Design and evaluation of novel glutaminase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.009

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved C log Ps, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

Full text of DOI:10.1016/j.bmc.2016.03.009

Accepted Manuscript
Design and Evaluation of Novel Glutaminase Inhibitors
Lee A. McDermott, Prema Iyer, Larry Vernetti, Shawn Rimer, Jingran Sun,
Melissa Boby, Tianyi Yang, Michael Fioravanti, Jason O’Neil, Liwei Wang,
William Katt, Qingqiu Huang, Richard Cerione
PII:
S0968-0896(16)30158-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.009
BMC 12858
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 December 2015
24 February 2016
3 March 2016
Please cite this article as: McDermott, L.A., Iyer, P., Vernetti, L., Rimer, S., Sun, J., Boby, M., Yang, T., Fioravanti,
M., O’Neil, J., Wang, L., Katt, W., Huang, Q., Cerione, R., Design and Evaluation of Novel Glutaminase Inhibitors,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.009
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Bioorganic & Medicinal Chemistry
Design and Evaluation of Novel Glutaminase Inhibitors
a,b
a,b
b
a
a
a
Lee A. McDermott,  Prema Iyer, Larry Vernetti, Shawn Rimer, Jingran Sun, Melissa Boby, Tianyi
a
a
a
a
c
d
Yang, Michael Fioravanti, Jason O’Neil, Liwei Wang, William Katt, Qingqiu Huang, and Richard
e
Cerione
a
University of Pittsburgh, Department of Pharmaceutical Sciences, Pittsburgh PA 15261, USA
University of Pittsburgh Drug Discovery Institute, Pittsburgh PA 15261, USA
Cornell University, Department of Molecular Medicine, Ithaca NY 14853, USA
Cornell University, Laboratory for Accelerator-based Sciences and Education, Ithaca NY 14853, USA
b
c
d
e
Cornell University, Department of Molecular Medicine and Department of Chemistry and Chemical Biology, Ithaca NY 14853, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel set of GAC (Kidney Glutaminase Isoform C) inhibitors able to inhibit the enzymatic
activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with
low nanomolar potency is described. Compounds in this series have a reduced number of
rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to
the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the
replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors
by heteroatom substituted heterocycloalkanes.
Available online
Keywords:
GAC
XXXX Elsevier Ltd. All rights reserved.
Novel glutaminase inhibitors
BPTES
CB-839
1. Introduction
In 1955, Eagle, in his paper “Nutrition needs of mammalian
cultured cells” reported that glutamine, a non-essential amino
reduction of tumor cell proliferation in vitro and in vivo and
suggests that glutaminase is a valid anticancer target.
14,15,16,17,18
acid, is quite essential for the growth of tumor cells in culture
1
media. Since that report, research in tumor cell metabolism has
revealed that high glutamine utilization and dependence, a
N
N
N
N
NH
O
O
N
H
S
property termed “glutamine addiction”, is a key attribute for a
S
S
2,3,4,5,6
BPTES
number of tumor cell lines.
The key step in glutamine processing in mitochondria is the
hydrolysis of the glutamine amide group by the enzyme
glutaminase. There are two main glutaminase isoforms, the
kidney isoform (KGA or GLS1) and the liver isoform (LGA or
GLS2). Starting with the 1969 paper of Knox et al., where it was
shown that kidney glutaminase activity is proportional to the
growth rate of tumors in rats, the evidence accumulated over the
years point to the fact that KGA and its splice variant kidney
glutaminase isoform C (GAC) particularly, is a target of interest
Me
N
OCF3
O
Me
N
N
O
Br
NH
S
NH
N
O
HN
N
N
CB-839
968
Figure 1. Glutaminase inhibitors
Among the selective small molecule GAC inhibitors, BPTES and
CB-839 are the most similar, having as a key feature the presence
of a lipophilic connecting chain (diethylthio in BPTES and n-
butyl in CB-839) between two heterocyclic aromatic moieties
7,8,9
for cancer therapy.
GAC upregulation is present in multiple
cancer cell lines, correlates with increased proliferative rates, and
it is linked to the dysregulation of a number of pathways,
including the dysregulation/amplification of the Myc
(
Figure 1). Being straight and lipophilic these chains contribute
10,11,12,13
to the relatively high ClogPs and the high number of rotatable
bonds (NRB) of these compounds (BPTES: ClogP=4.15,
NRB=12; CB-839: ClogP=4.74, NRB=13). Herein we describe a
oncogene.
Inhibition of GAC through antisense, siRNA and/or synthetic
molecules like BPTES, CB-839 and compound 968 leads to
—
——

Corresponding author. Tel.: +1-(732)-979-0403; e-mail: lam179@pitt.edu

novel and potent set of inhibitors with NRB values within the
that is inherently higher in molecules with a high number of
rotatable bonds, and as such it could lead to greater potency
inhibitors.
19
generally accepted drug-like range (≤10), improved ligand
20
21
25
efficiency (LE), lipophilic efficiency (LiPE/LLE) and/or
ClogPs when compared to the leading inhibitors.
As a means of maintaining the logP as low as possible, we
envisioned the use of saturated ring systems that contained other-
than-sulfur heteroatoms as surrogates for the conformation
assumed by the BPTES flexible chain. Ease of synthesis
considerations and our desire to have more than one heteroatom
present on the small to medium size ring systems that would not
clash with the walls of the binding pocket suggested to us that
heteroatom substituents on prospective saturated ring systems
should serve as connectors between the BPTES thiadiazoles
and/or their isosters, and not as stand-alone substituents. In that
regard, non-sulfur-containing ring systems such as 4-
hyrdoxypiperidine, 4-aminopiperidine, 3-amino azetidine, etc.
appeared as very suitable heteroatom containing rigid surrogates
for the flexible connector chains of BPTES/CB-839.
2. Results and Discussion
2.1 Design principles for new compounds
Catalytically active GAC units are tetrameric and recent
evidence suggests that in cells GAC may in fact operate as an
22
oligomer of tetramers.
With respect to structural information there are three crystal
structures of human GAC in complex with BPTES in the Protein
23,24
Data Bank (PDB), namely structures 3UO9, 3VOZ and 3VP1.
These structures show that BPTES binds in a stoichiometry of 2
molecules of inhibitor per GAC tetramer and at an allosteric
pocket that is formed at the interface between GAC dimers
B-factors in the 3UO9 x-ray structure suggest that one of the
(Figure 2).
23
BPTES phenyls is particularly flexible/mobile (Figure 2c). This
suggests that this phenyl moiety most likely does not contribute
significantly to binding. As such, this phenyl group and possibly
the whole phenylacetic acid moiety in that part of the molecule,
could be replaced by smaller groups or perhaps completely
eliminated from new compounds thus yielding compounds with
even better properties. A recent paper on a series of BPTES
analogs with flexible connector chains by the Tsukamoto group
suggested that removal of one of the two phenylacetic acid
26
moieties may indeed be viable.
.2 Chemistry
In order to assess the viability of replacing the flexible BPTES
2
side chain with heteroatom containing saturated rings, and to also
explore the possibility of replacing both of the phenyl moieties in
constrained analogs with smaller groups, we pursued the
synthesis of the “symmetrically” acylated compounds in Tables 1
and 2.
Figure 2. A & B: Binding of BPTES to glutaminase as appears in the 3UO9
x-ray structure. C: Heat map of B-factors for the BPTES atoms in the 3UO9
structure
S
Br
H2N
0.5 eq. Cy (5a-e)
S
Cy
S
H2N
NH2
N
N
a
N
N
N
N
Looking at the available BPTES/GAC crystal structures and
particularly the bent conformation assumed by the thiadiazole-
connecting diethylthio chain, it became apparent to us that this
flexible connector could be replaced by small to medium size
ring systems (Figure 3). Morphing this diethylthio chain
connector into a cyclic structure would be highly beneficial as it
would result in inhibitors with reduced number of rotatable
bonds, a property inversely related to the probability of good
4
6
a-e
NH2
a
NBoc
H2N
S
(±) 8
c
b
N
NH
H
N
N
S
H2N
d,e
b
7f
7a-e
7g,h
N
N
N
6f
H
9
S
N
NHBoc
NH2
19
N
absorption. An added benefit of this decrease in rotatable bonds
would be a reduction in the entropic energy penalty for binding,
H
N
H
NH2
NH2
NH2
N
Cy=
N
H
N
H
N
H
N
H
N
H
5a
5b
5c
5d
5e
Scheme 1. Synthesis of compounds 7a-h (Table 1). Reagents and
o
conditions: a) NaHCO
of PhCH COCl, Et N, DMF, rt.; c) 6d or 6e, 2 eq. of (Ac)
rt.; d) 30% TFA in CH
3
or Et
3
N or DIEA, EtOH, 80 C, sealed tube; b) 2 eq.
2
3
2
O, Et N, DMF,
3
o
2
2 3
Cl ; e) NaHCO , 4, EtOH 80 C.
Compounds 7a-e (Table 1) were prepared via the reaction of
-amino-5-bromothiadiazole (4) with 0.5 eq. of a cyclic diamines
a-e and then acylation of the corresponding constrained bis
2
5
thiadiazole intermediates 6a-e with phenylacetyl chloride
Scheme 1). Compounds 7g and 7h were prepared from bis-
thiadiazoles 6d and 6e after acylation with acetic anhydride
Scheme 1). Compound 7f was prepared from the known (±)-1-
N-Boc-1,3-diaminopentane 8 by a reaction with 2-amino-5-
bromothiadiazole to afford the N-Boc protected intermediate 9,
(
(
27
Figure 3. Design principles for new GAC inhibitors

H2N
ANBoc
Ph
H
S
ANBoc
S
N
ANBoc
a
b, c
Ph
N
b
O
L
Z
X
S
O
N
HO
N
Ph
N
O
N
L
L
10a-h
11a-h
12a-h
Y
N
O
N
N
O
O
X
Y
c,d
a
Z
N
i
N
Boc
Boc
d,e
Boc
X=N, Y=N, Z=CH, L=Cl: 19
X=N, Y=CH, Z=N, L=Br: 20
X=N, Y=N, Z=CH, L=Cl: 21
X=N, Y=CH, Z=N, L=Br: 22
23
24
O
ANBoc
S
O
Ph
H2N
e,f
HN
S
S
NH2
N
5a-d
N
N
O
A
j
N
N
N
1
O
N
N
f
N
H2N
N
NH
S
N
N
1
3a-h
N
O
d,e
Br
N
O
25
h
g
N
N
g
2
9
N
i
d
Br
16a-d
1
4a-h
14i
14j
Y
N
Ph
X
26a
N
Cl
N
Z
Ph
O
N
OH
O
N
OH
OH
OH
OH
i
h
c, d
OH HO
HO
HO
26b,c
ANBoc =
N
N
N
N
X
Y
N
N
N
N
N
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Z
HO
N
N
N
N
10a
10b
10c 10d
10e
10f
10g
10h
Ph
N
Cl
Cl
28
Ph
Scheme 2. Synthetic methods for preparation of compounds 14a-j and 16a-
d. Reagents and conditions: a) NaH, CS , MeI, THF, rt; b) NH NH MeOH,
rt; c) BrCN, Et N, MeOH, rt; d) 4N HCl in dioxane, rt; e) 4, Et N, EtOH or
DMF, 80 C, sealed tube.; (f) PhCH COCl, Et N, DMF, rt; g) 13d, (Ac) O,
Et N, DMF, rt.; h) 13d, EDCI, cyclopropylacetic acid, DMF, rt.; i) 12a-d,
PhCH COCl, Et N, DMF, rt.
27
X=N, Y=N, Z=CH: 30
X=N, Y=CH, Z=N: 31
2
2
2
3
3
o
2
3
2
Scheme 4. Synthesis of 26a-c. Reagents and conditions: a) 4-hydroxy-1-
N-Boc piperidine, NaH, THF or THF/DMSO 4:1, 50 C; b) 21,
benzophenone imine, (±)-BINAP, Pd
o
3
o
2
3
2
(dba)
3
, Cs
2
CO
3
, toluene, 90 C,
.
sealed tube c) NH
Et N, HATU, DMF; e) 4N HCl dioxane, rt,; f) 2-amino-5-
3
bromothiadiazole (4), Et N, DMF, 80 C; g) 19, Et N, DMSO, 4-
o
2
OH HCl, NaOAc, MeOH, rt.; d) phenylacetic acid,
followed by Boc deprotection, another reaction with 2-amino-5-
bromothiadiazole and then acylation with phenylacetyl chloride
Scheme 1).
3
o
3
o
hydroxypiperidine, 50 C; h) 19, NaH, THF, 50
benzophenone imine, (±)-BINAP, Pd (dba) , Cs CO , toluene, 90 C,
sealed tube, j) 22, 4N HCl, dioxane, then 20, Et N, DMSO, 50 C.
C i) 2-4 eq.
(
o
2
3
2
3
o
The preparation of compounds 14a-h (Table 2) involved first
3
the building of the 2-aminothiadiazole moiety on the N-Boc-
protected cyclic amino alcohols 10a-h to afford the
corresponding intermediates 12a-h, then Boc deprotection, a
reaction with 2-amino-5-bromothiadiazole and finally acylation
of the resulting intermediates 13a-h with phenylacetyl chloride
piperidine, respectively, via reaction with 2-amino-5-
bromothiadiazole, then acylation followed by Boc deprotection
and again reaction with 2-amino-5-bromothiazole (Scheme 3).
Compound 16g was prepared from 16f via acylation with
cyclopropyl acetic acid and HATU.
(Scheme 2).
H2N
Finally, with objective to expand our flexible-chain-
rigidification strategy to analogs in which one or both of the
BPTES thiadiazoles have bioisosterically been replaced by
pyridazine, pyridines or pyrazine we prepared compounds 26a-f
in Table 4.
NBoc
a, 17a
H
H
S
N
N
c,d
b
N
N
16e
O
NBoc
18a
H2N
Br
S
NBoc
NO2
NH2
H2N
NO2
H
a, 17b
N
N
N
N
4
N
H
S
f
N
N
c, d
NBoc
e
16f
16g
NO2
N
N
b
c
N
O
O
X
Y
a
N
O
1
8b
N
N
N
Scheme 3. Synthesis of compounds 16e-g (Table 3). Reagents and
Cl
NH2
NO2
o
X=N, Y=CH: 32
X=CH, Y=N: 33
OH
34
conditions: a) NaHCO
N HCl in dioxane, rt.; d) 4, NaHCO
phenylacetic acid, HATU, Et N, DMF, rt.; f) cyclopropyl acetic acid,
HATU, Et N, DMF, rt.
3
, EtOH, 80 C; b) PhCH
2 3
COCl, Et N, DMF, rt; c)
35
36
o
4
3
, EtOH, 80 C, sealed tube.; e)
e
3
d
3
g
O2N
N
N
OH
26d
NH2
Compounds 14i and 14j were prepared from their common bis
3
7
X
Y
thiadiazole intermediate 13d via acylation with either acetic acid
anhydride or cyclopropyl acetic acid and EDCI respectively.
To test if removal of one of the phenyl/phenacetyl moieties
from potent derivatives is tolerated we prepared the selected
analogs shown in Table 3.
Derivatives 16a-d were prepared from the corresponding N-
Boc intermediates 12a-d (Scheme 2) in three steps that involved
first the reaction of 12a-d with phenylacetyl chloride to afford
the corresponding N-phenylacetyl intermediates 15a-d, then Boc
deprotection and reaction with 2-amino-5-bromothiadiazole.
Compounds 16e and 16f were prepared from commercially
available 2-amino-1-N-Boc-azetidine and 4-amino-1-N-Boc-
NO2
f
N
N
O
d
h
26e,f
O2N
N
O
N
X
Y
NH2
X=N, Y=CH: 38
X=CH, Y=N: 39
X=N, Y=CH: 40
X=CH, Y=N: 41
Scheme 5. Synthesis of compounds 26d-f. Reagents and conditions: a) 32,
4-hydroxypiperidine, K CO , DMSO, 90 C; b) 34, 2-nitro-5-
o
2
3
o
hydroxypyridine, Ph
3
P, DIAD, THF, rt. c) Fe, NH
N, DMF, rt.; e) 33, 4-hydroxypiperidine,
3
N, EtOH, 90 C, sealed tube; f) 33, NaH, THF/DMSO (5:1), to afford
4
Cl aq. EtOH, 80 C d)
phenylacetic acid, HATU, Et
3
o
Et
8; g) 33, NaH, 50 C, THF to afford 39 h) H
1:1).
o
3
(
2
, 10% Pd/C, EtOH/EtOAc

Table 1. Properties and activity of “symmetrically” acylated bis-thiadiazoles with diamine containing saturated rings as surrogates for the flexible diethylthio
moiety of BPTES
Cmpd
a
c
d
Structure
MW
CLogP
NRB
GAC
IC50 (nM)
MDA-MB-231
IC50 (nM)
LE
LiPE
b
b
BPTES
CB-839
O

4.15
12
371
2,610
33
0.26
2.28
N
N
N
N
S
O
NH
HN
S
S
H
O
O
N
S
N
N
NH
O
F
F
F
e
f
f
N
N
571.57
534.66
4.74
4.31
13
8
180/25
0.23/0.24
1.99/2.86
N
H
O
H
N
N
S
S
g
g
g
7
a
O
>5,000
3,070
NT
NA
NA
N
N
N
N
N
N
N
N
H
H
O
N
N
S
N
S
N
g
7
b
N
O
520.63
534.66
520.63
520.63
4.25
3.80
3.74
3.74
8
9
9
9
NT
0.21
0.28
0.27
0.29
1.26
3.72
3.41
3.78
N
H
O
N
S
NH
N
N
O
S
29
70
30
70
7
c
N
N
N
N
H
O
H
N
S
H
N
O
7
d
1,200
1,570
S
N
N
N
N
NH
N
O
H
N
S
H
N
O
7
e
N
N
S
N
N
NH
N
H
N
H
N
H
S
N
O
O
N
S
N
7
f
N
534.66
3.74
10
50
320
0.28
3.56
N
N
H
O
H
O
H
N
S
N
S
7
7
g
NH
368.44
368.44
0.07
0.07
5
5
7,700
720
>3,000
>3,000
0.29
0.36
5.04
6.07
N
N
N
N
N
N
O
H
O
H
N
S
N
S
h
NH
N
N
N
N
a
b
c
d
e
f
Calculated using the ClogP module of the on-line/evaluation version of MavinSketch from ChemAxon
IC50 values are the result of a single experiment run in triplicate
LE= G/N = -1.4log(IC50)/N
LiPE=pIC50-logP
Reported in ref 16
Calculated using the literature reported IC50
NT: Not tested; NA: Not assessed
g
The synthesis of compounds 26a and 26c began with the
reaction of 2,6-dichloropyridazine (19) or 2,5-dibromopyrazine
20) with 4-hydroxy-1-N-Boc-piperidine, under aromatic
further to compounds 24d-f upon introduction of a second
nitropyridine moiety, followed by a nitro group reduction and
then acylation with phenylacetic acid and HATU (Scheme 5).
(
nucleophilic substitution conditions, to afford intermediates 21
and 22 which were then elaborated further to 26a and 26c as
described in Scheme 4. The synthesis of 26b was effected via the
initial reaction of 2,6-dichloropyridazine (19) with 4-
hydroxypiperidine to afford intermediate 27 followed by the
introduction of the second pyridazine moiety, amination and then
acylation with phenylacetic acid (Scheme 4). A key step in the
synthesis of compounds 26a-c was the Buchwald Pd-catalyzed
amination of intermediates 21, 28 and 29 with benzophenone
2.3 Evaluation of novel compounds
All novel analogs were evaluated in a biochemical assay
against recombinant GAC and against the MDA-MB-231 cancer
cell line, using BPTES and CB-839 as comparators.
An overview of the data in Tables 1-4 suggests that the
BPTES/CB-839 flexible chains can indeed be replaced by an
appropriate heteroatom substituted ring system. Focusing on
Tables 1 and 2 it is evident that small to medium size heteroatom
substituted ring moieties, such as 4-piperidinyl, 3-pyrrolidinyl or
2-azetidinyl are good surrogates for the conformation assumed by
the flexible BPTES connector chain. Compounds containing
those rings are up to 6-10 fold more potent than BPTES in the
GAC enzymatic assay (Tables 1 and 2). Larger rings or rings that
28
imine
.
Finally, compounds 26d-f were prepared from intermediates
5, 38 and 39, respectively (Scheme 5). These intermediates were
3
assessed via the reaction of 4-hydroxypiperidine with either 2-
nitro-5-chloropyridine or 2-chloro-5-nitropyridine, under
aromatic nucleophilic substitution conditions, and transformed

Table 2. Properties and activity of “symmetrically” acylated bis-thiadiazole with saturated hydroxysubstituted rings as surrogates for the flexible
diethylthio moiety of BPTES
a
GAC
IC50 (nM)
MDA-MB-231
IC50 (nM)
c
d
Cmpd
Structure
MW
CLogP
NRB
b
b
LE
LiPE
H
O
N
S
O
N
1
4a
507.59
4.04
9
60
>3,000
0.28
3.18
N
S
N
N
O
N
N
H
O
O
H
N
1
1
4b
4c
S
521.62
521.62
4.10
4.10
9
9
40
36
680
420
0.28
0.29
3.29
3.42
O
S
N
O
N
N
N
NH
N
H
N
S
O
S
N
O
N
N
N
NH
N
H
O
N
S
N
N
O
N
N
1
1
4d
4e
535.64
535.64
4.16
4.62
9
9
30
140
0.28
0.23
3.36
1.68
S
N
N
N
N
O
H
N
N
N
490
3,000
S
O
N
H
O
O
S
N
H
O
H
N
S
H
N
S
N
N
1
1
4f
N
O
535.64
535.64
4.22
4.22
10
10
30
230
0.28
0.23
3.30
1.88
N
O
N
O
H
N
S
H
N
S
N
N
4g
N
O
790
>3000
N
O
N
O
H
N
S
N
N
H
N
S
N
1
4h
O
549.67
4.63
10
190
>3000
0.25
2.09
N
O
N
H
O
N
S
O
N
1
1
4i
4j
N
S
383.45
463.58
0.50
2.43
5
9
1,005
157
>3,000
630
0.33
0.31
5.48
4.37
N
N
O
N
N
H
H
O
N
S
O
N
N
S
N
N
O
N
N
H
a
b
c
d
Calculated using the ClogP module of the on-line/evaluation version of MavinSketch from ChemAxon
IC50 values are the result of a single experiment run in triplicate
LE=G/N= -1.4log(IC50)/N
LiPE=pIC50-logP
cannot quite capture the flexible connector chain conformation
seen in the 3UO9 x-ray structure, for instance the rings in
compounds 7a, 7b (Table 1) and 14e (Table 2), lead to potency
loss.
smaller cyclopropyl ring is introduced as replacement for the
phenyl moiety.
Complete removal of only one of the phenylacetyl groups from
potent compounds leads to derivatives with reduced potency in
the GAC assay relative to that of their parent molecules (Tables
1-3). In this set, the piperidinyl and azetidinyl analogs suffered
the least potency loss.
To assess if introduction of a cyclopropylacetic acid moiety
may help in reversing the potency loss observed in analogs that
lack a phenylacetic acid moiety, in effect test the premise that
one phenyl ring may be replaced by a smaller group, and at the
same time assess the viability of mixed phenylacetic acid
cyclopropylacetic acid derivatives as lead optimization
candidates, we prepared analog 16g (Table 3). Compound 7c
The GAC activity of compounds 7d,e,g,h and 14b,c,g,f which
contain a pyrrolidine ring system (Tables 1 and 2), shows that
there is a chirality preference for one of the two enantiomers.
Comparison of the GAC activity of potent bis-phenyl analogs
and their direct bis-des-phenyl derivatives, for instance the a
comparison between the activity of derivatives 7d, 7e and 14d
with that of their bis-des-phenyl analogs 7g, 7h and 14i
respectively (Tables 1 and 2), shows that removal of both
phenyls from potent analogs leads to potency loss. However, as
the data with compounds 14d, 14i and 14j in Table 2 indicate,
this potency loss may be reversed to a great degree when the

Table 3. Properties and activity of constrained bis-thiadiazoles that lack one phenylacetic acid moiety
a
GAC
IC50 (nM)
MDA-MB-231
IC50 (nM)
c
d
Cmpd
Structure
MW
CLogP
NRB
b
b
LE
LiPE
O
S
S
NH2
NH2
O
N
1
6a
6b
389.46
2.14
6
540
>3,000
>3,000
>3,000
0.33
0.26
0.30
4.10
N
N
N
N
N
H
S
O
1
O
S
N
403.48
403.48
2.20
2.20
6
6
9,700
1,400
2.81
3.65
N
N
HN
N
N
NH2
O
S
1
6c
O
S
N
N
HN
N
N
N
O
O
S
1
6d
N
N
S
NH2
417.51
388.47
2.26
1.77
6
6
207
227
980
0.33
0.35
4.42
4.87
N
H
N
N
N
N
NH2
S
HN
S
N
O
N
1
6e
>3,000
N
N
H
N
H
O
N
S
e
e
e
e
1
6f
S
NH2
416.52
498.62
1.89
2.93
6
9
NT
NT
NA
NA
N
N
H
N
N
N
N
H
S
N
HN
N
O
1
6g
S
N
O
30
90
0.31
4.59
N
N
N
N
H
a
b
c
d
e
Calculated using the ClogP module of the on-line/evaluation version of MavinSketch from ChemAxon
IC50 values are the result of a single experiment run in triplicate
LE=G/N= -1.4log(IC50)/N
LiPE=pIC50-logP
NT: Not tested; NA: Not assessed
Table 4. Properties and activity of selected non bis-thiadiazole constrained derivatives
a
GAC
IC50 (nM)
MDA-MB-231
IC50 (nM)
c
d
Cmpd
Structure
MW
CLogP
NRB
b
b
LE
LiPE
O
H
N
HN
N
S
O
2
6a
6b
529.61
4.01
9
36
580
0.27
0.25
3.38
3.14
N
N
N
N
O
H
N
O
O
H
N
2
523.59
3.86
9
98
2,200
N
N
N
N
O
N
O
H
H
N
N
N
N
e
e
e
2
6c
N
O
O
523.59
521.61
3.38
4.63
9
9
>5,000
>5,000
NT
NA
NA
N
N
N
O
H
H
N
N
O
O
e
e
e
N
2
6d
N
NT
NA
NA
O
H
N
H
N
e
e
e
2
6e
521.61
521.61
4.63
4.63
9
9
>5,000
>5,000
NT
NA
NA
N
N
N
O
O
H
N
H
N
O
O
e
e
e
2
6f
N
N
NT
NA
NA
N
O
a
b
c
d
e
Calculated using the ClogP module of the on-line/evaluation version of MavinSketch from ChemAxon
IC50 values are the result of a single experiment run in triplicate
LE= G/N =-1.4log(IC50)/N
LiPE=pIC50-logP
NT: Not tested; NA: Not assessed

(Table 1) was the best overall lead in our compound sets, as such,
the most efficient way in answering this question was to
synthesize 16g and see if its activity could match or approximate
that of its parent 7c. What we found was the activity of 16g was
practically identical to that of the parent compound 7c.
The GAC potency values for compounds 26a and 26b in Table
4
suggest that our chain-rigidification strategy may be expanded
to derivatives in which one or both thiadiazole moieties have
been replaced by a pyridazine ring.
The data in Table 4 also suggest that pyrazine or pyridine rings
may not be ideal replacements for the thiadiazole/pyridazine ring
systems.
Overall, potent leads in this series, defined as compounds with
IC50≤100 nM in the GAC assay, have NRB ≤10 and better LE,
LiPE and/or logPs when compared to the leading inhibitors
BPTES and CB-839.
Figure 6. Overlay of binding modes of BPTES (green, PDB: 3UO9), 7d
orange) and 7e (azure) with GAC
(
of GAC in complex with 7d, 7e, and 14d (shown in Figures 5
29
and 6). An additional x-ray structure of GAC in complex with
1
4b was also solved and has been included in the supplemental
29
material. The crystal structures show that these leads bind in the
allosteric pocket BPTES binds. The ring systems of these
compounds occupy the same location the flexible connector
chain of BPTES occupies and through their conformation allow
for the projection of the thiadiazole groups to the same region the
BPTES thiadiazoles lie in the 3UO9 x-ray structure (Figures 5,
6). Crystal structure overlays and the variable orientation of the
phenylacetic acid moieties suggest that the BPTES binding
pocket has a degree of plasticity which makes the explanation of
the consistent potency difference observed for the R- and S-
pyrrolidynyl analogs challenging. As can be seen from the
overlay of the crystal structures of GAC with 7d and 7e (Figure
6
) both of these enantiomeric compounds appear to be
Figure 4. HLM stability of compounds 7c, 7f, 14c, 14d, 16g, BPTES
accommodated well in the BPTES allosteric pocket. Finally, our
crystal structures, and the 3VOZ/3VP1 BPTES-GAC x-ray
structures, suggest that the orientation of the thiadiazoles in the
binding pocket is not critical for activity.
and CB-839
With respect to cell potency, compounds with 4-
hydroxypiperidinyl and 4-aminopiperidinyl ring systems appear
to consistently outperform derivatives with other ring systems
which exhibit similar potency in the GAC enzyme assay (Tables
Conclusion
1
-3). This trend does not appear to be ClogP driven and at this
time is not quite understood.
In conclusion, a novel set of GAC inhibitors/leads was
synthesized with nanomolar range potency against GAC and
against the MDA-MB-231 cancer cell line. These leads have less
than 10 rotatable bonds, improved LE, LiPE and/or ClogPs when
compared to leading GAC inhibitors BPTES and CB-839. In
addition, in a microsomal stability assay, selected potent leads
had greater stability than their comparators. Property
improvements were the result of the replacement of the flexible
lipophilic connector chains, seen in the leading GAC inhibitors,
with heteroatom substituted cyclic ring systems that were chosen
as surrogates for the conformation assumed by the BPTES
flexible chain seen in the 3UO9 x-ray structure.
Selected potent analogs 7c, 7f, 14c, 14d and 16g were tested in a
human liver microsome (HLM) stability assay to ascertain if the
absence of a labile group, such as sulfur, and/or their improved
ClogP translates to better stability. Data from these experiments,
shown in Figure 4, denote that these compounds have indeed
improved microsomal stability over their comparators.
X-ray structures of GAC in complex with constrained derivatives
To confirm that our leads bind in the BPTES binding pocket
and possibly understand the potency differences among the chiral
derivatives with pyrrolidine rings we solved the crystal structure
Experimental Section
1
H NMR spectra were taken with a Bruker Avance 600 or
Bruker Avance III 400 MHz Spectrometer. MS spectra under EI
conditions were obtained with Shimadzu UFLC/Applied
Biosystems 2000 MS mass spectrometer. MS spectra under APCI
conditions were obtained using a Thar/Waters 3100 instrument.
Infrared spectra were taken with a Bruker Alpha attenuated total
reflectance (ATR) instrument. Column chromatography was
performed using a Teledyne Isco Combiflash Rx instrument.
Anhydrous solvents were obtained commercially and were used
without further drying. Cell culture media and serum were
obtained from Invitrogen (Carlsbad, CA). All other reagents
were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma
Aldrich (St. Louis, MO). MDA-MB-231 cells were purchased
Figure 5. Overlay of the BPTES (green, PDB: 3UO9) and 14d (brown)
binding pockets

from ATCC (Manassas, VA), and were cultured at 37°C, in 5%
CO , using RPMI-1640 media supplemented with 10% FBS.
Recombinant GAC was expressed in E. coli and purified.
Briefly, human GAC (residues 72-603) was cloned into the
6
pET28a vector from Novagen, and was expressed as a His -
peak areas, heights) at 30 min are referenced to the zero time-
point samples to determine the percentage of compound
remaining. Appropriate control compounds with known hepatic
clearance were included in each assay like for instance
metoprolol (control for moderate hepatic clearance), verapamil or
testosterone (high hepatic clearance) warfarin (control for low
hepatic clearance) etc.
2
tagged fusion protein in E. coli. It was then purified by ion
exchange and size exclusion chromatography
Biochemical assays: The GAC inhibitors were solvated in
DMSO. Assay vessels were charged with 1 L of inhibitor in
order to achieve the desired final concentration. To each vessel
was added 95 L of an aqueous solution containing 48 mM Tris-
acetate (pH 8.6), 21 mM glutamine, and 50 nM recombinant
GAC. Fifteen L of either water or 1 M potassium phosphate,
pH 8.2, were added to the mixture to begin the reaction. The
assay reagents were incubated for 10 minutes at room
temperature, at which point 10 L of ice-cold 2.4 M hydrochloric
acid was added to quench the enzymatic reaction. A second
reaction vessel contained 218 L of an aqueous solution
containing 114 mM Tris-HCl (pH 9.4), 0.35 mM ADP, 1.7 mM
Crystallization, data collection and structure determination:
Inhibitors 7d, 7e, 14b and 14d were dissolved in DMSO to make
inhibitor stock solutions (30mM). The GAC-inhibitor complex
was prepared by mixing 95l of GAC solution (20mg/ml, in
150mM NaCl, 5mM Tris-HCl, pH7.5) with 5l of inhibitor stock
solution (mole ratio 1:4) and incubated on ice for one hour.
Crystals were grown by the hanging drop vapor diffusion method
at 20°C. Typically, 1.2l of the complex solution was mixed with
1.2l of the reservoir solution consisting of 10% PEG6000 (w/v),
1.0M LiCl and 0.1M Tris-HCl buffer, pH8.5. Crystals were
observed after 24 hours, and reached a typical size of
3
100x100x200m , one week later. Crystals were pressurized at

-NAD, 238 mM hydrazine, and 1.3 units of glutamate
350MPa for 30 minutes and then frozen to liquid nitrogen
temperature using the high-pressure cryocooling method
described by Huang et al. (Journal of Applied Crystalography,
2015, in press) before data collection. Diffraction data were
collected at 100K at station A1 at MacCHESS. The diffraction
data were reduced using the HKL package (Otwinowski &
Minor, Methods in Enzymology, Volume 276: Macromolecular
Crystallography, part A, p.307-326, 1997, C.W. Carter, Jr. & R.
M. Sweet, Eds., Academic Press, New York). Crystal structures
of the GAC-inhibitor complexes have been determined by
molecular replacement with the program Phaser (McCoy et al.
Journal of Applied Crystalography, 2007, 40:658-674) using the
apo human GAC (PDB code 5D3O) as a search model. Four
molecules of GAC were observed in an asymmetric unit. The
model was examined and built in COOT (Emsley & Cowtan,
Acta Crystallographica 2004, D60, 2126-2132) and subsequent
refinement was carried out with Phenix_refine (Adams et al.,
Acta Crystallographica 2010, D66:213-221). Fitting of the
inhibitor and refinements were performed in COOT and Phenix
programs, respectively. Statistics of data collection and
processing and refinement statistics are given in the
supplementary material (Supplementary Table 1).
dehydrogenase. A third reaction vessel contained an identical
+
solution except that it lacked NAD . Forty L of the initial
reaction mixture was added to each of the second and third
vessels, which were then incubated at room temperature for one
hour. The absorbance of both the second and third reactions was
recorded at 340 nM. The third reaction was treated as a baseline,
and its absorbance was subtracted from that of the second
reaction prior to further data analysis. Dose curves and IC50
values were determined in Sigmaplot, using the built-in four
parameter logistic function.
Cell assays: MDA-MB-231 cells were cultured in RPMI-1640
media supplemented with 10% FBS. Prior to initiating the assay,
cells that were 70-80% confluent were trypsinized. These cells
were diluted, counted, and dispensed into 12-well culture plates
4
at a density of 2 X 10 cells per well. Each well was then brought
to 1 mL of media, total. The cells were allowed to adhere to the
wells overnight, at which point they were recounted (day 0 of the
assay). At this time, and every 48 hours thereafter, media was
exchanged for media containing either the indicated amount of a
given inhibitor, diluted from an appropriate DMSO stock, or an
equivalent amount of DMSO without inhibitor (0.33% DMSO by
volume). Cells were counted on the 6th day of culture. Cell
counting was performed by aspirating media, rinsing the cells
with room temperature PBS, and then incubating at 37°C for 5
minutes in 0.5 mL trypsin-EDTA solution. The culture plates
were then agitated to fully dissociate cells from the plate
surfaces, and 0.5 mL of RPMI-1640 complete media was added
to quench trypsin activity. Cells were then counted on a
hemocytometer, with 3 readings taken and averaged per sample.
All experiments were performed in triplicate. Dose curves and
IC50 values were determined in Sigmaplot, using the built-in four
parameter logistic function.
General procedure 1: Synthesis of compounds 6a-e
A mixture of 2-amino-5-bromothiadiazole (1 eq), desired
diamine 5 (0.5 eq) and Et
a sealed tube was heated at 75-80 C until consumption of 2-
amino-5-bromothiazole was complete. In case where Et N was
3
N (4 eq) or NaHCO (6 eq) in EtOH in
3
o
3
used as base the work up entailed solvent evaporation to small
volume and then addition of water, filtration and trituration of the
resulting solid with MeOH and drying to obtain the product. In
case were NaHCO
entailed cooling to rt, filtration of the NaHCO
the filtrate and then purification of the crude product with silica
column using a MeOH in CH Cl gradient as elusion system.
3
was used at the aprotic solvent the work-up
3
, evaporation of
2
2
Microsomal stability measurements: The method for human
liver microsome assays was adapted from Di et al. (Journal of
Biomolecular Screening, 2003, 453-462) and/or Xu et al. Journal
of the American Society for Mass Spectrometry, 2002, 155-165).
Briefly, test compounds (diluted to final 1 M (0.4% MeOH/
General procedure 2: Preparation of compounds 7a-f, 14a-h
A mixture of a desired bis-thiadiazole 6 or 13 (1 eq.) and Et
3
N
(3-4 eq) in DMF was treated with phenylacetyl chloride (2.1 eq
when thiadiazoles 6 were the starting material or 3-4 eq when
thiadiazoles 13 were the starting material) and the mixture was
stirred at room temperature until consumption of the starting
material was observed. Then followed concentration of the
mixture to a small volume and addition of excess of water to
precipitate the crude product. The crude product was purified by
0
.1% DMSO)) were tested for metabolic stability by incubation
against 1 mg protein/ml of pooled male human liver microsomes
for 0 and 30 minutes at 37º C in the presence and absence of 1
mM NADPH. The reactions were terminated by addition of
acetonitrile. Samples were centrifuged and the supernatant
fractions analyzed by MS/MS. The instrument responses (i.e.

trituration and/or column chromatography with a MeOH/CH
elution system.
Cl
2 2
and then quenched with water and partitioned between CH
and water. The water layer was extracted (X2) with CH Cl
the combined organic layer was then dried over Na SO , filtered
2
Cl
and
2
2
2
2
4
General procedure 3: Preparation of 7g, 7h and 14i
and concentrated to the crude product that was purified with
A mixture of the bis-thiadiazole precursors 6 or 13 (1 eq), Et
3
N
silica gel column and an EtOAc in hexanes gradient as eluent.
(3-4 eq) in DMF was treated with acetic anhydride (2 eq when 6
was starting material or 4 eq when 13 was starting material) and
the mixture was allowed to stir until consumption of the limiting
reagent material. The mixture was then concentrated to small
volume and then treated with water to yield a precipitate that was
filtered and then triturated with an organic solvent or washed
with water and then dried to afford the product.
General procedure 8: Preparation of derivatives 14j, 16g,
26a, 26d-f
A solution of the starting aromatic amine (13d or 16f or 25 or 36
or 40 or 41) (1 eq.) in DMF at room temperature was treated with
the desired acetic acid derivative (1 or 2 eq depending the
number of free amine moieties). The mixture was treated with
3
EDCI or excess of DIEA or Et N and then HATU stirred at room
General procedure 4: Preparation of compounds 11a-h
A solution of desired N-Boc-protected cyclic alcohol 10 (1 eq) in
THF was treated with NaH (60% suspension in mineral oil, 1.2
eq). The mixture was stirred at rt for approximately 20 min.
temperature until consumption of starting material was complete
and then evaporated. The residue was treated with water to afford
a suspension that was filtered to afford the crude product that was
and then purified by trituration or column chromatography.
2
Followed addition of CS (1.5 eq) and then approximately 5 min
later addition of MeI (1.2 eq). The mixture was stirred until
consumption of the limiting reagent was observed and then
General procedure 9: Preparation of intermediates 21, 22,
28, 38 and 39
partitioned between CH
was extracted with CH
organic layer was dried over Na
a residue that was chromatographed on a silica gel column with
an EtOAc in hexanes gradient to afford the product.
2
Cl
2
or EtOAc and water. The water layer
or EtOAc (X2) and the combined
1-NBoc-4-hydroxy piperidine (1 eq) or desired heterocyclic 4-
hydroxypyrolidine (27 or 34 or 37) (1 eq) in anhydrous THF or
anhydrous THF/DMSO mixture was treated with NaH (60%
dispersion in mineral oil, 1.5 eq) the mixture was stirred at room
temperature for 5 min and then followed addition of the
appropriate heterocycle (19 or 20 or 33) (1 eq). The mixture was
2
Cl
2
2 4
SO , filtered and concentrated to
o
General procedure 5: Preparation of compounds 12a-h
stirred at 50 C in a sealed vessel until consumption of the
A solution of desired xanthate 11 (1 eq) in MeOH was treated
starting materials was complete then cooled and partitioned
with 1.5 eq of H
2
NNH
2
at room temperature. When the starting
between EtOAc and NH
was extracted with EtOAc (X3) and combined organic layer was
dried over Na SO filtered and concentrated to the crude product
4
Cl saturated or water. Aqueous layer
xanthate was consumed the mixture was evaporated under
vacuum. The residue was re-dissolved in MeOH and the
resulting solution was evaporated again. After repeating this
dissolution and evaporation cycle one more time the residue was
2
4
that was purified with column chromatography.
taken up again in MeOH and the solution was treated with Et
3
N
General procedure 10: Preparation of benzophenone imines
23, 30, 31
(2 eq) and BrCN (1.2 eq). This mixture was stirred at room
temperature until consumption of the starting material was
complete. Followed solvent evaporation and partition of the
resulting residue between EtOAc and water. The water layer was
extracted with EtOAc (X2) and the combined organic layer was
A mixture of the appropriate halide starting material (21 or 28 or
29) (1 eq), benzophenone imine (2-4 eq), Cs
(±)-BINAP (0.3 eq) in toluene in a sealed tube was degassed via
bubbling for 5 min. The mixture was then treated with
2 3
CO (6-8 eq) and
N
2
o
dried over Na
2
SO
4
filtered and evaporated to the crude product.
Pd (dba) (0.15 eq) sealed and heated at 90 C (when starting
2 3
o
The crude product was purified via column chromatography
halide had a Boc group on) or at 120 C (when starting material
was a dihalogen compound). The mixture was stirred until
consumption of the starting material was observed, then cooled
and filtered. The solids were washed with EtOAc portions
thoroughly and the combined organic layer was evaporated to the
crude product residue that was purified via column
chromatography.
and/or precipitation out of CH
hexanes.
2 2 3
Cl (or CHCl ) with excess of
General procedure 6: Preparation of compounds 13a-h, 16a-
f, 25
To a solution desired intermediates 12 or 15 or 18 or 24 in a
small volume of dioxane was added 4N HCl in dioxane (2-3 eq).
The mixture was stirred until consumption of the starting
material was complete and then evaporated to dryness. The
General procedure 11: Preparation of compounds 24, 26b,
26c
residue was triturated with hot CH
and then dried to afford the corresponding intermediate HCl salt.
This salt in EtOH or DMF was treated with Et N (or NaHCO
2
Cl
2
fist and then hot hexanes
A mixture of starting benzophenone imine (23 or 30 or 31) in
MeOH was treated with NaOAc (3-5 eq) and hydroxylamine
hydrochloride (1.1 eq when 23 was used or 2.1 eq when 30or
31was used). The mixture was stirred at room temperature until
consumption of the starting material was complete, evaporated to
a solid residue which was chromatographed with a silica gel
column to afford the corresponding purified deprotected aromatic
amine intermediate. This intermediate was then dissolved in
3
3
when compounds 18 were starting materials) (4-5 eq) and 2-
amino-5-bromothiadiazole (1 eq) and the mixture was then
o
heated at 75-80 C in a sealed vessel until consumption of the
staring material was complete. Followed evaporation of the
solvent to a small volume and addition of excess of water to
precipitate the crude product which was then collected by
filtration, washed with water, dried and purified by trituration
and/or column chromatography.
3
DMF and treated at room temperature with Et N or DIEA (3-5
eq), phenylacetic acid (1.1 or 2.2 eq depending the number of
amine groups to be acylated) and HATU (1.1-2.2 eq). The
mixture was stirred at room temperature until consumption of the
starting material was complete and then partitioned between
EtOAc and water. The water layer was extracted with EtOAc
(X3) and the combined organic layer was evaporated to the crude
acylation product that was purified via column chromatography.
General procedure 7: Preparation of compounds 15a-d
A solution of desired thiadiazole 12 (1 eq) in CH
with Et N (3-4 eq) and phenyl acetyl chloride (1.5 eq). The
reaction was stirred until consumption of the starting material
2 2
Cl was treated
3

3.37 (m, 1H), 3.39-3.44 (m, 1H), 3.56 (dd, J=10.2, 6.0 Hz, 1H),
General procedure 12: Preparation of intermediates 27, 34,
4.18-4.20 (m, 1H), 6.31 (s, 2H), 6.32 (s, 2H), 7.10 (d, J=6Hz,
1H). ATR IR (cm ) 3252, 3136, 2920, 2860, 1606, 1563, 1492,
1470, 1334, 1299, 1235, 1188, 1117, 1028, 744. MS (ESI) m/z
-
1
3
7
Desired aromatic halide (19 or 32 or 33) (1eq), 4-
hydroxypiperidine (1 eq) an aprotic base such as Et N or K CO
2-3 eq) in DMSO or EtOH was heated in a sealed vessel at until
3
2
3
8 12 8 2
for C H N S calculated: 284.06, observed [M+H]: 285.0.
(
consumption of the starting material was observed. Work-up
consisted of either partial mixture evaporation, addition of water
and filtration of the resulting solid to afford the product (as in
case of 37) or partition of the partially evaporated mixture
between EtOAc and water, extraction of the aqueous layer with
portions of EtOAc until no UV absorption in the organic extract
and then evaporation of the combined organic layer to the crude
product that was purified via column chromatography.
2-N-[(3R)-1-(5-amino-1,3,4-thiadiazol-2-yl)pyrrolidin-3-yl]-
1,3,4-thiadiazole-2,5-diamine (6e)
Prepared according to general procedure 1 using NaHCO
aprotic base. The product was obtained as an off-white solid after
column chromatography with 0-30% MeOH in CH Cl gradient
3
was as
2
2
1
(48% yield). H NMR (600 MHz, DMSO-d6)  1.95 (m, 1H),
2.21 (m, 1H), 3.23 (dd, J=10.2, 3.6 Hz, 1H), 3.43 (distorted dd,
J= 16.8, 9.6 Hz, 1H), 3.56 (dd, J= 10.2, 6.0 Hz, 1H), 4.19 (m,
1
H), 6.32 (s, 2H), 6.33 (s, 2H), 7.10 (d, J= 6Hz, 1H). ATR IR
-1
General procedure 13: Preparation of diamines 40 and 41
A slurry of the desired dinitro compound (1 eq) in 1:1
EtOH/EtOAc and 10% Pd/C (0.1-0.2 eq based on Pd content)
(cm ) 3266, 3121, 2923, 2854, 1610, 1561, 1492, 1470, 1336,
1297, 1236, 1189, 1028, 743. MS (ESI) m/z for C H N S
calculated: 284.06, observed [M+H]: 285.0.
8
12
8 2
2
was hydrogenated under 1 atm of H until consumption of the
starting material was complete. Followed filtration and washing
of the solids with MeOH portions until no UV absorption was
detected in the solvent stream. Combined organic layer was then
evaporated to afford the crude product that was chromatographed
with column.
(±)-(anti)-3-[(5-Amino-[1,3,4]thiadiazol-2-ylamino)-
cyclopentyl]-carbamic acid tert-butyl ester (9)
To a stirred solution of (±)-(8) (478 mg, 2.38 mmol) in EtOH (12
ml) was added NaHCO (300 mg, 3.57 mmol) and 2-amino-5-
3
bromothiadiazole (515 mg, 2.86 mmol). The mixture was stirred
o
in a sealed vessel at 80 C until TLC showed consumption of the
5
-[4-(5-amino-1,3,4-thiadiazol-2-yl)-1,4-diazepan-1-yl]-1,3,4-
limiting reagent. The reaction mixture was then cooled and
evaporated to a residue that was partitioned between EtOAc and
water. The water layer was extracted with EtOAc (X2) and the
combined organic layer was dried over Na SO and evaporated.
The resulting residue was purified via column chromatography
with 0-30% methanol in methylene chloride gradient to yield the
thiadiazol-2-amine (6a)
Prepared according to general procedure 1 using Et N as aprotic
3
base. The product was isolated as a pinkish solid. (45% yield).
2
4
1
H NMR (600 MHz, DMSO-d6)  1.90 (apparent p, J=6.0 Hz,
2
H), 3.44 (t, J=6.0 Hz, 4H), 3.60 (s, 4H), 6.34 (s, 4H). ATR IR
-1
(cm ) 3272, 3111, 2945, 1624, 1571, 1500, 1437, 1383, 1357,
product,
(±)-(anti)-3-[(5-amino-[1,3,4]thiadiazol-2-ylamino)-
1
318, 1295, 1250, 1213, 1191, 1085, 1047, 1026, 952, 922, 878,
cyclopentyl]-carbamic acid tert-butyl ester (9) as an off-white/tan
solid (170 mg, 24% yield). H NMR (600 MHz, MeOD) δ 1.41
1
742, 687, 653. MS (ESI) m/z for C calculated: 298.08,
9
14
H N
S
8 2
observed [M+H]: 299.0.
(s, 9H), 1.47-1.54 (m, 2H), 1.85 (apparent t, J= 6.6 Hz, 2H),
2.03-2.09 (m, 1H), 2.11-2.19 (m, 1H), 3.95-4.06 (m, 2H). MS
(
3
ESI) m/z for C12 S calculated: 299.14, observed [M+H]:
00.1.
21 5 2
H N O
5
-[4-(5-amino-1,3,4-thiadiazol-2-yl)piperazin-1-yl]-1,3,4-
thiadiazol-2-amine (6b)
3
Prepared according to general procedure 1 using Et N as aprotic
(
±)-(anti)-2-N-[3-[(5-Amino-1,3,4-thiadiazol-2-
base. The product was isolated as an off-white solid (83% yield).
1
yl)amino]cyclopentyl]-1,3,4-thiadiazole-2,5-diamine (6f)
To a stirred solution of (±)-9 (170 mg, 0.57 mmol) in
dichloromethane (7 mL) was treated with trifluoroacetic acid (3
mL) at 0 C. The reaction mixture was then allowed to warm
slowly to room temperature stirred for 1 h and then evaporated.
The residue was dissolved in EtOH (3ml). Followed addition of
H NMR (600 MHz, DMSO d6)  3.34 (s, 8H), 6.55 (s, 4H).
-1
ATR IR (cm ) 3261, 3125, 2961, 2848, 1626, 1546, 1494, 1447,
1
375, 1323, 1273, 1234, 1150, 1065, 1048, 1023, 924, 762, 688.
MS (ESI) m/z for C calculated: 284.06, observed
M+H]: 284.9
o
8
12 8 2
H N S
[
NaHCO
3
(167 mg, 1.98 mmol) and 2-amino-5-bromo-thiadiazole
N2-(1-(5-amino-1, 3, 4-thiadiazol-2-yl) piperidin-4-yl)-1, 3, 4-
thiadiazole-2, 5-diamine (6c)
(
122 mg, 0.68 mmol). The mixture was stirred in a sealed vessel
o
at 80 C until consumption of the limiting reagent, then cooled
and evaporated. The residue was suspended in water then filtered,
dried and purified via column chromatography with a 0-20%
methanol in methylene chloride gradient to afford the product,
Prepared according to general procedure 1 using NaHCO
aprotic base. The product was obtained as a tan solid after
column chromatography with 0-35% MeOH in CH Cl gradient
) δ 1.43-1.529
m,2H), 1.95-2.01 (m, 2H), 3.01-3.08 (apparent triplet, 2H), 3.53-
.63 (m, 2H), 6.32 (s, 2H), 6.47 (s, 2H), 6.86 (s, 1H). ATR IR
3
was as
2
2
1
(
(
43% yield) H NMR (600 MHz, DMSO-d
6
(±)-(anti)-2-N-[3-[(5-amino-1,3,4-thiadiazol-2-
yl)amino]cyclopentyl]-1,3,4-thiadiazole-2,5-diamine (6f), (91
mg, 54% yield). H NMR (600 MHz DMSO-d6) δ 1.41-1.48 (m,
3
1
-1
(cm ) 3257, 3137, 2926, 1611, 1560, 1490, 1445, 1375, 1359,
2H), 1.83 (t, J=6.0 Hz, 2H), 2.02-2.08 (m, 2H), 3.91-3.96 (m,
1309, 1263, 1220, 1121, 1082, 1029, 979, 900, 841, 803, 747,
2H), 6.21 (s, 4H), 6.81 (d, J=6.6 Hz, 2H).
6
69. MS (ESI) m/z for C calculated: 298.08, observed
9
14 8 2
H N S
[M+H]: 299.0.
2
-Phenyl-N-{5-[4-(5-phenylacetylamino-[1,3,4]thiadiazol-2-
yl)-[1,4]diazepan-1-yl]-[1,3,4]thiadiazol-2-yl}-acetamide (7a)
Prepared according to general procedure 2. The compound was
obtained as an off white solid upon trituration of the crude
2
‐N‐[(3S)‐1‐(5‐amino‐1,3,4‐thiadiazol‐2‐yl)pyrrolidin‐3‐yl]‐1,3
,
4‐thiadiazole‐2,5‐diamine (6d)
Prepared according to general procedure 1 using NaHCO
aprotic base. The product was obtained as a tan solid after
column chromatography with a 0-30% MeOH in CH Cl gradient
) δ 1.93-1.98
m,1H), 2.19-2.24 (m,1H), 3.24 (dd, J= 10.2, 3.6Hz, 1H), 3.31-
3
was as
1
product with hot hexanes and MeOH. H NMR (600 MHz,
DMSO-d6)  1.92 (m, 2H), 3.54 (t, J= 6.0 Hz, 4H), 3.69 (s, 4H),
2
2
-1
1
3.74 (s, 4H), 7.19-7.40 (m, 10H), 12.23 (s, 2H). ATR IR (cm )
(97% yield). H NMR (600 MHz, DMSO-d
6
3172, 3111, 3063, 2940, 2923, 2811, 2769, 2720, 1679, 1573,
(

1
1
7
5
512, 1495, 1464, 1439, 1381, 1352, 1327, 1307, 1287, 1254,
217, 1181, 1151, 1084, 1049, 1033, 972, 943, 921, 809, 752,
1.89 (t, J=6 Hz, 2H), 2.07-2.20 (m, 2H), 3.70 (s, 4H), 4.04-4.11
(m, 2H), 7.23-7.35 (m, 10H), 7.40 (d, J=6Hz, 2H) 12.16 (s, 2H).
ATR IR (cm ) 3214, 3062, 3015, 1655, 1574, 1531, 1494, 1414,
1350, 1296, 1191, 1074, 1029, 970, 844, 802, 756, 707, 693. MS
-1
27, 693, 667. MS (ESI) m/z for C25
34.16, observed [M+H]: 534.9.
26
H N
8
O
S
2 2
calculated:
(ESI) m/z for C25
26 8 2 2
H N O S calculated: 534.16, observed [M+H]:
2
-Phenyl-N-(5-{4-[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
535.1.
yl]piperazin-1-yl}-1,3,4-thiadiazol-2-yl)acetamide (7b)
Prepared according to general procedure 2. The compound was
obtained as an off-white solid upon trituration of the crude
N-(5-{[(3S)-1-(5-acetamido-1,3,4-thiadiazol-2-yl)pyrrolidin-3-
yl]amino}-1,3,4-thiadiazol-2-yl)acetamide (7g)
Prepared according to general procedure 3. The compound was
1
product with hot MeOH (43% yield). H NMR (400 MHz,
DMSO-d6)  3.52 (s, 8H), 3.74 (s, 4), 7.24 (m, 2H), 7.33 (m,
isolated as an off-white/tan solid after washing of the crude
product with water (25% yield). H NMR (600 MHz, DMSO-d6)
δ 2.07 (multiple overlapping with s, 1H), 2.08 (s, 3H), 2.10 (s,
3H), 2.26-2.33 (m, 1H), 3.39 (dd, J=10.2, 3.6 Hz, 1H), 3.44-3.55
(m, 2H), 3.71 (dd, J=10.2, 6.0 Hz, 1H), 4.32-4.38 (m, 1H), 7.63
-1
1
8
1
9
5
H), 12.34 (s, 2H). ATR IR (cm ) 3172, 2858, 2802, 2726, 1681,
573, 1494, 1463, 1438, 1384, 1351, 1309, 1242, 1154, 1024,
24 8 2 2
24, 809, 732, 695. MS (ESI) m/z for C24H N O S calculated:
20.15, observed [M+H]: 521.2.
(d, J=6.0 Hz, 1H), 11.94 (s, 1H), 11.97 (s, 1H). MS (ESI), m/z
2
-Phenyl-N-(5-(4-((5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
for C12H N O S calculated: 368.08, observed [M+H]: 369.0.
16 8 2 2
yl)amino)piperidin-1-yl)-1,3,4-thiadiazol-2-yl)acetamide (7c)
Prepared according to general procedure 2. The compound was
obtained as a white solid upon column chromatography of the
crude product with column using 0-10% methanol in methylene
N-(5-{[(3R)-1-(5-acetamido-1,3,4-thiadiazol-2-yl)pyrrolidin-3-
yl]amino}-1,3,4-thiadiazol-2-yl)acetamide (7h)
Prepared according to general procedure 3. The compound was
1
chloride gradient (55% yield). H NMR (600 MHz, DMSO-d6) δ
isolated as an off-white/tan solid after washing of the crude
1
1.46-1.55 (m, 2H), 2.02-2.06 (m, 2H), 3.16-3.22 (m, 2H), 3.70
product with water (43% yield). H NMR (600 MHz, DMSO-d6)
(
s, 2H), 3.73 (s, 2H), 3.71-3.78 (m, 3H), 7.23-7.39 (m, 11H),
2.18 (s, 1H), 12.29 (s, 1H). ATR IR (cm ) 3389, 3243, 2852,
682, 1662, 1572, 1493, 1356, 1315, 1299, 1128, 967, 833, 811,
δ 2.07 (multiple overlapping with s, 1H), 2.08 (s, 3H), 2.09 (s,
3H), 2.27-2.33 (m, 1H), 3.40 (dd, J=10.2, 3.6 Hz,1H), 3.45-3.55
(m, 2H), 3.71 (dd, J=10.2, 6.0 Hz, 1H,), 4.32-4.38 (m, 1H), 7.63
(d, J=6.0 Hz, 1H), 11.95 (s, 2H). ATR IR (cm ) 3305, 3195,
3115, 2876, 2806, 2757, 1673, 1583, 1508, 1464, 1367, 1316,
-1
1
1
7
5
-1
57, 713, 693. MS (APCI), m/z for C25
26
H N
8
O
2
S
2
calculated:
34.16, observed [M+H]: 535.2.
1252, 1131, 1006, 962, 827, 696. MS (ESI) m/z for
2
1
‐Phenyl‐N‐(5‐{[(3S)‐1‐[5‐(2‐phenylacetamido)‐
,3,4‐thiadiazol‐2‐yl]pyrrolidin‐3‐yl]amino}‐1,3,4‐
thiadiazol‐2‐yl)acetamide (7d)
C
12
H
16
N
8
O
2
S
2
calculated: 368.08, observed [M+H]: 368.7
tert-Butyl
carboxylate (11a)
Prepared according to general procedure 4. The product was
obtained as a yellowish viscous oil after column chromatography
with 0-20% EtOAC in hexanes gradient (89% yield). H NMR
3-(((methylthio)carbonothioyl)oxy)azetidine-1-
Prepared according to general procedure 2. The compound was
obtained as an off-white solid after column chromatography of
the crude product with a 0-30% MeOH in CH
2
Cl
) δ 1.99-2.06 (m, 1H),
.24-2.32 (m, 1H), 3.35-3.39 (m, 1H), 3.42-3.54 (m, 2H), 3.71
apparent singlet overlapping with a multiplet, 5H), 4.30-4.36 (m,
2
gradient (41%
1
1
yield). H NMR (600 MHz, DMSO-d
2
(
6
(600 MHz, CDCl ) 1.47 (s, 9H), 2.61 (s, 3H), 4.05 (m, 2H),
3
-1
4.32 (m, 2H), 5.62 (m, 1H). ATR IR (cm ) 2975, 2929, 2882,
1698, 1477, 1455, 1389, 1365, 1295, 1254, 1204, 1135, 1103,
1056, 1019, 965, 927, 858, 769.
1
1
1
1
H), 7.22-7.26 (m, 2H), 7.27-7.34 (m, 8H), 7.65 (broad s, 1H),
-1
2.21 (s, 1H), 12.24 (s, 1H). ATR IR (cm ) 3188, 2861, 1681,
661, 1578, 1509, 1459, 1342, 1312, 1291, 1235, 1192, 1134,
140, 1073, 1030, 970, 833, 756, 718, 693. MS (ESI) m/z for
tert-Butyl
(3S)-3-{[(methylsulfanyl)methanethioyl]oxy}
C
24 24
H N
8
O
2
S
2
calculated: 520.15, observed [M-H]: 518.8.
pyrrolidine-1-carboxylate (11b)
Prepared according to general procedure 4. The product was
2
‐Phenyl‐N‐(5‐{[(3R)‐1‐[5‐(2‐phenylacetamido)‐
obtained as a colorless oil after column chromatography with 0-
20% ethyl acetate in hexanes gradient (92% yield). H NMR (600
1
1,3,4‐thiadiazol‐2‐yl]pyrrolidin‐3‐yl]amino}‐1,3,4‐
thiadiazol‐2‐yl)acetamide (7e)
Prepared according to general procedure 2. The compound was
obtained as an off-white solid after column chromatography of
MHz, CDCl
3
) δ 1.47 (s, 9H), 2.11-2.20 (m, 1H), 2.21-2.25 (m,
1H), 2.56 (s, 3H), 3.38-3.51 (m, 1H), 3.52-3.73 (m, 3H), 5.97
3 2
(apparent s, 1H). MS (ESI) m/z for C11H19NO S calculated:
the crude product with a 0-15% MeOH in CH
2
Cl
yield). H NMR (600 MHz, DMSO-d6) δ 2.00-2.06 (m, 1H),
.25-2.32 (m, 1H), 3.36-3.39 (m, 1H), 3.43-3.53 (m, 2H), 3.69
2
gradient (37%
277.08, observed [M+Na]: 300.1.
1
2
tert-Butyl
(3R)-3-{[(methylsulfanyl)methanethioyl]oxy}
(m, 1H), 3.71 (s, 2H), 3.72 (s, 2H), 4.32-4.37 (m, 1H), 7.23-7.34
pyrrolidine-1-carboxylate (11c)
Prepared according to general procedure 4. The product was
(m, 10H) 7.65 (d, J=6.0 Hz, 1H), 12.21 (s, 1H), 12.25 (s, 1H).
-1
ATR IR (cm ) 3339, 3186, 2868, 2735, 1682, 1662, 1577, 1512,
obtained as a yellowish oil after column chromatography with 0-
1
1
1
457, 1358, 1316, 1296, 1226, 1185, 1160, 1132, 1147, 1073,
029, 967, 842, 801, 755, 713, 693. MS (ESI) m/z for
20% ethyl acetate in hexanes gradient (94% yield). H NMR (600
MHz CDCl
1H), 2.56 (s, 3H), 3.40-3.51 (m, 1H), 3.52-3.73 (m, 3H), 5.96
apparent s, 1H). MS (ESI) m/z C11 calculated: 277.08,
observed [M+Na]: 300.1.
3
) δ = 1.46 (s, 9H) 2.11-2.21 (m, 1H), 2.22-2.28 (m,
24 24 8 2 2
C H N O S calculated: 520.15, observed [M+H]: 520.9.
(
3 2
H19NO S
(
[
[
±)-(anti)-2-Phenyl-N-{5-[3-(5-phenylacetylamino-
1,3,4]thiadiazol-2-ylamino)-cyclopentylamino]-
1,3,4]thiadiazol-2-yl}-acetamide (7f)
tert-Butyl
4-(((methylthio)carbonothioyl)oxy)piperidine-1-
Prepared according to general procedure 2. The compound was
carboxylate (11d)
obtained as an off-white solid after column chromatography of
Prepared according to general procedure 4. The product was
obtained as a colorless viscous oil after column chromatography
with 0-20% ethyl acetate in hexanes gradient (69% yield)
the crude product with 0-10% MeOH in CH
2 2
Cl gradient (5%
1
6
yield). H NMR (600 MHz, DMSO-d ) δ 1.46-1.53 (m, 2H),

1
HNMR (600 MHz, CDCl
m, 2H), 2.58 (s, 3H), 3.37 (ddd, J= 11.4, 7.8, 3.6 Hz, 2H), 3.69
broad m, 2H), 5.75 (apparent sep, J= 3.6 Hz). ATR IR (cm )
002, 2973, 2925, 2864, 1687, 1476, 1452, 1418, 1364, 1311,
3
)  1.49 (s, 9H), 1.84 (m, 2H), 1.99
tert-Butyl (3S)-3-[(5-amino-1,3,4-thiadiazol-2yl)oxy]
pyrrolidine-1-carboxylate (12b)
Prepared according to the general procedure 5. The compound
was obtained after purification of the crude product via column
chromatography with 0-5% MeOH in EtOAc gradient and a
(
(
3
-
1
1
273, 1237, 1209, 1162, 1129, 1049, 1008, 861, 767. MS (ESI)
m/z for C12
H
21NO
S
3 2
calculated: 291.1, observed [M+Na]: 314.1.
precipitation out of CHCl
3
with excess of hexanes as an off-white
solid (55% yield). H NMR (600 MHz, CDCl )  1.46 (s, 9H),
2.12-2.16 (m, 1H), 2.25-2.37 (m, 1H), 3.44-3.77 (m, 4H), 4.63 (s,
1
3
tert-Butyl
carboxylate (11e)
Prepared according to general procedure 4. The product was
obtained as a colorless oil after column chromatography with 0-
3-(((methylthio)carbonothioyl)oxy)piperidine-1-
-1
2H), 5.49 (s, 1H). ATR-IR (cm ) 3340, 3310, 3090, 2969, 2885,
1702, 1686, 1627, 1555, 1502, 1490, 1413, 1366, 1346, 1283,
1252, 1210, 1161, 1108, 1042, 968, 957, 930, 891, 875, 848, 765,
1
2
0% ethyl acetate in hexanes gradient (75% yield). H NMR (600
18 4 3
696. MS (ESI) m/z calculated for C11H N O S: 286.11, observed
MHz, CDCl
1
1
3
) δ 1.44 (s, 9H), 1.56 (s, brd, 1H), 1.81 (s,brd,1H),
.93 (s, brd, 2H), 2.53 (s, 3H), 3.18 (s, brd, 1H), 3.46 (s, brd,
H), 3.71 (s, brd, 1H), 3.92 (s, brd, 1H), 5.53 (broad s, 1H). MS
calculated: 291.1, observed [M+Na]:
[M-H]: 284.9.
tert-Butyl (R)-3-((5-amino-1,3,4-thiadiazol-2-yl)oxy)
pyrrolidine-1-carboxylate (12c)
(ESI) m/z for C12
H
21NO
S
3 2
314.1.
Prepared according to the general procedure 5. The compound
was obtained as an off-white solid after precipitation of the crude
tert-Butyl
(3S)-3-({[(methylsulfanyl)methanethioyl]oxy}
product out of CHCl
NMR (600 MHz, CDCl
2.26-2.35 (s, 1H), 3.40-3.79 (m, 4H), 4.63 (s, 2H), 5.49 (apparent
3
with excess of hexanes (48% yield). 1H
methyl)pyrrolidine-1-carboxylate (11f)
Prepared according to general procedure 4. The product was
3
) δ = 1.46 (s, 9H), 2.09-2.19 (m, 1H),
obtained as
chromatography with 0-20% ethyl acetate in hexanes gradient
70% yield). 1H NMR (600 MHz CDCl ) δ 1.47 (s, 9H), 1.69-
.79 (m, 1H), 2.05 (m, 1H), 2.57 (d, 3H, J=8.4 Hz), 2.66-2.76 (m,
H), 3.09-3.22 (m, 1H), 3.30-3.63 (m, 3H), 4.45-4.54 (m, 1H),
a
light yellow/brown oil after column
s, 1H). MS (ESI) m/z C11
[M-H]: 284.9.
18 4 3
H N O S calculated: 286.35, observed
(
3
1
1
4
1
tert-Butyl 4-((5-amino-1,3,4-thiadiazol-2-yl)oxy)piperidine-1-
carboxylate (12d)
Prepared according to the general procedure 5. The compound
-1
.55-4.63 (m, 1H). ATR IR (cm ) 2972, 2931, 2876, 1687, 1477,
453, 1400, 1363, 1212, 1166, 1130, 1058, 965, 921, 882, 770.
was isolated as a white powder after purification of the crude
product via column chromatography and 0-100% EtOAc in
1
tert-Butyl
(3R)-3-({[(methylsulfanyl)methanethioyl]oxy}
CH Cl
2 2
to 0-5% MeOH in EtOAc gradient (40% yield). H NMR
methyl)pyrrolidine-1-carboxylate (11g)
(600 MHz, CDCl )  1.48 (s, 9H), 1.80 (m, 2H), 2.06 (m, 2H),
3
Prepared according to general procedure 4. The product was
obtained as light yellow/brown oil after column chromatography
with 0-20% ethyl acetate in hexanes gradient (75% yield). 1H
3.31 (ddd, J=12.0, 8.4, 3.6 Hz, 2H), 3.73 (broad m, 2H), 4.71 (s,
-
1
2H), 5.13 (apparent sep, J= 4.2 Hz, 1H). ATR IR (cm ) 3383,
3255, 3086, 2973, 2935, 2872, 1657, 1558, 1496, 1427, 1365,
1280, 1243, 1228, 1165, 1125, 1074, 1052, 1024, 997, 901, 858,
843, 770, 749, 703, 675. MS (ESI) m/z for C H N O S
NMR (600 MHz CD
2
Cl
.10 (m, 1), 2.56 (s, 3H), 2.66-2.76 (m, 1H), 3.09-3.15 (m, 1H),
.28-3.45 (m, 1H), 3.40-3.46 (m, 1H), 3.51-3.56 (m, 1H), 4.49-
2
) δ 1.44 (s, 9H), 1.68-1.76 (m,1H), 2.01-
2
3
4
2
9
2
12
20
4
3
calculated: 300.13, observed [M+H]: 301.0.
-
1
.55 (m, 1H), 4.57-4.62 (m, 1H). ATR IR (cm ) 2972, 2931,
876, 1687, 1477, 1453, 1400, 1363, 1212, 1166, 1130, 1058,
65, 882, 770. MS (ESI) m/z for C12
tert-Butyl 3-((5-amino-1,3,4-thiadiazol-2-yl)oxy)piperidine-1-
carboxylate (12e)
3 2
H21NO S calculated:
91.09, observed [M+Na]: 314.1
Prepared according to the general procedure 5. The compound
was isolated as a white powder after purification of the crude
product via column and a 0-40% acetone in CH Cl gradient
tert-Butyl-4-({[(methylsulfanyl)methanethioyl]oxy}methyl)
piperidine-1-carboxylate (11h)
2
2
1
(51% yield). H NMR (600MHz, DMSO-d ) δ 1.30 (s, brd, 9H),
6
Prepared according to general procedure 4. The product was
obtained as a colorless oil after column chromatography with a 0-
1.42-1.44 (m, 2H), 1.65 (s, brd, 1H), 1.87(s, brd, 2H), 2.93 (s,
brd, 1H), 3.69(s, brd, 1H), 4.09 (s, brd, 1H), 4.77(m, 1H), 6.74 (s,
-1
5
0% EtOAc in hexanes gradient (82% yield).
2H). ATR IR (cm ) 3326, 3275, 3129, 2953, 2863, 1682, 1623,
1542, 1488, 1422, 1366, 1345, 1277, 1236, 1167, 1150, 1130,
1097, 1073, 1003, 956, 925, 884, 854, 816, 766, 687. MS (ESI)
m/z for C H N O S calculated: 300.13, observed [M+Na]:
1
H NMR (600 MHz, CDCl
3
) δ 1.22-1.29 (m, 2H), 1.46 (s, 9H),
1
.74 (d, J=12.6Hz, 2H), 1.98-2.05 (m, 1H), 2.56 (s, 3H), 2.72 (s,
-
brd, 2H), 4.14 (s, brd, 2H), 4.46 (d, J=6.6Hz, 2H). ATR IR (cm
12
20
4
3
1
)
2973, 2927, 2852, 1685, 1448, 1418, 1364, 1288, 1274, 1213,
165, 1139, 1060, 997, 970, 863, 768. MS (ESI) m/z calculated
: 305.11, observed [M+Na]: 328.3.
323.2
1
for C13
H
23NO
S
3 2
tert-Butyl (3S)-3-{[(5-amino-1,3,4-thiadiazol-2-yl)oxy]methyl}
pyrrolidine-1-carboxylate (12f)
tert-Butyl 3-((5-amino-1,3,4-thiadiazol-2-yl)oxy)azetidine-1-
carboxylate (12a)
Prepared according to the general procedure 5. The compound
Prepared according to the general procedure 5. The compound
was obtained as an off-white solid after purification of the crude
product via column chromatography and a 0-8% MeOH in
1
was obtained as an off-white solid after a precipitation of the
CH Cl gradient (62% yield). H NMR (600 MHz CDCl ) δ 1.46
2
2
3
1
crude product out CH
2
Cl
2
with excess of hexanes (81% yield). H
(s, 9H), 1.68-1.79 (m, 1H), 2.00-2.09 (m,1H), 2.64-2.73 (m, 1H),
3.10-3.25 (m, 1H), 3.30-3.61 (m ,3H), 4.31-4.38 (m, 1H), 4.22
NMR (600 MHz, DMSO-d6)  1.39 (s, 9H), 3.86 (broad s, 2H),
-
1
-1
4
3
1
8
2
.21 (broad s, 2H), 5.25 (m, 1H), 6.85 (s, 2H). ATR-IR (cm )
337, 3266, 3087, 2969, 2879, 1716, 1705, 1624, 1553, 1502,
488, 1401, 1368, 1349, 1296, 1262, 1180, 1136, 1108, 1043,
(dd, J= 10.2, 6.6 Hz, 1H), 4.66 (s, 2H). ATR IR (cm ) 3328,
3275, 3119, 2974, 2874, 1681, 1625, 1549, 1497, 1459, 1412,
1392, 1364, 1331, 1252, 1167, 1136, 1087, 969, 888, 877, 770,
58, 778, 691. MS (ESI) m/z for C10
16
H N
4
O
3
S calculated:
684. MS (ESI) m/z for C12
[M-H]: 298.9.
20 4 3
H N O S calculated: 300.13, observed
72.09, observed [M-H]: 270.8.

tert-Butyl (3R)-3-{[(5-amino-1,3,4-thiadiazol-2-yl)oxy]methyl}
pyrrolidine-1-carboxylate (12g)
Prepared according to the general procedure 5. The compound
was obtained as an off-white solid after purification of the crude
5-(4-((5-amino-1,3,4-thiadiazol-2-yl)oxy)piperidin-1-yl)-1,3,4-
thiadiazol-2-amine (13d)
Prepared according to general procedure 6. The compound was
product via column chromatography and a 0-5% MeOH in
obtained as a brown solid after suspension/trituration of the crude
1
1
CH
CDCl
.73 (m,1H), 3.10-3.20 (m,1H), 3.37-3.35 (m, 1H), 3.40-3.48
brd m, 1H), 3.52 (dd, J=10.8, 7,8 Hz, 1H), 4.29-4.33 (m, 1H),
.41 (dd, J=10.2, 6.6 Hz, 1H), 5.18 (very brd s, 2H). ATR IR
2
Cl
2
gradient as eluent (51% yield). H NMR (600 MHz
product in CH
2
Cl
2
(78% yield). H NMR (600 MHz, DMSO-d6)
3
) δ 1.43 (s, 9H), 1.68-1.76 (m,1H), 1.98-2.07 (m,1H), 2.65-
 1.77 (m, 2H), 2.09 (m, 2H), 3.20 (ddd, J=12.6, 9.0, 3.6 Hz,
2H), 3.48 (m, 2H), 4.94 (apparent sep, J=4.2 Hz, 1H), 6.49 (s,
2
(
-1
2H), 6.77 (s, 2H). ATR IR (cm ) 3276, 3116, 2950, 2858, 1625,
1550, 1489, 1451, 1356, 1307, 1245, 1217, 1122, 1095, 1017,
951, 922, 878, 756, 677. MS (ESI) m/z for C H N OS
4
-1
(cm ) 3324, 3280, 3105, 2970, 2878, 1684, 1623, 1555, 1495,
9
13
7
2
1
8
3
459, 1408, 1364, 1329, 1256, 1170, 1136, 1113, 992, 962, 939,
calculated: 299.06, observed [M-H]: 297.9.
85, 772, 687. MS (ESI) m/z for C12
20 4 3
H N O S calculated:
00.13, observed [M-H]: 298.9
5‐{3‐[(5‐amino‐1,3,4‐thiadiazol‐2‐yl)oxy]piperidin‐1‐
yl}‐1,3,4‐thiadiazol‐2‐amine (13e)
4
-(5-Amino-[1,3,4]thiadiazol-2-yloxymethyl)-piperidine-1-
Prepared according to general procedure 6. The compound was
obtained after purification of the crude product via column
chromatography and using 0-25% MeOH in CH Cl gradient as
carboxylic acid tert-butyl ester (12h)
Prepared according to the general procedure 5. The compound
was obtained as an off-white solid after purification of the crude
product via column chromatography and a 0-10% MeOH in
2
2
1
elution system (40% yield). H NMR (600 MHz, DMSO-d6) 
1
3
3
.55-1.63 (m, 1H); 1.77-1.87 (m, 2H), 1.96-2.02 (m, 1H), 3.07-
.12 (m, 1H), 3.19-3.23 (m, 1H), 3.45 (dd, J=12.6, 6.0Hz, 1H),
.63 (dd, 1H), 4.84-4.92 (m, 1H), 6.45 (s, 2H), 6.77 (s, 2H). MS
CH
excess of hexanes (31% yield). H NMR (600 MHz, DMSO-d6)
1.10(ddd, J= 24.6, 12.6, 4.2 Hz, 2H), 1.39 (s, 9H), 1.67 (d,
J=12.6 Hz, 2H), 1.92-1.94 (m, 1H), 2.70 (broad s, 2H), 3.95
2
Cl gradient and then a precipitation out of CH Cl with
2
2
2
1

9 13 7 2
(ESI) m/z for C H N OS calculated: 299.06, observed [M-H]:
297.9
-
(
)
broad s, 2H), 4.14 (d, J=6.6 Hz, 2H), 6.73 (s, 2H). ATR IR (cm
1
3296, 3103, 2974, 2928, 2875, 1680, 1625, 1559, 1513, 1498,
458, 1433, 1383, 1363, 1302,1262, 1233, 1175, 1132, 1092,
068, 998, 979, 931, 902, 867, 827, 764, 686. MS (ESI) m/z
5-[(3S)-3-{[(5-Amino-1,3,4-thiadiazol-2-yl)oxy]methyl}
pyrrolidin-1-yl]-1,3,4-thiadiazol-2-amine (13f)
1
1
Prepared according to general procedure 6. The compound was
isolated as an off-white/tan solid after suspension/trituration of
the crude product with boiling EtOH and then purification via
column chromatography and 0-25% methanol in methylene
22 4 3
calculated for C13H N O S: 314.14, observed [M+H]: 315.2.
5
-{3-[(5-amino-1,3,4-thiadiazol-2-yl)oxy]azetidin-1-yl}-1,3,4-
1
thiadiazol-2-amine (13a)
Prepared according to general procedure 6. The compound was
isolated as a tan solid after suspension of the crude product in hot
MeOH and CH

5
3
1
chloride gradient (50% yield). H NMR (600 MHz DMSO-d6) δ
1.78-1.85 (m, 1H), 2.04-2.12 (m, 1H), 2.73-2.82 (m, 1H), 3.13
(dd, J=9.6, 6.6 Hz, 1H), 3.25-3.32 (m, 1H), 3.34-3.42 (m, 1H),
3.46 (dd, J=9.6, 7.8, 1H), 4.25-4.36 (m, 2H), 6.31 (s, 2H), 6.75
1
2
Cl
2
(63% yield). H NMR (600 MHz, DMSO-d6)
-
1
3.96 (dd, J=9.0, 3.6 Hz, 2H), 4.29 (dd, J=9.0, 6.0 Hz, 2H),
(s, 2H). ATR IR (cm ) 3251, 3099, 2931, 2875, 1614, 1567,
1503, 1469, 1398, 1298, 1270, 1255, 1061, 1033, 1006, 989, 747,
682. MS (ESI) m/z for C H N OS calculated: 299.06, observed
-1
.41 (m, 1H), 6.56 (s, 2H), 6.86 (s, 2H). ATR-IR (cm ) 3256,
085, 2954, 2858, 2786, 1631, 1567, 1497, 1455, 1366, 1330,
237, 1174, 1113, 1060, 968, 760, 679. MS (ESI) m/z for
9
13
7
2
[M+H]: 299.9.
7 9 7 2
C H N OS calculated: 271.03, observed [M+H]: 272.0.
5
-[(3R)-3-{[(5-amino-1,3,4-thiadiazol-2-yl)oxy]methyl}
5
1
-[(3S)-3-[(5-amino-1,3,4-thiadiazol-2-yl)oxy]pyrrolidin-1-yl]-
,3,4-thiadiazol-2-amine (13b)
pyrrolidin-1-yl]-1,3,4-thiadiazol-2-amine (13g)
Prepared according to general procedure 6. The compound was
Prepared according to general procedure 6. The compound was
obtained as an off-white/tan solid after trituration of the crude
isolated as an off-white solid after suspension/trituration with
boiling ethyl acetate, methanol and hexanes (16% yield). H
1
product with boiling MeOH, boiling DCM, and boiling hexanes.
NMR (600 MHz DMSO-d6) δ 1.74-1.85 (m, 1H), 2.06-2.14 (m,
1H), 2.73-2.82 (m, 1H), 3.14 (dd, J=9.6, 6.6 Hz, 1H), 3.27-3.33
(m, 1H), 3.36-3.42 (m, 1H), 3.45 (dd, J= 9.6, 7.8 Hz, 1H), 4.25-
4.35 (m, 2H), 6.31 (s, 2H), 6.75 (s, 2H). MS (ESI) m/z for
C H N OS calculated: 299.06, observed [M+H]: 300.0.
1
H NMR (600 MHz, DMSO-d
6
) δ 2.21-2.35 (m, 2H), 3.38-3.44
(
m, 2H), 3.53 (d, J=12.0 Hz, 1H), 3.66 (dd, J=11.4, 4.8 Hz, 1H),
-1
5
3
1
7
.38-5.43 (m, 1H), 6.37 (s, 2H), 6.80 (s, 2H). ATR IR (cm )
275, 3106, 2952, 2859, 2775, 1626, 1577, 1545, 1498, 1474,
383, 1347, 1315, 1285, 1253, 1239, 1106, 1066, 1025, 890, 852,
9
13
7
2
54, 689. MS (ESI) m/z for C
8 11
H N
4
OS calculated: 285.05,
5-(4-{[(5-amino-1,3,4-thiadiazol-2-yl)oxy]methyl}piperidin-1-
yl)-1,3,4-thiadiazol-2-amine(13h)
observed [M-H] 283.7.
Prepared according to general procedure 6. The compound was
isolated as a tan solid after trituration of the crude product with
5
-[(3R)-3-[(5-amino-1,3,4-thiadiazol-2-yl)oxy]pyrrolidin-1-
1
yl]-1,3,4-thiadiazol-2-amine (13c)
boiling CH Cl and hexanes (26% yield). H NMR (600 MHz,
2
2
Prepared according to general procedure 6. The compound was
obtained as an off-white/tan solid after trituration of the crude
DMSO-d ) δ 1.34 (ddd, J= 25.2, 12.6, 4.8 Hz, 2H) 1.73 (d,
J=12.6 Hz, 2H), 1.93-2.02 (m, 1H), 2.88-2.95 (m, 2H), 3.63 (d,
6
product with boiling MeOH, boiling DCM, and boiling hexanes
J=13.2 Hz, 2H), 4.17 (d, J=6.0 Hz, 2H), 6.43 (s, 2H), 6.74 (s,
2H). ATR IR (cm ) 3260, 3112, 2946, 2843, 1616, 1551, 1492,
1
-1
(47% yield). H NMR (600 MHz, DMSO-d6) δ 2.21-2.26 (m,
1
3
2
1
1
H), 2.28-2.35 (m, 1H), 3.37-3.44 (m, 2H), 3.51-3.55 (m, 1H),
.63-3.68 (m, 1H), 5.39 (apparent s, 1H), 6.37 (s, 2H), 6.80 (s,
H). ATR IR (cm ), 3270, 3103, 2950, 2852, 2771, 1626, 1577,
543, 1503, 1469, 1380, 1346, 1312, 1286, 1250, 1238, 1107,
1457, 1384, 1328, 1291, 1262, 1215, 1049, 1000, 983, 959, 894,
834, 760, 685. MS (ESI) m/z for C H N OS calculated:
313.08, observed [M-H]: 312.0.
10
15
7
2
-
1
064, 1025, 890, 850, 752, 688. MS (ESI) m/z for C
8
11
H N
7
OS
2
2-Phenyl-N-[5-({1-[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
yl]azetidin-3-yl}oxy)-1,3,4-thiadiazol-2-yl]acetamide (14a)
calculated: 285.05, observed [M-H]: 283.7.

Prepared as described in general procedure 2. The compound was
obtained as an off-white solid after suspension of the crude
3063, 3029, 2850, 2752, 1684, 1574, 1503, 1454, 1355, 1315,
1252, 1199, 1142, 1073, 1008, 968, 927, 857, 807, 758, 716, 693.
product in hot CHCl
3
cooling and then addition of excess of
MS (ESI) m/z for C25
[M+H]: 536.1.
25 7 3 2
H N O S calculated: 535.15, observed
1
hexanes (54% yield). H NMR (600 MHz, DMSO-d6)  3.74 (s,
H), 3.77 (s, 2H), 4.14 (apparent broad d, J=9 Hz, 2H), 4.45
apparent t, J=7.8 Hz, 2H), 5.57 (m, 1H), 7.25-7.42 (m, 10H),
2
(
2-Phenyl-N-{5-[(3S)-3-({[5-(2-phenylacetamido)-1,3,4-
thiadiazol-2-yl]oxy}methyl)pyrrolidin-1-yl]-1,3,4-thiadiazol-
2-yl}acetamide (14f)
Prepared as described in general procedure 2. The compound
was obtained as an off-white solid after trituration of the crude
-
1
1
2
9
2.40 (s, 1H), 12.67 (s, 1H). ATR-IR (cm ) 3175, 3061, 3029,
869, 1687, 1570, 1494, 1455, 1173, 1137, 1095, 1056, 1030,
69, 813, 758, 720, 694. MS (ESI) m/z for C23
H
21
N
7
O
3
S
2
calculated: 507.11, observed [M+H]: 508.1.
2 2
product with boiling CH Cl , then EtOAc and then hexanes (39%
1
(S)-2-phenyl-N-(5-(3-((5-(2-phenylacetamido)-1,
3,
4-
yield). H NMR (600 MHz, DMSO-d6) δ 1.81-1.92 (m, 1H),
2.11-2.19 (m, 1H), 2.83-2.92 (m, 1H), 3.26 (dd, J=9.6, 6.6 Hz,
1H), 3.38-3.43 (m, 1H), 3.47-3.52 (m, 1H), 3.56 (dd, J= 9.6, 7.8
Hz, 1H), 3.72 (s, 2H), 3.76 (s, 2H), 4.41-4.51 (m, 2H), 7.23-7.36
(m, 10H) 12.24 (s, 1H), 12.57 (s, 1H). ATR IR (cm ) 3176,
3063, 3030, 2866, 1686, 1575, 1505, 1454, 1394, 1358, 1294,
1268, 1162, 1074, 1031, 968, 812, 753, 720, 694. MS (ESI) m/z
thiadiazol-2-yl) oxy) pyrrolidin-1-yl)-1, 3, 4-thiadiazol-2-yl)
acetamide (14b)
Prepared as described in general procedure 2. The compound
was obtained as an off-white solid after purification of the crude
product via column chromatography and a 0-20% MeOH in
-1
CH
2
Cl
2
gradient followed by a trituration with boiling hexanes
1
(21% yield). H NMR (400 MHz, DMSO-d
6
) δ 2.31-2.42 (m,
25 7 2
for C23H N OS calculated: 535.15, observed [M+H]: 536.0.
2
H), 3.48-3.57 (m, 2H), 3.68 (apparent d, 1H), 3.72 (s, 2H), 3.76
(s, 2H), 3.78 (dd, J=12.0, 4.4 Hz, 1H), 5.55-5.65 (m, 1H), 7.20-
2-Phenyl-N-{5-[(3R)-3-({[5-(2-phenylacetamido)-1,3,4-
thiadiazol-2-yl]oxy}methyl)pyrrolidin-1-yl]-1,3,4-thiadiazol-
2-yl}acetamide (14g)
-1
7
3
1
7
5
.40 (m, 10H), 12.27 (s, 1H), 12.59 (s, 1H). ATR IR (cm )
179, 3060, 3031, 2915, 2851, 2808, 2750, 1684, 1573, 1530,
495, 1454, 1360, 1298, 1256, 1198, 1147, 1080, 1028, 957, 857,
97, 758, 714, 694. MS (ESI) m/z for C24
21.13, observed [M+H] 522.2.
Prepared as described in general procedure 2. The compound
was obtained as an off-white solid after trituration of the crude
product with hot ethyl acetate, methanol, methylene chloride and
23 7 3 2
H N O S calculated:
1
then hexanes (37% yield). H NMR (600 MHz, DMSO-d6) δ
2
-phenyl-N-(5-{[(3R)-1-[5-(2-phenylacetamido)-1,3,4-
1.82-1.92 (m, 1H), 2.11-2.19 (m, 1H), 2.83-2.91 (m, 1H), 3.26
(dd, J=9.6, 6.6 Hz, 1H), 3.38-3.43 (m, 1H), 3.45-3.51 (m, 1H),
3.56 (dd, J=9.6, 7.8 Hz, 1H), 3.72 (s, 2H), 3.76 (s, 2H), 4.41-4.49
(m, 2H), 7.23-7.35 (m, 10H), 12.24 (brd s, 1H), 12.57 (brd s,
1H). ATR IR (cm ) 3178, 3065, 3032, 2918, 2850, 1685, 1573,
1505, 1452, 1394, 1354, 1290, 1315, 1253, 1159, 1074, 1030,
thiadiazol-2-yl]pyrrolidin-3-yl]oxy}-1,3,4-thiadiazol-2-
yl)acetamide (14c)
Prepared as described in general procedure 2. The compound
was obtained as an off-white solid after trituration of the crude
product with hot EtOH, then CH
-
1
2 2
Cl and then hexanes (26%
1
yield). H NMR (600 MHz, DMSO-d6) δ 2.31-2.40 (m, 2H),
966, 811, 750, 719, 692. MS (ESI) m/z for C25
25 7 2 2
H N O S
3
2
.48-3.56 (m, 2H), 3.68 (apparent d, 1H), 3.70 (s, 2H), 3.72 (s,
H), 3.80 (dd, J=12.0, 4.8 Hz, 1H), 5.55-5.59 (m, 1H), 7.23-7.34
calculated: 535.64, observed [M+H] 536.1.
-1
(m, 10H), 12.28 (s,1H) 12.60 (s,1H). ATR IR (cm ), 3174, 3059,
2-Phenyl-N-{5-[1-(5-phenylacetylamino-[1,3,4]thiadiazol-2-
3
1
6
030, 2870, 2810, 2750, 1685, 1573, 1530, 1496, 1454, 1360,
298, 1256, 1198, 1147, 1079, 1028, 958, 857, 798, 757, 714,
yl)-piperidin-4-ylmethoxy]-[1,3,4]thiadiazol-2-yl}-acetamide
(14h)
Prepared as described in general procedure 2. The compound was
94. MS (ESI) m/z for C24
H
23
N
7
O
3
S
2
calculated: 521.13,
observed [M+H]: 522.0.
obtained as an off-white solid upon purification of the crude
product via column chromatography and a 0-15% MeOH in
1
2
-Phenyl-N-{5-[1-(5-phenylacetylamino-[1,3,4]thiadiazol-2-
CH Cl
2 2
gradient (52% yield). H NMR (600 MHz, DMSO-d6) 
yl)-piperidin-4-yloxy]-[1,3,4]thiadiazol-2-yl}-acetamide (14d)
Prepared as described in general procedure 2. The compound was
obtained as an off-white solid after suspension of the crude
1.34-1.39 (m, 2H), 1.74-1.80 (m, 2H), 2.07-2.12 (m, 1H), 3.04 (t,
J=12 Hz, 2H), 3.72 (s, 2H), 3.75 (s, 2H), 3.79-3.83 (m, 2H),
4.27-4.30 (m, 2H), 7.24-7.34 (m, 10H), 12.27 (s, 1H), 12.55 (s,
1H). ATR IR (cm ) 3389, 3264, 3156, 2940, 2849, 1693, 1563,
1494, 1255, 969, 693. MS (ESI) m/z calculated for
-
1
product in boiling CHCl
3
then cooling and addition of excess of
hexanes (73% yield). H NMR (600 MHz, DMSO-d6)  1.84
m, 2H), 2.13 (m, 2H), 3.37 (m, 2H), 3.65 (m, 2H), 3.76 (s, 2H),
1
(
26 27 7 3 2
C H N O S : 549.16, observed [M+H]: 550.1.
3
1
1
1
.78 (s, 2H), 5.13 (m, 1H), 7.25 (m, 2H), 7.31 (m, 8H), 12.31 (s,
-1
H), 12.58 (s, 1H). ATR IR (cm ) 3176, 3060, 2955, 2856, 2809,
686, 1569, 1503, 1455, 1351, 1351, 1315, 1268, 1218, 1141,
087, 1016, 969, 947, 880, 830, 813, 758, 717, 695. MS (ESI)
N-{5-[1-(5-Acetylamino-[1,3,4]thiadiazol-2-yl)-piperidin-4-
yloxy]-[1,3,4]thiadiazol-2-yl}-acetamide (14i)
Prepared as described in general procedure 3.The product was
25 7 3 2
m/z for C25H N O S calculated: 535.15, observed [M+H]:
obtained as a tan solid after suspension of the crude product in
5
35.9.
3
hot CHCl then cooling and addition of excess of hexanes (70%
1
yield). H NMR (600 MHz, DMSO-d6)  1.85 (m, 2H), 2.11 (s,
2
-Phenyl-N-(5-(3-((5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
3H), 2.13 (s, 3H), 2.15 (m, 2H), 3.37 (m, 2H), 3.67 (m, 2H), 5.14
-1
yl)oxy)piperidin-1-yl)-1,3,4-thiadiazol-2-yl)acetamide (14e)
Prepared as described in general procedure 2. The compound was
obtained as an off-white solid after purification of the crude
(m, 1H), 12.04 (s, 1H), 12.30 (s, 1H). ATR-IR (cm ) 3181,
3099, 2876, 2806, 1686, 1574, 1503, 1448, 1368, 1308, 1267,
1249, 1113, 1096, 1009, 968, 947, 927, 881, 833, 816, 794, 696,
product via column chromatography and a 0-10% MeOH in
673. MS (ESI) m/z for C13
observed (M-H): 381.9.
17 7 3 2
H N O S calculated: 383.08,
1
CH
2
Cl
2
gradient (57% yield). H NMR (600 MHz, DMSO-d
6
)
1
2
.59-1.66 (m, 1H), 1.81-1.88 (m, 1H), 1.90-1.96 (m, 1H), 1.99-
.06 (m, 1H), 3.36-3.41 (m, 1H), 3.46-3.52 (m, 1H). 3.67-3.81
2-Cyclopropyl-N-(5-(4-((5-(2-cyclopropylacetamido)-1,3,4-
thiadiazol-2-yl)oxy)piperidin-1-yl)-1,3,4-thiadiazol-2-
yl)acetamide (14j).
(m, 2H), 3.71 (s, 2H), 3.75 (s, 2H), 5.03-5.09 (m, 1H), 7.23-7.35
-1
(m, 10H), 12.30 (s, 1H), 12.60 (s, 1H). ATR IR (cm ) 3177,

Prepared as described in general procedure 8 from 13d via
treatment with 4 eq of cyclopropyl acetic acid and EDCI (4 eq) as
coupling agent. The compound was obtained as an off-
1503, 1477, 1453, 1425, 1358, 1328, 1308, 1284, 1262, 1229,
1151, 1130, 1060, 1017, 895, 866, 830, 818, 769, 724, 693. MS
26 4 4
(ESI) m/z for C20H N O S calculated: 418.17, observed [M+H]:
white/beige solid after trituration of the crude product with
419.1.
1
MeOH, hexanes and then hot CHCl
3
(51% yield). H NMR (600
MHz, DMSO-d6)  0.18 (broad d, J=4.2 Hz, 4H), 0.47 (broad d,
J=3.6 Hz, 4H), 1.02 (m, 2H), 1.86 (m, 2H), 2.15 (m, 2H), 2.29
N-(5-((1-(5-amino-1, 3, 4-thiadiazol-2-yl) azetidin-3-yl) oxy)-1,
3, 4-thiadiazol-2-yl)-2-phenylacetamide (16a)
(
d, J=7.2 Hz, 2H), 2.31 (d, J=6.6 Hz, 2H), 3.39 (m 2H), 3.68 (m,
Prepared according to the general procedure 6. The compound
was obtained as a tan solid after purification of the crude product
-1
2
3
1
1
H), 5.15 (m, 1H), 11.98 (s, 1H), 12.25 (s, 1H). ATR IR (cm )
171, 3080, 2998, 2929, 2905, 2854, 2759, 1685, 1678, 1561,
498, 1458, 1450, 1386, 1375, 1324, 1263, 1250, 1208, 1187,
115, 1018, 951, 926, 901, 875, 830, 805, 776, 728, 691. MS
via column chromatography with a 0-20% MeOH in CH
2
Cl
2
1
gradient (25% yield). H NMR (600 MHz, DMSO-d
6
) δ 3.77 (s,
2H), 4.01 (dd, J=9.6, 3.6 Hz, 2H), 4.34 (dd, J=9.0, 6.6 Hz, 2H),
(
4
ESI) m/z for C19
64.0.
25
H N
7
O
S
3 2
calculated: 463.15, observed [M+H]:
5.52-5.54 (m, 1H), 6.56 (s, 2H), 7.25-7.35 (m, 5H), 12.66 (s, 1H).
-1
ATR IR cm 3408, 3277, 3137, 3089, 2919, 2868, 1686, 1567,
493, 1457, 1350, 1308, 1264, 1155, 1103, 1047, 975, 951, 881,
1
tert-Butyl
yl)oxy)azetidine-1-carboxylate (15a)
3-((5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
828, 748, 725, 692. MS (ESI) m/z for C15
389.07, observed [M+H]: 389.8.
15 7 2
H N O S2 calculated:
Prepared according to the general procedure 7. The compound
was obtained as a white solid after purification of the crude
N-(5-{[(3S)-1-(5-amino-1,3,4-thiadiazol-2-yl)pyrrolidin-3-
yl]oxy}-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (16b)
Prepared according to the general procedure 6. The compound
was obtained as a tan solid after purification of the crude product
product via column chromatography and 0-100% EtOAc in
1
hexanes gradient. (45% yield). H NMR (600 MHz, DMSO-d
6
) δ
1
5
.38 (s, 9H), 3.77 (s, 2H), 3.91 (broad s, 2H), 4.25 (broad s, 2H),
.35-5.37 (m, 1H), 7.25-7.34 (m, 5H), 12.65 (s, 1H). ATR IR
2 2
via column chromatography with a 0-10% MeOH in CH Cl
-1
1
(cm ) 3162, 3092, 2971, 2926, 2888, 2815, 1700, 1688, 1565,
gradient (32% yield). H NMR (600 MHz, DMSO-d6)  2.25-
2.33 (m, 1H), 2.35-2.39 (m, 1H), 3.43 (dd, J=9.0, 4.8Hz, 2H),
3.58 (d, J=12.0 Hz, 1H), 3.71 (dd, J=12.0, 4.8 Hz, 1H), 3.76 (s,
2H), 5.53-5.56 (m, 1H), 6.38 (s, 2H), 7.25-7.36 (m, 5H), 12.60 (s,
1504, 1480, 1453, 1392, 1349, 1308, 1273, 1176, 1137, 1089,
1022, 1005, 972, 948, 872, 857, 839, 813, 766, 723, 693. MS
(
[
ESI) m/z for C18
M+Na]: 413.0.
22 4 4
H N O S calculated: 390.14, observed
-
1
1H). ATR IR (cm ) 3400, 3263, 3157, 2926, 2847, 2653 1664,
562, 1494, 1470, 1365, 1351, 1331, 1313, 1284, 1252, 1217,
1190, 1157, 1096, 1078, 1055,1031,948, 916, 850, 790, 752,
1
tert-Butyl (3S)-3-{[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
yl]oxy}pyrrolidine-1-carboxylate (15b)
726, 713, 692. MS (ESI) m/z calculated for C16
17 7 2 2
H N O S :
Prepared according to the general procedure 7. The compound
was obtained as a white solid after purification of the crude
product via column chromatography with a 0-100% EtOAc in
403.09, observed [M+H]: 404.1.
N-(5-{[(3R)-1-(5-amino-1,3,4-thiadiazol-2-yl)pyrrolidin-3-
yl]oxy}-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (16c)
1
hexanes gradient. (76% yield). H NMR (600 MHz, DMSO-d6)

3
2
1
2
1
7
1.37-1.40 (two overlapping singlets, 9H), 2.12-2.21 (m, 2H),
.20-3.30 (m, 1H), 3.41-3.45 (m, 1H), 3.49-3.61 (m, 2H), 3.76 (s,
H), 5.43 (broad s,1H), 7.24-7.27 (m, 1H), 7.29-7.34 (m, 4H),
2.59 (s, 1H). ATR IR (cm ) 3168, 3093, 3060, 2974, 2928,
887, 2814, 2762, 2717, 1689, 1570, 1502, 1458, 1410, 1362,
328, 1306, 1262, 1174, 1148, 1120, 960, 885, 850, 771, 749,
Prepared according to the general procedure 6. The compound
was obtained as an off-white solid after purification of the crude
product via column chromatography with a 0-10% MeOH in
-1
1
CH Cl
2 2
gradient (40% yield). H NMR (600 MHz, DMSO-d
6
) δ
2.25-2.31 (m, 1H), 2.32-2.39 (m, 1H), 3.43 (dd, J= 9.0, 4.8 Hz,
2H), 3.58 (d, J=12.0 Hz, 1H), 3.71 (dd, J=12.0, 4.8 Hz, 1H), 3.76
(s, 2H), 5.52-5.57 (m, 1H), 6.37 (s, 2H), 7.22-7.35 (m, 5H), 12.60
24 4 4
25, 694. MS (ESI) m/z calculated for C19H N O S: 404.15,
-1
observed [M-H]: 402.9.
(s, 1H). ATR IR (cm ) 3397, 3271, 3160, 2918, 2848, 2650,
664, 1564, 1504, 1470, 1366, 1351, 1331, 1313, 1284, 1252,
1218, 1190, 1159, 1096, 1078, 1052, 1031, 949, 917, 850, 790,
1
tert-Butyl (3R)-3-{[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
yl]oxy}pyrrolidine-1-carboxylate (15c)
Prepared according to the general procedure 7. The compound
was obtained as an off-white solid after purification of the crude
752, 727, 714, 692. MS (ESI) m/z for C16
17 7 2 2
H N O S calculated:
403.09, observed [M-H]: 404.1.
product via column chromatography with a 0-100% EtOAc in
N-{5-[1-(5-Amino-[1,3,4]thiadiazol-2-yl)-piperidin-4-yloxy]-
[1,3,4]thiadiazol-2-yl}-2-phenyl-acetamide (16d)
1
hexanes gradient (71% yield). H NMR (600 MHz, CDCl
3
) δ
1
2
.46 (two overlapping singlets, 9H), 2.10-2.19 (brd m, 1H), 2.28-
.45 (brd m, 1H) 3.46-3.82 (cluster of m, 4H), 3.98 (s, 2H), 5.52
Prepared according to the general procedure 6. The compound
was obtained as a white solid after trituration of the crude product
with MeOH, and then boiling hexanes (44% yield). H NMR
1
(
(
s, 1H) 7.31 (distorted triplet, 1H), 7.36 (t, J=7.2, 2H), 7.43
distorted d, J=7.8, 2H), 12.05 (brd d, 1H). MS (ESI) m/z for
(600 MHz, DMSO-d6)  1.81 (m, 2H), 2.12 (m, 2H), 3.22 (m,
C
19 24
H N
4
O
4
S calculated: 404.15, observed [M-H]: 403.0.
2H), 3.51 (m, 2H), 3.76 (s, 2H), 5.10 (m, 1H), 6.49 (s, 2H), 7.24-
-
1
7
.36 (m, 5H), 12.55 (s, 1H). ATR IR (cm ) 3421, 3266, 3141,
4
1
-(5-Phenylacetylamino-[1,3,4]thiadiazol-2-yloxy)-piperidine-
-carboxylic acid tert-butyl ester (15d)
2959, 2834, 2747, 1679, 1591, 1558, 1508, 1497, 1463, 1367,
1357, 1299, 1253, 1226, 1194, 1116, 1016, 954, 909, 845, 797,
Prepared according to the general procedure 7. The compound
was obtained as a white solid after purification of the crude
781, 756, 708, 694, 687. MS (ESI) m/z for C17
calculated: 417.10, observed [M-H]: 416.0.
19 7 2 2
H N O S
product via column chromatography with a 0-70% EtOAc in
1
hexanes gradient (73% yield). H NMR (600 MHz, CDCl
1
3
) 
tert-butyl
yl)amino)azetidine-1-carboxylate (18a)
A mixture of 1-N-Boc-3-aminoazetidine (520 mg, 3.02 mmol), 2-
amino-5-bromothiadiazole (650 mg, 3.61 mmol) and NaHCO
(380 mg, 4.53 mmol) in EtOH (15 mL) in a sealed vessel was
3-((5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
.44 (s, 9H), 1.83 (broad m, 2H), 2.01 (broad m, 2H), 3.27 (ddd,
J=13.8, 10.2, 5.4 Hz, 2H), 3.67 (broad m, 2H), 3.90 (s, 2H), 5.14
m, 1H), 7.30 (m, 3H), 7.40 (d, J=7.2 Hz, 2H). 11.86 (brd s, 1H)
(
3
-1
ATR IR (cm ) 2967, 2950, 2914, 2882, 2799, 2729, 1684, 1567,

o
-1
heated at 80 C until consumption of the limiting reagent was
(m, 5H), 7.99 (d, J=6.6 Hz, 1H), 12.26 (s, 1H). ATR IR (cm )
indicated by TLC. The mixture was then cooled and partitioned
between EtOAC and water. The water layer was extracted twice
more with EtOAc and the combined organic layer was
evaporated to a residue that was chromatographed on a silica gel
column with 100% EtOAC to afford a solid that without further
characterization was dissolved in DMF (10 mL). This mixture
3413, 3357, 3261, 3078, 3062, 2920, 2851, 1666, 1622, 1576,
1351, 1492, 1462, 1311, 1293, 1258, 1180, 1134, 1028, 966, 917,
868, 827, 758, 721, 694. MS (ESI) m/z for C15
calculated: 388.09, observed [M+H]:388.9.
16 8 2
H N OS
was treated with Et
3
N (0.84 mL, 6.04 mmol) and phenylacetyl
N-(5-{[1-(5-Amino-1,3,4-thiadiazol-2-yl)piperidin-4-
chloride (560 mg, 3.63 mmol) and heated in a sealed vessel at 80
C for 48h. The reaction mixture was then cooled and partitioned
yl]amino}-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (16f)
Prepared according to the general procedure 6. The compound
o
between CH
twice more with CH
evaporated. The residue was chromatographed on a silica gel
2
Cl
2
and water. The aqueous layer was extracted
Cl and the combined organic layer
was obtained as an orange solid after purification of the crude
2
2
product via column chromatography and a 0-15% MeOH in
1
CH Cl
2 2
gradient (57% yield). H NMR (600 MHz, DMSO-d6) δ
column with 0-100% EtOAc in hexanes to afford the
1.45-1.53 (m, 2H), 1.98-2.03 (m, 2H), 3.02-3.09 (m, 2H), 3.54-
3.60 (m, 2H), 3.69-3.75 (singlet overlapping with m, 3H), 6.46
(s, 2H), 7.23-7.37 (m, 6H), 12.17 (s, 1H). ATR IR cm 3306,
3240, 3140, 3050, 3027, 2934, 2914, 2843, 2739, 2693, 1649,
1567, 1527, 1489, 1467, 1441, 1386, 1317, 1244, 1218, 1195,
1124, 1098, 1046, 1018, 971, 922, 884, 843, 814, 789, 756, 721,
intermediate
tert-butyl
3-((5-(2-phenylacetamido)-1,3,4-
-
1
thiadiazol-2-yl)amino)azetidine-1-carboxylate (18a) as a grey
1
solid (154 mg, 0.39 mmol, 13% yield). H NMR (600 MHz,
CDCl
3
)  1.42 (s, 9H), 3.83 (dd, J=9.0, 4.8 Hz, 2H), 3.92 (s,
H), 4.27 (dd, J=9.0, 7.8 Hz, 2H), 4.45 (bm, 1H), 5.45 (bs, 1H),
.26-7.36 (m, 3H), 7.42 (apparent d, J=7.8 Hz, 2H), 12.23 (broad
2
7
693.
20 8 2
MS (ESI) m/z for C17H N OS calculated: 416.12,
s, 1H).
observed [M+H]: 417.8.
tert-Butyl
4-{[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
2-Cyclopropyl-N-[5-(4-{[5-(2-phenylacetamido)-1,3,4-
thiadiazol-2-yl]amino}piperidin-1-yl)-1,3,4-thiadiazol-2-
yl]acetamide (16g)
yl]amino}piperidine-1-carboxylate (18b)
A solution of 4-amino-1-N-Boc-piperidine (200 mg, 1.00 mmol,)
in EtOH (11 mL) was treated with NaHCO
mmol,) and 2-amino-5-bromothiadiazole (180 mg, 1.00 mmol)
and the resulting mixture was stirred in a sealed vessel at 80
until consumption of the starting materials was indicated by TLC.
The mixture was then cooled and evaporated to dryness. The
residue was treated with water to yield a suspension that was then
filtered. The yellow solid obtained from this operation was
washed with water, dried and chromatographed with column and
3
(336 mg, 4.00
Prepared according to general procedure 8 from 16f by treatment
with 1 eq of cyclopropyl acetic acid, DIEA (3 eq) and HATU
(1.5 eq). The compound was obtained as an off-white solid after
ꢀC
purification of the crude product via column chromatography
1
with a 0-15% MeOH in CH
Cl
2 2
gradient (30%). H NMR (600
MHz, DMSO-d6) δ 0.15-0.19 (m, 2H), 0.44-0.48 (m, 2H), 0.99-
1.05 (m, 1H), 1.48-1.55 (m, 2H), 2.02-2.08 (m, 2H), 2.28 (d,
J=6.6Hz, 2H), 3.15-3.23 (m, 2H), 3.70 (s, 2H), 3.73-3.79 (m,
-
0
-15% MeOH in CH
Cl
2 2
gradient to afford the intermediate
3H), 7.23-7.37 (m, 6H), 11.95 (s, 1H), 12.18 (s, 1H). ATR IR cm
1
thiadiazole addition product as a pale-yellow solid (135mg, 45%
3330, 2950, 2925, 2837, 2769, 1671, 1561, 1507, 1460, 1380,
1
yield). H NMR (600 MHz, DMSO-d6) δ 1.21-1.29 (m, 2H),
1325, 1288, 1255, 1190, 1126, 1104, 967, 927, 831, 752, 695.
1
3
1
.39 (s, 9H), 1.87-1.92 (m, 2H), 2.81-2.98 (broad s, 2H), 3.51-
.58 (m, 1H), 3.76-3.83 (m, 2H), 6.24 (s, 2H), 6.75 (d, J=7.2 Hz,
H). This addition product (118 mg, 0.39 mmol) was dissolved
26 8 2 2
MS (ESI) m/z for C22H N O S calculated: 498.16, observed
[M+H]: 499.6.
in anhydrous DMF (1.5 ml) and then treated with phenylacetic
acid (59 mg, 0.43 mmol,), DIEA (0.21 mL, 1.18 mmol) and
HATU (225 mg, 0.59 mmol). This mixture was stirred at room
temperature until consumption of the limiting reagent was
complete and evaporated to a residue to which water was added.
The suspension formed was filtered to afford a reddish-yellow
solid that was dried and chromatographed with column and 0-
4-(6-Chloro-pyridazin-3-yloxy)-piperidine-1-carboxylic acid
tert-butyl ester (21)
Prepared according to general procedure 9 from 1-NBoc-4-
hydroxypiperidine and 3, 6‐dichloropyridazine using THF/
DMSO (4/1) as solvent. The compound was obtained after
purification of the crude product with silica gel column and 0-
1
100% EtOAc in hexanes as a white solid (73 % yield). H NMR
1
00% EtOAc in hexanes gradient as eluent to give the product,
(600 MHz DMSO-d6) δ 1.41 (s, 9H), 1.58-1.63 (m, 2H), 1.98-
2.02 (m, 2H), 3.15-3.26 (m, 2H), 3.67-3.71 (m, 2H), 5.31-5.34
(m, 1H), 7.32 (d, J=9.6 Hz, 1H), 7.80 (d, J=9.6 Hz, 1H). ATR
tert-butyl 4-{[5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl]
amino}piperidine-1-carboxylate (18b), as a pale-white solid (101
mg, 62% yield). H NMR (600 MHz, DMSO-d6) δ 1.23-1.29
1
-1
IR (cm ) 3051, 3004, 2967, 2927, 2876, 1672, 1581, 1480, 1421,
(m, 2H), 1.39 (s, 9H), 1.90-1.95 (m, 2H), 2.89 (broad s, 2H),
1363, 1293, 1274, 1231, 1165, 1127, 1075, 1022, 938, 914, 858,
3
6
2
1
8
.63-3.69 (m, 1H), 3.70 (s, 2H), 3.76-3.84 (m, 2H), 7.23-7.34 (m,
764, 715, 673. MS (ESI) m/z calculated for C14
313.12, observed [M+Na]: 336.0.
3 3
H20ClN O :
-1
H), 12.16 (s, 1H). ATR IR (cm ) 3192, 3109, 3007, 2974,
929, 2849, 1692, 1647, 1581, 1558, 1508, 1452, 1414, 1361,
319, 1297, 1272, 1239, 1176, 1136, 1090, 1028, 1005, 971, 943,
4-[6-(Benzhydrylidene-amino)-pyridazin-3-yloxy]-piperidine-
1-carboxylic acid tert-butyl ester (23)
74, 844, 802, 754, 712. MS (ESI) m/z for
20 27 5 3
C H N O S
calculated: 417.18, observed [M+H]: 418.9.
Prepared according to general procedure 10 using 21 as starting
material. The compound was obtained as a yellow solid after
chromatography of the crude product with column and 0-100%
N-(5-{[1-(5-amino-1,3,4-thiadiazol-2-yl)azetidin-3-yl]amino}-
,3,4-thiadiazol-2-yl)-2-phenylacetamide (16e)
1
1
EtOAc in hexanes (74% yield). H NMR (600 MHz DMSO-d6) δ
Prepared according to the general procedure 6. The compound
was obtained after purification of the crude product via column
1.39 (s, 9H), 1.49-1.52 (m, 2H), 1.92-1.96 (m, 2H), 3.10-3.12 (m,
2H), 3.68-3.70 (m, 2H), 5.19-5.20 (m, 1H), 7.02 (d, J=9.0 Hz,
1H), 7.10 (d, J=9.0 Hz, 1H), 7.14-7.18 (m, 2H), 7.34-7.38 (m,
3H), 7.51 (t, J=7.2 Hz, 2H), 7.59 (t, J=7.2 Hz, 1H) 7.70 (d,
J=7.8Hz, 2H). ATR IR (cm ) 3058, 2976, 2961, 2935, 2850,
1686, 1624, 1597, 1578, 1476, 1425, 1363, 1331, 1293, 1269,
chromatography with 0-10% MeOH in CH
trituration/suspension in hot CH Cl as an off-white solid. H
NMR (600 MHz, DMSO-d6)  3.71 (s, 2H), 3.75 (m, 2H), 4.20
apparent t, J=7.8 Hz, 2H), 4.57 (m, 1H), 6.52 (s, 2H), 7.22-7.40
2
Cl
2
and
1
2
2
-1
(

1
237, 1163, 1132, 1091, 1027, 957, 842, 818, 773, 697, 658. MS
835, 766, 745, 670. MS (ESI) m/z for C
213.07, observed [M+H]: 214.0.
9
H
12ClN
3
O calculated:
(ESI) m/z calculated for C27 : 458.23, observed [M+H]:
30 4 3
H N O
459.3
3
-Chloro-6-{4-[(6-chloropyridazin-3-yl)oxy]piperidin-1-
yl}pyridazine (28)
Prepared as per the general procedure
4
-(6-Phenylacetylamino-pyridazin-3-yloxy)-piperidine-1-
9
from 3,6-
carboxylic acid tert-butyl ester (24)
dichloropyridazine and 27 using THF as the reaction solvent. The
compound was obtained as a white solid after purification of the
crude product via column chromatography and a 0-100% EtOAc
Prepared according to general procedure 11 from 23. The
purified aromatic amine intermediate from the first step was
obtained after column chromatography with a 0-10% MeOH in
1
in hexanes gradient (82% yield). H NMR (600 MHz, DMSO-
2 2
CH Cl gradient. The final product (compound 24) was obtained
as a white solid after column chromatography of the crude
d6)  1.76 (m, 2H), 2.14 (m, 2H), 3.50 (ddd, J=13.2, 9.0, 3.6 Hz,
2H), 4.04 (m, 2H), 5.45 (apparent sep, J=4.2 Hz, 1 H), 7.34 (d,
J=9.6 Hz, 1H), 7.46 (d, J=10.2 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H),
7.82 (d, J=9.0 Hz, 1H). ATR IR (cm ) 3051, 2973, 2958, 2938,
2920, 2858, 1583, 1529, 1440, 1415, 1369, 1330, 1301, 1246,
1236, 1188, 1167, 1150, 1121, 1082, 1047, 1030, 972, 946, 919,
acylation product from the second step with a 0-5% MeOH in
1
CH Cl
2 2
gradient (68% yield). H NMR (600 MHz DMSO-d6) δ
-1
1.41 (s, 9H) 1.55-1.62 (m, 2H) 1.98-2.03 (m, 2H) 3.21 (broad s,
2H) 3.64-3.73 (m, 2H) 3.74 (s, 2H), 5.27-5.33 (m, 1H), 7.19 (d,
J=9.6Hz, 1H), 7.23-7.45 (m, 5H), 8.20 (d, J=9.6Hz, 1H), 11.12
-1
(
s, 1H). ATR IR (cm ) 3191, 3130, 3061, 3006, 2977, 2962,
848, 827, 764, 700, 675. MS (ESI) m/z for C13
2 5
H13Cl N O
2
1
8
931, 2861, 1687, 1609, 1568, 1522, 1477, 1423, 1379, 1364,
344, 1302, 1268, 1235, 1165, 1134, 1103, 1074, 1022, 970, 936,
64, 844, 814, 768, 713, 695. MS (ESI) m/z calculated for
calculated: 325.05, observed [M+H]: 325.9.
N-(diphenylmethylidene)-6-[4-({6-[(diphenylmethylidene)
amino]pyridazin-3-yl}oxy)piperidin-1-yl]pyridazin-3-amine
30)
22 28 4 4
C H N O : 412.21, observed [M+H]: 413.3.
(
N-{6-[1-(5-Amino-[1,3,4]thiadiazol-2-yl)-piperidin-4-yloxy]-
pyridazin-3-yl}-2-phenyl-acetamide (25)
Prepared according to general procedure 6. The compound was
Prepared from 28 according to general procedure 10. The
compound was obtained as an off-white solid after purification of
the crude product via column chromatography with a 0-100%
obtained as a tan solid from the crude product via column
EtOAc in CH precipitation of the
2
Cl
2
gradient and
a
chromatography with a 0-20% MeOH in CH
yield). H NMR (600 MHz DMSO-d6) δ 1.70-1.82 (m, 2H) 2.05-
2
Cl
2
gradient (85%
chromatographed product out of CH Cl with excess of hexanes
2 2
1
1
(30% yield). H NMR (600 MHz, DMSO-d6)  1.62 (m, 2H),
2.04 (m, 2H), 3.25 (apparent t, J=9.6H, 2H), 3.95 (m, 2H), 5.26
(broad m, 1H), 6.88 (d, J=9.6 Hz, 1H), 7.04 (d, J=9.0 Hz, 1H),
7.12 (d, J=9.0 Hz, 1H), 7.16 (m, 4H), 7.21 (d, J=9.6 Hz, 1H),
7.36 (m, 6H), 7.52 (m, 4H), 7.60 (m, 2H), 7.71(dd, J=13.2, 7.2
2
5
.13 (m, 2H), 3.20-3.30 (m,2H) 3.52-3.56 (m, 2H), 3.74 (s, 2H),
.32-5.37 (m, 1H), 6.48 (s,2H), 7.21-7.38 (m, 6H), 8.21
-1
(d,J=10.2Hz, 1H) 11.13 (s, 1H). ATR IR (cm ) 3452, 3306,
3
1
1
8
231, 3166, 3131, 3063, 3018, 2992, 2959, 2896, 2822, 2992,
695, 1593, 1566, 1521, 1485, 1464, 1435, 1380, 1339, 1306,
266, 1255, 1223, 1193, 1142, 1119, 1101, 1032, 963, 942, 895,
57, 813, 770, 756, 722, 697, 668. MS (ESI) m/z calculated for
-1
Hz, 4H). ATR IR (cm ) 3062, 2987, 2963, 2940, 2917, 1625,
1594, 1571, 1534, 1475, 1444, 1421, 1308, 1297, 1250, 1231,
1176, 1155, 1122, 1097, 1074, 1018, 995, 958, 906, 836, 780,
C
19 21
H N
7
O
2
S: 411.15, observed [M+H]: 412.3.
763, 734, 695, 650. MS (ESI) m/z for C39
15.27, observed [M+H]: 616.2.
33 7
H N O calculated:
6
2
-Phenyl-N-{6-[1-(5-phenylacetylamino-[1,3,4]thiadiazol-2-
yl)-piperidin-4-yloxy]-pyridazin-3-yl}-acetamide (26a)
Prepared according to general procedure 8 from 25 by treatment
2-Phenyl-N-{6-[1-(6-phenylacetylamino-pyridazin-3-yl)-
piperidin-4-yloxy]-pyridazin-3-yl}-acetamide (26b)
with phenylacetic acid (1.3 eq), Et
3
N (4 eq) and HATU (1.2 eq.)
Prepared from 30 according to general procedure 11. The
purified aromatic diamine intermediate from the first step was
obtained after column chromatography with 0-15% MeOH in
The compound was obtained as an off-white solid after
suspension/trituration of the crude product with water (42 %
1
yield). H NMR (600 MHz DMSO-d6) δ 1.75-1.83 (m, 2H),
2 2
CH Cl gradient. The final product (compound 26b) was
2
2
7
1
2
1
7
5
.09-2.13 (m, 2H), 3.35-3.42 (m, 2H), 3.63-3.68 (m, 2H), 3.72 (s,
H), 3.73 (s, 2H), 5.34-5.37 (m, 1H), 7.19-7.25 (m, 3H), 7.28-
.35 (m, 8H), 8.19 (d, J=9.6 Hz, 1H), 11.12 (s, 1H), 12.29 (s,
H). ATR IR (cm ) 3364, 3179, 3088, 3063, 3029, 2976, 2955,
930, 2838, 2798, 2838, 2798, 2746, 1688, 1580, 1499, 1437,
385, 1350, 1320, 1296, 1257, 1181, 1129, 1100, 1033, 962, 861,
obtained as a white solid after purification of the crude acylation
product from the second step via column chromatography with a
1
0-100% EtOAc in CH
Cl
2 2
gradient. H NMR (600 MHz,
-1
DMSO-d6)  1.72 (m, 2H), 2.11 (m, 2H), 3.40 (apparent t,
J=10.2 Hz, 2H), 3.71 (s, 2H), 3.75 (s, 2H), 3.97 (m, 2H), 5.39
(m, 1H), 7.21 (d, J=9.6 Hz, 1H) 7.24 (m, 2H), 7.34 (m, 8H), 7.39
(d, J=9.6 Hz, 1H), 8.01 (d, J=9.6 Hz, 1H), 8.20 (d, J=9.6 Hz,
59, 712, 693. MS (ESI) m/z calculated for C27
29.19, observed [M+H]: 530.1.
27 7 3
H N O S:
-
1
1H), 10.92 (s, 1H), 11.13 (s, 1H). ATR IR (cm ) 3348, 3221,
3
192, 3102, 3059, 3028, 2949, 2919, 2839, 1685, 1509, 1421,
1
-(6-Chloro-pyridazin-3-yl)-piperidin-4-ol (27)
1356, 1313, 1295, 1258, 1234, 1227, 1137, 1119, 1095, 1020,
997, 951, 907, 864, 834, 813, 773, 731, 713, 692, 654. MS (ESI)
m/z for C H N O calculated: 523.23, observed [M+H]: 524.1.
Prepared according to general procedure 12 from 3,6-
dichloropyridazine and 4-hydroxypiperidine using Et N as
3
2
9
29
7
3
aprotic base and DMSO as solvent. The compound was obtained
as a white solid after purification of the crude product via silica
tert‐butyl
gel column chromatography with a 0-100% EtOAc in hexanes
4‐[(5‐bromopyrazin‐2‐yl)oxy]piperidine‐1‐carboxylate (22)
Prepared according to general procedure 9 from 1-NBoc-4-
hydroxypiperidine and 2,5-dibromopyrazine using THF as the
reaction solvent. The compound was obtained as a white solid
after purification of the crude product via column
1
gradient (89% yield). H NMR (600 MHz, CD
3
OD)  1.54 (m,
2
1
1
2
1
H), 1.94 (m, 2H), 3.28 (m, 2H), 3.89 (apparent sep, J= 4.2 Hz,
H), 4.11 (apparent dt, J=13.2, 4.8 Hz, 2H), 7.32 (d, J=9.6 Hz,
H), 7.4 (d, J=9.6 Hz, 1H). ATR IR (cm ) 3199, 3077, 3008,
991, 2941, 2847, 1583, 1530, 1429, 1362, 1312, 1298, 1257,
237, 1224, 1184, 1167, 1148, 1107, 1061, 1032, 1012, 981, 921,
-1
chromatography with a 0-30% EtOAc in hexanes gradient. (74%
1
yield). H NMR (600 MHz, CDCl ) δ 1.73 (broad m, 2H), 1.96
3
(broad m, 2H), 3.28 (m, 2H), 3.76 (broad m, 2H), 5.13 (m, 1H),

-
1
7
3
1
1
6
.98 (d, J=0.6 Hz, 1H), 8.15 (d, J=0.6 Hz, 1H). ATR IR (cm )
001, 2976, 2933, 2858, 1687, 1560, 1525, 1476, 1452, 1437,
413, 1363, 1330, 1300, 1265, 1234, 1189, 1160, 1129, 1106,
086, 1029, 1008, 970, 943, 920, 893, 858, 816, 771, 759, 694,
1550, 1510, 1454, 1408, 1376, 1354, 1268, 1251, 1224, 1164,
1122, 1064, 1041, 1020, 965, 928, 886, 846, 767, 710, 692, 662.
MS (ESI) m/z for C29
[M+H]: 523.8
29 7 3
H N O calculated: 523.23, observed
66. MS (ESI) m/z calculated for C14
H20BrN
3
O
3
:
357.07,
observed [M+H]: 357.8.
1-(6-Nitropyridin-3-yl)piperidin-4-ol (34)
Prepared according to general procedure 12 from 2-nitro-5-
2
‐Bromo‐5‐{4‐[(5‐bromopyrazin‐2‐yl)oxy]piperidin‐1‐
yl}pyrazine (29)
A solution of tert‐butyl 4‐[(5‐bromopyrazin‐2‐yl)oxy]piperidine-
‐carboxylate (22) (540 mg, 1.51 mmol) in dioxane (3 mL) was
chloropyridine and 4 hydroxypiperidine using K CO as the
2 3
o
aprotic base, DMSO as solvent and 90 C as the reaction
temperature. The compound was obtained as a yellow solid upon
purification of the crude product via column chromatography
1
1
treated with 4N HCl in dioxane (1 mL) and the mixture was
with a 30-100% EtOAc in hexanes gradient (65% yield). H
stirred at rt for 6 hr. The solvent was then evaporated to a white
NMR (600 MHz, DMSO-d6)  1.42 (m, 2H), 1.81 (m, 2H), 3.24
(ddd, J= 13.2, 9.6, 3.0 Hz, 2H), 3.75 (m, 1H), 3.83 (m, 2H), 4.78
(d, J=4.2 Hz, 1H), 7.45 (dd, J=9.0, 3.0 Hz, 1H), 8.11 (d, J=9.0
Hz, 1H), 8.23 (d, J=3.0 Hz, 1H). ATR IR (cm ) 3393, 3321,
3107, 3088, 2947, 2930, 2855, 1569, 1507, 1490, 1420, 1382,
1324, 1270, 1260, 1219, 1173, 1101, 1072, 1016, 1003, 962, 901,
residue which was suspended in CH
mL) of hexanes. Followed filtration of the solids, wash again
X2) with hexanes and drying to afford the intermediate
dihydrochloride salt. This salt without delay was added to a 1:1
mixture of THF/DMSO (12 mL). Followed addition of Et
2 2
Cl (2 mL) and excess (10
-1
(
3
N
(
5
0.84 mL, 6.02 mmol), 2,5-dibromopyrazine and then stirring at
0 C for 24 h. The solvents were then evaporated and the residue
824, 745, 692, 664. MS (ESI) m/z for C10
223.10, observed [M+H]: 224.1.
13 3 3
H N O calculated:
o
was partitioned between EtOAc and water. The water layer was
extracted with EtOAc once more and the combined organic layer
was evaporated. The residue was chromatographed with a
column and 0-30% EtOAc in hexanes gradient as elution system
to afford the product 2‐bromo‐5‐{4‐[(5‐bromopyrazin‐2‐yl)-
2‐Nitro‐5‐{4‐[(6‐nitropyridin‐3‐yl)oxy]piperidin‐1‐yl}pyridine
(35)
A solution of 1-(6-nitropyridin-3-yl)piperidin-4-ol (34) (110 mg,
0.49 mmol), 2-nitro-5-hydroxypyridine (69 mg, 0.49 mmol) and
Ph P (136 mg, 0.52 mmol) in THF (7 mL) was treated with
3
diisopropyl azodicarboxylate, (0.10 mL, 0.52 mmol) and stirred
oxy]piperidin‐1‐yl}pyrazine as an off-white solid (110 mg, 18%
1
yield) H NMR (600 MHz, CDCl
3
)  1.88 (m, 2H), 2.10 (m, 2H),
3
1
8
1
1
8
.51 (ddd, J=12.6, 7.8, 3.6 Hz, 2H), 3.91 (m, 2H), 5.25 (m, J=
0.8, 7.2, 3.6 Hz, 1H), 7.92 (s, 1H), 8.01 (s, 1H), 8.14 (s, 1H),
.17 (s, 1H). ATR IR (cm ) 3064, 2985, 2945, 2841, 1554, 1519,
505, 1473, 1450, 1437, 1392, 1334, 1313, 1278, 1245, 1234,
187, 1160, 1122, 1101, 1056, 1028, 1009, 996, 970, 933, 890,
at room temperature for 4 h. The mixture was then partitioned
2 2
between CH Cl and water. The organic layer was evaporated and
-
1
the residue was chromatographed with a silica gel column and 0-
1
2 2
20% acetone in CH Cl gradient to afford the product. H NMR
(600 MHz, DMSO-d6)  1.78 (dddd, J=16.2, 12.0, 7.8, 3.6 Hz,
2H), 2.14 (m, 2H), 3.48 (ddd, J= 12.6, 9.0, 3.6 Hz, 2H), 3.89 (m,
2H), 5.01 (ddd, J= 12.0, 7.8, 4.2 Hz, 1H), 7.55 (dd, J= 9.0, 3.0
Hz, 1H), 7.85 (dd, J= 9.0, 3.0 Hz, 1H), 8.17 (d, J=9.0 Hz, 1H),
8.32 (d, J= 3.0 Hz, 1H), 8.35 (d, J= 9.0 Hz, 1H), 8.38 (d, J= 3.0
2 5
66, 772, 755, 694, 667. MS (ESI) m/z for C13H13Br N O
calculated: 414.95, observed [M+H]: 415.9
N‐(Diphenylmethylidene)‐5‐[4‐({5‐[(diphenylmethylidene)ami
no]pyrazin‐2‐yl}oxy)piperidin‐1‐yl]pyrazin‐2‐amine (31)
Prepared from 29 according to the general procedure 10. The
compound was obtained as a yellow solid after purification of the
15 5 5
Hz, 1H) MS (ESI) m/z for C15H N O calculated: 345.11,
observed [M+H]: 346.0.
crude product via column chromatography with a 0-100%
5-{4-[(6-Aminopyridin-3-yl)oxy]piperidin-1-yl}pyridin-2-
amine (36)
2-nitro-5-{4-[(6-nitropyridin-3-yl)oxy]piperidin-1-yl}pyridine
(35) (210 mg, 0.61 mmol), was dissolved in EtOH (10 mL). To
1
EtOAc in CH
CDCl
H), 3.33 (ddd, J= 12.6, 8.4, 3.0 Hz, 2H), 3.82 (m, 2H), 5.09
ddd, J=11.4, 7.8, 3.6 Hz, 1H), 7.16 (distorted t, 4H), 7.31 (m,
H), 7.42 (m, 5H), 7.48 (m, 3H), 7.55 (d, J=0.6 Hz, 1H), 7.78 (t,
2
Cl
2
gradient (34% yield). HNMR (600 MHz,
3
-d6)  1.79 (dddd, J=16.8, 12.6, 8.4, 4.2 Hz, 2H), 2.02 (m,
2
(
this solution was added Fe (680 mg, 12.17 mmol), aq. saturated
o
5
4
NH Cl (4 mL) and the mixture was heated for 15 min at 80 C
-
J=7.8 Hz, 4H), 7.87 (s, 1H), 7.91 (d, J=0.6 Hz, 1H). ATR IR (cm
then cooled and partitioned between EtOAc and water. The water
layer was extracted with EtOAC until no UV absorption could be
detected in the organic extract. The combined organic layer was
1
)
3055, 2957, 2921, 2849, 1619, 1604, 1594, 1568, 1480, 1456,
1
1
7
442, 1391, 1341, 1316, 1297, 1266, 1252, 1236, 1156, 1116,
073, 1059, 1031, 1015, 975, 955, 916, 877, 843, 815, 781, 769,
38, 720, 690. MS (ESI) m/z for C39H N O calculated: 615.27,
33 7
then dried over Na
was chromatographed with a silica gel column and 0-10% MeOH
in CH Cl gradient to afford the product an off-white/tan solid.
45 mg, 32% yield). H NMR (600 MHz, DMSO-d6)  1.68 (m,
2 4
SO , filtered, concentrated and the residue
observed [M]: 615.3
2
2
1
(
2
‐Phenyl‐N‐[5‐({1‐[5‐(2‐phenylacetamido)pyrazin‐2‐
2H), 1.97 (m, 2H), 2.77 (ddd, J= 12.0, 9.6, 3.0 Hz, 2H), 3.22 (m,
2H), 4.19 (apparent sep, J= 3.6 Hz, 1H), 5.39 (s, 2H), 5.51 (s,
2H), 6.39 (d, J= 8.4 Hz, 1H), 6.41 (d, J=8.4 Hz, 1H), 7.16 (dd,
J=8.4, 3.0 Hz, 1H), 7.18 (dd, J=9.0, 3.0 Hz, 1H), 7.63 (d, J=3
Hz, 1H), 7.67 (d, J= 3Hz, 1H). ATR IR (cm ) 3414, 3398, 3307,
3167, 3038, 2944, 2817, 1639, 1566, 1489, 1463, 1400, 1372,
1312, 1259, 1232, 1221, 1183, 1162, 1138, 1114, 1062, 1039,
yl]piperidin‐4‐yl}oxy)pyrazin‐2‐yl]acetamide (26c)
Prepared from 31 according to the general procedure 11. The
purified aromatic diamine intermediate, from the first step, was
obtained after column chromatography with 0-20% MeOH in
CH Cl gradient. The final product (compound 26c) was obtained
2 2
as a white solid after purification of the crude acylation product,
-1
from the second step, via column chromatography with a 0-90%
EtOAc in CH
1012, 979, 912, 822, 787, 743, 669.
MS (ESI) m/z for
1
2
Cl
2
gradient. (38%yield). H NMR (400 MHz,
15 19 5
C H N O calculated: 285.16, observed [M+H]: 286.0.
DMSO-d6)  1.67 (m, 2H), 2.04 (m, 2H), 3.34 (m, 2H), 3.68 (s,
H), 3.72 (s, 2H), 3.94 (m, 2H), 5.18 (m, 1H), 7.25 (m, 2H), 7.32
m, 8H), 8.08 (d, J=1.2 Hz, 1H), 8.13 (d, J=1.2 Hz, 1H), 8.76 (s,
H), 8.83 (d, J=1.2 Hz, 1H), 10.53 (s, 1H), 10.77 (s, 1H). ATR
2
(
1
2-Phenyl-N-[5-(4-{[6-(2-phenylacetamido)pyridin-3-
yl]oxy}piperidin-1-yl)pyridin-2-yl]acetamide (26d)
Prepared according to general procedure 8 from 36 using
-1
3
phenylacetic acid (4 eq), Et N (6 eq) and HATU (3 eq.). The
IR (cm ) 3301, 3087, 3062, 3023, 2925, 2850, 2831, 1670, 1588,

compound was obtained as an off-white solid after purification of
Prepared from 38 according to general procedure 13. The
the crude product by trituration with MeOH and then EtOAc
compound was obtained as an off-white solid after purification of
1
(67% yield). H NMR (600 MHz, DMSO-d6)  1.73 (m, 2H),
the crude product via column chromatography and 0-10% MeOH
1
2
2
3
1
.04 (m, 2H), 3.05 (m, 2H), 3.48 (m, 2H), 3.66 (s, 2H), 3.68 (s,
H), 4.56 (m, 1H), 7.24 (m, 2H), 7.33 (m, 8H), 7.41 (dd, J=9.0,
.0 Hz, 1H), 7.48 (dd, J=9.0, 3.0 Hz, 1H), 7.90 (d, J=9.6 Hz,
H), 7.99 (d, J=9.0 Hz, 1H), 8.04 (d, J=3 Hz, 1H), 8.07 (d, J=3
in CH Cl
2 2
gradient as eluent (69% yield). H NMR (400 MHz,
DMSO-d6)  1.68 (m, 2H), 2.02 (m, 2H), 2.80 (ddd, J=12.4, 9.6,
2.8 Hz, 2H), 3.23 (m,2H), 4.72 (broad s, 2H), 4.87 (apparent sep,
J= 4.0 Hz, 1H), 5.37 (broad s, 2H), 6.40 (dd, J=8.8, 0.4 Hz), 6.52
(dd, J=8.8, 0.8 Hz, 1H), 7.00 (dd, J=8.8, 3.2 Hz, 1H), 7.19 (dd,
J=8.8, 2.8 Hz, 1H), 7.50 (dd, J=2.8, 0.4 Hz, 1H), 7.64 (d, J=2.8
-
1
Hz, 1H), 10.46 (s, 1H), 10.59 (s, 1H). ATR IR (cm ) 3392, 3238,
3
1
7
084, 3059, 3026, 2927, 2825, 1666, 1583, 1508, 1493, 1464,
390, 1344, 1297, 1278, 1217, 1139, 1030, 962, 910, 830, 764,
20, 693. MS (ESI) m/z for C31H N O calculated: 521.24,
31 5 3
19 5
Hz, 1H). MS (ESI) m/z for C15H N O calculated: 285.16,
observed [M+H]: 286.1.
observed [M+H]: 522.0.
6
‐{4‐[(5‐aminopyridin‐2‐yl)oxy]piperidin‐1‐
1
-(5-Nitropyridin-2-yl)piperidin-4-ol (37)
Prepared according to general procedure 12 from 2-chloro-5-
nitropyridine and 4-hydroxypiperidine using Et N as the aprotic
base, EtOH as solvent and 70 C as the reaction temperature. The
product was obtained as yellow solid after water
suspension/trituration (80% yield). H NMR (600 MHz, CDCl
1.54 (d, J= 4.2 Hz, 1H) 1.64 (m, 2H), 1.99 (m, 2H), 3.50 (ddd,
yl}pyridin‐3‐amine (41)
Prepared from 39 according to general procedure 13. The
compound was obtained as an off-white solid after purification of
the crude product via column chromatography and 0-5% MeOH
3
o
1
a
in CH Cl gradient as eluent (77% yield) H NMR (600 MHz,
2
2
1
3
)
DMSO-d6)  1.57 (m, 2H), 1.96 (m, 2H), 2.98 (ddd, J=13.2,
10.2, 3 Hz, 2H), 3.74 (m, 2H), 4.55 (broad s, 2H), 4.72 (broad s,
2H), 4.92 (apparent sep, J= 4.2 Hz, 1H), 6.50 (d, J=9 Hz, 1H),
6.65 (d, J=9.0 Hz, 1H), 6.90 (dd, J=9.0, 3.0 Hz, 1H), 6.98 (dd,
J=8.4, 2.4 Hz, 1H), 7.48 (d, J=2.4 Hz, 1H), 7.59 (d, J=3 Hz,
1H). MS (ESI) m/z for C H N O calculated: 285.16, observed

J= 12.6, 8.4, 3.0 Hz, 2H), 4.06 (m, 1H), 4.16 (m, 2H), 6.60 (d,
J= 11.4 Hz, 1H), 8.19 (dd, J=9.6, 3.0 Hz, 1H), 9.03 (d, J=2.4,
-1
1
1
1
7
H). ATR IR (cm ) 3463, 3104, 3084, 2942, 2916, 2873, 1589,
571, 1514, 1478, 1433, 1338, 1328, 1285, 1243, 1213, 1160,
129, 1115, 1067, 1016, 995, 976, 950, 933, 919, 819, 761, 744,
1
5
19
5
[M+H]: 286.1.
13 3 3
22. MS (ESI) m/z for C10H N O calculated: 223.10, observed
[
M+H]:224.1.
2-Phenyl-N-[6-({1-[6-(2-phenylacetamido)pyridin-3-
yl]piperidin-4-yl}oxy)pyridin-3-yl]acetamide (26e)
Prepared from 40 according to the general procedure 8 using
2
-Nitro-5-{4-[(5-nitropyridin-2-yl)oxy]piperidin-1-yl}pyridine
(
38)
phenylacetic acid (2.5 eq), Et N (6 eq) and HATU (2 eq.) The
compound was obtained as a white solid after purification of the
3
Prepared from 37 and 2-chloro-5-nitropyridine according to
general procedure 9 using a mixture of anhydrous THF and
DMSO (5:1) as solvent. The compound was obtained as a yellow
solid after purification of the crude product via column
chromatography with 0-80% EtOAc in CH
yield). H NMR (600 MHz, DMSO-d6)  1.81 (m, 2H), 2.14
crude product via column chromatography and 0-100% EtOAc in
1
hexanes (65% yield). H NMR (600 MHz, DMSO-d6)  1.74
(m, 2H), 2.06 (m, 2H), 3.03 (apparent t, J= 10.2 Hz, 2H), 3.50
(m, 2H), 3.63 (s, 2H), 3.67 (s, 2H), 5.09 (m, 1H), 6.77 (d, J=9.0
Hz, 1H), 7.25 (m, 2H), 7.32 (m, 8H), 7.41 (broad d, J=9.0 Hz,
1H), 7.90 (m, 2H), 8.04 (s, 1H), 8.34 (s, 1H), 10.20 (s, 1H), 10.46
2
Cl
2
gradient (73%
1
(
(
m, 2H), 3.50 (ddd, J= 12.6, 9.0, 3.0 Hz, 2H), 3.90 (m, 2H), 5.44
apparent sep, J= 4.2 Hz 1H), 7.05 (d, J=9.0 Hz, 1H), 7.54 (dd,
-
1
(s, 1H). ATR IR (cm ) 3268, 3030, 2934, 2816, 2794, 1659,
1611, 1578, 1522, 1482, 1452, 1410, 1391, 1360, 1340, 1295,
1279, 1248, 1231, 1185, 1144, 1119, 1049, 1034, 1019, 980, 972,
916, 826, 748, 726, 704, 692, 657. MS (ESI) m/z for C H N O
3
J=9.6, 3.0 Hz, 1H), 8.17 (d, J=7.2 Hz, 1H), 8.31 (d, J=2.4 Hz,
1
IR (cm ) 3058, 2963, 2921, 2854, 1684, 1600, 1569, 1502, 1469,
H), 8.50 (dd, J=9.0, 2.4 Hz, 1H), 9.11 (d, J=3.0 Hz, 1H). ATR
-
1
31
31
5
1
1
6
418, 1399, 1343, 1311, 1283, 1270, 1222, 1182, 1170, 1108,
090, 1021, 1000, 971, 948, 911, 829, 760, 746, 720, 693, 681,
58. MS (ESI) m/z for C15H N O calculated: 345.11, observed
15 5 5
calculated: 521.24, observed [M+H]:521.9.
[M+H]: 345.9.
2-Phenyl-N-[6-(4-{[5-(2-phenylacetamido)pyridin-2-
yl]oxy}piperidin-1-yl)pyridin-3-yl]acetamide (26f)
5
-Nitro-2-{4-[(5-nitropyridin-2-yl)oxy]piperidin-1-yl}pyridine
Prepared from 41 according to general procedure 8 using
(39)
phenylacetic acid (2.5 eq), Et N (6 eq) and HATU (2 eq.). The
3
Prepared from 37 and 2-chloro-5-nitropyridine according to
general procedure 9 using THF as solvent. The compound was
obtained as a yellow solid after purification of the crude product
compound was obtained as an off-white solid after purification of
the crude product by trituration with MeOH and then with
1
EtOAc. H NMR (600 MHz, DMSO-d6) 1.61 (m, 2H), 2.01
via column and 0-100% EtOAc in hexanes gradient as eluent
(m, 2H), 3.23 (m, 2H), 3.60 (s, 2H), 3.63 (s, 2H), 3.93 (m, 2H),
5.15 (m, 1H), 6.76 (d, J=9.0 Hz, 1H), 6.85 (d, J=9.0 Hz, 1H),
7.26 (m, 2H), 7.33 (m, 8H), 7.77 (dd, J=9.0, 2.4 Hz, 1H), 7.89
(dd, J=9.0, 3.0 Hz, 1H), 8.28 (d, J=3.0 Hz, 1H), 8.34 (d, J=2.4
1
(
2
4
47% yield). H NMR (600 MHz, DMSO-d6)  1.74 (m, 2H),
.11 (m, 2H), 3.67 (m, 2H), 4.16 (m, 2H), 5.45 (apparent sep, J=
.2 Hz, 1H), 7.01 (d, J=9.6 Hz, 1H), 7.03 (d, J=9.0 Hz, 1H), 8.21
-
1
(
dd, J=9.6, 3.0 Hz, 1H), 8.48 (dd, J=9.6, 3.0 Hz, 1H), 8.96 (d,
Hz, 1H), 10.01 (s, 1H), 10.20 (s, 1H). ATR IR (cm ) 3214, 3167,
3062, 3028, 2971, 2942, 2844, 1663, 1636, 1600, 1549, 1529,
1484, 1453, 1402, 1387, 1367, 1355, 1303, 1270, 1241, 1225,
1199, 1152, 1116, 1058, 1038, 1015, 983, 967, 930, 908, 844,
817, 767, 735, 710, 692. MS (ESI) m/z for C H N O
3
-1
J=2.4 Hz, 1H), 9.09 (d, J=3.0 Hz, 1H). ATR IR (cm ) 3097,
3
1
1
6
080, 3050, 2967, 2923, 2869, 1594, 1574, 1508, 1471, 1460,
429, 1399, 1342, 1313, 1291, 1268, 1226, 1165, 1139, 1113,
095, 1024, 993, 954, 925, 867, 833, 805, 759, 742, 720, 681,
31
31
5
59. MS (ESI) m/z for C15
15
H N
5
O
5
calculated: 345.11, observed
calculated: 521.24, observed [M+H]: 522.0.
[M+H]: 346.0.
Acknowledgments
5
‐{4‐[(5‐Aminopyridin‐2‐yl)oxy]piperidin‐1‐yl}pyridine‐2‐ami
ne (40)
Funding support was provided by the University of Pittsburgh
Medical Center Competitive Medical Research Fund (CMRF).

CHESS is supported by the NSF & NIH/NIGMS via NSF award
DMR-1332208 and the MacCHESS resource is supported by
NIGMS award GM-103485.
1
6. Gross, M. I.; Demo, S. D.; Dennison, J. B.; Chen, L.; Rogan-
Chernov, T.; Goyal, B.; Janes, J. R.; Laidig, G. J.; Lewis, E.
R.; Li, L.; MacKinnon, A. L.; Parlati, F.; Rodriguez, M. L M.;
Shwonek, P. J.; Sjogren, E. B.; Stanton, T. F.; Wang, T.;
Yang, J.; Zhao, F.; Bennett, M. K. “Antitumor activity of
glutaminase inhibitor CB-839 in triple negative breast cancer”
Mol. Cancer Ther. 2014, 13, 890-901
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29. Crystal structure coordinates have been deposited in the PDB
for the crystals of GAC in complex with compounds 7d

(PDB: 5FI2), 7e (PDB: 5FI6), 14b (PDB: 5FI7) and 14d
(PDB: 5I94).

Graphical Abstract
Design and Evaluation of Novel Glutaminase
Inhibitors
Leave this area blank for abstract info.
a,b
a,b
b
a
a
a
Lee A. McDermott , Prema Iyer, Larry Vernetti, Shawn Rimer, Jingran Sun, Melissa Boby, Tianyi
a
a
a
a
a
c
d
Yang, Michael Fioravanti, Jason O’Neil, Liwei Wang, Dylan Drakes, William Katt, Qingqiu Huang, and
e
Richard Cerione
a
b
University of Pittsburgh, Department of Pharmaceutical Sciences, Pittsburgh PA 15261, USA; University of Pittsburgh Drug Discovery Institute,
c
d
Pittsburgh PA 15261, USA; Cornell University, Department. of Molecular Medicine, Ithaca NY 14853, USA; Cornell University, Laboratory for
Accelerator-based Sciences and Education, Ithaca NY 14853, USA; Cornell Univeristy, Department of Molecular Medicine and Department of Chemistry
and Chemical Biology, Ithaca NY 14583, USA
GAC IC50: 371 nM
LE: 0.26
LiPE: 2.28
GAC IC50: 30 nM
LE: 0.31
LiPE: 4.59
H
S
N
O
O
HN
N
N
N
N
N
N
S
O
S
N
O
N
HN
NH
N
ClogP: 4.15
S
S
H
N
ClogP: 2.93
Lipinski/Veber violations: 2
BPTES
16g
Lipinski/Veber violations: NONE