.01131
0.09212
8b
8c
8d
0.2
0.2
0.2
93.7
0.00501
0.1222
97.0
70.5
0.06691
0.5152
8e
8f
0.2
0.2
96.5
89.0
0.007951
0.04891
0.09352
8g
8h
Et
H
Et
H
-
ND
36
2.072
19.22
10
8i
H
0.2
10
37
ND
8j
H
82.5
ND
8k
H
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78.5
ND
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Conc.
(μM)
Compound
R1
R2
% Inhib IC50 (μM)
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8
9
8l
H
0.2
40.5
ND
ND
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8m
H
-
5.622
8n
8o
Me
-
ND
0.1122
0.003091
0.4632
0.2
98.0
8p
8q
10
26.5
93.0
ND
0.02151
0.08512
0.2
0.02921
0.09222
8r
8s
0.2
0.2
87.5
86.8
0.0331
0.9972
8t
0.2
0.2
51.5
37.0
ND
0.3271
0.9832
8u
8v
0.2
28.0
ND
1 Results from assay performed at MDS; 2 Results from assay performed at Dart NeuroScience; See experimental
section for more details on the two assays
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Table 4. Impact of N-1 versus N-2 Alkylation on MAO-B Inhibitor Potency
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R3
%
Inhib
IC50 (μM)
Compound
R1
R2
Conc (nM)
200
N1
N2
0.01131
0.09212
0.3361
0.1242
0.04891
0.09352
0.1351
8b
Me
-
93.7
47.0
8'b
-
Me
200
8f
Me
-
200
200
89.0
52.0
8'f
Me
1 Results from assay performed at MDS; 2 Results from assay performed at Dart NeuroScience; See experimental
section for more details on the two assays
One of the most promising leads identified in the SAR campaign was compound 8b (Table 3).
The IC50 against MAO-B was 15 nM with >3000 fold selectivity of MAO-A. An initial estimate
of metabolism was provided by incubation of 8b in human hepatocytes, which showed that
>70% of the parent drug remained after 60 minutes of incubation. Based on a favorable in vitro
profile of 8b in a number of additional assay relevant to the ability to further develop 8b (e.g. a
panel screen, CYP450 inhibition, hERG inhibition, Ames, and Caco-2 cell permeability), the in
vivo evaluation of the compound for efficacy in memory models was undertaken. Compound 8b
was evaluated in a mouse cFC assay and was shown to be efficacious in the dose range of 1-10
mg/kg when dosed orally (Figure 6).
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Figure 6. Compound 8b Increases Freezing in Contextual Fear Conditioning in Mice; Mice were administered
0 (vehicle only), 1, 5, or 10 mg/kg oral 8b. Training was 30 minutes after oral administration. An unconditioned
stimulus, shock, was administered in a training chamber. For weak training, the unconditioned stimulus was
delivered 2 times with a 1-minute inter-trial interval between shocks. After 24 hours, the mouse is returned to the
training chamber and freezing behavior, as a measure of memory, was evaluated for 3 minutes
While 8b was orally efficacious in rodent, pharmacokinetic analyses in rat revealed rapid
metabolism. To better assess the PK profile of 8b in multiple species, the in vitro metabolism
was examined in hepatocytes isolated from a number of mammalian species, including mouse,
rat, monkey, and dog. Results from these assay dramatically demonstrated that, while 8b was
stable in human hepatocytes, it was rapidly cleared in all other species, particularly rodent. A
comprehensive LC/MS analyses of the major metabolites was performed, and M4 was identified
as a major metabolite in all species at 60 minutes (Table 5). Using LC/MS/MS, the mass of the
major metabolites were determined, and the structures of the metabolites were confirmed, if
possible, through comparison of retention time and mass spectra with synthetically prepared
compounds (Figure 7). This study definitively confirmed M4 was the hydroxylated metabolite
of 8b, which corresponded to compound 8f (Table 3). These results suggested that the efficacy
noted in the rodent studies with 8b might actually be mediated by the presence of high levels of
8f in the CNS and not 8b.
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Table 5. Metabolite profile of Compound 8b in different mammalian hepatocytes*
Control
Mouse
Rat
Dog
Monk.
Human
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7
8
9
97.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.4
45.4
19.5
27.6
0.0
3.0
0.0
31.8
40.9
12.2
3.0
13.2
0.0
7.0
7.8
0.0
22.2
44.1
70.7
0.0
5.9
7.3
0.9
4.8
0.6
4.4
Parent
M2
M3
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33.0
0.0
9.3
9.8
M4
M5
M6
M7
M8
17.1
3.2
0.0
4.4
4.7
3.3
1.6
*Percent ionization peak area after 60 min incubation
Figure 7. Major metabolites identified for 8b.
To explore this possibility, the selectivity of compound 8f was determined against a similar
battery of in vitro screens to those evaluated with 8b, including a receptor panel screen, hERG,
Ames assay, and CYP enzymes (see supplementary material). The results of these assays
suggest that 8f is a viable candidate for efficacy studies with good selectivity towards MAO-B
over MAO-A (72 nM vs 19% inhibition of MAO-A at 50 μM concentration of 8f), no significant
interactions with the enzymes and receptors evaluated in the panel screen, and no obvious safety
concerns. The metabolic stability of 8f was determined in a number of mammalian hepatocyte
species, and, unlike 8b, compound 8f was shown to be very stable in all species evaluated (Table
6). In vivo pharmacokinetic analyses (Table 7) showed that 8f had modest, but acceptable,
bioavailability in rat (%F = 34) and good brain penetration (brain-to-plasma ratio = 1.75).
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Table 6. Metabolic Stability of Compound 8f in Pooled Hepatocytes
% Remaining at 60
Intrinsic Clearance
Species
Half-life (minutes)
Minutes
90
(µmol/min/106 cells)
9
Mouse
Rat
Rabbit
Monkey
Mini-pig
Human
>60
>60
53.2
>60
>60
>60
<11
<11
13.0
<11
<11
<11
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81
49
79
82
93
Table 7. Pharmacokinetic Parameters for Compound 8f in Rats
Route of Administration
Mean Parameter
Dose (mg/kg)
Intravenous
10
Oral
100
4070 (322)
--
4960 (256)
0.3 (0)
4260 (387)
1.4 (0.1)
--
C0 or Cmax (ng/mL)
Tmax (h)
AUC(inf) (ng•h/mL)
T1/2 (h)
CL/F (mL/min/kg)
Vss (L/kg)
MRT (h)
F(inf) (%)
13904 (556)
0.23 (0.0)
132 (46.0)
2.25 (0.0766)
0.31 (0.11)
--
--
--
33.8 (3.1)
1.75 (0.15)
--
Brain:Plasma Ratio (at 1 hour)
For oral administration, 3 animals were dosed in a 2% CMC suspension at 10 mg/mL. Intravenous dosing was
performed on 3 animals using a 33.3 mg/mL of 8f dissolved in NMP. Numbers in parentheses are the standard
deviations of the calculated PK parameters.
Given its satisfactory drug properties, 8f was assessed for cognitive enhancement in the cFC and
NOR assays in both mice and rats (Figure 8); results from these behavioral tests showed that 8f
was efficacious in both memory paradigms.
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Figure 8. Improved memory demonstrated by mice in a cFC assay and rats in NOR assay after
administration of Compound 8f; a) Mice were administered 0 (vehicle), 1, 3, or 10 mg/kg 8f p.o. Training was
30 minutes after oral administration. An unconditioned stimulus, shock, was administered in a training chamber. For
weak training, the unconditioned stimulus was delivered 2 times with a 1-minute inter-trial interval between shocks.
After 24 hours, the mouse is returned to the training chamber and freezing behavior, as a measure of memory, was
evaluated for 3 minutes; b) A rat was placed into the training box containing two identical objects and allowed to
explore these objects. Animals were trained for 15 minutes; 0 (vehicle), 0.1, 0.3, 1 and 3 mg/kg of 8f was
administered p.o. 30 min before training (i.p. administration of 8f at 0.01 mg/kg was used as a positive control). To
test for memory retention 24 hours later, the rat was presented with two objects, one ‘familiar’ and ‘novel’ and
observed for 10 minutes an object exploration preference was measured.
To minimize the potential that the observed efficacy of 8f was confounded by behavioral side
effects, 8f was evaluated in two additional assays, a tail-flick test and an elevated plus maze (see
Supplementary Materials for graphical results). The tail-flick test evaluates the pain response of
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a rodent to heat being applied to its tail; an analgesic effect due to the administration of a drug
would be indicated if there is an increased latency to move the tail from the heat source. The
relevance of this assay is that a proper, and uncompromised, perception of pain is necessary for a
memory response in the cFC assay. Results from the tail-flick test with 8f indicated that there is
no impact on pain perception when dosed in the efficacy range for memory (up to 10 mg/kg),
though there is a slight, but significant, decrease in latency at 30 mg/kg, indicative of an increase
perception of pain (Figure 2A in Supplementary Materials).
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Another potential biological result that could confound a fear based memory assay is an increase
or decrease in anxiety. To evaluate the impact of 8f on anxiety, mice were evaluated in an
elevated plus maze; an increase in anxiety would be indicated if mice spent more time in the
closed arm while an increase in the time spent in the open arm would suggest a decrease in
anxiety. There was no significant change in the time spent walking in either arm of the maze at
any dose tested (Figure 2B in Supplementary Materials suggesting that 8f has no detectable
impact on anxiety at the doses that showed improvement for memory.
CONCLUSIONS
Using a phenotypic high throughput screen that utilized a CRE-Luci SK-N-MC cell line, we
identified a number of small molecules hits including HT-0411. Follow-on studies suggested that
CREB activation by HT-0411 was attributed to its specific and selective inhibition of MAO-B.
Lead optimization of the series derived from HT-0411 resulted in 5-(1-methyl-5-
(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-carboxamides as novel, potent and selective
inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of these
analogs identified a number of potential clinical development candidates; ultimately, compound
8f was identified as a candidate molecule with high selectivity towards MAO-B over MAO-A
and an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic
properties in rodents and dogs, and efficacy in multiple rodent memory models.
Compound 8f enhanced long-term memory in contextual fear and object recognition tasks in
both mice and rats. No significant behavioral side effects, which could potentially confound the
interpretation of the memory tests, were found in mice at dose levels that enhance memory
formation. Compound 8f showed no increase in anxiogenesis in an elevated plus maze and an
increased pain sensitivity in the tail flick assay, but at dose levels at least 1000 times greater than
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the efficacious dose, suggesting a sufficient therapeutic index for further development. The
overall selectivity of 8f for MAO-B over MAO-A and other proteins, its acceptable
pharmacokinetic profile, and good physicochemical properties support the conclusion that 8f is a
memory-enhancing compound with pro-cognitive potential at doses well below those associated
with behavioral side effects. For these reasons, 8f was progressed into IND toxicology studies to
enable future clinical studies.
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METHODS
CRE-Luciferase assay. CRE-Luciferase (CRE-Luci) expressing SK-N-MC cells were seeded
the day before compound treatment at 20,000 cells/well in 96-well white plates. Compounds
were typically added 1 hour before forskolin (5 µM final conc.) addition, and luciferase reagents
(BriteLite from Perkin Elmer, Shelton, CT) were added 6 hours after compound addition.
Luminescence (expressed in relative luminescence unit, RLU) was detected with a Victor V plate
reader (Perkin Elmer, Shelton, CT). Results were expression as TIR (TIR = RLU(cpd +
forsk)/RLUforsk).
MAO-A and MAO-B assays (MDS-Taiwan). Human recombinant MAO-A and MAO-B
expressed in insect cells (Sigma, St. Louis, MO) are used. Test compound and/or vehicle is
preincubated with ~13 µg/ml enzyme in 100 mM phosphate buffer pH 7.4 for 15 minutes at
37 oC. The reaction is initiated by addition of 50 µM kynuramine for 60 minute incubation
period and terminated by further addition of 6 N NaOH. Determination of the amount of 4-
hydroxyquinoline formed is read spectrofluorimetrically at 325 nm/465 nm
MAO-B assay (Dart NeuroScience, LLC). Human recombinant MAO-B expressed in insect
cells (Sigma, St. Louis, MO) is used along with Promega’s (Madison, WI) MAO-Glo kit. Test
compound and/or vehicle is pre-incubated with 0.1 µg enzyme/reaction in 100 mM phosphate
buffer pH 7.4 for 15 minutes at 37 oC. The reaction is initiated by addition of 0.4 µM substrate
(Km = 0.4 µM) for 60 minute incubation period and terminated by further addition of 10 µL and
luciferin detection reagent. After a further incubation of 10 min at room temperature,
luminescence was measured using a Victor 5 plate reader (Perkin Elmer, Shelton, CT).
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Pharmacokinetic Analysis in Sprauge Dawley Rats
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Test Article Formulation
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Oral formulation (p.o): A 2% carboxymethyl cellulose (CMC) suspension was prepared by
slowly adding 1.2 g of CMC to 60mL of distilled water while stirring. A 119.15 mg sample of
compound 8f was dissolved in 3.575 mL of DMSO to form a clear stock solution. A total of 8.34
mL of 2% CMC was added to the 3.575 mL 8f stock solution, 2 mL at a time. The dosing
solution was vortexed until the solution became homogenous. The final concentration in the
dosing mixture was 10 mg/mL.
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Intravenous formulation (i.v.): A 38.64 mg sample of compound 8f was dissolved in 1.161 mL
of N-methyl-2-pyrrolidone (NMP) after mixing the solution by vortex until the solution became
clear. The solution was filtered through 0.2 μm filter under aseptic conditions. The final
concentration of the dosing solution was 33.3 mg/mL.
Dose formulations were assayed in duplicate by LCMS/MS to conform their final
concentrations.
In vivo Assessment
Three animals were dosed in each arm (p.o and i.vi) of the pharmacokinetic study. The p.o. dose
was 100 mg/kg and the i.v. dose was 10 mg/kg.
Blood samples were collected at the following target times after each dose administration:
 p.o. : predose, 15, 30 min, 1, 2, 4, 6, and 24 hr post dose
Brain samples were collected Plasma samples were evaluated using LCMS/MS. The lower limit
of quantification (LLOQ) of compound 8f was 1 ng/mL using a 0.05 mL plasma aliquot.
A separate group of 3 animals were dosed p.o. to access the brain-to-plasma ratio. Dosing
solutions were similarly prepared as above. Samples of brain and plasma were taken 1 hour post
dose and evaluated by LCMS/MS.
Data Analysis
Plasma concentration versus time data was analyzed by non-compartmental approaches using the
WinNonlin software program (version 5.0.1, Pharsight, Mountain View, CA). The area under the
plasma concentration-time curve (AUC) was determined using the linear-log trapezoidal rules.
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The plasma clearance (CL) was determined as the ratio of dose/AUC. The volume of distribution
at steady state (Vss) was estimated as CL*MRT. Half-life estimates were obtained from fitting
the data to one compartmental model using the WinNonlin program. Mean calculations of all
pharmacokinetic parameters and their associated statistics were generated from un-rounded
numbers and may differ slightly from those values which would have been determined using
rounded numbers.
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Contextual Fear Conditioning (cFC) assay in mice. Young-adult (10-12 weeks old) C57BL/6
male mice and Sprague Dawley male rats of 250-300 g (Taconic, NY) were used. Mice were
group-housed (5 mice) in standard laboratory cages while rats were housed in pairs and
maintained on a 12:12 light-dark cycle. The experiments were always conducted during the light
phase of the cycle. With the exception of testing times, the mice had ad lib access to food and
water. The experiments were conducted according with the Animal Welfare assurance #A3280-
01 and animals were maintained in accordance with the animal Welfare Act and Department of
Health and Human Services guide.
On the training day, the mouse was placed into the conditioning chamber (Med Associates, Inc.,
VA) for 2 minutes before the onset of unconditioned stimulus (US), 0.5 mA, of 2 sec foot shock.
The unconditioned stimulus (US) was repeated two times with a 1 min inter-trial interval
between shocks. Training was performed by automated software package (Med Associates,
Inc.,VA). After the last training trial, the mice were left in the conditioning chamber for another
30 sec and were then placed back in their home cages. Twenty-four (24) hours after training, the
mouse was placed into the same training chamber and contextual memory was assessed by
scoring freezing behavior (‘freezing’ serves as memory score). Freezing was defined as the
complete lack of movement in intervals of 5 seconds.6, 35, 36, 37,38 Total testing time lasted 3
minutes. After each experimental subject, the experimental apparatus was thoroughly cleaned
with 75% ethanol, water, dried, and ventilated for a few minutes.
All experiments were designed and performed in a balanced fashion, meaning that (i) for each
experimental condition (e.g. a specific dose-effect) an equal number of experimental and control
mice was used; and (ii) each experimental condition was replicated 2-3 independent times, and
replicate days were added to generate final number of subjects. The proceeding of each
experiment was filmed. In each experiment, the experimenter was unaware (blind) to the
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treatment of the subjects during training and testing. Data were analyzed by Student’s unpaired
t-test using a software package (Statview 5.0.1; SAS Institute, Inc). All values in the text and
figures are expressed as mean + SEM.
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Compounds were dissolved in 1% DMSO/PBS and administered i.p., in a volume of 8 mL/kg, 20
min before training. Control animals received vehicle alone (1% DMSO/PBS). For oral
administration the compounds were dissolved in 30% DMSO/70% CMC and animals were dosed
30 minutes prior to training. Consequently, control animals received 30% DMSO/70%
CMC. For each training and drug procedure, an experimentally naïve group of animals were
used.
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Object recognition assay in Rodents. Prior to initiation of training, animals were handled for 3-
5 minutes for 5 days. Training and testing were performed identically for mice and rats with an
exception of training apparatus dimensions (for mice: a Plexiglas box of L=48 cm; W=38 cm and
H=20 cm; for rats: a Plexiglas box of L=70 cm; W=60 cm and H=35 cm). The day before
training, an individual animal was placed into a training apparatus located in a dimly lit room
and allowed to habituate to the environment for 15 minutes.39, 40 Training was initiated 24h hours
after habituation. An animal was placed back into the training box, which contained two identical
objects (e.g. a small conus-shape object), and was allowed to explore these objects. The objects
were placed into the central area of the box and the spatial position of objects (left-right sides)
was counterbalanced between subjects. Animals were trained for 15 minutes. To test for
memory retention, animals were observed for 10 minutes 24 hours after training. A rodent was
presented with two objects, one of which was used during training, and thus was ‘familiar’ and
the other of which was novel (e.g. a small pyramid-shape object). To insure that the
discrimination targets do not differ in smell, after each experimental subject, the apparatus and
the objects were thoroughly cleaned with 90% ethanol, dried and ventilated for a few minutes.
The experiments were videotaped via an overhead video camera system. Tapes were then
reviewed by a blinded observer and the following behavioral parameters were determined: time
of exploration of an each object; the total time of exploration of the objects; number of
approaches to the objects; and time (latency) to first approach to an object. The discrimination
index – memory score - was determined as described previously.40, 41. This Data was analyzed
by Student’s unpaired t test using a software package (Statview 5.0.1; SAS Institute, Inc). All
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values in the text and figures are expressed as mean + SEM.
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Evaluation of Potential Behavioral Side Effects of 8f
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Anxiety. An elevated maze was used to evaluate expression of anxiety.42 The apparatus
manufactured by Coulbourn Instruments (Allentown, PA, USA) was made of plastic and
consisted of two opposed open arms (50 cm × 10 cm), two opposed enclosed arms with no roof
(50 cm × 10 cm × 30 cm) and an open square (10 cm × 10 cm) in the center. The maze was
elevated 55 cm above the floor. There was no raised edge or margin along the open arms, and the
ends of the closed arms also were open. Behavioral tests were conducted under low light
conditions (35 lux in the center). Each test lasted 5 min. A mouse was placed into the center of
the maze facing one of the open arms and was observed using EthoVision (Noldus Inc.,
Wageningen, Netherlands) tracking software with an overhead video camera. The ambulation
distance in open arms and the closed arms was quantified. After each subject was tested, the
apparatus was thoroughly cleaned with 70% ethanol in water, dried and ventilated Total
distanced traveled, total distance traveled in open arms, and total distance traveled in closed arms
were recorded during a 5-minute test. Five groups of mice were evaluated: 0 (vehicle only), 0.01,
0.1, 1, and 10 mg/kg; i.p.
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Pain sensation. To assess the possible effects of 8f on pain sensation behavior in a tail flick test
was utilized.43 In this version of the tail flick assay the latency mouse to flick its tail out of a hot
water bath was measured. A mouse was scruffed and its tail was immersed 3/8” into the water.
A hand held stop watch was used in the other hand to record the latency for the mouse to respond
to the hot water stimulus. A full tail flick or partial tail movement was sufficient to be considered
a positive response. If a mouse did not respond in 10 sec it was removed from the hot water bath
and was given a latency of 10 sec. All mice were measured for baseline tail flick latency and
then injected with vehicle or drug immediately after. The subsequent tail flick testing was
performed 20 min post-injection. The evaluation was run with 6 groups of mice by: 0 (vehicle
only), 0.01, 0.1, 1, 10 and 30 mg/kg i.p.
CHEMISTRY
General Methods: Solvents and reagents were purchased and used without further purification
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unless otherwise noted. All H, F, and C NMR spectra were recorded on Varian (400 MHz)
instrument. Chemical shifts are reported in ppm relative to residual solvent peaks as an internal
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standard set to δH 7.27 or δC 77.0 (CDCl3), δH 2.50 or δC 40.45 (DMSO-d6), and unreferenced
for 19F. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, br = broad, m = multiplet), coupling constant (Hz), integration. Analytical thin layer
chromatography was performed on Merck KGaA/Millipore silica gel 60 F254 plates. Visualization
was accomplished with UV light. Flash column chromatography (FCC) was performed with
Selecto 60Å, 63 to 200µm particle size silica gel. Melting points were obtained on either an
Electrothermal IA-9100 apparatus or a Mel-Temp II Melting Point Apparatus. Melting points are
uncorrected. Elemental and Karl Fisher analyses were obtained at Robertson Microlit Laboratories
(Ledgewood, New Jersey, USA). Single crystal x-ray diffraction was obtained at the UCSD
Crystallography Facility (San Diego, California, USA).
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Analytical HPLC was performed on a Waters 2695 Separations Unit with 2487 Dual Absorbance
Detector and Micromass ZQ fitted with ESI Probe. Materials were dissolved in acetonitrile and
diluted with equal volume water.
LC Protocol: Observed, 254 nm. Solvent system, acetonitrile (0.1 % formic acid) and water (0.1
% formic acid). Column, XTerra MS C-18 3.5 uM (2.1 x 50 mm), 30°C oven temperature. Run
time, 10 min. Flow rate 0.3 mL/min.
Preparation of 1-methyl-5-(trifluoromethyl)pyrazol-3-ol (3):
Ammonia gas was introduced via dispersion tube into neat ethyl 4,4,4-trifluoroacetoacetate (1,
37.37 g, 203 mmol) with heating (85◦ C) for 3 h to give 2,12 which was crystallized from hot
hexanes to afford 23.5 g (63.2 %) light yellow needles (mp = 25◦ C). Methyl hydrazine 6.13 g,
133 mmol) was added to cold neat 2 (21.59 g, 118 mmol), the solution was stirred 1 h then
heated in an oil bath at 85◦ C overnight to form a 85:15 mixture of the 3-, and 5-hydroxy
regioisomers (3 being the major product).13 The desired 3-hydroxy isomer (3) was isolated by
trituration of the concentrated reaction mixture with aqueous saturated NaHCO3 followed by
filtration. Note: the undesired 5-hydroxy isomer dissolves readily in aqueous saturated NaHCO3,
while the desired 3-hydroxy isomer (3) remains insoluble. The title compound was obtained as a
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white solid (9.45 g, 48 % yield). H NMR (DMSO-d6) 10.27 (br.s, 1 H), 6.01 (s, 3 H), 3.71 (s. 3
H). 19F NMR (DMSO-d6) -59.45 ppm). m/z 167.0 [M+H]+.
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