Discovery of Aromatic Carbamates that Confer Neuroprotective Activity by Enhancing Autophagy and Inducing the Anti-Apoptotic Protein B-Cell Lymphoma 2 (Bcl-2)

Article abstract of DOI:10.1021/acs.jmedchem.7b01199

Neurodegenerative diseases share certain pathophysiological hallmarks that represent common targets for drug discovery. In particular, dysfunction of proteostasis and the resultant apoptotic death of neurons represent common pathways for pharmacological intervention. A library of aromatic carbamate derivatives based on the clinically available drug flupirtine was synthesized to determine a structure-activity relationship for neuroprotective activity. Several derivatives were identified that possess greater protective effect in human induced pluripotent stem cell-derived neurons, protecting up to 80% of neurons against etoposide-induced apoptosis at concentrations as low as 100 nM. The developed aromatic carbamates possess physicochemical properties desirable for CNS therapeutics. The primary known mechanisms of action of the parent scaffold are not responsible for the observed neuroprotective activity. Herein, we demonstrate that neuroprotective aromatic carbamates function to increase the Bcl-2/Bax ratio to an antiapoptotic state and activate autophagy through induction of beclin 1.

Full text of DOI:10.1021/acs.jmedchem.7b01199

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Article
Discovery of Aromatic Carbamates that Confer Neuroprotective
Activity by Enhancing Autophagy and Inducing the
Anti-Apoptotic Protein B-cell lymphoma 2 (Bcl-2)
Nihar Kinarivala, Ronak Patel, Rose-Mary Boustany, Abraham J. Al-Ahmad, and Paul C. Trippier
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Nov 2017
Downloaded from http://pubs.acs.org on November 7, 2017
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Page 1 of 55
Journal of Medicinal Chemistry
Discovery of Aromatic Carbamates that Confer Neuroprotective Activity by Enhancing Autophagy and
Inducing the AntiꢀApoptotic Protein Bꢀcell lymphoma 2 (Bclꢀ2)
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a
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a, c
Nihar Kinarivala , Ronak Patel , RoseꢀMary Boustany , Abraham AlꢀAhmad and Paul C. Trippier*
a
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences
Center, Amarillo, Texas 79106, United States.
b
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Department of Biochemistry and Molecular Genetics, American University of Beirut Medical Center, Beirut,
Lebanon.
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Center for Chemical Biology, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock,
Texas 79409, United States.
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ABSTRACT
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Neurodegenerative diseases share certain pathophysiological hallmarks that represent common targets
for drug discovery. In particular, dysfunction of proteostasis and the resultant apoptotic death of neurons,
represent common pathways for pharmacological intervention. A library of aromatic carbamate derivatives
based on the clinically available drug flupirtine was synthesized to determine a structureꢀactivity relationship for
neuroprotective activity. Several derivatives were identified that possess greater protective effect in human
induced pluripotent stem cellꢀderived neurons, protecting up to 80% of neurons against etoposideꢀinduced
apoptosis at concentrations as low as 100 nM. The developed aromatic carbamates possess physicochemical
properties desirable for CNS therapeutics. The primary known mechanisms of action of the parent scaffold are
not responsible for the observed neuroprotective activity. Herein, we demonstrate that neuroprotective
aromatic carbamates function to increase the Bclꢀ2/Bax ratio to an antiꢀapoptotic state and activate autophagy
through induction of beclin 1.
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Journal of Medicinal Chemistry
INTRODUCTION
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Neurodegenerative diseases are a group of disorders characterized by the loss of structure and/or
function of neurons. At the molecular level, neurodegenerative diseases possess several similarities, such as
1
abnormal deposition of proteins (which in many disorders are misfolded), mitochondrial stress leading to the
2
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formation of reactive oxygen species (ROS), microglial activation and neuroinflammation, dysregulation of
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proteostasis involving the ubiquitinꢀproteasome pathway and autophagyꢀlysosome pathway, programmed cell
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death, including apoptosis and dysregulation of receptors involved in synaptic plasticity, memory, learning and
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other functions.
These features are evident in the most prevalent neurodegenerative diseases including
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS) and
1, 5, 7, 8
Batten disease, the most common (yet rare) neurodegenerative disease of childhood.
Current treatment
options for all of these disorders are symptomatic and do not slow or reverse disease progression.
Pathophysiological similarities at the molecular level suggest that lessons learned from one neurodegenerative
disease may be applicable to other diseases, leading to the design of pharmacological interventions with broad
9
utility.
Drug repurposing, employing a clinically approved agent for one indication in a different indication, has
10
become a rich source of lead compounds. To this end, flupirtine (1, Figure 1), a nonꢀopioid analgesic
1
1
approved in the European Union for pain following surgery, has been shown to possess a range of additional
pharmacological activities. The antiꢀseizure activity of 1 has been ascribed to its indirect antagonism of the Nꢀ
12
methylꢀDꢀaspartate (NMDA) receptor. The compound is known to act as a potassium K 7 channel (KCNQ)
v
opener and reduces intracellular levels of calcium, which is related to its activity as an indirect NMDA receptor
13, 14
antagonist.
Neuroprotective activity in a variety of neurodegenerative disease models has been reported
14
for 1, with mechanisms of action including upregulation of the antiꢀapoptotic protein Bꢀcell lymphoma 2 (Bclꢀ
1
1
2) and antioxidant activity via increased glutathione levels. Overall in vitro, in vivo and/or clinical trials have
demonstrated protective properties in Alzheimer’s disease, Parkinson’s disease, epilepsy, CreutzfeldtꢀJakob
disease, glaucoma, ageꢀrelated macular degeneration, ischemic stroke, prion disease, HIV related
11, 13ꢀ15
neuroinfection, multiple sclerosis and Batten disease.
However, chronic use of 1, as would be required
to treat neurodegenerative diseases, may lead to mild hepatotoxicity that has led to restricted use in the
16
European Union and precludes its use as a potential therapeutic for neurodegenerative disease.
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Retigabine (2, Figure 1), the phenyl bioisostere of 1, is approved by the Food and Drug Administration
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(
FDA) for the treatment of seizures and is currently in Phase II clinical trials for the treatment of ALS. In
comparison with 1, 2 possesses greater potency as a NMDA receptor antagonist in neurons differentiated from
19
ALS patient induced pluripotent stem cells (iPSCs). Like 1, 2 acts as KCNQ opener, and thus an indirect
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antagonist at the NMDA receptor and also possesses antioxidant properties.
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Figure 1. Structures of (1) and (2).
Given the noted neuroprotective effects of 1 across several neurodegenerative diseases, the recent
progression of repurposing 2 to clinical trial for ALS and the currently illꢀdefined molecular targets of the two
agents, we instigated a campaign to fully probe the unexplored structureꢀactivity relationships (SARs) that
define the neuroprotective properties of the aromatic carbamate scaffold. Even though libraries of both 1 and 2
12
derivatives have been reported in the literature, the focus was solely on NMDA antagonistic activity. Herein,
we demonstrate the neuroprotective SAR of 1 violates its SAR for NMDA antagonism and directly show no
correlation between neuroprotective activity and NMDA antagonism or antioxidant activity. We demonstrate the
translational neuroprotective activity of designed aromatic carbamate derivatives in neurons differentiated from
a human iPSC line, a model considered highly translatable and widely accepted for drug screening as well as
19, 22
repurposing molecules for neurodegenerative drug discovery.
Further, we disclose the neuroprotective
mechanism of action of structurally novel aromatic carbamates to be multiꢀmodal; acting to significantly
upregulate the antiꢀapoptotic protein Bclꢀ2 and induce autophagy by elevation of beclin 1.
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Journal of Medicinal Chemistry
CHEMISTRY
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The synthetic route employed to access derivatives of 1 was adapted from Seydel et al. Commercially
available 2,6ꢀdichloroꢀ3ꢀnitropyridine (3) underwent selective amination at the 2ꢀposition to provide 6ꢀchloroꢀ3ꢀ
nitropyridinꢀ2ꢀamine (4) in good yield (Scheme 1). Pyridine (4) underwent nucleophilic substitution with a
suitably substituted benzylamine to afford the 6ꢀaminoꢀ2ꢀbenzylaminoꢀ5ꢀnitropyridine intermediate (5) which
was subsequently reduced to provide triaminopyridine intermediate (6). Regioselective addition of a suitably
functionalized chloroformate to the 3ꢀamino position proceeded in good yield to afford the targeted aromatic
carbamate of general structure (7).
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To access the corresponding ether bioisostere, commercially available 4ꢀtrifluoromethyl benzyl alcohol
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and pyridine intermediate (4) underwent a Williamson ether synthesis to afford the respective pyridine ether
derivative (SI, Scheme 1). This intermediate was subjected to the same synthetic transformations as described
in Scheme 1 to yield the 4ꢀtrifluorophenylmethyl ether analogue (14) (Table 2). The morpholino analogue (15)
(
Table 2) was synthesized based on the same synthetic route (SI, Scheme 2).
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To achieve excision of the 2ꢀamino moiety of 1 we employed either commercially available 2ꢀchloroꢀ5ꢀ
nitropyridine (16a) or its 4ꢀmethyl counterpart (16b) as starting material and followed the same reaction
sequence as delineated in Scheme 1 to obtain analogues (19, 21ꢀ23) and an analogue with a 4ꢀmethyl
substitution in the pyridine ring (20) (Scheme 2).
Synthetic access to structurally diverse derivatives of 2 was achieved by reductive amination of
commercially available 2ꢀnitroꢀ1,4ꢀphenyldiamine (24) with a suitably functionalized benzaldehyde (Scheme 3).
The secondary amine was found to be more reactive compared to the primary amine and was thus protected
as the benzylcarbamate (26). Addition of a suitably functionalized chloroformate afforded dicarbamate (27).
Subsequent exposure of (27) to palladium on charcoal under a hydrogen atmosphere achieved concomitant
benzylcarbamate deprotection and reduction of the 2ꢀnitro moiety to the respective amine (2, 28ꢀ30) in one
24
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1
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RESULTS AND DISCUSSION
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Figure 2. Schematic representation of screening strategy to identify neuroprotective aromatic carbamates.
Screening. Our screening strategy (Figure 2) was designed to allow rapid initial evaluation of
neuroprotective activity, and identification and exclusion of toxic derivatives. To begin to understand the
structural features of aromatic carbamates required for neuroprotective activity and to assign an initial SAR we
25
assayed their protective activity in ‘neuronꢀlike’ PC12 cells induced to apoptosis by exposure to etoposide.
Etoposide is an anticancer agent that works as a topoisomerase II inhibitor and is widely used to induce
26
apoptosis in cells for screening of neuroprotective molecules. To ensure that screened compounds do not
simply act to displace or block etoposide from its binding site we conducted a secondary screen on selected
derivatives to determine protective effect to ameliorate apoptotic cell death in PC12 cells exposed to serum
2
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starvation. Identical trends in activity were observed across both external insults; derivatives with greater
protective effect than 1 to ameliorate etoposideꢀinduced apoptosis also demonstrated greater protective effect
to ameliorate serum starvationꢀinduced apoptosis and vice versa (SI, Table S1). To rapidly assign a SAR we
compared protective activity at an aromatic carbamate concentration of 3 ꢁM, with all compounds screened at
1
, 3, 10 and 30 ꢁM concentrations. The 3 ꢁM concentration is the value at which hit compound 1 provided
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greatest protective activity in the primary etoposideꢀbased assay and is an achievable value for translation to
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free brain concentration in vivo. Compounds which indicated greater protective activity than 1 were furthered
screened at 0.1 and 0.3 ꢁM to determine doseꢀdependent effects. (SI, Table S2ꢀS5).
To determine protective effect in human cells, aromatic carbamates that showed greater activity than 1
in PC12 cells (and selected inactive compounds, compared to 1, to act as controls) were screened to
determine their ability to ameliorate etoposideꢀinduced apoptosis in human SHꢀSY5Y neuroblastoma cells. To
determine the translational potential of aromatic carbamates that show protective effect in both PC12 and SHꢀ
SY5Y cells we screened the ability of these analogues (and selected inactive controls compared to 1) to
ameliorate etoposideꢀinduced apoptosis in human neurons differentiated from iPSCs. These iPSCꢀderived
human neurons possess several advantages over immortalized neuronal cell lines and primary mouse neurons
as they require less specialist isolation, can be produced in large quantities, and have no specialist licensing
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requirements.
StructureꢀActivity Relationships. To provide a diverse library of aromatic carbamates, the structure of
1
was dissected in to three classes; analogues at the carbamate position (class I, Table 1), analogues of the
benzylamine group (class II, Table 2) and analogues of the ring system (class III, Table 3). Further, hybrid
analogues (Table 4) were also generated where modifications were made at more than one site.
Multiparameter optimization (MPO) score was calculated for all synthesized analogues to allow further
comparison of derivatives and identify predicted bloodꢀbrain barrier (BBB) penetration, an essential parameter
28
29
for neuroprotective compounds. An MPO score ≥4 is predictive of brain penetration, therefore we designed
our analogues such that the majority possess an MPO score ≥4 (Table 1ꢀ4, SI Table 6).
Within class I, a range of substituents could be tolerated at the carbamate moiety including electronꢀ
donating and electronꢀwithdrawing groups (Table 1). To explore the effect of homologation within the
carbamate moiety, analogues with 1, 3, 4 and 6 methylene groups, including linear and branched chains, were
synthesized. The methyl (8a) and hexyl (8e) carbamate analogues demonstrated protective activity (cell
viability of 97% and 78% respectively). However, the hexyl derivative suffered a reduced MPO score <4. The
isobutyl carbamate derivative (8c) showed a reduction in activity (63%) while the propyl (8b) and tertꢀbutyl (8d)
carbamates displayed no activity above vehicle control, demonstrating protection of cell viability at 47% and
42% respectively, suggesting that branching alters binding to the target receptor and reduces activity, a
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Journal of Medicinal Chemistry
commonly observed effect in medicinal chemistry, coupled with low levels of toxicity. The allyl carbamate (8f)
was well tolerated (85%) while the alkyne carbamate (8g) displayed reduced activity (62%). Addition of
electronꢀwithdrawing groups influence protective effect based on their regiochemistry; 1ꢀchloroethyl (8h) and
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2,2,2ꢀtrichloroethyl (8j) analogues were well tolerated (89% and 77% respectively) but with a reduced MPO
score in the case of (8j), while the 2ꢀchloroethyl analogue (8i) lost activity (54%), probably due to the
accessible electrophilic site imparting toxicity. Phenyl (8k) and fluorenylmethyloxycarbonyl (Fmoc) (8m)
derivatized carbamates also showed a reduction in activity (59% and 56% respectively), further supporting that
nonꢀlinear steric bulk in this region of the scaffold engenders loss of activity. Interestingly, the benzyl analogue
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(
8l) was equally tolerated when compared with allyl carbamate (8f) despite the clear steric differences between
these two structures (81% and 85% respectively).
Table 1. Protective effect of carbamate analogues of 1 to ameliorate etoposideꢀinduced apoptosis in PC12 cells. n=3, percentage cell
viability represented as mean ± SEM.
Compound
Vehicle
1
Z
% Viability
3 ꢁM)
52.13 ±
.13
79.50 ±
.22
96.64 ±
.97
46.66 ±
.49
62.91 ±
.72
MPO
Score v2
(
1
4.50
3
8a
4.50
4.41
4.22
5
8b
2
8
c
d
1
8
42.17 ±
.46
4.32
2
8
e
78.46 ±
6.52
3.73
4.48
4.50
8
f
84.87 ±
7
.95
61.87 ±
.01
8g
4
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h
88.94 ±
4.42
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8.77
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i
53.92 ±
.68
4.50
3.71
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8
j
77.25 ±
.79
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k
58.98 ±
.97
4.02
3
8l
81.21 ±
.49
3.93
2.82
5
8m
56.42 ±
.19
5
Within class II derivatives the benzylamine moiety was found to be pharmacophoric with minor changes
in substitution significantly affecting activity (Table 2). Substitution of chlorine at the para position (9a) reduced
activity to 64% viable cells while hydrogen (9b) retains activity with 79% viable cells, suggesting this effect may
be due to size rather than electronegativity. Fluorine and hydrogen have widely different electronegativities but
comparable Van der Waals radii. The 2ꢀtrilfuoromethyl derivative (9c) retains activity, providing cell viability of
8
3%, while 3ꢀtrifluoromethyl (9d) and 4ꢀtrifluoromethyl (9e) both decrease activity to 68%. However, the 4ꢀ
trifluoromethyl analogue (9e) at just 0.1 ꢁM concentration, increased cell viability to 84% (Figure 3), which was
comparable to 1 at 3 ꢁM concentration (80%). Bioisoteric replacement to incorporate a 3ꢀ(aminomethyl)ꢀ6ꢀ
(
trifluoromethyl)pyridine moiety (9f) showed similar cell viability to 1 at 3 ꢁM concentration (82%). Similar to 4ꢀ
trifluoromethyl (9e), 3ꢀ(aminomethyl)ꢀ6ꢀ(trifluoromethyl)pyridine (9f) also showed activity at concentrations as
low as 0.1 ꢁM with 74% viable cells. 4ꢀtrifluoromethoxy benzylamine (9g) and its bioisostere 4ꢀ
thio(trifluoromethyl) benzylamine (9h) retain activity at 81% and 79% respectively. However, their respective
MPO scores are <4 at 3.67 and 3.56 respectively. Reduced concentration of 9g (0.1 ꢁM) displays 77% cell
viability, similar to 1 at 3 ꢁM concentration. In comparison, 9h at 0.3 ꢁM concentration increases cell viability to
9
3%. The 4ꢀmethyl analogue (9i) increases cell viability to 136%, suggesting an effect to enhance cell
proliferation which would be advantageous in the treatment of neurodegenerative diseases. However, the
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Journal of Medicinal Chemistry
derivative degrades in solvent upon storage for four days. The 3ꢀmethyl derivative (9j) retains activity
comparable to 1 (82%) but is less active than it’s 4ꢀsubstituted counterpart. The 4ꢀmethoxy derivative (9k)
retains activity at 80% while the 3ꢀmethoxy derivative (9l) increases activity to 87%. This suggests that both
electronꢀwithdrawing and electronꢀdonating groups are well tolerated around the ring. Derivatives with electronꢀ
withdrawing 3,5ꢀdisubstitution (9m, 9n) enhance activity compared to their monoꢀsubstituted counterpart (9d).
Derivatives with 2,4ꢀdisubstitution (9o, 9p) retain activity, this is particularly appreciable when electronꢀ
donating methyl groups are employed (88% cell viability). Adding steric bulk at the paraꢀposition in the form of
a phenyl (9q) or thiophene (9r) reduces activity and induces toxicity at higher concentrations, along with
reducing MPO score. Increasing steric bulk in the form of a napthyl group (9s) reduces activity below that of 1.
This effect is also seen when the carbon chain length is increased from methylene to ethylene (9t, 9u). These
results combined, may suggest that the benzylamine ring and its substitutions interact with a specific binding
pocket of the protein target(s), and adding bulk or increasing linker length may disrupt this interaction. Adding
an αꢀmethyl group (9v) inactivates the molecule and imparts toxicity at higher concentrations. Bioisosteric
replacement of the benzylamine with benzylether (14) results in reduced activity (70%) and interestingly, the
morpholino derivative (15) increases activity to 89%, which suggests that the amine may not be playing a role
as a hydrogen bond donor.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Protective effect of benzylamine analogues of 1 to ameliorate etoposideꢀinduced apoptosis in PC12 cells. n=3, percentage cell
viability represented as mean ± SEM.
Compound
Vehicle
1
R
% Viability
3 ꢁM)
52.13 ±
.13
79.50 ±
.22
MPO
Score v2
(
1
4.50
4.44
4.50
3
9
a
b
64.32 ±
.47
2
9
78.63 ±
.27
6
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Page 12 of 55
9
c
d
82.76 ±
4.30
4.30
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2.15
9
68.17 ±
3.05
9e
68.38 ±
.67
4.30
4.10
3.67
3.56
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
f
81.57 ±
.54
80.59 ±
.90
6
9g
9h
9i
1
79.24 ±
3.63
1
135.53 ±
2.27
4.48
4.48
1
9
j
82.28 ±
12.82
9k
79.68 ±
4.22
4.22
6
.13
87.49 ±
.13
9
l
6
9m
72.94 ±
5.41
4.14
3.41
4.50
9n
75.70 ±
3.69
9o
78.04 ±
7.51
9
p
q
87.89 ±
4.66
4.23
3.90
9
48.88 ±
3.91
9
r
76.08 ±
7.37
3.96
4.24
9s
57.59 ±
3.67
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Journal of Medicinal Chemistry
9
t
59.29 ±
4.50
4.50
4.50
4.62
5.00
1
2
3
4
5
6
7
8
9
5.17
9u
49.96 ±
3.71
9v
66.17 ±
2.76
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
4
5
69.94 ±
4.27
1
88.74 ±
.53
5
To understand the effect of substitutions on the pyridine ring (class III), ring analogues were
synthesized (Table 3). The carbocyclic bioisostere of 1, 2 demonstrates similar protective activity (74% cell
viability). Several analogues of 1 and 2 possessing the same substitution pattern were synthesized to further
understand the role of the central ring in the activity of the molecule. Analogues of 2 were either equipotent
with their 1 counterparts (1 vs 2, 8b vs 29) or less active (8e vs 30, which shows a decrease in cell viability
protection by 20%). Excision of the 2ꢀposition amine (19) resulted in increased activity to 110% cell viability.
Addition of a 4ꢀmethyl group to the central pyridine ring (20) resulted in no protective activity above vehicle
control.
Table 3. Protective effect of ring analogues of 1 to ameliorate etoposideꢀinduced apoptosis in PC12 cells. n=3, percentage cell viability
represented as mean ± SEM.
Compound
Structure
% Viability MPO Score
3 ꢁM) v2
(
Vehicle
1
52.13 ± 1.13
79.50 ± 3.22 4.50
2
73.92 ± 8.03 4.50
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1
9
0
109.51 ±
5.00
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.90
2
53.80 ± 1.14 4.79
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A number of hybrid analogues were synthesized to evaluate the cumulative effect on protective activity
upon structural modification at more than one site (Table 4). The hybrid derivatives combining class I and class
II analogues, demonstrated an additive effect on their activity (11 vs 9d & 8f; 12 vs 9a & 8f and 13 vs 9c & 8j).
As excision of the 2ꢀposition amine (19) resulted in enhanced cell proliferation we sought to determine if further
increases in cell viability can be achieved through combination with substituents that show high activity in other
derivatives (21ꢀ23). However, all demonstrated reduced protective activity over their 2ꢀamine counterpart.
Table 4. Protective effect of hybrid analogues of 1 to ameliorate etoposideꢀinduced apoptosis in PC12 cells. n=3, percentage cell
viability represented as mean ± SEM.
Compound
Vehicle
1
Structure
% Viability
3 ꢁM)
MPO
(
Score v2
52.13 ±
1.13
79.50 ±
4.50
4.48
3.22
10
41.28 ±
.84
5
11
107.25 ±
1.33
4.06
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Journal of Medicinal Chemistry
1
2
3
79.65 ±
.06
4.25
2.99
1
2
3
4
5
6
7
8
9
3
1
107.50 ±
.23
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21
63.99 ±
.21
4.68
4.87
5.00
3.71
4.39
3.73
3
O
O
22
23
28
29
30
64.27 ±
7.19
NH
N
N
H
67.50 ±
7.60
91.62 ±
6.35
55.56 ±
4.28
60.13 ±
.59
6
A number of aromatic carbamates have been identified that possess significant protective activity to
rescue PC12 cells from etoposideꢀinduced apoptosis in our primary assay and demonstrate predicted (MPO
score ≥4) BBB penetration. To determine the translational effect a representative set of the most active
compounds from all three classesꢀ 1, 2, allyl carbamate (8f), benzyl carbamate (8l), 4ꢀ
trifluoromethylbenzylamine (9e), 4ꢀtrifluoromethoxybenzylamine (9g), 4ꢀthio(trifluoromethyl)benzylamine (9h)
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Page 16 of 55
and two less active control compounds 4ꢀchlorobenzylamine (9a) and benzylether (14) were screened in
secondary assays. It should be noted that three compounds (8l, 9g and 9h) possess MPO scores of 3.93, 3.67
and 3.56 respectively. However, these were included in this subꢀset of compounds due to their much higher
protective activity at concentrations as low as 0.1 ꢁM (Figure 3). Further synthetic modifications to optimize
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
30
BBB penetration are possible once lead compounds are identified.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Summary of protective activity of selected aromatic carbamates to ameliorate etoposideꢀinduced apoptosis in PC12 cells at
low concentrations. TwoꢀWay ANOVA, Tukey Test, 95% Confidence Interval. *ꢀ p<0.05, **ꢀ p<0.01, ***ꢀ p<0.001, ****ꢀ p<0.0001. n=3,
%
viability represented as mean ± SEM.
The ability of the representative set of compounds (Figure 3) to ameliorate etoposideꢀinduced apoptosis
in human neuroblastoma SHꢀSY5Y cells was determined to evaluate if activity is retained upon transitioning
from rat to human ‘neuronꢀlike’ cells. A similar trend in protective activity was observed in SHꢀSY5Y cells as in
PC12 cells (Figure 4). The overall cell viability protection was lower in SHꢀSY5Y cells compared to PC12 cells
(
69% cell viability in SHꢀSY5Y for 1 at 3 ꢁM vs 80% in PC12 cells) which may be attributed to different
expression levels of the protein target(s) of our developed compounds between cell types.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Selected aromatic carbamates are protective to ameliorate etoposideꢀinduced apoptosis in human SHꢀSY5Y cells. Cell
viability measured using PrestoBlue dye after treatment with 5 ꢁM etoposide for 12 hours. TwoꢀWay ANOVA, Tukey Test, 95%
Confidence Interval. *ꢀ p<0.05, **ꢀ p<0.01, ***ꢀ p<0.001, ****ꢀ p<0.0001. n=3, % viability represented as mean ± SEM.
Protective Effect in Human iPSCꢀDerived Neurons. To determine the translational activity of
selected analogues of 1 in the most representative model of terminally differentiated human neurons, aromatic
22, 31
carbamates were screened in neurons differentiated from IMR90ꢀc4 iPSCs.
These cells represent the best
currently available in vitro model of human neurons for translational drug screening. The parent scaffold 1,
benzyl carbamate analogue (8l) and 4ꢀtrifluoromethoxy analogue (9g) were active at 3, 10 and 30 ꢁM
concentrations, while 2 was active only at 10 and 30 ꢁM concentrations (active defined as protective ability
above that of vehicle control). Interestingly aromatic carbamate 9a, inactive in the PC12 cell line, demonstrated
low protective activity in the iPSCꢀderived neurons at 10 and 30 ꢁM concentration, but less so than 1 at these
concentrations. Gratifyingly, the 4ꢀtrifluoromethyl benzylamine analogue (9e) retained significant protective
activity from just 0.1 ꢁM concentration through 3 ꢁM (Figure 5). The electronegativity of the trifluoromethyl
substituent may also have a role to deactivate the benzylamine, preventing formation of the quinone diimine
16
reactive intermediate postulated to be responsible for the hepatotoxicity of 1. The ether bioisostere (14)
provided 65% cell viability compared with 1 (70%) at 3 ꢁM concentration. While this does represent a slight
reduction in protective activity, the ether analogue is incapable of being metabolized to the quinone diimine
reactive intermediate toxicophore, thus potentially offering an avenue to explore aromatic carbamates devoid
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Journal of Medicinal Chemistry
Page 18 of 55
of liver toxicity. This assay supports the translational potential of our preliminary screen as a similar trend in
activity is observed in neurons differentiated from human iPSCs and rat ‘neuronꢀlike’ PC12 cells.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Selected aromatic carbamates ameliorate etoposideꢀinduced apoptosis in human IMR90ꢀc4 iPSCꢀderived neurons. Cell
viability measured using PrestoBlue dye after treatment with 15 ꢁg/mL etoposide for 36 hours. TwoꢀWay ANOVA, Tukey Test, 95%
Confidence Interval. *ꢀ p<0.05, **ꢀ p<0.01, ***ꢀ p<0.001, ****ꢀ p<0.0001. n=3, % viability represented as mean ± SEM.
Mechanism of Action Determination. We sought to understand the mechanism by which the
developed aromatic carbamates exert their neuroprotective activity to aid in future design of more active
compounds. As antioxidant mechanisms of action are widely known to confer neuroprotective effect and both 1
32
and 2 are known to act to reduce reactive oxygen species (ROS) levels, we employed CellROX green dye
which, by immunocytochemistry, correlates a proportional green intensity to the quantity of ROS present in a
cellular system.
Exposure of PC12 cells to 10 ꢁM of 1, 2, 8f or 8l and staining with CellROX green dye indicated no
amelioration of ROS upon treatment with the neuroprotective derivatives 8f or 8l, while 2 and 1 do ameliorate
ROS induced by H O as expected (Figure 6A). To further validate these results, PC12 cells were treated with
2
2
aromatic carbamates in the concentration range of 1ꢀ30 ꢁM. Introduction of ROS was generated using H O
2
2
and cell viability was measured after four hours using PrestoBlue dye. Protection against cell death induced by
H O was observed with 1 and 2 while the more active analogues 8f, 8l and 9e failed to show any activity
2
2
above vehicle control (Figure 6B). Analogue 9a which was less active compared to 1 was used as a negative
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Journal of Medicinal Chemistry
control and showed no protective activity. Thus, synthesized aromatic carbamate derivatives with greater
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
neuroprotective activity than 1 and 2 do not act as antioxidants.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Antioxidant activity is demonstrated by 1 and 2. However, equipotent or greater potency protective derivatives 8f, 8l and 9e
do not act as antioxidants. (A) PC12 cells stained with CellROX and DAPIꢀblue nuclear stain. Greater intensity of green fluorescence
indicates greater levels of ROS. Visualized through a fluorescence microscope (40x magnification, bar = 100 ꢂm). (B) Measurement of
cell viability using PrestoBlue dye after treating PC12 cells with 400 ꢁM H
2 2
O . TwoꢀWay ANOVA, Tukey Test, 95% Confidence Interval.
*ꢀ p<0.05, **ꢀ p<0.01, ***ꢀ p<0.001, ****ꢀ p<0.0001. n=2, % viability represented as mean ± SEM.
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Molecules acting as KCNQ openers, including 1 and 2, are known indirect antagonists of the NMDA
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
33
receptor. We next sought to determine the activity of analogues of 1 to ameliorate NMDA induced
34
excitotoxicity. NMDA receptors are expressed in PC12 cells, but the receptors are not functional. Thus, a
mechanism of action involving NMDA receptor antagonism cannot be responsible for the observed protective
35
activity. Human neurons differentiated from iPSCs however, do express functional NMDA receptors and we
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
have shown translational protective effect of our aromatic carbamates against etoposideꢀinduced toxicity
(
Figure 5). Thus, we employed neurons differentiated from human iPSCs (IMR90ꢀc4) to evaluate our designed
35
compounds in an NMDA challenge assay (Figure 7). No protective effect was observed with treatment of
selected aromatic carbamates. However, a trend was observed where 1 and 2, at a higher concentration of 30
ꢂM, protected against NMDA insult. The 4ꢀtrifluormethyl analogue (9e), active in the human iPSCꢀderived
neuron assay at a low concentration of 0.1 ꢂM, failed to show any NMDA antagonistic activity at all
concentrations screened.
Figure 7. Selected aromatic carbamates lack NMDA antagonistic activity when screened in human IMR90ꢀc4 iPSCꢀderived neurons.
Cell viability measured using PrestoBlue against 500 ꢂM NMDA treatment. n=1, 4 technical replicates. % viability represented as mean
±
SEM.
Neuroprotective Derivatives Induce Bclꢀ2 and Increase Bclꢀ2/Bax ratio. Upregulation of the antiꢀ
36
apoptotic protein Bclꢀ2 is reported as a mechanism of action of 1, albeit at high concentration, and thus we
sought to understand the relationship of neuroprotective aromatic carbamates to the degree of Bclꢀ2 induction
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Journal of Medicinal Chemistry
in PC12 cells. The ratio of Bclꢀ2 to Bclꢀ2ꢀassociated X protein (Bax), a proꢀapoptotic protein, determines cell
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
37
survival or cell death and thus the ratio of Bclꢀ2 to Bax was determined. Cells were pretreated with the
aromatic carbamates for four hours before the addition of etoposide. After 24 hours of treatment, 1 and 2 at 3
ꢂM showed 68% and 64% increase in the Bclꢀ2/Bax ratio. Upon treatment with 8f at 3 ꢂM a 150% increase in
the Bclꢀ2/Bax ratio was observed while 9a, the reduced activity control, showed a 44% reduction. The 4ꢀ
trifluoromethyl analogue (9e) at 3 ꢂM and 0.1 ꢂM showed 232% and 159% increase in the Bclꢀ2/Bax ratio
respectively. This was determined to be a transient effect as no change in the Bclꢀ2/Bax ratio was observed
after 48 hours of treatment (Figure 8A, B).
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Figure 8. Effect of selected aromatic carbamates to modulate the Bclꢀ2/Bax ratio in PC12 cells after 24 and 48 hours of treatment. (A)
Quantified results. n=2, values represented as mean ± SEM. (B) Representative immunoblot for Bclꢀ2 and Bax.
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Neuroprotective Derivatives Induce Autophagy. Mutations in genes regulating autophagy contribute
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to the pathogenesis of a wide range of neurodegenerative disorders. Activation of autophagy has been shown
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8, 39
to be protective in several models of neurodegeneration.
This effect results from enhanced clearance of
Thus, we evaluated the effect of analogues of 1 to induce
autophagy. Induction of autophagy in PC12 cells was measured by monodansylcadaverine (MDC) staining
38, 40
abnormal or misfolded proteins from neurons.
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using flow cytometry after 48 hours of treatment. Rapamycin, which induces autophagy by mTOR inhibition,
was employed as a positive control; at 500 nM concentration a twoꢀfold increase in MDC staining was
observed over vehicle. At 10 ꢂM concentration, 1 showed an 8ꢀfold increase in MDC staining while 1, 2 and 8f
at 3 ꢂM show approximately 2ꢀfold increase in MDC staining (Figure 9A). Derivative 8l at 3 ꢂM resulted in
approximately threeꢀfold increase in MDC staining. A doseꢀdependent induction in autophagy by MDC staining
was observed with 9a, 9e, 9g and 9h. Interestingly the inactive neuroprotective compound 9a, included as a
negative control, demonstrated a significant effect to enhance autophagy, with a >fiveꢀfold increase at 3 ꢂM
concentration and >12ꢀfold at 10 ꢂM, suggesting that autophagy modulation alone is not sufficient for the
aromatic carbamates to engender neuroprotective effect. Combination of Bclꢀ2 induction and autophagy
activation is required (9a suppressed Bclꢀ2 expression (Figure 8A, B). Compound 9h (71% protection of cell
viability of human iPSCꢀderived neurons from apoptosis) demonstrated eightꢀfold increase in autophagy at 3
ꢂM but comparison to the more protective derivative 8l (79% protection) at 3 ꢂM reveals a threeꢀfold increase
in autophagy. Perhaps suggesting a threshold value of autophagy modulation is required for protective activity
that both compounds exceed to provide broadly similar activity, with the greater protective effect of 8l being
attributed to slightly greater induction of Bclꢀ2 (Figure 8A).
A second experiment to determine autophagy modulation was conduted by measuring beclin 1
expression levels in PC12 cells by immunoblotting (Figure 7B). Activation of autophagy correlates with
43
increased expression of beclin 1. Similar trends were observed to the MDC experiment. A 32% increase in
beclin 1 expression was observed upon treatment with 1 at 3 ꢂM. Interestingly, by comparison, aromatic
carbamate 9a showed a reduced level of beclin 1 expresion (20%) at 3 ꢂM, although this did increase to 104%
at 10 ꢂM. A doseꢀdependant increase of beclin 1 expression was observed with aromatic carbamates 9e, 9g
and 14. The most protective derivative identified (9e) which confers 75% protection of human iPSCꢀderived
neurons at 0.1 uM concentration increases beclin 1 expression by 18% at this concentration (Figure 9B, C).
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Journal of Medicinal Chemistry
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Figure 9. Effect of selected aromatic carbamates to modulate autophagy (A) Effect of derivatives of 1 to induce autophagy as observed
by MDC staining. OneꢀWay ANOVA, Dunnett Test, 95% Confidence Interval. *ꢀ p<0.05, **ꢀ p<0.01, ***ꢀ p<0.001, ****ꢀ p<0.0001. (B)
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Effect of aromatic carbamates on beclin 1 expression. Quantified results. n=2, values represented as Mean ± SEM. (C) Representative
immunoblot for beclin 1.
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Upregulation of antiꢀapoptotic Bclꢀ2 family proteins or down regulation of proꢀapoptotic proteins of the
Bclꢀ2 family has been widely shown in the literature as an effective strategy for neuroprotection. Small
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molecule inhibitors targeting Bax exert a neuroprotective effect. Likewise, modulation of autophagy by small
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molecules such as rapamycin results in neuroprotective effect in several neurodegenerative diseases including
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Parkinson’s disease, where rapamaycin enhances clearance of the pathological protein αꢀsynuclein.
Rapamycin functions to induce autophagy by binding FK506 binding protein 12 and then inhibiting
phosphorylation of mTOR. In addition to inducing autophagy, rapamycin downregulates overall protein
47, 48
synthesis and cell proliferation due to its action on mTOR.
Furthermore, rapamycin has poor BBB
penetration making it unsuitable as a therapeutic agent for neurodegenerative disease, making the
identification of novel autophagy inducers an important pursuit.
CONCLUSIONS
The clinically available agent 1 has been known for years to possess moderate neuroprotective activity.
However, the low propensity for neuroprotective effect even at high concentration, compounded by the known
liver toxicity precludes the therapeutic use of 1 for neurodegenerative diseases. Herein, we disclose the first
detailed neuroprotective SAR of derivatives of 1, identifying a library of aromatic carbamates with enhanced
neuroprotective effect. We disclose derivatives of 1 that engender protective activity to ameliorate etoposideꢀ
and serum starvationꢀinduced apoptosis in ‘neuronꢀlike’ PC12 and SHꢀSY5Y cell lines. Further, we have
identified a number of aromatic carbamates that demonstrate translational protective activity to ameliorate
etoposideꢀinduced apoptosis in human iPSCꢀderived neurons.
Mechanism of action determination shows that the antioxidant, NMDA antagonistic and potassium
channel opening activity of parent compounds 1 and 2 do not correlate to neuroprotective activity. We identify
the mechanism of action of our developed aromatic carbamate derivatives of 1 to be multiꢀmodal; inducing Bclꢀ
2, increasing the Bclꢀ2/Bax ratio to favor an antiꢀapoptotic state, and increasing autophagy. Both of these
actions are required to take place to confer neuroprotective effect with aromatic carbamate 9a increasing
autophagy by fiveꢀfold at 3 ꢂM yet reducing Bclꢀ2/Bax ratio by 44% which results in a compound with little
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Journal of Medicinal Chemistry
neuroprotective effect in human iPSCꢀderived neurons above vehicle control. The most active derivative of 1,
ꢀtrifluoromethane (9e), confers 76% cell viability in human iPSCꢀderived neurons at 0.1 ꢂM concentration.
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This derivative engenders a 159% increase in the Bclꢀ2/Bax ratio and an approximate 18% increase in beclin 1
expression indicating induction of autophagy at the same concentration, with both parameters increasing in
intensity in a doseꢀdependent manner.
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The correlation of neuroprotective activity to both induction of Bclꢀ2 and induction of beclin 1, used
herein as a quantification of autophagy, may highlight another potential mechanism of action of the aromatic
carbamates as disruptors of the Bclꢀ2:beclin 1 proteinꢀprotein interaction (PPI). The Bclꢀ2 protein is known to
49
form a complex with beclin 1 where it functions to regulate autophagy by exerting an inhibitory effect. Thus,
expression levels of Bclꢀ2 exert multiple influences on proteostasis and cell death in neurodegenerative
40
diseases. Further studies to employ aromatic carbamates as chemical probes to investigate the target
protein(s) are underway.
EXPERIMENTAL SECTION
General Synthetic Procedures: All reactions were carried out in ovenꢀ or flameꢀdried glassware under
positive nitrogen pressure unless otherwise noted. Reaction progress was monitored by thinꢀlayer
chromatography (TLC) carried out on silica gel plates (2.5 cm x 7.5 cm, 200 ꢀm thick, 60 F254) and visualized
by using UV (254 nm) or by potassium permanganate and/or phosphomolybdic acid solution as indicator.
Flash column chromatography was performed with silica gel (40ꢀ63 ꢀm, 60 Å) using the mobile phase
indicated or on a Teledyne Isco (CombiFlash R 200 UV/Vis). Commercial grade solvents and reagents were
f
purchased from Fisher Scientific (Houston, TX) or Sigma Aldrich (Milwaukee, WI) and were used without
further purification except as indicated. Anhydrous solvents were purchased from Across Organics and stored
under an atmosphere of dry nitrogen over molecular sieves.
1
13
H and C NMR spectra were recorded in the indicated solvent on a Bruker 400 MHz Avance III HD
1
13
spectrometer at 400 and 100 MHz for H and C respectively with TMS as an internal standard. Multiplicities
are indicated by s (single), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), br (broad).
Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J), in hertz. Highꢀresolution
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mass spectroscopy was performed on a LC/MS ITꢀTOF (Shimadzu) using an ESI source conducted at the
University of Texas at Arlington, Shimadzu Center for Advanced Analytical Chemistry. Lowꢀresolution mass
spectroscopy was performed on a LC/MS Transmission Quadrupole Analyzer (Varian 1200) using an ESI
source. Highꢀpressure liquid chromatography was performed on a Gilson HPLC system with 321 pumps and
155 UV/Vis detector using Trilution software v.2.1 with an ACE Equivalence 3 (C18, 3 mm, 4.6*150 mm)
column. All samples were determined to possess >95% purity, except where indicated otherwise.
1
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Ethyl (2ꢀaminoꢀ6ꢀ((4ꢀfluorobenzyl)amino)pyridinꢀ3ꢀyl)carbamate (1). To
a
solution of N ꢀ(4ꢀ
fluorobenzyl)pyridineꢀ2,3,6ꢀtriamine (265 mg, 1.14 mmol,) in 1,4ꢀdioxane (10 mL) was added ethyl
chloroformate (123 mg, 1.14 mmol). The reaction was stirred at room temperature overnight protected from
light. The resultant precipitate was collected and washed with 1:1 dioxane:diethyl ether (40 mL), diethyl ether
1
(
50 mL) and air dried at room temperature to afford the the title compound as a blueꢀgreen solid. (82%). H
NMR (400 MHz, DMSOꢀd ) – δ : 1.21 (3H, t, J= 4.8 Hz, CH CH ), 4.06 (2H, q, J=7.0 Hz, CH CH ), 4.53 (2H,
6
H
2
3
2
3
br. s, CH Ph), 5.92 (1H, d, J= 8.6 Hz, ArH), 7.20 (2H, t, J= 8.8 Hz, ArH,), 7.34 (1H, br. s, NH), 7.43ꢀ7.50 (3H,
2
13
m, ArH), 8.15 (1H, br. s, NH), 8.52 (1H, br. s, NH,), 13.06 (1H, br. s, NH). C NMR (100 MHz, DMSOꢀd ) – δ :
6
C
14.93, 44.94, 60.97, 94.23, 107.16, 115.73 (d, J= 21.30 Hz), 130.04 (d, J= 8.21 Hz), 134.19 (d, J= 2.96 Hz),
143.26, 149.48, 155.50, 160.73, 163.14. HPLCꢀ Retention time: 5.94 min; Purity: 99%. m/z (ESI) – 305.1
+
(
100%) [MH ]. HRMS (ESI): C H N O F requires: 305.1408. Found: 305.1413.
15
17
4
2
Ethyl (2ꢀaminoꢀ4ꢀ((4ꢀfluorobenzyl)amino)phenyl)carbamate (2). To
a
solution of benzyl (4ꢀ
(
(ethoxycarbonyl)amino)ꢀ3ꢀnitrophenyl)(4ꢀfluorobenzyl)carbamate (180 mg, 0.39 mmol) in MeOH (5 mL) was
added Pd/C (4 mg, 0.004 mmol) and stirred for 2 hours at room temperature under an atmosphere of
hydrogen. The reaction mixture was filtered through celite® washing with MeOH (50 mL) and evaporated in
1
vaccuo to provide the title compound as a purple solid. (Quant). H NMR (400 MHz, CDCl ) – δ : 1.31 (3H, t,
3
H
J= 7.24 Hz, CH CH ), 3.61 (2H, br. s, NH ), 4.21 (2H, q, J= 7.12 Hz, CH CH ), 4.27 (2H, br. s, CH Ph), 6.02ꢀ
2
3
2
2
3
2
13
6.09 (3H, m), 6.94 (1H, d, J= 8.40 Hz, ArH), 7.04 (2H, t, J= 8.68 Hz, ArH), 7.33 (2H, t, J= 8.61 Hz, ArH).
C
NMR (100 MHz, CDCl ) – δ : 14.54, 29.69, 61.58, 105.05, 107.94, 115.40 (d, J= 21.42 Hz), 117.74, 127.45,
3
C
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1
29.87 (d, J= 7.50 Hz), 132.85, 139.37, 155.77, 160.99, 163.44. HPLCꢀ Retention time: 6.79 min; Purity: 99%.
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+
m/z (ESI) – 305.1 (100%) [MH ]. HRMS (ESI): C H N O F requires: 304.1456. Found: 304.1454.
16
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3
2
12
6ꢀChloroꢀ3ꢀnitropyridinꢀ2ꢀamine (4). To a solution of 2,6ꢀdichloroꢀ3ꢀnitropyridine (3) (1 mol equiv.) in MeOH
(5 mL) was added a solution of 7N ammonia in MeOH (1 mol equiv.) and the reaction was stirred overnight at
0
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room temperature. The reaction mixture was diluted with water (20 mL), the insoluble portion was collected by
filtration and crystallized from ethanol to obtain the title compound. (Based on Recovery of Starting Materialꢀ
1
65%). H NMR (400 MHz, CDCl ) – 6.77 (1H, d, J= 8.57 Hz, ArH); 8.25 (2H, br. s, NH ), 8.39 (1H, J= 8.56 Hz,
3
2
13
ArH). C NMR (100 MHz, DMSOꢀd6) – 112.44, 126.57, 138.85, 153.92, 155.44. m/z (ESI) ꢀ 174.1 (100%)
+
[MH ]; 176.0 (40%).
General synthetic procedure for 6ꢀaminoꢀ2ꢀbenzylaminoꢀ5ꢀnitropyridine intermediates (5): To a
suspension of 6ꢀchloroꢀ3ꢀnitropyridinꢀ2ꢀamine (4) (1.73 mmol) in IPA (5 mL) was added appropriately
substituted benzylamine (2.07 mmol ) and triethylamine (2.50 mmol ) and the mixture refluxed for 3 hours. The
reaction mixture was allowed to cool to room temperature and water (30 mL) was added slowly with stirring to
provide a yellow solid. Extraction was performed using ethyl acetate (3 x 20 mL), dried (Na SO ), filtered and
2
4
evaporated in vaccuo. Purification by column chromatography provided the title compound.
General synthetic procedure for triaminopyridine intermediate (6) Method A: SnCl .2H O (4.56 mmol)
2
2
o
was dissolved in conc. HCl (10 mL) and heated at 70 C for 30 mins until a colorless solution was formed. 6ꢀ
aminoꢀ2ꢀbenzylaminoꢀ5ꢀnitropyridine intermediate (5) (1.14 mmol) was added portionwise and the solution
o
heated at 70 C for 3 hrs with a condenser equipped with subba seal and empty balloon. Water was added and
the orange solution was allowed to cool to room temperature. The solution was basified to pH 12 (2M NaOH)
and extracted with CH Cl (3 x 20 mL), the organic layer was washed with brine (50 mL), dried (Na SO ),
2
2
2
4
filtered and evaporated in vaccuo to provide the title compound. The title compound was taken forward without
further purification.
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General synthetic procedure for triaminopyridine intermediate (6) Method B: To a solution of 6ꢀaminoꢀ2ꢀ
benzylaminoꢀ5ꢀnitropyridine intermediate (5) (1 mol equiv.) in 1,4ꢀdioxane (10 mL per mol equiv.) was added
1
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Raney Nickel (AlꢀNi 50:50 wt%) (10x weight of 5) and hydrazine hydrate (10x volume of 5) and the suspension
®
allowed to stir at room temperature overnight. The suspension was filtered through celite washing with CH Cl
2
2
(100 mL) and evaporated in vaccuo to provide the title compound. The title compound was taken forward
without further purification.
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General synthetic procedure for pyridine carbamate derivatives (7): To a solution of triaminopyridine (6)
(
1.14 mmol) in 1,4ꢀdioxane (10 mL) was added the respective chloroformate (1.14 mmol). The reaction was
stirred at room temperature overnight protected from light. The resultant precipitate was collected and washed
with 1:1 dioxane:diethyl ether (40 mL), diethyl ether (50 mL) and air dried at room temperature to afford the title
compound.
Methyl 6ꢀ(4ꢀfluorobenzylamino)ꢀ2ꢀaminopyridinꢀ3ꢀylcarbamate (8a). According to the general procedure for
1
pyridine carbamates, the title compound was obtained as a blueꢀgreen solid. (85%). H NMR (400 MHz,
DMSOꢀd ) – δ : 3.61 (3H, s, CH ), 4.52 (2H, d, J= 5.16 Hz, CH Ph), 5.91 (1H, d, J= 8.77 Hz, ArH), 7.21 (2H, t,
6
H
3
2
J= 8.93 Hz, ArH), 7.34 (1H, br.s ), 7.37ꢀ7.47 (3H, m, ArH), 8.17 (1H, br. s), 8.57 (1H, br. s, NH), 13.03 (1H, br.
13
s, NH). C NMR (100 MHz, DMSOꢀd ) – δ : 44.95, 52.40, 94.10, 107.10, 115.77 (d, J= 21.32 Hz), 130.01 (d,
6
C
19
J= 8.16 Hz), 134.19 (d, J= 2.80 Hz), 134.21, 143.42, 149.68, 155.96, 160.74, 163.15. F NMR (376 MHz,
+
DMSOꢀd ) – δ : ꢀ115.20. HPLCꢀ Retention time: 6.08 min; Purity: 99%. m/z (ESI) – 291.1 (100%) [MH ].
6
F
Propyl (2ꢀaminoꢀ6ꢀ((4ꢀfluorobenzyl)amino)pyridinꢀ3ꢀyl)carbamate (8b). According to the general procedure
1
for pyridine carbamates, the title compound was obtained as a blueꢀgreen solid (98%). H NMR (400 MHz,
DMSOꢀd ) – δ : 0.91 (3H, br. s, CH CH ), 1.59 (2H, br. s, CH ), 4.00 (2H, t, J= 6.43 Hz, CH Ph), 4.56 (2H, m,
6
H
2
3
2
2
CH ), 5.97 (1H, t, J= 8.45 Hz, ArH), 7.19 (2H, t, J= 8.89 Hz, ArH), 7.42ꢀ7.52 (4H, m), 8.13 (1H, br. s, NH), 8.44
2
13
(
1H, br. s, NH), 8.58 (1H, br. s, NH). C NMR (100 MHz, DMSOꢀd ) – δ :10.74, 22.31, 44.93, 66.52, 94.29,
6 C
95.49, 107.19, 115.73 (d, J= 21.32 Hz), 130.06 (d, J= 6.74 Hz), 134.22 (d, J= 2.99 Hz), 149.49, 160.73,
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1
63.14. F NMR (376 MHz, DMSOꢀd ) – δ : ꢀ115.23. HPLCꢀ Retention time: 5.18 min; Purity: 99%. m/z (ESI)
6 F
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
–
319.6 (100%) [MH ].
Isobutyl 6ꢀ(4ꢀfluorobenzylamino)ꢀ2ꢀaminopyridinꢀ3ꢀylcarbamate (8c). According to the general procedure
1
for pyridine carbamates,
t
he title compound was obtained as a blueꢀgreen solid (34%). H NMR (400 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DMSOꢀd ) – δ : 0.92 (6H, m, CH ), 1.88 (1H, br. s, CH), 3.79 (2H, d, J= 6.35 Hz, CH ), 4.53 (2H, d, J= 5.11
6
H
3
2
Hz, CH Ph), 5.92 (1H, d, J= 8.88 Hz, ArH), 7.20 (2H, t, J= 8.98 Hz, ArH), 7.36 (1H, br. s, NH), 7.43ꢀ7.49 (3H,
2
13
m), 8.19 (1H, br. s, NH), 8.54 (1H, br. s. NH), 13.12 (1H, br. s, NH). C NMR (100 MHz, DMSOꢀd ) – δ :
6
C
19.42, 27.98, 44.94, 66.81, 70.95, 94.09, 107.29, 115.77 (d, J= 21.30 Hz), 129.99 (d, J= 8.35 Hz), 134.20
19
(
d, J= 2.53 Hz), 149.57, 160.73, 163.14. F NMR (376 MHz, DMSOꢀd ) – δ : ꢀ115.23. HPLCꢀ Retention time:
6
F
+
5
.42 min; Purity: 99%. m/z (ESI) – 333.1 (100%) [MNa ].
tertꢀButyl 6ꢀ(4ꢀfluorobenzylamino)ꢀ2ꢀaminopyridinꢀ3ꢀylcarbamate (8d). According to the general procedure
for pyridine carbamates,
t
he title compound was obtained as a yellowꢀbrown solid (25%). R = 0.24 (Mobile
f
1
phaseꢀ 1:1= hexane/EtOAc). H NMR (400 MHz, DMSOꢀd ) – δ : 1.42 (9H, s, CH ), 4.35 (2H, d, J= 6.11 Hz,
6
H
3
CH Ph), 5.13 (2H, br. s, NH), 5.68 (1H, d. J= 8.53 Hz, ArH), 6.50 (1H, t, J= 6.53 Hz, NH), 7.01 (1H, d, J= 7.35
2
1
3
Hz, ArH), 7.11 (2H, t, J= 8.97 Hz, ArH), 7.35 (2H, t, J= 6.67 Hz, ArH), 7.96 (1H, br. s, NH). C NMR (100 MHz,
DMSOꢀd ) – δ : 28.65, 44.10, 66.81, 78.71, 95.71, 107.48, 115.19 (d, J= 23.64 Hz), 129.49 (d, J= 8.02 Hz)
,
6
C
1
37.75 (d, J= 3.03 Hz), 153.25, 154.73, 155.82, 160.20, 162.60. HPLCꢀ Retention time: 5.82 min; Purity:
+
9
9%. m/z (ESI) – 333.1 (100%) [MH ].
Hexyl (2ꢀaminoꢀ6ꢀ((4ꢀfluorobenzyl)amino)pyridinꢀ3ꢀyl)carbamate (8e). According to the general procedure
1
for pyridine carbamates,
t
he title compound was obtained as a blueꢀgreen solid (61%). H NMR (400 MHz,
DMSOꢀd ) – δ : 0.87 (3H, br. s, CH ), 1.25ꢀ1.34 (6H, m, CH ), 1.58 (2H, br. s, CH ), 4.00 (2H, q, J= 5.89 Hz,
6
H
3
2
2
CH CH ), 4.54 (2H, d, J= 4.72 Hz, CH Ph), 5.92 (1H, d, J= 8.80 Hz, ArH), 7.20 (2H, t, J= 8.90 Hz, ArH), 7.38ꢀ
2
3
2
13
7.47 (4H, m), 8.24 (1H, br. s, NH), 8.53 (1H, br. s, NH), 13.22 (1H, br. s, NH). C NMR (100 MHz, DMSOꢀd ) –
6
δC: 14.36, 22.49, 25.50, 28.90, 31.39, 44.93, 65.06, 94.11, 107.28, 115.77 (d, J= 21.32 Hz), 130.00 (d, J=
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