Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl linker

Article abstract of DOI:10.1016/j.bmc.2020.115698

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl ([sbnd]SCH₂CH₂[sbnd]) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC₅₀ value of 50 nM. Interestingly, the corresponding derivative with an n-propyl ([sbnd]CH₂CH₂CH₂[sbnd]) linker showed substantially lower inhibitory potency (IC₅₀ = 2.3 μM) while the derivative with a dimethylsulfide ([sbnd]CH₂SCH₂[sbnd]) linker showed no inhibitory activity at concentrations up to 100 μM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.

Full text of DOI:10.1016/j.bmc.2020.115698

Journal Pre-proofs
Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl
linker
Bridget Duvall, Sarah C. Zimmermann, Run-Duo Gao, Ajit G. Thomas, Filip
Kalčic, Vijayabhaskar Veeravalli, Amira Elgogary, Rana Rais, Camilo Rojas,
Anne Le, Barbara S. Slusher, Takashi Tsukamoto
PII:
S0968-0896(20)30528-9
https://doi.org/10.1016/j.bmc.2020.115698
BMC 115698
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 April 2020
16 July 2020
30 July 2020
Please cite this article as: B. Duvall, S.C. Zimmermann, R-D. Gao, A.G. Thomas, F. Kalčic, V. Veeravalli, A.
Elgogary, R. Rais, C. Rojas, A. Le, B.S. Slusher, T. Tsukamoto, Allosteric kidney-type glutaminase (GLS)
inhibitors with a mercaptoethyl linker, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.bmc.2020.115698
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Allosteric kidney-type glutaminase (GLS) inhibitors
with a mercaptoethyl linker
Bridget Duvall,† Sarah C. Zimmermann,†,‡ Run-Duo Gao,† Ajit G. Thomas,† Filip Kalčic,†
Vijayabhaskar Veeravalli,†,‡ Amira Elgogary,∥ Rana Rais,†,‡ Camilo Rojas,†,§ Anne Le,∥ Barbara
S. Slusher,†,‡ and Takashi Tsukamoto*,†,‡
†Johns Hopkins Drug Discovery, ‡Department of Neurology, and §Department of Molecular and
Comparative Pathobiology, ∥Department of Pathology, Johns Hopkins University, Baltimore,
MD 21205, USA
CORRESPONDING AUTHOR *Phone: (410) 614-0982. Fax: (410) 614-0659. E-mail:
ttsukamoto@jhmi.edu.

Abstract. A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a
mercaptoethyl (-SCH2CH2-) linker were synthesized in an effort to further expand the structural
diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl
sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl
linker between the two thiadiazole rings was found to potently inhibit GLS with an IC50 value of
50 nM. Interestingly, the corresponding derivative with an n-propyl (-CH2CH2CH2-) linker
showed substantially lower inhibitory potency (IC50 = 2.3 μM) while the derivative with a
dimethylsulfide (-CH2SCH2-) linker showed no inhibitory activity at concentrations up 100 μM,
underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to
the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and
tested as GLS inhibitors to further explore SAR within this scaffold including derivatives
possessing a pyridazine as a replacement for one of the two thiadiazole moiety.

1. Introduction
Given the central importance of glutamine in reprogrammed energy metabolism of malignant
cells, kidney-type glutaminase (GLS) has gained growing interest as a therapeutic target for the
2
treatment of cancer.1, Since the discovery of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-
yl)ethyl sulfide (BPTES, Fig. 1),3 the first reported allosteric inhibitor of GLS,4 extensive
structural optimization efforts have been collectively made by a number of research groups with
a focus towards clinical translation.5-7 This led to the discovery of CB-839 (Fig. 1),8 a first-in-
class GLS inhibitor, currently investigated in clinical trials.
H
N
H
N
S
S
S
N
N
N N
O
O
BPTES (IC50 = 0.19, 0.58 M)
H
N
N
OCF3
N
H
N
S
O
N
N
N
O
CB-839 (IC50 < 0.05 M)*
H
N
H
S
S
N
N
N
N N
O
O
O
O
1
(1S,3S)- (IC50 < 0.1 M)
H
N
H
N
S
S
X
N
N
N N
2a
2b
(X = CH2, IC50 = 0.59 M)
(X = S, IC50 > 20 M )
H
N
H
S
S
S
N
N
N
N N
O
O
3a
(IC50 = 0.05 M)
Figure 1. Representative allosteric GLS inhibitors and compound 3a containing a mercaptoethyl
linker. The IC50 values are taken from the original publications3, 8-10 except for compound 3a.
*After 60-min preincubation.
Examination of the SAR data for BPTES-derived compounds accumulated to date suggests
that the allosteric sites of GLS tolerate many forms of linker that connect the two heterocycles.

For instance, while BPTES has a diethylsulfide (-CH2CH2SCH2CH2-) linker connecting the two
thiadiazole rings, two heterocycles in CB-839 are connected by an n-butyl (-CH2CH2CH2CH2-)
linker devoid of the central sulfide group. Furthermore, incorporation of a cyclic system into the
linker created further diversity in the structure of GLS inhibitors derived from BPTES,5
including compound (1S,3S)-1 containing a 1,3-cyclohexane linker.10 The co-crystal structure of
GLS in complex with BPTES (PDB: 3UO9) suggests that the acyclic diethylsulfide (-
CH2CH2SCH2CH2-) linker of BPTES is somewhat compressed to fit the allosteric binding site of
GLS.11 This explains why compound (1S,3S)-1 with a substantially shorter linker can retain high
GLS inhibitory potency. Indeed, the co-crystal structure of GLS in complex with (1S,3S)-1
(PDB: 5JYP)12 displayed the same network of hydrogen bonds as seen with BPTES (PDB:
3UO9). Subsequently, other cyclic systems have also been found to serve as effective linkers in
GLS inhibitors.13, 14 Potent inhibition shown by compound (1S,3S)-1 indicates that an n-propyl (-
CH2CH2CH2-) linker can also be very effective. Indeed, the corresponding BPTES analog 2a
(Fig. 1) was found to inhibit GLS with submicromolar potency (IC50 = 0.59 μM).9 In contrast,
compound 2b (Fig. 1) possessing a dimethylsulfide (-CH2SCH2-) linker showed substantially
lower inhibitory activity (IC50 > 20 μM).9 These findings prompted us to explore other short
acyclic linkers in the pursuit of a new series of GLS inhibitors derived from BPTES. To our
surprise, BPTES analog 3a containing a mercaptoethyl (-SCH2CH2-) linker was found to
potently inhibit GLS with an IC50 value of 0.05 μM, providing a new scaffold for the
development of potent GLS inhibitors. Herein, we report the discovery of a series of
mercaptoethyl-linked derivatives of BPTES as a new subclass of allosteric GLS inhibitors.
2. Results and discussion

As shown in Scheme 1, some of the mercaptoethyl-linked compounds were synthesized using
5-amino-1,3,4-thiadiazole-2-thiol 4 as a starting material. Reaction of 4 with tert-butyl acrylate
in the presence of trimethylamine gave the Michael adduct 5. After HATU-mediated N-acylation
of 5 with phenylacetic acid, the tert-butyl ester of the resulting compound 6 was hydrolyzed by
TFA, providing S-substituted 3-mercatopropionic acid 7. The carboxylic acid of 7 was converted
into a 2-amino-1,3,4-thiadiazole moiety by treating with thiosemicarbazide and POCl3 to give
compound 8. N-Acylation of 8 with phenylacetic acid mediated by HATU gave compound 3a, a
mercaptoethyl-linked derivative of BPTES. Intermediate 8 was also coupled with 2-(pyridin-2-
yl)acetic acid, 2-(3-(trifluoromethoxy)phenyl)acetic acid, and 2-(4-(dimethylamino)phenyl)acetic
acid, affording 3b, 3c, and 3d, respectively.
Scheme 1. Synthesis of compounds 8 and 3a-3d a
H
H
S
S
S
S
H2N
S
O
N
N
S
O
S
OH
H2N
SH
a
b
c
N
N
N
S
N
6
N N
7
N
4
N
O
O
O
O
O
5
H
H
H
S
N
S
S
N
S
NH2
N
S
N
d
e
Aryl
N
N
N
N
N
N N
O
O
O
8
3a
3c
3d
3b
(Aryl = pyridin-2-yl)
(Aryl = 3-(trifluoromethoxy)phenyl)
(Aryl = 4-(dimethylamino)phenyl)
(Aryl = phenyl),
aReagents and conditions: (a) triethylamine, tert-butyl acrylate, MeOH, rt to 65 °C, 20%; (b)
diisopropylethylamine, phenylacetic acid, HATU, DMF, 0 °C to rt, 100%; (c) trifluoroacetic
acid, DCM, rt, 99%; (d) thiosemicarbazide, POCl3, 90 °C, 93%; (e) diisopropylethylamine,
arylacetic acid, HATU, DMF, 0 °C to rt, 45% for 3a, 28% for 3b, 79% for 3c, and 42% for 3d.
As shown in Scheme 2, various acyl groups can be also introduced to the 5-amino-1,3,4-
thiadiazol-2-yl)mercapto side of the scaffold by starting from 3-(benzyloxy)propanenitrile 9. The
nitrile group of 9 was converted into a 2-amino-1,3,4-thiadiazole moiety by treating with
thiosemicarbazide in TFA to give compound 10. N-Acylation with phenylacetyl chloride gave
compound 11. After removal of the benzyl group with BBr3, the resulting primary alcohol 12

was transformed to the corresponding mesylate ester 13, which was subsequently reacted with 5-
amino-1,3,4-thiadiazole-2-thiol 4 to give compound 14. Coupling of 14 with (pyridin-2-yl)acetic
acid was most effectively achieved by using propylphosphonic anhydride (T3P) and
trimethylamine to afford the desired product 15a. 3-(Trifluoromethoxy)phenylacetamide
derivative 15b was synthesized by HATU-mediated coupling of 14 and 3-
(trifluoromethoxy)phenylacetic acid.
Scheme 2. Synthesis of compounds 14 and 15a-15ba
H
H
N
S
S
S
BnO
NH2
H2N
BnO
N
HO
c
f
a
b
d
BnO
MsO
CN
N
10
N
N
N
N N
O
O
9
11
12
H
H
H
H
S
S
S
S
S
N
S
N
N
S
N
e
Aryl
N
N
13
N
N
N
14
N
N
N
N N
O
O
O
15a
O
(Aryl = pyridin-2-yl)
15b
(Aryl = 3-(trifluoromethoxy)phenyl)
aReagents and conditions: (a) thiosemicarbazide, TFA, 65 °C, 57%; (b) triethylamine,
phenylacetyl chloride, DCM, rt, 100%; (c) boron tribromide, DCM, rt; (d) mesyl chloride, DCM,
0 °C, 65% from 11; (e) 5-amino-1,3,4-thiadiazole-2-thiol, K2CO3, DMF, rt, 52%; (f) (pyridin-2-
yl)acetic acid or 3-(trifluoromethoxy)phenylacetic acid, T3P, triethylamine, DMF, 0 °C, 23% for
15a, 26% for 15b.
We also synthesized derivatives in which one of the two thiadiazole rings was replaced by a
pyridazine ring. As shown in Scheme 3, copper-catalyzed coupling of (6-iodopyridazin-3-yl)-2-
phenylacetamide 16 with 1715 produced compound 18, which was subsequently reacted with
phenylacetyl chloride to afford compound 19.
Scheme 3. Synthesis of compounds 18 and 19a

S
S
I
HS
NH2
S
NH2
O
O
a
+
N N
N N
N
N
N
N
N
N
17
18
16
H
H
H
S
S
N
O
b
N N
N
O
N
H
N
19
aReagents and conditions: (a) CuI, K2CO3, HO(CH2)2OH, i-PrOH, 80 °C, 40% yield; (b)
triethylamine, phenylacetyl chloride, DCM, rt, 66% yield.
The other thiadiazole ring was also successfully replaced by a pyridazine ring (Scheme 4).
Palladium-catalyzed Suzuki coupling of 16 with allylboronic acid pinacol ester 20 provided
compound 21. Ozonolysis of 21 followed by treatment with sodium borohydride afforded
primary alcohol 22. Compound 22 was converted into the corresponding mesylate ester 23,
which was subsequently reacted with 5-amino-1,3,4-thiadiazole-2-thiol 4 to give compound 24.
N-Acylation of 24 with phenylacetic acid mediated by HATU gave the desired product 25.
Scheme 4. Synthesis of compounds 24 and 25a
HO
MsO
O
B
O
O
O
O
a
b
c
N
N
N
N
N
N
N
N
N
20
H 21
H 22
H 23
H
S
S
H2N
S
N
S
O
O
e
d
N
N
N N
N
O
N
N
N
N
N
24
25
H
H
aReagents and conditions: (a) 16, Pd(Ph3)4, potassium carbonate, dioxane, 120 °C, 42%; (b) (i)
O3, DCM, -78 °C; (ii) sodium borohydride, -78 to 0 °C, 14%; (c) mesyl chloride, triethylamine,
DCM, 0 °C, (d) 4, potassium carbonate, ACN, 50 °C; (e) phenylacetic acid,
diisopropylethylamine, HATU, DMF, 0 °C to rt, 11% (from 22).
All new synthesized compounds were tested for their ability to inhibit GLS using L-[3H]-
glutamine as substrate and human kidney-type glutaminase (hKGA124-669).16 Since IC50 values

of GLS inhibitors are known to be highly assay-dependent, we used some of the representative
inhibitors shown in Fig. 1 as controls in our assay, enabling a head-to-head comparison with the
new compounds tested. In our assay, BPTES and CB-839 inhibited GLS with IC50 values of 3.3
and 0.05 µM, respectively. Compounds (±)-1 and 2a exhibited IC50 values of 0.28 and 2.3 µM,
respectively, while compound 2b showed no inhibitory activity at concentrations up to 100 µM.
Compound 8 containing a mercaptoethyl linker was found to inhibit GLS with an IC50 value of
1.6 µM. As seen with compound 3a, incorporation of an additional phenylacetyl group into 8
resulted in substantial improvement of GLS inhibitory potency with an IC50 value of 50 nM.
It is worth noting that the corresponding analog containing an n-propyl (compound 2a) linker
showed a nearly 50-fold reduction in inhibitory potency and compound 2b displayed negligible
activity despite its structural similarity to compound 3a. Although the precise reason for the high
inhibitory potency of 3a remains to be investigated, it is conceivable that noncovalent sulfur
interaction plays an important role in the binding of 3a to the allosteric binding site of GLS.
Among various forms of noncovalent sulfur interactions,17 the sulfur-aromatic interaction
appears to be most relevant to our case given that Tyr394 and/or Phe322 is anticipated to be in
the vicinity of the sulfide moiety of 3a bound to the allosteric site of GLS based on its co-crystal
structures with various BPTES-derived GLS inhibitors.11-13, 18 Computational model studies have
indicated that certain configurations for the complexes of dimethylsulfide with aryl rings lead to
greater stabilization compared to the corresponding propane-containing complexes.19 Although
speculative, these findings may explain the much higher GLS inhibitory potency of compound
3a containing a mercaptoethyl linker as opposed to compound 2a with an n-propyl linker.
Besides the potent inhibitory GLS activity, compound 3a exhibited excellent phase I metabolic

stability (87% remaining after 60-min incubation) in mouse liver microsomes fortified with
NADPH, providing a promising molecular template for further structural optimization.
Additional analogs containing a mercaptoethyl-linked bis(thiadiazole) consistently showed
submicromolar inhibitory potency ranging from 0.1 to 0.2 μM. One exception is compound 14
lacking one phenylacetyl group, which inhibited GLS with an IC50 value of 3.0 µM, similar to
that of compound 8 devoid of a phenylacetyl group on the other side of compound 3a.
As demonstrated by CB-839, a pyridazine ring is one of the most effective alternatives to the
thiadiazole ring found in many GLS inhibitors.8 Compounds 18 and 19 possess a pyridazine ring
on the sulfur end of the mercatoethyl linker. These compounds displayed substantially weaker
potency toward GLS compared to their thiadiazole counterparts 8 and 3a. In contrast,
compounds 24 and 25 containing a pyridazine ring on the carbon side of the mercaptoethyl linker
showed GLS inhibitory potency comparable to those of compounds 8 and 3a. These findings
suggest that 2-mercapto-1,3,4-thiadiazole moiety is uniquely essential for the potent GLS
inhibitory activity shown by our new series of GLS inhibitors.
Table 1. Inhibition of GLS by representative GLS inhibitors and compounds containing a
mercaptoethyl linker
Cmpd
Structure
IC50 (µM)*
3.3 ± 0.7
H
H
N
S
S
N
S
BPTES
N N
N N
O
O
H
N
N
OCF3
N
H
N
CB-839
(±)-1
0.05 ± 0.01
0.28 ± 0.04
S
O
N N
N
O
H
N
H
S
S
N
N N
N N
O
O

H
N
H
N
S
S
2a
2b
8
2.3 ± 0.9
>100
N N
N N
O
O
O
O
H
N
H
N
S
S
S
N N
N N
H
N
S
S
S
NH2
1.6 ± 0.1
0.05 ± 0.03
0.11 ± 0.02
0.14 ± 0.02
N N
S
N N
O
H
N
H
N
S
S
3a
3b
3c
N N
N N
O
O
O
H
N
H
N
S
S
S
N
N N
N N
O
H
H
N
S
S
N
S
OCF3
N N
S
N N
S
O
O
H
H
N
N
S
S
3d
0.20 ± 0.08
N N
N N
O
O
N(CH3)2
H
N
S
S
H2N
14
3.0 ± 0.2
0.10 ± 0.03
0.08 ± 0.02
N N
N N
O
H
N
H
N
S
S
N
S
15a
15b
N N
N N
O
O
H
N
H
S
N N
S
F3CO
S
S
N
N N
S
O
O
NH2
O
18
19
24
25
>100
14 ± 1
N N
N
N
H
N
S
H
N
S
O
N N
N
O
O
N
N
S
H
S
H2N
O
6.8 ± 1.2
0.03 ± 0.01
N N
N
N
N
H
H
N
S
S
N N
O
N
N
N
H
*Values are mean ± SD of at least three experiments.

Because of its highly lipophilic molecular properties, BPTES is known to have very poor
aqueous solubility.16 Given its overall structural similarity to BPTES, compound 3a was
expected to have an equally poor solubility. Indeed, our attempt to assess the antiproliferative
effects of compound 3a at final concentrations of 1-10 μM failed due to precipitation that
occurred upon addition of the stock solution of 3a to cell culture media. Our efforts to
incorporate a pyridazine ring as an alternative to one of the two thiadiazole rings in compound 3a
were motivated by its solubility-enhancing effect observed in other cases.20 Disappointingly,
however, there was no improvement in solubility of compound 25 with a saturation
concentration below 1 μM despite the presence of a pyridazine ring.
3. Conclusion
Allosteric GLS inhibitors derived from BPTES show a great deal of structural diversity as a
result of continuous medicinal chemistry efforts made by many during the past decade. Structural
modification of the linker component of GLS inhibitors was found to be particularly well
tolerated, generating many variations in this region. The mercaptoethyl moiety represents
another promising linker that can be utilized in BPTES-derived GLS inhibitors. As demonstrated
by our SAR studies, GLS inhibitors containing the mercaptoethyl linker consistently showed
potent GLS inhibitory activity as long as the mercapto side is attached to a 1,3,4-thiadiazole ring.
One major challenge to be addressed is poor aqueous solubility which limits further
pharmacological characterization. It should be noted that BPTES-derived inhibitors can
incorporate a variety of acyl groups to both sides without substantial loss in GLS inhibitory
potency. As such, introduction of a soluble moiety to these sites might represent one way to
improve aqueous solubility while maintaining the potent inhibitory activity within this series of
GLS inhibitors. Another interesting direction for further structural medications would be to

replace the 1,3,4-thiadiazole with a 1,3,4-selenadiazole ring, which was reported to serve as a
very effective moiety in GLS inhibitors.21
4. Experimental
4.1. Chemical synthesis
4.1.1. General
All solvents were reagent grade or HPLC grade. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further purification. The preparation of
5,5'-thiobis(methylene)bis(1,3,4-thiadiazol-2-amine) has been previously reported.9 Melting
1
points were obtained on a Mel-Temp apparatus and are uncorrected. H NMR spectra were
recorded at 400 or 500 MHz. 13C NMR spectra were recorded at 100 or 125 MHz. In some cases,
signals of quaternary carbons in 13C NMR were not observable. Ozone was obtained from
oxygen in a GE60/FM100 tabletop generator (Ozone Services). The HPLC solvent system
consisted of deionized water and acetonitrile, both containing 0.1% formic acid. Preparative
HPLC purification was performed on an Agilent 1200 series HPLC system equipped with an
Agilent G1315D DAD detector using a Phenomenex Luna 5 μm C18 column (21.2 mm × 250
mm, 5 μm). Analytical HPLC was performed on an Agilent 1200 series HPLC system equipped
with an Agilent G1315D DAD detector (detection at 220 nm) and an Agilent 6120 quadrupole
MS detector. The analytical HPLC conditions involve either (A) 20% acetonitrile/80% water for
0.25 min followed by gradient to 85% acetonitrile/15% water over 1.5 min and continuation of
85% acetonitrile/15% water for 2.25 min with an Agilent Eclipse Plus C18 column (2.1 mm × 50
mm, 3.5 μm) at a flow rate of 1.25 mL/min; or (B) 5% acetonitrile/95% water for 0.25 min
followed by gradient to 40% acetonitrile/60% water over 1.5 min and continuation of 85%
acetonitrile/15% water for 2.25 min with an Agilent Eclipse Plus C18 column (2.1 mm × 50 mm,

3.5 μm) at a flow rate of 1.25 mL/min. Unless otherwise noted, all final compounds biologically
tested were confirmed to be of ≥ 95% purity by the HPLC methods described above.
4.1.2.
(2b)
N,N'-(5,5'-Thiobis(methylene)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)
To a solution of 5,5'-thiobis(methylene)bis(1,3,4-thiadiazol-2-amine)9 (1.20 g, 4.61 mmol) in
DMA (20 mL) was added triethylamine (2.43 g, 24.0 mmol) and phenylacetyl chloride (1.40 g,
9.06 mmol), and the mixture was stirred overnight at rt. The solvent was removed in vacuo and
the resultant residue was triturated with 10% NaHCO3 (75 mL), with hot MeOH (75 mL), and
then dried to afford 900 mg of 2b as an off-white solid (40% yield); 1H NMR (DMSO-d6) δ 3.81
13
(s, 4H), 4.13 (s, 4H), 7.25-7.28 (m, 2H) 7.31 (m, 8H), 12.76 (s, 2H). C NMR (DMSO-d6) δ
28.6, 41.5, 126.9, 128.4, 129.3, 134.5, 159.3, 162.0, 169.5. Analytical HPLC (Method A);
retention time 1.91 min, m/z 497.1 [M + H]+.
4.1.3. tert-Butyl 3-(5-amino-1,3,4-thiadiazol-2-ylthio)propanoate (5)
To a solution of 5-amino-1,3,4-thiadiazole-2-thiol 4 (17.5 g, 131 mmol) in MeOH (160 mL)
was added triethylamine (15.9 g, 157 mmol) and tert-butyl acrylate (20.2 g, 158 mmol) by
addition funnel. The mixture was stirred at rt for 1 h and then stirred at 65 °C for 18 h. The
solvent was removed in vacuo and the residue was partitioned between DCM (200 mL) and H2O
(100 ml). The organic layer was dried over MgSO4, filtered, and concentrated to ~10 mL volume
when precipitation occurs. The resultant solid was collected by vacuum filtration and washed
with Et2O to afford 7.01 g of 5 as an off-white solid (20% yield); 1H NMR (DMSO-d6) δ 1.40 (s,
9H), 2.63 (t, J = 6.8 Hz, 2H), 3.19 (t, J = 6.8 Hz, 2H), 7.33 (brs, 2H).
4.1.4. tert-Butyl 3-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-ylthio)propanoate (6)

To a solution of tert-butyl 3-(5-amino-1,3,4-thiadiazol-2-ylthio)propanoate 5 (7.00 g, 26.8
mmol) in DMF (100 mL) was added DIEA (13.8 g, 107 mmol) and phenylacetic acid (4.01 g,
29.5 mmol). HATU (11.2 g, 29.5 mmol) was added at 0 °C and the resulting mixture was
warmed to room temperature overnight. The solvent was removed in vacuo, and the residue was
taken up in DCM (200 mL) and washed with 10% KHSO4 (100 mL), sat’d NaHCO3 (100 mL),
and brine (100 mL). The organic layer was then dried over MgSO4 and concentrated. The
resultant solid was triturated with 20% ethyl acetate/hexanes and then Et2O to afford 10.2 g of 6
as a white solid (100% yield); 1H NMR (CDCl3) δ 1.42 (s, 9H), 2.75 (t, J = 7.0 Hz, 2H), 3.45 (t,
J = 7.0 Hz, 2H), 4.01 (s, 2H), 7.28-7.45 (m, 5H), 12.32 (s, 1H).
4.1.5. 3-(5-(2-Phenylacetamido)-1,3,4-thiadiazol-2-ylthio)propanoic acid (7)
To a solution of 6 (10.2 g, 27.0 mmol) in DCM (50 mL) was added TFA (50 mL). The mixture
was stirred at rt for 2 h and then concentrated to give 8.68 g of 7 as an off-white solid that was
used in the subsequent step without further purification (99% yield). 1H NMR (DMSO-d6) δ 2.70
(t, J = 6.7 Hz, 2H), 3.36 (t, J = 6.7 Hz, 2H), 3.82 (s, 2H), 7-26-7.35 (m, 5H), 12.90 (brs, 1H).
4.1.6.
N-(5-(2-(5-Amino-1,3,4-thiadiazol-2-yl)ethylthio)-1,3,4-thiadiazol-2-yl)-2-
phenylacetamide (8)
To a solution of 3-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-ylthio)propanoic acid 7 (8.68 g,
26.8 mmol) in POCl3 (50 mL) was added thiosemicarbazide (7.34 g, 80.5 mmol), and the
mixture was heated at 90 °C for 1.5 h. The reaction was cooled to room temperature, poured into
100 mL of ice and stirred vigorously. The resultant solid was washed with copious amounts of
1
water to afford 9.43 g of 8 as a white solid (93% yield); H NMR (DMSO-d6) δ 3.24 (t, J = 6.8
Hz, 2H), 3.54 (t, J = 6.8 Hz, 2H), 3.82 (s, 2H), 7.21-7.35 (m, 5H), , 12.91 (brs, 1H). 13C NMR

(DMSO-d6) δ 29.6, 32.3, 41.4, 127.0, 128.5, 129.3, 134.4, 155.5, 158.1, 158.8, 168.8, 169.6.
HPLC (Method A); retention time 1.41 min, m/z 379.1 [M + H]+
.
4.1.7.
2-Phenyl-N-(5-(2-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)ethylthio)-1,3,4-
thiadiazol-2-yl)acetamide (3a)
To a solution of N-(5-(2-(5-amino-1,3,4-thiadiazol-2-yl)ethylthio)-1,3,4-thiadiazol-2-yl)-2-
phenylacetamide 8 (770 mg, 2.03 mmol) in DMF (30 mL) was added phenylacetic acid (304 mg,
2.24 mmol) and diisopropylethylamine (1.05 g, 8.14 mmol). The mixture was cooled to 0 °C and
HATU (773 mg, 2.03 mmol) was added. The reaction was allowed to warm to rt overnight, and
the solvent was removed in vacuo. The resultant residue was taken up in DCM (100 mL) and
washed with 10% KHSO4, (50 mL), sat’d NaHCO3 (50 mL), and brine (50 mL). The organic
layer was dried over MgSO4, filtered, and concentrated. The residual product was triturated with
1
20% ethylacetate/hexanes to afford 450 mg of 3a as a tan solid (45% yield); H NMR (DMSO-
d6) δ 3.41 (t, J = 7.0 Hz, 2H), 3.60 (t, J = 7.0 Hz, 2H), 3.80 (s, 2H), 3.81 (s, 2H), 7.24-7.45 (m,
13
10H), 12.72 (brs, 1H), 12.87 (brs, 1H). C NMR (DMSO-d6) δ 29.1, 32.4, 41.4, 41.5, 126.9,
127.0, 128.4, 129.2, 129.3, 134.4, 134.6, 157.9, 158.7, 158.8, 161.4, 169.4, 169.6. HPLC
(Method A); retention time 1.98 min, m/z 497.1 [M + H]+.
4.1.8. 2-Phenyl-N-(5-(2-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)ethylthio)-1,3,4-
thiadiazol-2-yl)acetamide (3b)
Compound 3b was prepared from 8 as was described for 3a except 2-(pyridin-2-yl)acetic acid
hydrochloride was used in place of phenylacetic acid. The solvent was removed in vacuo, and
the residual material was triturated with sat’d NaHCO3, water, and then methanol to give 3b as a
1
tan solid (28% yield); H NMR (DMSO-d6) δ 3.42 (t, J = 6.8 Hz, 2H), 3.61 (t, J = 6.8 Hz, 2H),
3.81 (s, 2H), 4.01 (s, 2H), 7.26-7.33 (m, 6H), 7.39 (d, J = 8.0 Hz, 1H), 7.77 (td, J = 7.6, 2.0 Hz,

13
1H). 8.48-8.50 (d, J = 4.0 Hz, 1H), 12.76 (s, 1H), 12.92 (s, 1H). C NMR (DMSO-d6) δ 29.1,
32.4, 41.4, 44.1, 122.2, 124.2, 127.0, 128.5, 129.3, 134.5, 136.8, 149.2, 154.9, 158.0, 158.7,
158.9, 161.5, 168.6, 169.7. HPLC (Method A); retention time 1.93 min, m/z 498.1 [M + H]+.
4.1.9.
2-Phenyl-N-(5-(2-(5-(2-(3-(trifluoromethoxy)phenyl)acetamido)-1,3,4-thiadiazol-2-
yl)ethylthio)-1,3,4-thiadiazol-2-yl)acetamide (3c)
Compound 3c was prepared from 8 as was described for 3a except 2-(3-
(trifluoromethoxy)phenyl)acetic acid was used in place of phenyl acetic acid. The solvent was
removed in vacuo, and the residual material was triturated with sat’d NaHCO3 and then water to
afford the crude product which was then chromatographed with 2% to 15% MeOH/DCM to
afford 3c as a white solid (79% yield); 1H NMR (DMSO-d6) δ 3.42 (t, J = 7.2 Hz, 2H), 3.61 (t, J
= 7.2 Hz, 2H), 3.81 (s, 2H), 3.90 (s, 2H), 7.26-7.36 (m, 8H), 7.45-7.49 (m, 1H), 12.78 (brs, 1H),
12.91 (brs, 1H). 13C NMR (DMSO-d6) 29.1, 32.4, 40.9, 41.4, 120.0 (q, J = 258 Hz), 119.4,
121.9, 126.9, 128.4, 128.6, 129.3, 130.3, 134.4, 137.2, 148.3, 157.9, 158.9, 161.5, 168.9, 169.6.
HPLC (Method A); retention time 2.65 min, m/z 581.1 [M + H]+.
4.1.10.
2-(4-(Dimethylamino)phenyl)-N-(5-(2-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-
ylthio)ethyl)-1,3,4-thiadiazol-2-yl)acetamide (3d)
Compound 3d was prepared from 8 as was described for 3a except 2-(4-
(dimethylamino)phenyl)acetic acid was used in place of phenylacetic acid. The solvent was
removed in vacuo, and the residual material was triturated with sat’d NaHCO3, water, and then
DCM to afford 8 as a tan solid (42% yield); 1H NMR (DMSO-d6) δ 2.85 (s, 6H), 3.41 (t, J = 6.8
Hz, 2H), 3.60 (t, J = 6.8 Hz, 2H), 3.63 (s, 2H), 3.81 (s, 2H), 6.67 (d, J = 8.8 Hz, 2H), 7.12 (d, J =
13
8.4 Hz, 2H), 7.24-7.35 (m, 5H), 12.62 (brs, 1H), 12.85 (brs, 1H). C NMR (DMSO-d6) δ 29.1,

32.4, 40.2, 40.7, 41.4, 112.5, 121.9, 126.9, 128.5, 129.3, 129.7, 134.5, 149.5, 157.9, 158.8, 158.9,
161.4, 169.6, 170.1. HPLC (Method A); retention time 1.69 min, m/z 540.3 [M + H]+.
4.1.11. 5-(2-(Benzyloxy)ethyl)-1,3,4-thiadiazol-2-amine (10)
A solution of 3-(benzyloxy)propanenitrile 9 (12.8 g, 79.4 mmol) and thiosemcarbazide (8.74
g, 96.0 mmol) in TFA (50 mL) under an atmosphere of nitrogen was heated at 65 °C for 3 h. The
reaction was concentrated in vacuo, the residue was taken up in DCM (300 mL), washed with
sat’d NaHCO3 (100 mL). The organic layer was dried over MgSO4, filtered, and concentrated.
1
Trituration of the crude material provided 10.7 g of 10 as a white solid (57% yield); H NMR
(CDCl3) δ 3.23 (t, J = 6.1 Hz, 2H), 3.78 (t, J = 6.1 Hz, 2H), 4.56 (s, 2H), 5.01 (s, 2H), 7.34-7.39
(m, 5H).
4.1.12. N-(5-(2-(Benzyloxy)ethyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (11)
To a solution of 5-(2-(benzyloxy)ethyl)-1,3,4-thiadiazol-2-amine 10 (1.20 g, 5.10 mmol) in
DCM (75 mL) was added phenylacetyl chloride (867 mg, 5.60 mmol) and triethylamine (774
mg, 7.65 mmol) at room temperature. After 1 h, the reaction was partitioned between DCM (75
mL) and sat’d NaHCO3 (25 mL). The organic layer was washed with sat’d NaCl (75 mL), dried
over MgSO4, and filtered to afford 1.80 g of 11 as a white solid (100% yield); 1H NMR (CDCl3)
δ 3.33 (t, J = 5.93 Hz, 2H), 3.81 (t, J = 5.9 Hz, 2H), 4.04 (s, 2H), 4.58 (s, 2H), 7.29-7.45 (m,
10H).
4.1.13. N-(5-(2-Hydroxyethyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (12)
To a solution of N-(5-(2-(benzyloxy)ethyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide 11 (540
mg, 1.53 mmol) in DCM at rt was added BBr3 (1.15 g, 4.58 mmol). After 0.5 h, the reaction was
quenched by the addition of H2O (20 mL). The reaction mixture partitioned between DCM (25
mL) and H2O (25 mL). The organic layer was dried over MgSO4, filtered, and concentrated. The

crude material was triturated with Et2O and afforded 300 mg of 12 as a crude product which was
used in the next step without further purification or characterization.
4.1.14. 2-(5-(2-Phenylacetamido)-1,3,4-thiadiazol-2-yl)ethyl methanesulfonate (13)
To a solution of N-(5-(2-hydroxyethyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide 12 (300 mg,
1.14 mmol) in DCM (20 ml) at 0 °C was added triethylamine (172 mg, 1.70 mmol) followed by
methanesulfonyl chloride (131 mg, 1.14 mmol). After 45 min, the solution was partitioned
between DCM (50 mL) and H2O (25 mL), the organic layer was removed and dried over MgSO4,
filtered, and concentrated. The crude material was purified by trituration with Et2O and afforded
340 mg of 13 as a white solid (65% yield from 11); 1H NMR (CDCl3) δ 3.03 (s, 3H), 3.49 (t, J =
6.4 Hz, 2H), 3.97 (s, 2H), 4.62 (t, J = 6.4 Hz, 2H), 7.29-7.40 (m, 5H), 10.95 (s, 1H).
4.1.15.
(14)
N-(6-(2-(5-Amino-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-yl)-2-phenylacetamide
To a solution of 2-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)ethyl methanesulfonate 13
(340 mg, 1.00 mmol) in DMF (5 mL) was added potassium carbonate (276 mg, 2.00 mmol) and
5-amino-1,3,4-thiadiazole-2-thiol (160 mg, 1.2 mmol). After 30 min, the DMF was removed the
material was taken up in DCM (50 mL), washed with H2O (25 mL), dried over MgSO4, filtered,
and concentrated. The crude material was purified by trituration with MeOH/EtOAc to afford
1
200 mg of 14 as a white solid (52% yield); H NMR (DMSO-d6) δ 3.34 (m, 2H), 3.44 (m, 2H),
13
3.80 (s, 2H), 7.24-7.32 (m, 5H). C NMR (DMSO-d6) δ 29.3, 33.2, 41.6, 126.9, 128.5, 129.3,
134.7, 149.2, 158.7, 161.6, 169.5, 169.7. HPLC (Method B); retention time 2.35 min, m/z 378.9
[M + H]+.
4.1.16. 2-Phenyl-N-(5-(2-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-ylthio)ethyl)-1,3,4-
thiadiazol-2-yl)acetamide (15a)

To
a
solution of N-(6-(2-(5-amino-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-yl)-2-
phenylacetamide 14 (50 mg, 0.13 mmol) and 2-(pyridin-2-yl)acetic acid (20.0 mg, 0.15 mmol) in
anhydrous DMF at 0 °C was added propylphosphonic anhydride (T3P,~50% solution in DMF,
84 mg, 0.13 mmol), followed by triethylamine (35 mg, 0.34 mmol). The solution was stirred at
rt for 4 h, was quenched with ice water (2 mL) and concentrated. The material was purified by
RP HPLC to afford 15 mg of 15a as a white solid (23% yield); 1H NMR (DMSO-d6) δ 3.42 (t, J
= 6.8 Hz, 2H), 3.61 (t, J = 6.8 Hz, 2H), 3.80 (s, 2H), 4.02 (s, 2H), 7.24-7.35 (m, 5H), 7.40 (d, J =
8.0 Hz, 1H), 7.78 (td, J = 8.0, 2.0 Hz, 1H), 8.48-8.50 (m, 1H), 12.75 (brs, 1H), 12.94 (brs, 1H).
1H NMR (DMSO-d6) δ 29.1, 32.4, 41.5, 44.0, 122.2, 124.2, 126.9, 128.5, 129.3 134.6, 136.8,
149.1, 154.8, 157. 9, 158.7, 158.8, 161.5, 168.8, 169.4. HPLC (Method A); retention time 2.47
min, m/z 497.9 [M + H]+.
4.1.17.
ylthio)ethyl)-1,3,4-thiadiazol-2-yl)acetamide (15b)
To solution of N-(6-(2-(5-amino-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-yl)-2-
2-Phenyl-N-(5-(2-(5-(2-(3-(trifluoromethoxy)phenyl)acetamido)-1,3,4-thiadiazol-2-
a
phenylacetamide 14 (50 mg, 0.13 mmol) in DMF (3 mL) at 0 °C was added 2-(3-
(trifluoromethoxy)phenyl)acetic acid (34.9 mg, 0.16 mmol), DIEA (68.2 mg, 0.53 mmol) and
then HATU (60.3 mg, 0.16 mmol). The reaction was allowed to warm to rt overnight, was
concentrated, partitioned between CHCl3 (25 mL) and H2O (15 mL). The organic layer was
washed with sat’d NaHCO3 (15 mL) and then 10% KHSO4 (15 mL), dried over MgSO4, filtered,
concentrated, and then purified by RP HPLC to afford 20 mg of 15b as a white solid (26%
1
yield); H NMR (DMSO-d6) δ 3.42 (t, J = 6.8 Hz, 2H), 3.60 (t, J = 6.8 Hz, 2H), 3.80 (s, 2H),
3.90 (s, 2H), 7.24-7.35 (m, 8H), 7.47 (t, J = 8.0 Hz, 1H), 12.75 (brs, 1H), 12.94 (brs, 1H). 13C
NMR (DMSO-d6) δ 29.1, 32.4, 40.8, 41.5, 119.4, 120.1 (q, J = 255 Hz), 122.0, 126.9, 128.4,

128.6, 129.2, 130.3, 134.6, 137.1, 148.3 (q, J = 1.5 Hz), 157. 9, 158.7, 159.0, 161. 5, 169.2,
169.4. HPLC (Method A); retention time 2.12 min, m/z 580.9 [M + H]+.
4.1.18.
(18)
N-(6-(2-(5-Amino-1,3,4-thiadiazol-2-yl)ethylthio)pyridazin-3-yl)-2-phenylacetamide
To a solution of S-(2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl) ethanethioate (110 mg, 0.54 mmol)
in MeOH-water (2.5 mL:5 mL) was added NaOH (43 mg, 1.08 mmol) at rt. The mixture was
stirred at rt for 2 h and MeOH was removed under vacuum. The water layer was lyophilized to
afford compound 17, which was directly used in next step without further purification. Cu(I)
iodide (10.3 mg, 0.054 mmol), potassium carbonate (149.3 mg, 1.08 mmol), N-(6-iodopyridazin-
3-yl)-2-phenylacetamide 16 (183 mg, 0.54 mmol), and compound 17 were added to a flask. 2-
Propanol (5.0 mL), and ethylene glycol (67 mg, 1.08 mmol) were added by syringes at room
temperature. The mixture was stirred at 80 ˚C for 24 h. The reaction mixture was then allowed to
reach room temperature. The reaction mixture was then filtered and concentrated. The crude
product was purified by flash column chromatography on silica gel to afford 80 mg of 18 as a
1
yellow solid (40% yield); H NMR (DMSO-d6) δ 3.22 (t, J = 7.2 Hz), 3.55 (t, J = 7.2 Hz), 3.78
(s, 2H), 7.10 (brs, 2H), 7.24-7.36 (m, 5H), 7.61 (d, J = 9.2 Hz), 8.15 (d, J = 9.2 Hz), 11.30 (brs,
1H). 13C NMR (DMSO-d6) δ 28.69, 29.43, 42.86, 118.88, 126.71, 127.96, 128.37, 129.27,
135.41, 153.48, 156.01, 156.64, 168.67, 170.80. HPLC (Method A); retention time 1.17 min, m/z
373.1 [M + H]+.
4.1.19. 2-Phenyl-N-(6-(2-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)ethylthio)pyridazin-3-
yl)acetamide (19)
To
a
solution of
N-(6-(2-(5-amino-1,3,4-thiadiazol-2-yl)ethylthio)pyridazin-3-yl)-2-
phenylacetamide 18 (20 mg, 0.053 mmol) in DCM (5 mL) was added triethylamine (16 mg, 0.16

mmol) and phenylacetyl chloride (9.1 mg, 0.06 mmol). After being stirred at rt overnight,
phenylacetyl chloride (4.5 mg, 0.03 mmol) was added and the mixture was stirred for another 1
h. The solvent was removed in vacuo and the residue was purified by silica gel chromatography
1
(100% DCM to 10% MeOH/DCM) and afforded 18 mg 19 as a white solid (66% yield); H
NMR (DMSO-d6) δ 3.39 (t, J = 6.8 Hz), 3.62 (t, J = 6.8 Hz), 3.77 (s, 2H), 3.80 (s, 2H), 7.23-7.36
(m, 10H), 7.62 (d, J = 9.2 Hz), 8.14 (d, J = 9.6 Hz), 11.30 (brs, 1H), 12.72 (brs, 1H) . 13C NMR
(DMSO-d6) δ 28.8, 29.0, 41.6, 42.8, 118.9, 126.7, 126.9, 128.0, 128.4, 128.5, 129.3, 134.6,
135.4, 153.5, 156.4, 158.7, 162.0, 169.4, 170.8. HPLC (Method A); retention time 1.94min, m/z
491.2 [M + H]+.
4.1.20. N-(6-Allylpyridazin-3-yl)-2-phenylacetamide (21)
To a solution of 16 (1.10 g, 3.2 mmol) in anhydrous dioxane was added potassium carbonate
(1.10 g, 8.0 mmol), allylboronic acid pinacol ester 20 (790 mg, 4.7 mmol), and palladium-
tetrakis(triphenylphosphine) (180 mg, 0.16 mmol). The mixture was heated in a sealed tube at
120 °C for 14 h. The reaction mixture was cooled and the solvent was removed in vacuo. The
residue was taken up in EtOAc (100 mL) and washed with H2O (50 mL). The organic layer was
washed with sat’d NH4Cl, dried over MgSO4, filtered, concentrated and then purified by silica
gel chromatography (20% EtOAc/hex to 80% EtOAc/hex) to afford 330 mg of 21 as a yellow
solid (42% yield);1H NMR (CDCl3) δ 3.72 (dt, J = 6.8, 1.3 Hz, 2H), 3.90 (s, 2H), 5.18 (m, 2H),
6.02 (m, 1H), 7.33.-7.43 (m, 6H), 8.45 (d, J = 9.2 Hz, 1H), 8.95 (s, 1H).
4.1.21. N-(6-(2-Hydroxyethyl)pyridazin-3-yl)-2-phenylacetamide (22)
To a solution of N-(6-allylpyridazin-3-yl)-2-phenylacetamide 21 (280 mg, 1.1 mmol) in
anhydrous DCM at -78 °C was bubbled O3 (at a rate of ~ 31 ml/min) for 15 min. The solution
changed color from dark to light yellow at this time. The solution was flushed with N2 for 5 min

and sodium borohydride (43 mg, 1.14 mmol) was added to the mixture at this same temperature.
The reaction was maintained at this temperature for 60 min, and then warmed and maintained at
0 °C for 60 min. The mixture was partitioned between DCM and water. The DCM layer was
dried over MgSO4, filtered, and concentrated in vacuo. The residual material was purified by
silica gel chromatography (50% EtOAc/hex to 100% EtOAc to 30% MeOH/EtOAc) and
afforded 40 mg of 22 as a light yellow solid (14% yield); 1H NMR (CDCl3) δ 3.13 (t, J = 5.6 Hz,
2H), 3.88 (s, 2H), 4.07 (4.07, t, J = 5.6 Hz, 2H), 7.34-7.42 (m, 6H), 8.47 (d, J = 9.0 Hz, 1H), 8.93
(s, 1H).
4.1.22. 2-(6-(2-Phenylacetamido)pyridazin-3-yl)ethyl methanesulfonate (23)
To a solution of N-(6-(2-hydroxyethyl)pyridazin-3-yl)-2-phenylacetamide 22 (50 mg, 0.19
mmol) in DCM (10 mL) at 0 °C was added triethylamine (29 mg, 0.29 mmol) followed by
methanesulfonyl chloride (25 mg, 0.21 mmol). After 2 h, additional triethylamine (29 mg, 0.29
mmol) and methanesulfonyl chloride (25 mg, 0.22 mmol) was added and the mixture was stirred
for additional 6 h. The reaction mixture was partitioned between DCM (25 mL) and H2O (15
mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residual
material was used in the subsequent step without further purification or characterization.
4.1.23.
(24)
N-(6-(2-(5-Amino-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-yl)-2-phenylacetamide
To a solution of 2-(6-(2-phenylacetamido)pyridazin-3-yl)ethyl methanesulfonate 23 (65 mg,
0.19 mmol) in acetonitrile (30 mL) was added 5-amino-1,3,4-thiadiazole-2-thiol (28 mg, 0.21
mmol) and potassium carbonate (54 mg, 0.39 mmol). The mixture was heated at 50 °C for 7.5 h
and the solvent was removed in vacuo. A small portion of the crude material was purified by RP
HPLC to obtain 24 as a colorless oil for the in vitro screening. 1H NMR (CD3OD w/ two drops of

DMSO-d6) δ 3.34 (t, J = 7.0 Hz, 2H), 3.54 (t, J = 7.0 Hz, 2H), 3.82 (s, 2H), 7.29 (t, J = 7.0 Hz,
1H), 7.35-7.41 (m, 4H), 7.62 (d, J = 9.0 Hz, 1H), 8.35-8.37 (m, 1H). 13C NMR (CD3OD w/ two
drops of DMSO-d6) δ 35.1, 36.3, 44.5, 120.7, 128.3, 129.8, 130.6, 136.59, 136.63, 153.2, 156.0,
159.4, 173.1. HPLC (Method A); retention time 2.29 min, m/z 373.1 [M + H]+. The remaining
material was taken to the next step without further purification.
4.1.24. 2-Phenyl-N-(6-(2-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-
yl)acetamide (25)
To
a
solution of N-(6-(2-(5-amino-1,3,4-thiadiazol-2-ylthio)ethyl)pyridazin-3-yl)-2-
phenylacetamide 24 (70 mg, 0.19 mmol) in DMF (1.0 mL) at 0 °C, was added phenylacetic acid
(33 mg, 0.24 mmol), diisopropylethylamine (146 mg, 1.13 mmol), and HATU (92 mg, 0.24
mmol). The mixture was warmed to rt overnight, concentrated in vacuo, taken up in EtOAc (40
mL), washed with sat’d NaHCO3 (15 mL) and 10% KHSO4, dried over MgSO4, and
concentrated in vacuo. Purification by RP HPLC afforded 10 mg of 25 as a light pink oil (11%
yield from 22). 1H NMR (CDCl3) δ 3.39 (t, J = 6.5 Hz, 2H), 3.69 (t, J = 6.5 Hz, 2H), 3.93 (s,
13
2H), 4.06 (s, 2H), 7.29-7.39 (m, 11H), 8.54 (d, J = 9.0 Hz, 1H), 10.7 (s, 1H), 12.7 (s,1H). C
NMR (CDCl3) δ 34.3, 36.3, 42.7, 44.2, 119.6, 127.4, 127.5, 128.8, 128.9, 129.4, 129.5, 130.1,
133.7, 134.3, 154.6, 157.1, 159.5, 161.3, 169.6, 171.2. HPLC (Method B); retention time 3.08
min, m/z 491.1 [M + H]+.
4.2. in vitro GLS assay
A 96-well microplate-based glutaminase assay was used to determine the activity of various
glutaminase inhibitors.16 The hKGA∆1 construct was prepared from the hKGA cDNA26 by
deleting the sequence encoded in exon 1 and cloning into pET15b as described for the rat
KGA∆1 construct.22 Purified human kidney glutaminase (hKGA124−669; 250 nM) was used as

the source of enzyme and radiolabeled glutamine (L-[3H]-glutamine, Perkin Elmer, Boston,
MA); 2 mM and at a specific activity of 0.91 μCi/μmol) was used as the substrate. The assay was
conducted in the presence and absence of inhibitors, at room temperature (45 min incubation), in
45 mM phosphate buffer (pH 8.2). At the end of the reaction period, the assay was terminated
upon the addition of 20 mM imidazole buffer (pH 7). 96-Well spin columns packed with strong
anion ion-exchange resin (Bio-Rad, Hercules, CA) were used to separate the substrate and the
reaction product. Unreacted [3H]-glutamine was removed by washing with imidazole buffer.
[3H]-Glutamate, the reaction product, was then eluted with 0.1 M HCl and analyzed for
radioactivity using Perkin-Elmer’s TopCount instrument in conjunction with their 96-well
LumaPlates (Waltham, MA).
4.3. Metabolic stability assessment
The metabolic stability of compound 3a was evaluated using mouse liver microsomes as we
have previously described.23 The reaction was carried out in 100 mM potassium phosphate
buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM
glucose 6-phosphate, 3.3 mM MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50 µM
sodium citrate). Reactions in triplicate were initiated by addition of mouse liver microsomes to
the incubation mixture (compound final concentration was 1 µM; 0.2 mg/mL protein). Control
reaction was carried out in the absence of NADPH to measure CYP-independent metabolism of
the compound. After 60 min of incubation, aliquots of the mixture were removed and the
reaction was quenched by addition of two times the volume of ice cold acetonitrile spiked with
the internal standard. Compound disappearance was monitored over time using liquid
chromatography and tandem mass spectrometry (LC/MS/MS) as described in section 4.4.
4.4. Aqueous solubility assessment

The aqueous solubility of compound 25 was determined by the shake flask method.24 Compound
25 was dissolved in PBS buffer (pH = 7.4) at 5 mg/mL concentration and incubated for 24 h at
37 °C on a thermomixer. Samples were subjected to constant mixing at a shaking speed of
1000 rpm to ensure saturation. After 24 hours the solution was filtered (0.45 μm PTFE) and the
filtrate was diluted appropriately and solubility in the filtrate samples was determined against a
calibration curve ranging 5-10000 nM. Samples were analyzed on a Dionex ultra-high-
performance LC system coupled with Q Exactive Focus orbitrap mass spectrometer (Thermo
Fisher Scientific Inc., Waltham MA). The separation of analytes was achieved using the Agilent
Eclipse Plus column (100 × 2.1 mm i.d.; maintained at 35 ºC) packed with a 1.8 µm C18
stationary phase. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid
in acetonitrile. Pumps were operated at a flow rate of 0.3 mL/min for 8 min using gradient
elution. The mass spectrometer controlled by Xcalibur software 4.0.27.13 (Thermo Scientific)
was operated with a heated electrospray ionization (HESI) ion source in positive ionization
mode. The [M + H]+ ion transitions of 25 at m/z 491.1318 and that of the internal standard
(losartan) at m/z 423.1695 were monitored in Full MS scan mode. Data were acquired and
quantified with Xcalibur 2.2.
Acknowledgement
The authors of this manuscript have been supported by NIH grants P30MH075673 (B.S.S.),
R01CA193895 (B.S.S.), R21NS074151 (T.T.), and F32CA200278 (S.C.Z.). The authors are also
grateful for the support provided by the Bloomberg-Kimmel Institute for Cancer Immunotherapy
at Johns Hopkins School of Medicine.
Appendix A. Supplementary Data

Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bmc.2020.#####
References
1. Katt WP, Cerione RA. Glutaminase regulation in cancer cells: a druggable chain of events.
Drug Discov Today. 2014;19:450-457.
2. Li L, Meng Y, Li Z, et al. Discovery and development of small molecule modulators targeting
glutamine metabolism. Eur J Med Chem. 2019;163:215-242.
3. Newcomb RW. Selective inhibition of glutaminase by bis-thiadiazoles. 2002. US 6,451,828
B1.
4. Robinson MM, McBryant SJ, Tsukamoto T, et al. Novel mechanism of inhibition of rat
kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide
(BPTES). Biochem. J. 2007;406:407-414.
5. Zimmermann SC, Duvall B, Tsukamoto T. Recent Progress in the Discovery of Allosteric
Inhibitors of Kidney-Type Glutaminase. J Med Chem. 2019;62:46-59.
6. Song M, Kim SH, Im CY, et al. Recent Development of Small Molecule Glutaminase
Inhibitors. Curr Top Med Chem. 2018;18:432-443.
7. Xu X, Meng Y, Li L, et al. Overview of the Development of Glutaminase Inhibitors:
Achievements and Future Directions. J Med Chem. 2019;62:1096-1115.
8. Gross MI, Demo SD, Dennison JB, et al. Antitumor Activity of the Glutaminase Inhibitor CB-
839 in Triple-Negative Breast Cancer. Mol. Cancer Ther. 2014;13:890-901.
9. Li J, Chen L, Goyal B, et al. Heterocyclic Inhibitors of Glutaminase. 2013. WO 2013078123
A1.
10. Lemieux RM, Popovici-muller J, Salituro FG, et al. Compounds and their methods of use.
2015. U.S. Pat. Appl. 20150291576.
11. DeLaBarre B, Gross S, Fang C, et al. Full-length human glutaminase in complex with an
allosteric inhibitor. Biochemistry. 2011;50:10764-10770.
12. Ramachandran S, Pan CQ, Zimmermann SC, et al. Structural basis for exploring the
allosteric inhibition of human kidney type glutaminase. Oncotarget. 2016;7:57943-57954.
13. McDermott LA, Iyer P, Vernetti L, et al. Design and evaluation of novel glutaminase
inhibitors. Bioorg Med Chem. 2016;24:1819-1839.
14. McDermott L, Koes D, Mohammed S, et al. GAC inhibitors with a 4-hydroxypiperidine
spacer: Requirements for potency. Bioorg Med Chem Lett. 2019;29:126632.
15. Reddy RK, Glinka T, Totrov M, et al. Boronic acid derivatives and therapeutic uses thereof.
2019. US 10,206,937 B2.
16. Shukla K, Ferraris DV, Thomas AG, et al. Design, Synthesis, and Pharmacological
Evaluation of Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl Sulfide 3 (BPTES) Analogs
as Glutaminase Inhibitors. J. Med. Chem. 2012;55:10551-10563.
17. Beno BR, Yeung KS, Bartberger MD, et al. A Survey of the Role of Noncovalent Sulfur
Interactions in Drug Design. J Med Chem. 2015;58:4383-4438.
18. Thangavelu K, Pan CQ, Karlberg T, et al. Structural basis for the allosteric inhibitory
mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk
signaling in cancer cell metabolism. Proc. Natl. Acad. Sci. U.S.A. 2012;109:7705–7710.

19. Valley CC, Cembran A, Perlmutter JD, et al. The methionine-aromatic motif plays a unique
role in stabilizing protein structure. J Biol Chem. 2012;287:34979-34991.
20. Walker MA. Improvement in aqueous solubility achieved via small molecular changes.
Bioorg Med Chem Lett. 2017;27:5100-5108.
21. Chen Z, Li D, Xu N, et al. Novel 1,3,4-Selenadiazole-Containing Kidney-Type Glutaminase
Inhibitors Showed Improved Cellular Uptake and Antitumor Activity. J Med Chem.
2019;62:589-603.
22. Kenny J, Bao Y, Hamm B, et al. Bacterial expression, purification, and characterization of rat
kidney-type mitochondrial glutaminase. Protein Expr Purif. 2003;31:140-148.
23. Slack RD, Ku TC, Cao J, et al. Structure-Activity Relationships for a Series of (Bis(4-
fluorophenyl)methyl)sulfinyl Alkyl Alicyclic Amines at the Dopamine Transporter:
Functionalizing the Terminal Nitrogen Affects Affinity, Selectivity, and Metabolic Stability. J
Med Chem. 2020;63:2343-2357.
24. Zhou L, Yang L, Tilton S, et al. Development of a high throughput equilibrium solubility
assay using miniaturized shake-flask method in early drug discovery. J Pharm Sci.
2007;96:3052-3071.
TOC Graphic
H
N
H
N
S
S
N N
N N
O
O
O
O
O
O
2a
(IC50 = 2.3 M)
H
N
H
N
S
S
S
N N
N N
2b
(IC50 > 100 M )
H
N
H
N
S
S
S
N N
N N
3a
(IC50 = 0.05 M)

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: