Identification of the minimum PAR4 inhibitor pharmacophore and optimization of a series of 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazoles

Article abstract of DOI:10.1016/j.bmcl.2016.10.020

This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490–525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by ～50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate.

Full text of DOI:10.1016/j.bmcl.2016.10.020

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5481–5486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of the minimum PAR4 inhibitor pharmacophore and
optimization of a series of 2-methoxy-6-arylimidazo[2,1-b][1,3,4]
thiadiazoles
Kayla J. Temple a,b,y, Matthew T. Duvernay b,y, Jae G. Maeng b, Anna L. Blobaum a,b, Shaun R. Stauffer b,c
,
Heidi E. Hamm b, Craig W. Lindsley a,b,c,
⇑
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490–525
and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic
liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo
[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and
Received 2 September 2016
Revised 7 October 2016
Accepted 8 October 2016
Available online 11 October 2016
c-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydro-
gen bond donors/acceptors by ꢀ50%. This minimum PAR4 pharmacophore, with competitive inhibition,
versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired
functional groups to engender optimal DMPK properties towards a preclinical candidate.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
PAR4
Platelet aggregation
Minimum pharmacophore
Structure–Activity Relationship (SAR)
Protease Activated Receptor 4 (PAR4) is a G Protein-Coupled
Receptor (GPCR) essential for the thrombin-induced procoagulant
effect on platelets, and as such, has garnered a great deal of interest
as a target for anti-platelet therapy to treat thrombosis without
bleeding.1–3 Historically dominated by antibody therapy, small
molecule PAR4 antagonists are only now emerging as in vivo tool
compounds and clinical candidates.1–4 Until recently, the PAR4
antagonists in the primary literature suffered from poor DMPK
AP and c-thrombin that could then be optimized with more favor-
able DMPK properties. This exercise led to the discovery that the
most basic core of 2, a 6-(benzofuran-2-yl)-2-methoxyimidazo
[2,1-b][1,3,4]thiadiazole 3 as a potent PAR4 inhibitor (PAR4 AP
IC50 = 1.69 nM, PAR4 c-thrombin IC50 = 58.8 nM), as the minimum
pharmacophore (MW = 271) of 2.9 However, the potential liabili-
ties of an unsubstituted benzofuran, as in 3, raised metabolic
stability concerns. Thus, in this Letter, we describe efforts to survey
alternative 6-position substituents on the 2-methoxyimidazo[2,
1-b][1,3,4]thiadiazole core to identify a minimum PAR4 pharma-
cophore that could then be further optimized with functional
groups that would engender desirable DMPK properties.
properties and a lack of activity upon
Our lab recently divulged a new series of small PAR4 inhibitors 1
c
-thrombin activation.4–7
with an improved DMPK profile and weak activity upon c-throm-
bin activation.4–7 Bristol-Myers-Squibb (BMS) also disclosed a
novel series of PAR4 inhibitors, represented by 2 (an analog of
BMS986120), with exquisite potency against both activating pep-
In the initial SAR campaign, we elected to survey functionalized
aryl moieties in the 6-position and assess if these simple analogs
were sufficient to elicit PAR4 inhibition. Analogs 4, or 3, could be
readily accessed in two steps from commercial materials
(Scheme 1), and enabled the evaluation of multiple regions of
tide (AP) and c-thrombin mediated PAR4 stimulation, but with vir-
tually no free drug levels (rat and human fu < 0.001).8 Both 1 and 2
(Fig. 1) are high molecular weight compounds (490–510), with
high cLogPs (>4) and the noted high plasma protein binding.4,8
Thus, we deconstructed 2 in an attempt to identify a minimum
pharmacophore that retained potent PAR4 inhibition against both
the heterobiarylcore.10 Here, commercial
a
-bromo ketones 5 were
condensed with 5-bromo-1,3,4-thidiazol-2-amine 6 to provide
2-bromo-6-arylimidazo[2,1-b][1,3,4]thiadiazoles in 65–86%
7
yield. An SNAr reaction with methoxide under mild conditions
delivered analogs 4 in yields ranging from 72–80%.
SAR for select analogs 4 are highlighted in Table 1, all with
molecular weights less than 325. The most basic analog, 4f with
an unsubstituted phenyl ring, was a PAR4 antagonist with a
⇑
Corresponding author.
E-mail addresses: heidi.hamm@vanderbilt.edu (H.E. Hamm), craig.lindsley@
vanderbilt.edu (C.W. Lindsley).
y
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.10.020
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

5482
K. J. Temple et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5481–5486
OCF3
N
N
OMe
N
N
S
Alternate aryl, heterocycles
Cl
OH
Alternate alkoxy
amino, etc...
1, VU0661259
PAR4 AP IC50 = 179 nM
PAR4 γ-thrombin IC50 = 4.35 μM
N
S
N
minimum
pharmacophore
OMe
O
N
MW = 508
3
O
S
PAR4 APIC50 = 1.69 nM
PAR4 -thrombin IC50 = 58.8 nM
N
N
S
N
γ
OMe
Ph
MW = 271
O
N
MeO
2, BMS-3
PAR4 AP IC50 = 4.3 pM
PAR4 γ-thrombin IC50 = 22.9 pM
MW = 490
Figure 1. Structures of reported PAR4 antagonists 1 and 2, and the minimum pharmacophore of 2, fragment 3, and plans to further optimize 3 into a more desirable fragment
core.
O
PAR4 IC50 against the AP of 757 nM. Simple substitution of the phe-
nyl ring afforded robust SAR in terms of PAR4 inhibition against the
Br
Ar
5
+
N
S
b
a
N
S
AP, with a significant right-shift in PAR4 potency upon activation
with -thrombin. In general, substitutions at the 4-postion of the
N
N
Ar
Br
Ar
OMe
c
N
N
N
N
phenyl ring afforded the most potent analogs. Moreover, lipophilic,
electron-withdrawing moieties proved optimal. For example, the
4-CF3 derivative (4c) was a 15.6 nM PAR4 inhibitor against the
7
4
Br
H2N
S
6
AP, and displayed an IC50 of 348 nM against c-thrombin activation.
Scheme 1. Synthesis of aryl analogs 4. Reagents and conditions: (a) CH3CN/IPA,
80 °C, 18 h, 65–86%; (b) NaOMe, DCM/MeOH (3:1), 1 h, rt 72–80%.
This result is quite significant considering the reduced, minimum
pharmacophore and activities relative to 1 and 2. Likewise, the 4-
OCF3 congener (4n) was a 27.7 nM PAR4 inhibitor against the AP,
Table 1
and displayed an IC50 of 246 nM against
The 4-Cl analog (4k) proved also to inhibit PAR4 upon AP activation
(IC50 = 39.5 nM) as well as -thrombin (IC50 = 621 nM). Interest-
c-thrombin activation.
Structures and activities of analogs 4
Rn
N
S
c
N
OMe
ingly, the difluoromethoxy congener 4v, of 4n, remained potent
against the AP (IC50 = 83.4 nM), but loss of a single fluorine atom
N
4
led to no activity against
c-thrombin activation. Finally, sub-
stituents in the 2-position were generally detrimental to potency,
and electron donating moieties, such as 4-OCH3 (4g) and 4-OH
(4l), led to significant diminution in potency.
Compd
R
PAR4-AP PAC-1
IC50 (nM)a
(pIC50 SEM)
PAR4 c-thrombin PAC-1
IC50 (nM)a
(pIC50 SEM)
Concentration response curves (CRCs) for 4c, 4n and 4k against
AP and c-thrombin mediated activation are shown in Figure 2A–F,
and all three proved to be competitive inhibitors of PAR4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
2-CF3
3-CF3
4-CF3
2,6-diF
2,4-diF
H
4-OCH3
4-CH3
2-Cl
3-Cl
4-Cl
4-OH
4-CN
4-OCF3
4-SO2CH3
3,5-diCF3
2-F
3-F
4-F
3,4-diCl
4-NO2
4-OCHF2
3-CF3,4-F
2-NO2
3-NO2
1330 (5.88 0.06)
36.3 (7.44 0.10)
15.6 (7.81 0.07)
1300 (5.89 0.05)
175 (6.76 0.03)
757 (6.12 0.07)
592 (6.23 0.11)
57.2 (7.24 0.04)
437 (6.36 0.02)
72.8 (7.14 0.07)
39.5 (7.40 0.04)
>10,000 (>5)
937 (6.03 0.06)
27.7 (7.57 0.09)
1,200 (5.92 0.27)
52.3 (7.28 0.04)
526 (6.28 0.04)
675 (6.17 0.14)
406 (6.39 0.03)
28.5 (7.55 0.06)
207 (6.68 0.04)
83.4 (7.08 0.05)
68.8 (7.16 0.05)
4840 (5.32 0.24)
>10,000 (>5)
ND
1310 (5.88 0.24)
348 (6.46 0.06)
ND
ND
ND
ND
ND
ND
>10,000 (>5)
621 (6.21 0.15)
ND
ND
246 (6.64 0.19)
ND
5620 (5.25 0.08)
ND
(Fig. 2G–I). This is in sharp contrast to 1, which is a non-
competitive inhibitor of PAR49, and
2 which has a mixed
competitive/non-competitive profile.4 These results are exciting
as until now small molecule tools with diverse modes of pharma-
cology and PAR4 antagonism to potentially assess in vivo effects of
PAR4 inhibition have not existed.
While PAR4 activity against AP and c-thrombin, coupled with a
novel competitive mode of inhibition generated enthusiasm for
these low molecular weight PAR4 antagonists 4c, 4n and 4k, their
in vitro DMPK profiles were suboptimal, and reminiscent of 1.4,8,9
While cLogPs were acceptable (3.2–3.7), experimental LogPs were
>4 and PSAs were <40. These physiochemical properties correlated
with high plasma protein binding (human and rat fus between
0.006 and 0.014) and moderate to high intrinsic clearance in
hepatic microsomes (human CLheps 13.6 to 19.9 mL/min/kg and
rat CLheps 46.3 to 58.3 mL/min/kg).
These data led to second and third generation libraries aimed at
replacing the 6-aryl moiety with heterocycles and surveying
alternative ethers and amine substituents for the 2-methoxy group
in 4c, 4n and 4k. Overall, heterocyclic replacements for the 6-aryl
ND
ND
4s
4t
1460 (5.84 0.39)
ND
2540 (5.60 0.21)
1090 (5.96 0.15)
ND
ND
4u
4v
4w
4x
4y
a
Average of three independent determinations upon PAR4 activation with either
AP or
-thrombin.4,9
c

K. J. Temple et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5481–5486
5483
Figure 2. Molecular pharmacology profile of 4c, 4n and 4k. (A–C) PAR4 antagonist CRCs (n = 3) against 200
lM PAR4-AP showing equivalent inhibition of both PAC-1 and P-
selectin. n = 3, mean SEM; D–F) PAR4 antagonist CRCs (n = 3) against 100 nM -thrombin showing equivalent inhibition of both PAC-1 and P-selectin. n = 3, mean SEM;
c
(G–I) Progressive fold-shift experiments showing a parallel right-ward shift of the CRC (competitive mode of PAR4 inhibition) with 4c, 4n and 4k. Schild EC50 log DR-1 versus
log [antagonist] plot slopes: 4c (0.95 0.09), 4k (1.09 1.3), 4n (0.94 0.08).4,9
moiety (e.g., 2-, 3- and 4-pyridyl, thienylthiazolyl) lost 10- to
50-fold activity relative to the unsubstituted phenyl comparator
4f. Alternatives for the 2-methoxy group were equally steep. 2-
Ethoxy congeners displayed activity (IC50s 600–900 nM), but lost
ꢀ20-fold activity relative to 4c, 4n and 4k. Larger, branched and
cyclic ethers lost all PAR4 inhibitory activity. In an attempt to
improve the physiochemical properties of this series, we
performed SNAr reactions on cores 7 with various primary and sec-
ondary amines. SAR was steep, with the vast majority of 2-amino
congeners studied possessing no PAR4 inhibitory activity. As
shown in Figure 3, only a single N(CH3)2 analog 8 was active
the N-benzyl moiety to a simple N-Me, would afford a small mole-
cule that aligns with fragment 3 (Fig. 4A). Indeed, this proved suc-
cessful, generating indole 5, a 20 nM PAR4 inhibitor against AP
(ꢀ9-fold more potent than 1) with a 25% reduction in molecular
weight. While activity against
and efficacious (IC50 = 1.0
ence in potency was noted between inhibition of PAR4-AP and
thrombin mediated stimulation (Fig. 4B), yet 5 retained selectivity
versus PAR1 (IC50 > 10 M) and off-target effects on the collagen
receptor were eliminated (Fig. 5). Although 5 maintains -throm-
c
-thrombin with 5 was more potent
l
M) than that of 1, an ꢀ50-fold differ-
c-
l
c
bin stimulated antagonism within four-fold of 4n, fragments 4 rep-
(PAR4-AP IC50 = 3.45
tive. None of the non-2-OCH3 derivatives displayed any PAR4 activ-
ity against -thrombin mediated activation. Clearly, the 2-methoxy
l
M), whereas the NHCH3 derivative was inac-
resented the best path forward towards improved PAR4 inhibitors
based on physiochemical properties and
c-thrombin ligand effi-
c
ciency metrics (5 LE = 0.33 vs. 4n LE = 0.43) with competitive
moiety is an essential element of the PAR4 pharmacophore, at least
with the context of the imidazo[2,1-b][1,3,4]thiadiazole bicyclic
scaffold.
We envisioned that deletion of the 2-hydroxy methyl moiety
and transposition of the imidazo[2,1-b][1,3,4]thiadiazole bicycle
from the 3- to the 2-position, while simultaneously truncating
inhibition.
We previously reported on the potency and selectivity of exam-
ple 1 noting >100 fold difference in IC50 values comparing PAR1-AP
and PAR4-AP.4 However, when used at micromolar concentrations
that are effective against the tethered ligand (c-thrombin medi-
ated activation) significant off-targets against collagen I induced
aggregation were noted (Fig. 5). This was surprising considering
the weak effects on the primary anti-target PAR1. This was also
an alarming revelation since collagen I-mediated platelet
activation is intimately involved in hemostasis and thrombosis.
Therefore, before focusing solely on the simplified pharma-
cophores 4, we elected to re-evaluate 14 in an effort to further
minimize the key components required for PAR4 inhibition and
address off-target effects. Fragment 3 is devoid of collagen receptor
F
F
CH3
CH3
N
S
H
N
S
N
N
N
N
N
N
CH3
F
F
8
9
PAR4-AP IC50 = 3.4 μM
PAR4-AP IC50 >10 μM
Figure 3. Structures and PAR4 activity of 2-amino congeners 8 and 9.

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K. J. Temple et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5481–5486
Figure 4. Identification and pharmacological profile of a minimum pharmacophore of 1. (A) Strategy to mimick the minimum PAR4 pharmacophore 3 of 2 within the indole
series, and identification of 5. (B) Pharmacological profile of 5 against PAR4 -thrombin (100 nM) PAC-1 (IC50 = 1.0 M, pIC50 = 6.01 0.05), PAR4 -thrombin P-selectin
(IC50 = 1.0 M, pIC50 = 6.00 0.04), PAR4-AP PAC-1(IC50 = 21 nM, pIC50 = 7.68 0.04), PAR4-AP (200 M) P-selectin (IC50 = 20 nM, pIC50 = 7.70 0.03) and PAC-AP (PAC1 and
P-selectin, IC50s > 10 M). n = 3, mean SEM.
c
l
c
l
l
l
inhibitors. Here, platelet aggregation was significantly inhibited
at doses (316 nM to 1 M against AP and 1 M to 3.16 M against
-thrombin) of the PAR4 antagonists that abolished PAC1 binding.
l
l
l
c
This was a pivotal finding, as this is the first demonstration that
competitive PAR4 inhibitors are efficacious in this pharmacody-
namics assay.
Furthermore, we wanted to assess the ability to inhibit platelet
aggregation with 5 (Fig. 7), as there was a large disconnect
between
icantly inhibited at doses (316 nM to 1
10 M against -thrombin) of the PAR4 antagonist 5 that abolished
c
-thrombin and AP. Here, platelet aggregation was signif-
l
M against AP and only at
l
c
PAC1 binding. Thus, excellent correlation once again between the
FACS and aggregation assays, but poor correlation between activity
against PAR4-AP and
c-thrombin induced PAR4 activation. c-
Thrombin cleavage of PAR4 generates an intramolecular tethered
ligand which is covalently attached to the receptor, while PAR4-
AP is a soluble surrogate for the tethered ligand. It is impossible
to measure the concentration of the tethered ligand proximal to
the binding pocket because it is covalently attached to the recep-
tor. Theoretically, an infinite concentration of ligand is available
due to its inability to diffuse away from the receptor. Thus, one
would predict that it would take higher concentrations of an
orthosteric antagonist to compete with the tethered ligand as com-
pared to the soluble AP. Indeed, we have extensively documented
this phenomenon. If this was the sole distinguishing factor
Figure 5. Identification of a minimum pharmacophore of 1 devoid of off-target
effects. Washed human platelets were incubated with the indicated concentration
of antagonist (1, 5) for 20 min prior to activation with either (A) 20
lM PAR1-AP or
(B) 10 g/mL collagen I (Coll I). Platelets were allowed to aggregate for 10 min.
l
Shown are representative tracings of three independent experiments.
off-target effects as are fragment inhibitors 4, suggesting the
hydroxymethyl moiety of 1 might be suspect in this series.
The PAR4 IC50s from the flow cytometry assays were encourag-
ing, but would these novel, competitive PAR4 antagonists (4c, 4k,
and 4n) inhibit human platelet aggregation? As shown in Figure 6,
a standard platelet aggregation assay11 demonstrated excellent
correlation and consistent data with the three competitive PAR4
between
be possible to predict
However, this is not the case. Proportional differences between
-thrombin and PAR4-AP IC50s are highly variable, which is why
c-thrombin and PAR4-AP, IC50s should correlate. It should
c-thrombin IC50s based on PAR4-AP IC50s.
c
we chose to track SAR with both ligands. Based on the data
presented here we can’t explain this lack of correlation, however

K. J. Temple et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5481–5486
5485
Figure 6. Activity of fragments 4 in platelet aggregation. Washed human platelets were incubated with the indicated concentration of antagonist for 20 min prior to
activation with either 40 nM -thrombin A (4c), C (4k), E (4n) or 200 M PAR4-AP B (4c), D (4k), F (4n). Platelet aggregation was monitored for 10 min at which point final
c
l
reported % aggregation values were taken. Shown are the means SEM for at least three independent experiments. Values were compared to controls and significance
determined by paired t-test. *p-Value <0.05, **p-value <0.005, ***p-value <0.0005.
Figure 7. Activity of 5 in platelet aggregation. Washed human platelets were incubated with the indicated concentration of antagonist 5 for 20 min prior to activation with
either (A) 40 nM
c-thrombin or (B) 200 lM PAR4-AP Platelet aggregation was monitored for 10 min at which point final reported % aggregation values were taken. Shown are
the means SEM for at least three independent experiments. Values were compared to controls and significance determined by paired t-test. ***p-Value <0.0005.
it is important to keep in mind that one is an enzyme (
c
-thrombin)
donors/acceptors by ꢀ50%. This simplified core represents the
minimum pharmacophore for PAR4 inhibition in a competitive
manner. While initial analogs did not address the DMPK liabilities
of 1 and 2 (though fu was slightly improved), the most basic core,
4h, can serve as a sub-micromolar lead from which to build in
more optimal DMPK and physiochemical properties. Similarly, 5
was identified as a minimum PAR4 pharmacophore of indole series
and the other is a peptide (PAR4-AP). Thrombin interacts with
PAR412,13 and its heterodimer partner PAR114 proximal to but out-
side the binding pocket. c-Thrombin binding to PAR4 outside the
binding picket, or the reorganization of the N-terminus after cleav-
age could induce conformational states in the receptor that the AP
is incapable of inducing since it interacts only with binding pocket.
This could create two distinct receptor states and therefore two
distinct lines of SAR for antagonist. In support of this hypothesis
our lab has previously published on functional selectivity between
PAR1-AP and thrombin induced endothelial cell signaling.15
In summary, we identified a greatly simplified 2-methoxy-6-
arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that affords nanomo-
1, but with an unexpected 50-fold loss in activity at c-thrombin,
yet nanomolar potency against the AP. Finally, we disclose the first
example demonstrating that competitive PAR4 inhibitors signifi-
cantly inhibit human platelet aggregation. Further optimization
efforts around these fragments are in progress, as well as the eval-
uation of alternate phenyl bioisosteres beyond the imidazo[2,1-b]
[1,3,4]thiadiazole bicyclic scaffold, and these results will be
reported in due course.
lar PAR4 inhibition of AP as well as
c-thrombin, while reducing
both molecular weight and the number of hydrogen bond

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K. J. Temple et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5481–5486
10. Example experimental (4h). A mixture of commercially available 2-bromo-1-(p-
tolyl)ethan-1-one (2.35 mmol) and 5-bromo-1,3,4-thiadiazol-2-amine
Acknowledgments
(3.52 mmol) were dissolved in CH3CN/IPA (1:1; 9.4 mL) in a microwave vial
that was sealed and heated to 80 °C for 18 h. Next, the vial was placed in a
Microwave for 30 min at 150 °C. The solvent was evaporated and the mixture
was re-suspended in DCM (20 mL), washed with saturated NaHCO3 (20 mL),
brine, dried over magnesium sulfate and filtered. The organic layer was
concentrated under reduced pressure onto celite and loaded onto a 3 inch silica
plug and purified using Teledyne ISCO Combi-Flash system (solid loading, 100%
DCM, 10 min run) to afford 2-bromo-6-(p-tolyl)imidazo[2,1-b][1,3,4]
thiadiazole (1.70 mmol) in 72% yield. LC–MS [M+H] = 294/296, RT = 1.174; 1H
NMR (400 MHz, CDCl3) d 7.98 (s, 1H), 7.68–7.70 (d, J = 8.16 Hz, 2H), 7.21–7.23
(d, J = 8.03 Hz, 2H), 2.38 (s, 3H). A solution of 2-bromo-6-(p-tolyl)imidazo[2,1-
b][1,3,4]thiadiazole (1.02 mmol) in a mixture of DCM/MeOH (3:1; 20 mL) was
treated at 22 °C with a 25 wt.% solution of NaOMe (2.04 mmol) in MeOH and
the reaction was stirred for 1 h at room temperature. Upon completion as
determined by LCMS, the reaction mixture was quenched by addition of 1% HCl
(10 mL) followed by addition of saturated NaHCO3 (10 mL). The aqueous layer
was extracted with DCM (3 ꢁ 15 mL), dried over magnesium sulfate and
filtered. The organic layer was concentrated under reduced pressure onto silica
gel and purified using Teledyne ISCO Combi-Flash system (solid loading, 12G,
100% DCM, 15 min run) to afford 2-bromo-6-(p-tolyl)imidazo[2,1-b][1,3,4]
thiadiazole, 4h (0.737 mmol) in 72% yield. LC–MS [M+H] = 246, RT = 1.113; 1H
NMR (400 MHz, CDCl3) d 7.78 (s, 1H), 7.66–7.68 (dt, J = 8.21 Hz, 2H), 7.19–7.21
(d, J = 8.03 Hz, 2H), 4.18 (s, 3H), 2.37 (s, 3H).
We thank the NIH for funding (NS081669 and NS082198.) We
also thank William K. Warren, Jr. and the William K. Warren Foun-
dation who funded the William K. Warren, Jr. Chair in Medicine (to
C.W.L.).
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