Targeting the Warburg Effect in cancer; Relationships for 2-arylpyridazinones as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3)

Article abstract of DOI:10.1016/j.bmc.2013.12.041

High-throughput screening of a small-molecule library identified a 5-triazolo-2-arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3) Such inhibitors are of interest due to PFKFB3's

Full text of DOI:10.1016/j.bmc.2013.12.041

Accepted Manuscript
Targeting the Warburg Effect in cancer; relationships for 2-arylpyridazinones
as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bi-
sphosphatase 3 (PFKFB3)
Darby G. Brooke, Ellen M. van Dam, Colin K.W. Watts, Amanda Khoury, Marie
A. Dziadek, Hilary Brooks, Lisa-Jane K. Graham, Jack U. Flanagan, William
A. Denny
PII:
S0968-0896(13)01049-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.12.041
BMC 11303
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 October 2013
5 December 2013
17 December 2013
Please cite this article as: Brooke, D.G., van Dam, E.M., Watts, C.K.W., Khoury, A., Dziadek, M.A., Brooks, H.,
Graham, L.K., Flanagan, J.U., Denny, W.A., Targeting the Warburg Effect in cancer; relationships for 2-
arylpyridazinones as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3
(PFKFB3), Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.12.041
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Targeting the Warburg Effect in cancer; relationships for 2-arylpyridazinones as
inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-
bisphosphatase 3 (PFKFB3)
Darby G. Brooke,1 Ellen M. van Dam,2 Colin K. W. Watts,2 Amanda Khoury,2 Marie A.
Dziadek,2 Hilary Brooks,2 Lisa-Jane K. Graham,2 Jack U. Flanagan,1,3 William A. Denny*,1,3
1Auckland Cancer Society Research Centre, School of Medical Sciences, The University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand, 2Garvan Institute of Medical
Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia, and 3Maurice
Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand. mds.
Abbreviations: BCA, bicinchoninic acid assay; ECAR, extracellular acidification rate;
PFKFB3, 6-phosphofructo-2-kinase/2,6-bisphosphatase 3; F6P, fructose-6-phosphate; PFK-1,
6-phosphofructo-1-kinase; F1,6-BP, fructose-1,6-bisphosphate; F2,6-BP, fructose-2,6-
bisphosphate; RIPA, radioimmunoprecipitation assay; TEMPO, (2,2,6,2-
tetramethylpiperidin-1-yl)oxy.
Keywords: glycolysis; PFKFB3; pyridazinone; Warburg effect

Abstract
High-throughput screening of a small-molecule library identified a 5-triazolo-2-
arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-
kinase/2,6-bisphosphatase 3 (PFKFB3). Such inhibitors are of interest due to PFKFB3‟s
control of the important glycolytic pathway used by cancer cells to generate ATP. A series of
analogues was synthesized to study structure-activity relationships key to enzyme inhibition.
Changes to the triazolo or pyridazinone rings were not favoured, but limited-size
substitutions on the aryl ring provided modest increases in potency against the enzyme.
Selected analogues and literature-described inhibitors were evaluated for their ability to
suppress the glycolytic pathway, as detected by a decrease in lactate production, but none of
these compounds demonstrated such suppression at non-cytotoxic concentrations.
Corresponding author. Tel +64 9 373 7599: fax +64 9 373 7502
E-mail address: b.denny@auckland.ac.nz
1. Introduction
It is well known that, even in the presence of oxygen, many cancer cells generate ATP from
glucose primarily via glycolysis, rather than via the more efficient process of mitochondrial
oxidative phosphorylation (respiration). This switch of energy metabolism in cancer cells to
this process of „aerobic glycolysis‟ (c.f. normal cells favouring of glycolysis only under
anaerobic conditions) is known as the Warburg Effect1 and has been described as an
„emerging hallmark‟ of cancer.2

Despite the high rates of glycolysis maintained by malignant cells, which is exploited for 18F-
fluorodeoxyglucose-based PET imaging of tumours,3 it nevertheless seems paradoxical that
this significantly less efficient process (generating 2 ATP molecules per cycle) is preferred
over oxidative phosphorylation (generating 36 ATP molecules per cycle). Current thinking,
based on a 55 year-old hypothesis,4 holds that this process enables the utilization of
glycolytic metabolites as feedstock for other biosynthetic pathways, generating the organelles
and macromolecules necessary for new cell formation, as well as antioxidants that are used to
neutralize reactive oxygen species created during rapid cell proliferation.5
The rate-limiting enzyme in this process of conversion of glucose to pyruvate, the precursor
of anaerobic ATP production, is considered to be 6-phosphofructo-1-kinase (PFK-1),6 which
converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6-BP). The activity of
this enzyme is negatively regulated by ATP, but this regulation can be abrogated by the
allosteric activator fructose-2,6-bisphosphate (F2,6-BP),7,8 which is synthesised from F6P by
the family of 6-phosphofructo-2-kinase/2,6-bisphosphatase (PFKFB) bifunctional enzymes.
Of the four isoforms of this family, PFKFB3 is the most important in this context, with its
kinase/phosphatase ratio of about 740:1 being the highest.6,9 PFKFB3 (iPFK-2, PRG1) was
originally identified as a progestin-regulated gene,10 and is overexpressed in many cancer
types including colon, prostate, pancreatic , breast, thyroid, and leukemia.11 It is also induced
by hypoxia, which is common in the tumour environment, through functional HIF (hypoxia-
inducible factor) binding sites in the gene promoter.12 PFKFB3 activity is also regulated by
protein phosphorylation which occurs by a variety of signalling pathways relevant to cancer
e.g., AMPK, PKA, PKC and PI3K/AKT.13

PFKFB3 has thus been of interest as a target for cancer therapy, and this has been validated
by antisense and si/shRNA studies which show decreased glycolysis in knock-down cell lines
and tumor xenografts and inhibition of their proliferation.14-16 A number of compounds have
been evaluated as potential inhibitors. Perhaps the most well-studied is 3-(3-pyridinyl)-1-(4-
pyridinyl)-2-propen-1-one (3-PO; 1), which has been reported as a modest inhibitor of
isolated PFKFB3 enzyme (IC50 25±9 µM),7 but also to suppress glycolytic flux, decrease the
cellular concentrations of F2,6-BP and lactate, and to be cytostatic in both syngeneic and
xenograft mouse models.7 A more potent analogue (PFK-015) (2) has recently been reported
and extensively evaluated,17 although lactate secretion or other glycolytic endpoints were not
determined. A series of benzo[e]indoles (e.g., 3; PFK-095) has also been reported,18 but only
cell line inhibition data have been published on these.
A crystal structure of the human enzyme at 2.1 Å resolution showed the active pocket of the
6-phosphofructo-2-kinase domain existed in a rather rigid conformation, allowing
independent binding of both F6P and ATP.19 Computational screening of compounds using a
2.1Å resolution crystal structure of PFKFB3 identified the chromenone (4) as a competitive
inhibitor (IC50 2.97 μM) of F6P, with a Ki of 1.29 μM.20 A similarity search around 4 led to
the 5-fold more potent chromene analogue 5 (IC50 0.67 μM), which in HeLa cells resulted in
a 40% decrease in F2,6-BP levels and a >30% decrease in lactate secretion on exposure at the
IC50 for 8 h.20 A combined pharmacophore screening/structure-based docking approach
selected some compounds from the NCI Diversity Set II as likely actives,21 and a recent
patent22 described a number of 4-sulfonamido-2-hydroxybenzoic acids, reported to inhibit
PFKFKB3 with IC50s from 0.2 to 11 µM; e.g., 6 (KAN-222); IC50 (Kinase-Glo) 0.49 µM
(Figure 1).

In a search for drug-like inhibitors of PFKFB3, we conducted a high-throughput screen of
87,500 compounds (see section 4.2.3 for details) and identified the
triazolophenylpyridazinone 7 (Figure 1), which showed low micromolar activity (IC50 12
µM; Table 1) in a Kinase-Glo® assay for the enzyme. The synthesis of 7 has been
published,23 but no biology has been reported. In this paper, we report the synthesis of range
of analogues of 7, and their structure-activity relationships as inhibitors of PFKFB3.
2. Results and Discussion
2.1. Chemistry
The compounds of Tables 1-3 were prepared by the synthetic route outlined in Scheme 1. The
starting materials 4,5-dibromopyridazin-3(2H)-one (28),24 4,5-diiodopyridazin-3(2H)-one
(29)25 and 2-phenyl-4,5-dibromopyridazin-3(2H)-one (30)26 were known compounds, and 2-
phenyl-4,5-dichloropyridazin-3(2H)-one (31) was commercially available. The other required
analogues were accessed by one of three methods: (i) reaction of 4,5-dibromopyridazin-
3(2H)-one (28) and substituted benzyl bromides 32-34, using the conditions of Ryabtsova et
al.26 to give compounds 35-37 (Scheme 1A); (ii) reaction of the appropriate 4,5-
dihalopyridazin-3(2H)-ones 28 and 29 with aryl boronic acids 38 and 39, based on the
method of Chang et al.27 to give compounds 40, 41 Scheme 1B); or (iii) reaction of
appropriate arylhydrazines 42-44 with mucobromic acid (45) using the procedures of Devraj
et al.28 to give compounds 46-48, Scheme 1C). Finally, 2-benzyl-5-bromopyridazin-3(2H)-
one (50) was prepared23 from 2-benzyl-4,5-dibromopyridazin-3(2H)-one (49), whilst

Grignard treatment26 of 2-phenyl-4,5-dibromopyridazin-3(2H)-one (30) gave 2-phenyl-4-
isopropyl-5-bromopyridazin-3(2H)-one (51) (Scheme 1D).
The 2-substituted halopyridazin-3(2H)-ones were then converted to the corresponding azides
with NaN3 in DMF, using the conditions of Qian et al.29 and these were sufficiently clean to
be used without purification for the preparation of the compounds of Tables 1-3 (Scheme 2).
2.2. Structure-Activity Relationships
The ability of the compounds to inhibit human recombinant PFKFB3 was determined using a
Kinase-Glo® assay (Promega), which measures enzyme activity by quantifying the amount of
ATP remaining in solution following the kinase reaction. The presence of the GST tag had no
effect on the kinase activity (data not shown). The known compound 3-PO (1) had an IC50 of
26±1 µM in this assay, comparable with published data.7 However, our IC50 value for 2
(>1000 µM) is not in agreement with the published18 inhibitory data of 26% inhibition at 250
nM, possibly due to its very low solubility. Our IC50 value for 6 (27 µM) is also at variance
with the published19 value of 0.49 µM.
To explore the structure-activity relationships around compound 7, we first examined the
requirement for the acetyltriazole, which provides four polar atoms. Compounds 8-12
explored a number of alternatives for this moiety, including monocyclic (8), bicyclic (9, 10)
and acyclic (11, 12) units with various polar substituents as replacement for the ketone, but
all were inactive (Table 1).

Attention then turned to replacement of the bromine, which was seen as a potential metabolic
liability (Table 2). Analogues with the smaller chloro (13) and larger iodo (14) halogens were
about two-fold less active and equally active respectively (IC50s 26 and 13 µM), suggesting
the size of the substituent is relevant, which may indicate it contributes a halogen bond.30
Interestingly, the isopropyl- (15) and ethoxy- (16) substituted analogues were much less
active. Both of these substituents have similar bulk to an iodo group (the molar refractivities
of I, OEt and iPr are 13.9, 12.5 and 17.1, respectively), but very different electronic
properties (the Hammett sigma values for I, OEt and iPr being 0.18, -0.24 and -0.45,
respectively),31 suggesting that electron-donation to the pyridazone ring is not favoured. The
moderately electron-donating spiromorpholine substituent in 17 did give better (albeit low)
activity (IC50 55 µM). However, this compound has a significantly different substitution
pattern on the triazole ring (X = CO2Et, not COMe and Y = H, not Me), so that direct
comparison is difficult.
The compounds in Table 3 explore variations on the phenyl ring. The results for compounds
18-20 and the diaza analogue 21 show that an electron-deficient phenyl ring (possessing
electron-withdrawing groups or aza ring substitution) allows retention of potency. The
activity of the benzyl analogue 22 (IC50 3.4 µM) prompted the synthesis of the analogues 23-
25. The complete loss of activity with the des-bromo analogue 23 confirmed the results of
Table 2 above, in that a sizeable substituent in this position is required for activity. The 4-
iodo compound 24 retained activity, suggesting there is further bulk-tolerance in this region.
Compound 27, with a solubilising side chain at the 4-position, was thus prepared, but this was
100-fold less potent than 23. To evaluate whether this loss of potency related to steric or
polar attributes of this side chain, the phenethyl analogue 25 was prepared. This, with an IC50
of 2.6 µM, was the most potent compound prepared in the series, providing evidence of

considerable bulk-tolerance for lipophilic substituents in this position. However, the propyl
analogue 26 was 4-fold less potent than 25, suggesting there are steric limits to this binding
region.
To gain some insight into how 7 might interact with PFKFB3, a potential binding site and
mode was predicted using molecular docking along with that for the recently crystallised 4.20
When challenged with a docking cavity that covered both the kinase ATP and F6P binding
sites, the pose for 4 top ranked by the ChemPLP scoring function was located in the F6P site
with the acetyltriazole phenyl moiety in close proximity to the side chain of Arg98 consistent
with published data.20 The top ranked mode predicted for 7 was also located in the F6P site
(Figure 2), with the terminal acetyltriazole group in a similar location to that predicted for the
chromone-phenol of 4, while its terminal phenyl group was placed in channel accessing bulk
solvent defined by residues Thr48, Asn69, Arg75, Lys168, Ser171 and Tyr424.
The literature compounds 1 (3-PO) and 6 (KAN222), together with several of the more active
PFKFB3 inhibitors in the 2-arylpyridazinone series (compounds 7, 19, 20, 21 and 26) were
evaluated for their ability to inhibit glycolysis (and thus lactate production) in MDA-MB-231
cells at a concentration of 10 µM, using the Seahorse Bioscience XF24 cellular bioenergetics
analyzer platform. In this system, inhibition of the glycolytic pathway is characterised by a
decrease in the extracellular acidification rate (ECAR).32 At 10 M, neither 1 nor 7 showed
any effect on ECAR (Figure 3). In contrast, iodoacetate, which inhibits the glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) decreased ECAR rapidly by ~75%
(Figure 3) and concomitantly increased the oxygen consumption rate (OCR, suggesting the
stimulation of mitochondrial respiration) (data not shown). Some of the other 2-
arylpyridazinones effected a small decrease in ECAR (~30% after 60 min), but there was also

clear evidence of cytotoxicity (as shown by cell detachment) at this time. This could be due
to an off-target effect, as iodoacetate and other glycolytic inhibitors showed no signs of
cellular toxicity in the same time-frame.
3. Conclusions
The results show that the triazolophenylpyridazinone 7 is a modest inhibitor of PFKFB3, the
key rate-controlling enzyme in the glycolytic pathway whereby cancer cells generate ATP.
The structure-activity relationships for analogues of 7 show the presence of the triazolo and
pyridazinone rings is key to their inhibitory effect, and that modification on the pendant
phenyl ring could significantly improve this activity. However, none of these analogues, or
the known inhibitors 1 and 3, showed evidence of being able to decrease lactate production in
cells at non-cytotoxic concentrations. We are thus unable to conclude that the kinase-
inhibitory activities of any of these compounds are related to inhibition of cellular PFKFB3,
or whether the undoubted in vivo activity7,17 of the literature compounds 1 and 6 is due to a
different mechanism. However, only short-term effects of the compounds on glycolysis, i.e.
within 6 h of administration of the compound, were measured. Depending on the half-life of
F2,6BP, longer exposure to PFKFB3 inhibitors may be needed to measure an effect on the
glycolytic pathways in MDA-MB-231 cells.
4. Experimental Section
4.1. Chemistry
Unless otherwise stated, crude product mixtures were dissolved in a small volume of MeOH
and/or EtOAc, and this mixture was then adsorbed on a small amount of silica gel, prior to
purification by column chromatography on silica gel. Warming was often required to

dissolve azide starting materials in dioxane. The regiochemistry of final products was
confirmed by 1H NMR observation of the nOe interaction between the pyridazinonyl H and
triazolyl methyl (or analogous) groups. Experimental characterization data are provided for
all new compounds, and for any literature compounds previously lacking full preparative
and/or characterization data.
4.1.1 4,5-Dibromo-2-(4-iodobenzyl)pyridazin-3(2H)-one (35) (Scheme 1A). 4,5-
Dibromopyridazin-3(2H)-one (28) (7.753 g, 30.54 mmol, 1.00 equiv.), 4-iodobenzyl bromide
(70) (12.23 g, 41.19 mmol, 1.35 equiv.), K2CO3 (11.150 g, 80.67 mmol, 2.64 equiv.) and (n-
Bu)4NBr (310 mg, 0.96 mmol, 0.03 equiv.) were stirred overnight at 20 oC in CH3CN (100
mL), based on published conditions.26 After ~24 h the reaction mixture was concentrated to
dryness under reduced pressure, and the residue was re-suspended in CH2Cl2, filtered twice
through a pad of Celite® and the filtrate was concentrated to dryness under reduced pressure.
The product was re-precipitated from EtOAc:hexanes, to afford 35 (11.52 g, 80%) mp 144-
146 oC; 1H NMR (CDCl3)  7.79 (s, 1H), 7.67 (m, 2H), 7.19 (m, 2H), 5.24 (s, 2H; HRESIMS
81
calc. for C11H8 Br2IN2O m/z [M + H]+ 472.8002, found 472.8004, calc. for
81
79
C11H8 Br79BrIN2O m/z [M + H]+ 470.8022, found 470.8025, calc. for C11H8 Br2IN2O for
m/z [M + H]+ 468.8043, found 468.8044; TLC Rf = 0.49 (20% EtOAc:hexanes).
4.1.2. 4,5-Dibromo-2-phenethylpyridazin-3(2H)-one (36). Similar reaction of 28 (5.217 g,
20.55 mmol, 1.00 equiv.) and (2-bromoethyl)benzene (33) (4.00 mL, 29.29 mmol, 1.43
equiv.) for 24 h and workup gave a crude product that was re-precipitated from EtOAc to
give 36 (1.529 g, 21%) as a microcrystalline white solid: mp 125-126 °C; 1H NMR (CDCl3):
s, 1H), 7.29 (m, 2H), 7.23 (m, 3H), 4.39 (m, 2H), 3.10 (m, 2H)HRESIMS calc. for
C12H1081Br2N2NaO m/z [M + Na]+ 382.9012, found 382.9026, calc. for C12H1081Br79BrN2NaO

m/z [M + Na]+ 380.9032, found 380.9051, calc. for C12H1079Br2N2NaO for m/z [M + Na]+
378.9052, found 378.9066; TLC Rf = 0.58 (20% EtOAc:hexanes).
4.1.3. 4,5-Dibromo-2-(3-phenylpropyl)pyridazin-3(2H)-one (37). Similar reaction of 28
(5.845 g, 23.02 mmol, 1.00 equiv.) and 1-bromo-3-phenylpropane (34) (5.00 mL, 32.90
mmol, 1.43 equiv.) for 43 h and workup gave a product that was purified by column
chromatography on silica gel, eluting with 0–15% EtOAc:hexanes, to give 37 (6.177 g, 72%)
as a pale yellow oil: 1H NMR (CDCl3): s, 1H), 7.28 (m, 2H), 7.18 (m, 3H), 4.21 (m,
2H), 2.70 (m, 2H), 2.16 (m, 2H)HRESIMS calc. for C13H1281Br2N2NaO m/z [M + Na]+
396.9168, found 396.9177, calc. for C13H1281Br79BrN2NaO m/z [M + Na]+ 394.9189, found
394.9205, calc. for C13H1279Br2N2NaO for m/z [M + Na]+ 392.9209, found 392.9221; TLC Rf
= 0.52 (20% EtOAc:hexanes).
4.1.4. 4,5-Dibromo-2-(pyrimidin-5-yl)pyridazin-3(2H)-one (40) (Scheme 1B). Following
a published method,27 28 (10.123 g, 39.87 mmol, 1.00 equiv.), pyrimidine-5-boronic acid
(37) (24.90 g, 200.97 mmol, 5.40 equiv.), TEMPO (18.50 g, 118.40 mmol, 2.97 equiv.),
Cu(OAc)2.H2O (4.20 g, 21.04 mmol, 0.53 equiv) and powdered 4Å molecular sieves (6.7 g)
were combined in CH2Cl2 (300 mL). To this mixture was added pyridine (3.40 mL, 42.21
mmol, 1.06 equiv), and the resulting khaki suspension was stirred vigorously at 20 oC under
N2 for 15 min, and then under atmospheric air (via three needles in one of the septa capping
the reaction vessel). A further quantity of pyridine (15.00 mL, 186.22 mmol, 4.67 equiv.)
was then added, causing an immediate change in the colour of the reaction mixture to a blue
suspension, which became a green suspension after stirring overnight. The rate of stirring was
decreased and the mixture was stirred at 20 oC for 8.5 days. The reaction was then filtered
through a pad of Celite,® and the filtrate was diluted, washed with saturated aqueous NaOAc,

and extracted with CH2Cl2 (x 4). The combined organic extracts were washed (saturated
aqueous NaHCO3, dried, and concentrated to dryness under reduced pressure. The resulting
viscous red semi-solid was triturated with 20% EtOAc:hexanes to give 40 (9.993 g) as a ~1:1
mixture with an unidentified impurity (probably starting material judged by 1H NMR), and
was used without further purification: 1H NMR (CDCl3): 10.77 (v br s, 1H), 9.23 (s, 1H),
81
9.16 (s, 2H), 8.03 (s, 1H), 7.84 (s, 1H)HRESIMS calc. for C8H5 Br2N4O m/z [M + H]+
81
334.8784, found 334.8801, calc. for C8H5 Br79BrN4O m/z [M + H]+ 332.8804, found
79
332.8820, calc. for C8H5 Br2N4O m/z [M + H]+ 330.8825, found 330.8832; TLC Rf = 0.18,
0.20 (20% EtOAc:hexanes).
4.1.5. 4,5-Diiodo-2-phenylpyridazin-3(2H)-one (41). Similar reaction of 4,5-
diiodopyridazin-3(2H)-one (29) (5.211 g, 14.98 mmol, 1.00 equiv.) and phenylboronic acid
(38) (3.67 g, 30.06 mmol, 2.01 equiv) in CH2Cl2 (150 mL) for 9 days, then filtered through a
pad of Celite®. The filtrate was diluted and washed with saturated aqueous NH4OAc, and the
resulting mixture was then extracted with EtOAc (x 4), and the combined organics were
washed, dried and evaporated to give an orange oil. This was re-precipitated from
EtOAc:hexanes to afford 41 (1.752 g, 28%) as a yellow semi-crystalline solid: mp 122-124
°C; 1H NMR (CDCl3): 7.73 (s, 1H), 7.56 (m, 2H), 7.49 (m, 2H), 7.43 (m, 1H)HRESIMS
calc. for C10H7I2N2O m/z [M + H]+ 424.8642, found 424.8653; TLC Rf = 0.52 (20%
EtOAc:hexanes).
4.1.6. 4-(4,5-Dibromo-6-oxopyridazin-1(6H)-yl)benzonitrile (46) (Scheme 1C). A
suspension of 4-hydrazinylbenzonitrile hydrochloride (42) (9.360 g, 55.19 mmol, 1.25 equiv.)
in 6M HCl (220 mL) was added to a solution/suspension of mucobromic acid (45) (11.430 g,
44.32 mmol, 1.00 equiv.) in 6M HCl (300 mL), and the resulting suspension was stirred at

65-95°C for 18 h, when TLC analysis indicated the reaction was complete.28 The viscous
canary yellow suspension was cooled to 20 oC and the solid was collected by filtration and
washed sequentially with water and hexanes. The dried crude product was triturated with
MeOH to furnish 46 (10.047 g, 64%) as an amorphous off-white solid: mp 263-265 °C; 1H
NMR (DMSO-d6): s, 1H), 8.02 (d, J = 8.53 Hz, 2H), 7.80 (d, J = 8.53 Hz,
81
2H)HRESIMS calc. for C11H5 Br2N3NaO m/z [M + Na]+ 379.8651, found 379.8649, calc.
81
for C11H5 Br79BrN3NaO m/z [M + Na]+ 377.8671, found 377.8661, calc. for
79
C11H5 Br2N3NaO for m/z [M + Na]+ 375.8692, found 375.8694; TLC Rf = 0.38 (20%
EtOAc:hexanes).
4.1.7. 4,5-Dibromo-2-(4-(trifluoromethoxy)phenyl)pyridazin-3(2H)-one (47). Similar
reaction of a suspension of (4-(trifluoromethoxy)phenyl)hydrazine hydrochloride (43)
(15.330 g, 67.06 mmol, 1.21 equiv.) in 6M HCl (100 mL) and 45 (14.300 g, 55.45 mmol,
1.00 equiv.) in 6M HCl (360 mL) for 30 min. More 6M HCl (250 mL), H2O (100 mL) and a
portion of cHCl (50 mL) were then added and the reaction was continued for a further 16 h at
85-100°C, when TLC showed the reaction was compete. The cooled mixture was filtered to
give a dark orange solid that was washed with Et2O and triturated with hexanes to give 47
(4.327 g) as an amorphous white solid: mp 138-140 °C. Further workup of the Et2O layer
gave more 47 (5.375 g) for an overall yield of 35%.; 1H NMR (CDCl3): s, 1H), 7.66
81
(m, 2H), 7.33 (m, 2H)HRESIMS calc. for C11H5 Br2F3N2NaO2 m/z [M + Na]+ 438.8522,
81
found 438.8556, calc. for C11H5 Br79BrF3N2NaO2 m/z [M + Na]+ 436.8542, found 436.8568,
79
calc. for C11H5 Br2F3N2NaO2 m/z [M + Na]+ 434.8562, found 434.8586; TLC Rf = 0.56
(20% EtOAc:hexanes).

4.1.8. 4,5-Dibromo-2-(4-chlorophenyl)pyridazin-3(2H)-one (48). Similar reaction of (4-
chlorophenyl)hydrazine hydrochloride (44) (9.80 g, 54.74 mmol, 1.20 equiv.) in 6M HCl
(250 mL) and 45 (11.763 g, 45.61 mmol, 1.00 equiv.) in 6M HCl (300 mL) for 32 h and
workup gave a solid that was collected by filtration and triturated with Et2O to give 48
(11.797 g, 71%) as an apricot-tan solid: mp 181-182 °C; 1H NMR (CDCl3): s, 1H),
81
7.70 (m, 2H), 7.45 (m, 2H)HRESIMS calc. for C10H5 Br2ClN2NaO m/z [M + Na]+
81
388.8308, found 388.8274, calc. for C10H5 Br79BrClN2NaO m/z [M + Na]+ 386.8328, found
79
386.8303, calc. for C10H5 Br2ClN2NaO m/z [M + Na]+ 384.8349, found 384.8324; TLC Rf =
0.59 (20% EtOAc:hexanes).
4.1.9. 2-Phenyl-4-isopropyl-5-bromopyridazin-3(2H)-one (51) (Scheme 1D). Using a
published method,26 a solution of 2-phenyl-4,5-dibromopyridazin-3(2H)-one (30) (1.043 g,
3.16 mmol, 1.00 equiv.) in THF (23 mL) was cooled to –20 °C and treated with one portion
of (i-Pr)MgCl (2M in THF, 1.58 mL, 3.16 mmol, 1.00 equiv.) to give a very dark red-brown
solution. After exactly 3 min the reaction was quenched by addition of water (0.570 uL,
31.61 mmol, 10.00 equiv.), allowed to warm to 20 oC over 145 min, then poured into
saturated aqueous NH4Cl solution. The mixture was extracted with CH2Cl2 (x 4), and the
combined extracts were dried (MgSO4) and concentrated to dryness under reduced pressure.
The dark orange residue was purified by column chromatography on silica gel, eluting with
0–30% EtOAc:hexanes, to give 51 (0.229 g, 25%) as a yellow oil: 1H NMR (CDCl3): 7.91 (s,
1H), 7.54 (m, 2H), 7.47 (m, 2H), 7.39 (m, 1H), 3.48 (sept, J = 6.9 Hz, 1H), 1.40 (d, J = 7.0
Hz, 6H); LRMS (APCI+): m/z [M + H]+ 295.4 (81Br, 80%), 293.4 (79Br, 100%); TLC Rf =
0.70 (20% EtOAc:hexanes).
4.1.10. General procedure for the preparation of azides (Scheme 1)

Based on a published method,29 pyridazinones and NaN3 were combined in dry DMF, and the
resulting solution stirred at 20 oC under air (via two needles in the septum capping the
reaction vessel). When analysis of an aliquot of the reaction by TLC (azides were generally
more polar on TLC than their bromide precursors) and LRMS indicated the reaction was
complete – within a few hours (typically after 2-3 h) – the reaction mixture was concentrated
to dryness under reduced pressure, and the resulting oily residue triturated with water to give
an amorphous solid. This material was collected by filtration, washed with hexanes, and
finally dried under high vacuum to give the azide as an amorphous solid. Azides were stored
in the dark under N2 at ~4 °C, and used as soon as possible. Due to safety concerns, melting-
point measurements of these compounds were not obtained. In this way were prepared:
4.1.11. 5-Azido-4-bromo-2-phenylpyridazin-3(2H)-one (52) as an amorphous tan solid,
91% yield; 1H NMR (CDCl3):  7.74 (s, 1H), 7.56 (m, 2H), 7.48 (m, 2H), 7.41 (m, 1H);
LRMS (APCI+): m/z [M + H]+ 294.3 (81Br, 40%), 292.3 (79Br, 40%); TLC Rf = 0.36 (20%
EtOAc:hexanes).
4.1.12. 5-Azido-4-chloro-2-phenylpyridazin-3(2H)-one (53) as an amorphous yellow-
orange solid, 92% yield; 1H NMR (CDCl3):  7.75 (s, 1H), 7.57 (m, 2H), 7.48 (m, 2H), 7.42
(m, 1H); LRMS (APCI+): m/z [M + H]+ 250.3 (81Br, 35%), 248.3 (79Br, 100%); TLC Rf =
0.38 (20% EtOAc:hexanes).
4.1.13. 5-Azido-4-iodo-2-phenylpyridazin-3(2H)-one (54) as an amorphous beige-yellow
solid, ~ 100% yield; 1H NMR (CDCl3): δ 7.66 (s, 1H), 7.56 (m, 2H), 7.47 (m, 2H), 7.41 (m,
1H); LRMS (APCI+): m/z [M + H]+ 340.4 (65%); TLC Rf = 0.35 (20% EtOAc:hexanes).

4.1.14. 5-azido-4-isopropyl-2-phenylpyridazin-3(2H)-one (55) as a pale orange oil, 52%
yield; 1H NMR (CDCl3): δ 7.83 (s, 1H), 7.55 (m, 2H), 7.46 (m, 2H), 7.38 (m, 1H), 3.39 (sept,
J = 7.0 Hz, 1H), 1.32 (d, J = 7.0 Hz, 6H); LRMS (APCI+): m/z [M + H]+ 256.5 (30%); TLC
Rf = 0.50 (20% EtOAc:hexanes).
4.1.15. 4-(4-Azido-5-bromo-6-oxopyridazin-1(6H)-yl)benzonitrile (56) as an amorphous
dull yellow solid, ~ 100% yield; 1H NMR (CDCl3):  7.87 (m, 5H); LRMS (APCI+): m/z [M
+ H]+ 319 (81Br, 10%), 317 (79Br, 10%), m/z [(M – N3 + OH]+ 293.3 (81Br, 90%), 291.3 (79Br,
100%); TLC Rf = 0.11 (20% EtOAc:hexanes), 0.70 (50% EtOAc:hexanes)
4.1.16. 5-Azido-4-bromo-2-(4-(trifluoromethoxy)phenyl)pyridazin-3(2H)-one (57) as an
amorphous dull yellow solid, 71% yield; 1H NMR (CDCl3): δ 7.75 (s, 1H), 7.65 (m, 2H),
7.32 (m, 2H); LRMS (APCI+): m/z [M + H]+ 378.3 (81Br, 80%), 376 (79Br, 80%); TLC Rf =
0.35 (20% EtOAc:hexanes).
4.12.17. 5-Azido-4-bromo-2-(4-chlorophenyl)pyridazin-3(2H)-one (58) as an amorphous
yellow-tan solid, 94% yield; 1H NMR (CDCl3): δ 7.74 (s, 1H), 7.55 (m, 2H), 7.44 (m, 2H);
LRMS (APCI+): m/z [M + H]+ 328.3 (81Br, 60%), 326 (79Br, 40%); TLC Rf = 0.39 (20%
EtOAc:hexanes).
4.1.18. 5-Azido-4-bromo-2-(pyrimidin-5-yl)pyridazin-3(2H)-one (59) as an amorphous
solid, 80% yield (estimated as ~70% pure by comparison of 1H NMR integral ratios); 1H
NMR (CDCl3): δ 9.22 (s, 1H), 9.15 (s, 2H), 7.82 (s, 1H); LRMS (APCI+): m/z [M + H]+ 296
(81Br, 55%), 294 (79Br, 60%); TLC Rf = 0.03 (20% EtOAc:hexanes), 0.21 (40%
EtOAc:hexanes).

4.1.19. 5-Azido-2-benzyl-4-bromopyridazin-3(2H)-one (60) as an amorphous butter
yellow solid, 76% yield; 1H NMR (CDCl3): δ 7.61 (s, 1H), 7.44 (m, 2H), 7.32 (m, 3H), 5.33
(s, 2H); LRMS (APCI+): m/z [M + H]+ 308 (81Br, 50%), 306.2 (79Br, 55%); TLC Rf = 0.39
(20% EtOAc:hexanes).
4.1.20. 5-Azido-2-benzylpyridazin-3(2H)-one (61) as a crystalline yellow solid, ~100%
yield (estimated as ~65% pure by comparison of 1H NMR integral ratios) and was used
without further purification. TLC Rf = 0.24 (20% EtOAc:hexanes).
4.1.21. 5-Azido-4-bromo-2-(4-iodobenzyl)pyridazin-3(2H)-one (62) as an amorphous
beige-yellow solid, 76% yield; 1H NMR (CDCl3): δ 7.66 (m, 2H), 7.60 (s, 1H), 7.19 (m, 2H),
5.26 (s, 2H); LRMS (APCI+): m/z [M + H]+ 434 (81Br, 100%), 432.5 (79Br, 100%); TLC Rf =
0.35 (20% EtOAc:hexanes).
4.1.22. 5-Azido-4-bromo-2-phenethylpyridazin-3(2H)-one (63) as an off-white amorphous
solid, ~100% yield; 1H NMR (CDCl3): δ 7.57 (s, 1H), 7.30 (m, 2H), 7.23 (m, 3H), 4.42 (m,
2H), 3.10 (app t, J = 7.8 Hz, 2H); LRMS (APCI+): m/z [M + H]+ 322.4 (81Br, 100%), 320
(79Br, 100%); TLC Rf = 0.38 (20% EtOAc:hexanes).
4.1.23. 5-Azido-4-bromo-2-(3-phenylpropyl)pyridazin-3(2H)-one (64) as a yellow
crystalline solid, ~100% yield; 1H NMR (CDCl3): δ 7.58 (s, 1H), 7.28 (m, 2H), 7.18 (m, 3H),
4.23 (app t, J = 7.2 Hz, 2H), 2.69 (app t, J = 7.6 Hz, 2H), 2.15 (app pent., J = 7.4 Hz, 2H);
LRMS (APCI+): m/z [M + H]+ 336 (81Br, 90%), 334.5 (79Br, 100%); TLC Rf = 0.34 (20%
EtOAc:hexanes).

4.1.24. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-phenylpyridazin-3(2H)-
one (7) (Table 1). Using a literature general method,23 2,4-pentanedione (0.33 mL, 3.21
mmol, 0.99 equiv.) and triethylamine (0.45 mL, 3.23 mmol, 1.00 equiv.) were combined, and
cooled to 0 °C (bath temperature). To the resulting solution was added, dropwise, a solution
of azide 52 (944 mg, 3.23 mmol, 1.00 equiv.) in dry dioxane (4.00 mL), and the resulting
mixture was stirred at 0 °C for 9 min, after which it became too viscous. The reaction
temperature was maintained between 5-10 °C for the 1 h, then warmed to 20 oC, diluted with
Et2O, and then filtered. The dried solid was purified by column chromatography on silica
gel, eluting with 0–50% EtOAc:hexanes, followed by re-precipation from EtOAc:hexanes, to
give 7 (366 mg, 30%) as a white microcrystalline solid: mp 208-210 °C (lit.19) 206-207 °C);
1H NMR (CDCl3):  7.89 (s, 1H), 7.66 (m, 2H), 7.52 (m, 3H), 2.79 (s, 3H), 2.66 (s, 3H);
HRESIMS calcd. for C15H1281BrN5NaO2 m/z [M + Na]+ 398.0047, found 398.0043; calc. for
C15H1279BrN5NaO2 m/z [M + Na]+ 396.0067, found 396.0064; TLC Rf = 0.10 (20%
EtOAc:hexanes), 0.67 (50% EtOAc:hexanes); HPLC purity: 99.9%.
4.1.25. 4-Bromo-5-morpholino-2-phenylpyridazin-3(2H)-one (8). Following a general
literature method,33 4,5-dibromo-2-phenylpyridazin-3(2H)-one (30) (1.067 g, 3.23 mmol,
1.00 equiv.) and morpholine (0.590 mL, 6.75 mmol, 2.09 equiv.) were heated under reflux in
dry EtOH (33.0 mL) for 19 h. The reaction was cooled to 20 oC and partially concentrated
under reduced pressure to give 8 (677 mg, 62%) as off-white crystals which was collected by
filtration: mp 157-158 °C (lit.27 151-152 °C); 1H NMR (CDCl3):  7.66 (s, 1H), 7.57 (m, 2H),
7.46 (m, 2H), 7.38 (m, 1H), 3.89 (m, 4H), 3.47 (m, 4H); HRESIMS calcd. for
C14H1481BrN3NaO2 m/z [M + Na]+ 360.1047, found 360.1067, calcd. for C14H1479BrN3NaO2

m/z [M + Na]+ 358.0162, found 358.0183; TLC Rf = 0.27 (40% EtOAc:hexanes); HPLC
purity: 99.9%.
4.1.26. 2-(5-Bromo-6-oxo-1-phenyl-1,6-dihydropyridazin-4-yl)-1,2,3,4-
tetrahydroisoquinoline-5-carbonitrile (9). A similar reaction of 30 (1.048 g, 3.18 mmol,
1.00 equiv.) and 1,2,3,4-tetrahydroisoquinoline-5-carbonitrile (0.58 g, 3.69 mmol, 1.16
equiv) under reflux in EtOH (34 mL)for 16 h. Filtration while hot and purification of the
precipitate by re-precipitation from MeOH gave 9 (41 mg). Further concentration of the
filtrate and purification gave additional material (180 mg total, 14%): mp 172-174 °C; 1H
NMR (CDCl3):  8.08 (s, 1H), 7.75 (m, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.50 (m, 4H), 7.43 (m,
2H), 4.75 (s, 2H), 3.84 (t, J = 5.8 Hz, 2H), 3.17 (t, J = 5.7 Hz, 2H); HRESIMS calcd. for
C20H1581BrN4NaO m/z [M + Na]+ 431.0302, found 431.0263, calcd. for C20H1581BrN4NaO
m/z [M + Na]+ 429.0321, found 429.0279; TLC Rf = 0.29 (40% EtOAc:hexanes); HPLC
purity: 98.0%.
4.1.27. 4-Bromo-2-phenyl-5-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pyridazin-3(2H)-one
(10). Following a general literature method,34 a solution of 30 (540 mg, 1.64 mmol, 1.00
equiv.) in DMF (50 mL) was treated with anhydrous Cs2CO3 (824 mg, 2.51 mmol, 1.53
equiv.) and 2-oxa-6-azaspiro[3.3]heptan-6-ium oxalate35 (720 mg, 2.50 mmol, 1.53 equiv.)
and the mixture was stirred at 50 °C for 16 h, then at 100 °C for 4 h. The cooled reaction
mixture was diluted with brine and extracted with EtOAc (x 5). The combined organic
extracts were washed with water (x 3) and brine, dried (MgSO4) and evaporated under
reduced pressure. The residue was triturated with EtOAc/hexanes to give 10 (246 mg, 45%
yield) as fine off-white crystals: mp 214-217 °C; 1H NMR (CDCl3):  7.54 (m, 2H), 7.43 (m,
2H), 7.34 (m, 1H), 7.23 (s, 1H), 4.85 (s, 4H), 4.62 (s, 4H); HRESIMS calcd. for

C15H1481BrN3NaO2 m/z [M + Na]+ 372.0412, found 372.0155; calcd. for C15H1479BrN3NaO2
m/z [M + Na]+ 370.0162, found 370.0157. TLC Rf = 0.05 (50% EtOAc:hexanes); HPLC
purity: 98.1%.
4.1.28. 2-((5-Bromo-6-oxo-1-phenyl-1,6-dihydropyridazin-4-yl)amino)acetamide (11).
Similar reaction of 30 (1.005 g, 3.05 mmol, 1.00 equiv.), glycinamide hydrochloride (0.70 g,
6.31 mol, 2.07 equiv), and pyridine (0.51 mL, 6.31 mmol, 2.07 equiv.) was heated under
reflux in a mixture of EtOH (41 mL) and DMF (5 mL). After 48 h starting material remained,
so more glycinamide hydrochloride (2.98 equiv.) and NEt3 (2.60 mL, 18.65 mmol, 6.12
equiv.) were added and the mixture was heated under reflux for a further 16 h. The cooled
mixture was filtered through a pad of Celite® and the filtrate was diluted with brine, extracted
with CH2Cl2 (x 3) and the combined extracts were washed with brine, dried (MgSO4) and
evaporated under reduced pressure. The residue was re-precipitated from MeOH:EtOAc to
give 11 (77 mg, 8%) as an amorphous off-white solid: mp 216-219 °C; 1H NMR (CDCl3): 
7.75 (s, 1H), 7.56 (br s, 1H), 7.47 (m, 4H), 7.39 (m, 1H), 7.24 (br s, 1H), 6.71 (m, 1H), 4.05
(d, J = 6.20 Hz, 2H); HRESIMS calcd. for C12H1181BrN4NaO2 m/z [M + Na]+ 346.9938,
found 346.9947, calcd. for C12H1179BrN4NaO2 m/z [M + Na]+ 344.9958, found 344.9963;
TLC Rf = 0.22 (10% MeOH:EtOAc); HPLC purity: 98.7%.
4.1.29. 4-Bromo-2-phenyl-5-(((tetrahydrofuran-2-yl)methyl)amino)pyridazin-3(2H)-one
(12). Similar reaction of 30 (1.073 g, 3.25 mmol, 1.00 equiv.) and tetrahydrofurfuryl amine
(0.70 mL, 6.78 mmol, 2.09 equiv.) were heated under reflux in EtOH (32 mL) for 16 h, then
cooled and purified by column column chromatography on silica gel, eluting with 0–60%
EtOAc:hexanes. Re-precipitation of the major product from EtOAc:hexanes gave 12 (785
mg, 67%) as an off-white crystalline solid: mp 133-134 °C; 1H NMR (CDCl3):  7.70 (s, 1H),

7.57 (m, 2H), 7.44 (m, 2H), 7.35 (m, 1H), 5.23 (m, 1H), 4.14 (m, 1H), 3.93 (m, 1H), 3.83 (m,
1H), 3.57 (ddd, J = 9.8, 6.2, 3.5 Hz, 1H), 3.37 (m, 1H), 2.08 (m, 1H), 1.98 (m, 2H), 1.67 (m,
1H); HRESIMS calcd. for C15H1681BrN3NaO2 m/z [M + Na]+ 374.0298, found 374.0299,
calcd. for C15H1679BrN3NaO2 m/z [M + Na]+ 372.0318, found 372.0319; TLC Rf = 0.18 (50%
EtOAc:hexanes); HPLC purity: 99.7%.
4.1.30. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-chloro-2-phenylpyridazin-3(2H)-
one (13) (Table 2). Using a literature general method,23 2,4-pentanedione (0.72 mL, 7.01
mmol, 1.00 equiv.), triethylamine (0.98 mL, 7.03 mmol, 1.00 equiv.) and azide 53 (1.746 g,
7.05 mmol, 1.00 equiv.) in dry dioxane (8.8 mL) were reacted at between 5-10 oC for 60 min,
then warmed to 20 oC, filtered and washed with Et2O. Purification by column column
chromatography on silica gel, eluting with 0–30% EtOAc:hexanes, gave 13 as a an
amorphous white solid (0.612 g, 26%): mp 213-216 °C; 1H NMR (CDCl3):  8.01 (s, 1H),
7.66 (m, 2H), 7.55 (m, 2H), 7.49 (m, 1H), 2.78 (s, 3H), 2.65 (s, 3H); HRESIMS calcd. for
C15H1237ClN5NaO2 m/z [M + Na]+ 354.0543, found 354.0555; calc. for C15H1235ClN5NaO2
m/z [M + Na]+ 352.0572, found 352.0581; TLC Rf = 0.28 (30% EtOAc:hexanes); HPLC
purity: 99.7%.
4.1.31. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-iodo-2-phenylpyridazin-3(2H)-one
(14). 2,4-Pentanedione (0.33 mL, 3.21 mmol, 1.01 equiv.), triethylamine (0.44 mL, 3.16
mmol, 0.99 equiv.) and azide 54 (1.077 g, 3.18 mmol, 1.00 equiv.) were similarly reacted in
dry dioxane (35.0 mL) at 5-10 oC for 155 min, then filtered through a pad of Celite® and the
filtrate concentrated to dryness under reduced pressure. The residue was dried and then
triturated with cold MeOH to afford a solid that was collected by filtration and washed with
hexanes to give 14 (0.517 g, 40%) as a semi-crystalline cream solid: mp 218-220 °C; 1H

NMR (CDCl3):  7.70 (s, 1H), 7.65 (m, 2H), 7.54 (m, 2H), 7.49 (m, 1H), 2.79 (s, 3H), 2.67
(s, 3H); HRESIMS calcd. for C15H12IN5NaO2 m/z [M + Na]+ 443.9928, found 443.9931; calc.
for C15H13IN5O2 m/z [M + H]+ 422.0108, found 422.0129; TLC Rf = 0.10 (20%
EtOAc:hexanes); HPLC purity: 97.1%.
4.1.32. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-isopropyl-2-phenylpyridazin-
3(2H)-one (15). 2,4-Pentanedione (0.46 mL, 4,5 mmol, 11 equiv.), triethylamine (0.20 mL,
1.40 mmol , 2.5 equiv.) and azide 55 (103 mg, 0.40 mmol, 1.00 equiv.) in dry dioxane (20.0
mL) were similarly reacted at 75 oC for 10 days, then evaporated to dryness under reduced
pressure. The residue was purified by column chromatography on silica gel, eluting with 0–
40% EtOAc:hexanes. The resulting product was further purified by trituration with cold
MeOH and filtration of the resulting suspension through a pad of Celite®, followed by
concentration of the filtrate to dryness under reduced pressure, yielding 15 (86 mg, 63%) as
an amorphous brittle solid: mp 76-79 °C; 1H NMR (CDCl3):  7.69 (s, 1H), 7.63 (m, 2H),
7.52 (m, 2H), 7.45 (m, 1H), 2.78 (s, 3H), 2.64 (sep, J = 6.9 Hz, 1H), 2.61 (s, 3H), 1.30 (d, J =
6.9 Hz, 6H); HRESIMS calcd. for C18H19N5NaO2 m/z [M + Na]+ 360.1431, found 360.1428;
calc. for C18H20N5O2 m/z [M + Na]+ 338.1612, found 338.1623; TLC Rf = 0.61 (40%
EtOAc:hexanes); HPLC purity: 96.6%.
4.1.33. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-ethoxy-2-phenylpyridazin-3(2H)-
one (16). Based on a literature general method,36 2,4-pentanedione (0.21 mL, 2.06 mmol,
1.00 equiv.), azide 53 (511 mg, 2.06 mmol, 1.00 equiv.), and K2CO3 (903 mg, 6.53 mmol,
3.17 equiv.) were combined in dry EtOH (7.7 mL) and heated at 70-75°C for 60 min, then
concentrated to dryness under reduced pressure. The residue was neutralized with 10%
aqueous HCl solution, and the resulting mixture was extracted with EtOAc (x 4). The

combined organic extracts were dried (MgSO4), and evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel, eluting with 0–60%
EtOAc:hexanes, followed by re-precipitation of product from EtOAc:hexanes, to give 16 (8
mg, 1%) as an amorphous white solid: mp 100-102 °C; 1H NMR (CDCl3):  7.97 (s, 1H),
7.61 (m, 2H), 7.53 (m, 2H), 7.46 (m, 1H), 4.72 (q, J = 7.1 Hz, 2H), 2.77 (s, 3H), 2.59 (s, 3H),
1.27 (t, J = 7.0 Hz, 3H); HRESIMS calcd. for C17H17N5NaO3 m/z [M + Na]+ 362.1224, found
362.1221; TLC Rf = 0.67 (50% EtOAc:hexanes); HPLC purity: 99.6%.
4.1.34. Ethyl 1-(6-oxo-1-phenyl-5-(2-oxa-6-azaspiro[3.3]heptan-6-yl)-1,6-
dihydropyridazin-4-yl)-1H-1,2,3-triazole-4-carboxylate (17). Based on a general literature
method,37 ethyl propynoate (0.20 mL, 1.98 mmol, 1.20 equiv.), N,N-diisopropylethylamine
(0.60 mL, 3.44 mmol, 2.01 equiv.) and CuI (16 mg, 0.085 mmol, 0.05 equiv.) were added to a
solution of azide 53 (0.407 g, 1.64 mmol, 1.00 equiv.) in CH3CN (4.00 mL), and the resulting
dark orange-brown solution was stirred at 20 oC for 110 min, then concentrated to dryness
under reduced pressure. The residue was washed with hot MeOH, and the insoluble yellow
solid collected by filtration. MS analysis confirmed this material contained the desired
triazole product, whilst 1H NMR analysis showed it comprised a ~3:1 mixture of isomers,
favouring the desired product.
A solution of crude triazole (182 mg, 0.53 mmol, 1.00 equiv.) in DMF (17.00 mL) was
treated successively with N,N-diisopropylethylamine (0.40 mL, 2.30 mmol, 4.37 equiv.) and
2-oxa-6-azaspiro[3.3]heptan-6-ium oxalate34 (311 mg, 1.08 mmol, 2.05 equiv.) and the
resulting mixture was stirred at 20 oC for 16 h, then concentrated to dryness under reduced
pressure. The residue was re-suspended in MeOH, filtered through a pad of Celite®, and the
filtrate was purified by column column chromatography on silica gel, eluting with 0–50%

EtOAc:hexanes followed by re-precipitation (twice) from EtOAc:hexanes, to give 17 (51 mg,
24%) as an amorphous white solid: mp 238-240 °C; 1H NMR (CDCl3):  8.30 (s, 1H), 7.50
(m, 5H), 7.41 (m, 1H), 4.67 (br s, 4H), 4.51 (q, J = 7.15 Hz, 2H), 4.31 (br s, 4H), 1.47 (t, J =
7.14 Hz, 3H); HRESIMS calcd. for C20H20N6NaO4 m/z [M + Na]+ 431.1438, found 431.1433,
calcd. for C20H21N6O4 m/z [M + H]+ 409.1619, found 409.1622; TLC Rf = 0.13 (50%
EtOAc:hexanes); HPLC purity: 97.1%.
4.1.35. 4-(4-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-5-bromo-6-oxopyridazin-1(6H)-
yl)benzonitrile (18) (Table 3). Similar reaction of 2,4-pentanedione (0.26 mL, 2.53 mmol,
0.99 equiv.), triethylamine (0.36 mL, 2.58 mmol, 1.01 equiv.) and azide 56 (811 mg, 2.56
mmol, 1.00 equiv.) in dry dioxane (45.2 mL) for 50 min, followed by filtration through
Celite®, evaporation of the filtrate and purification of the residue by column chromatography
on silica gel, eluting with 0–50% EtOAc:hexanes, followed by re-precipitation from
EtOAc:hexanes, gave 18 (126 mg total, 12%) as an amorphous yellow solid: mp 222-224 °C;
1H NMR (CDCl3):  7.94 (s, 1H), 7.90 (m, 2H), 7.84 (m, 2H), 2.79 (s, 3H), 2.67 (s, 3H);
HRESIMS calcd. for C16H1181BrN6NaO2 m/z [M + Na]+ 422.9999, found 422.9992, calcd. for
C16H1179BrN6NaO2 m/z [M + Na]+ 421.0019 found 421.0007; TLC Rf = 0.40 (40%
EtOAc:hexanes); HPLC purity: 98.7%.
4.1.36. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(4-
(trifluoromethoxy)phenyl)-pyridazin-3(2H)-one (19). Similar reaction of 2,4-pentanedione
(0.38 mL, 3.70 mmol, 1.02 equiv.), triethylamine (0.51 mL, 3.66 mmol, 1.00 equiv.) and
azide 57 (1.370 g, 3.64 mmol, 1.00 equiv.) in dry dioxane (22.0 mL) for 90 min, followed by
filtration through Celite®, evaporation of the filtrate and trituration of the residue with cold
MeOH gave 19 (0.338 g, 20%) as semi-crystalline yellow solid: mp 158-160 °C; 1H NMR

(CDCl3):  7.91 (s, 1H), 7.75 (m, 2H), 7.38 (m, 2H), 2.79 (s, 3H), 2.66 (s, 3H); HRESIMS
calcd. for C16H1181BrF3N5NaO3 m/z [M + Na]+ 481.9870, found 481.9857, calcd. for
C16H1179BrF3N5NaO3 m/z [M + Na]+ 479.9890, found 479.9875; TLC Rf = 0.20 (20%
EtOAc:hexanes); HPLC purity: 100%.
4.1.37. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(4-
chlorophenyl)pyridazin-3(2H)-one (20). 2,4-Pentanedione (0.32 mL, 3.12 mmol, 0.97
equiv.), triethylamine (0.44 mL, 3.16 mmol, 1.01 equiv.) and azide 58 (1.022 g, 3.13 mmol,
1.00 equiv.) in dry dioxane (22.0 mL) were reacted at 5-10 oC for 60 min, then warmed to 20
oC and filtered through a pad of Celite®. The filtrate was evaporated under reduced pressure
and the residue was purified by column chromatography on silica gel, eluting with 0–40%
EtOAc:hexanes, followed by re-precipitation from EtOAc:hexanes, to give 20 (0.056 g, 4%)
as an amorphous white solid: mp 188-190 °C; 1H NMR (CDCl3):  7.89 (s, 1H), 7.65 (m,
37
2H), 7.51 (m, 2H), 2.78 (s, 3H), 2.66 (s, 3H); HRESIMS calcd. for C15H11N5O2 Cl81BrNa
37
m/z [M + Na]+ 433.9628, found 433.9622, calcd. for C15H11N5O2 Cl79BrNa and
35
C15H11N5O2 Cl81BrNa m/z [M + Na]+ 431.9656, found 431.9642, calcd. for
35
C15H11N5O2 Cl79BrNa m/z [M + Na]+ 429.9677, found 429.9664; TLC Rf = 0.42 (30%
EtOAc:hexanes); HPLC purity: 99.5%.
4.1.38. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(pyrimidin-5-
yl)pyridazin-3(2H)-one (21). Similar reaction of 2,4-pentanedione (1.32 mL, 12.86 mmol,
1.00 equiv.), triethylamine (1.80 mL, 12.91 mmol, 1.00 equiv.) and azide 59 (3.793 g, 12.90
mmol, 1.00 equiv.) in dry dioxane (23 mL) for 105 min gave a heterogeneous mixture that
was filtered. The filtrate was diluted with hexanes, concentrated to half its original volume,
then filtered through Celite®. The filtrate was evaporated and purified by column

chromatography on silica gel, eluting with 0-50% EtOAc:hexanes, followed by re-
precipitation from EtOAc:hexanes, to give 21 (0.870 g, 12%) as an amorphous yellow solid:
mp 174-177 °C; 1H NMR (CDCl3):  9.29 (s, 1H), 9.24 (s, 2H), 7.99 (s, 1H), 2.79 (s, 3H),
2.67 (s, 3H); HRESIMS calcd. for C13H1081BrN7NaO2 m/z [M + Na]+ 399.9952, found
399.9941, C13H1079BrN7NaO2 m/z [M + Na]+ 397.9972, found 397.9958; TLC Rf = 0.20
(50% EtOAc:hexanes); HPLC purity: 98.2%.
4.1.39. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-2-benzyl-4-bromopyridazin-3(2H)-
one (22). Similar reaction of 2,4-pentanedione (0.34 mL, 3.31 mmol, 1.02 equiv.),
triethylamine (0.45 mL, 3.23 mmol, 0.99 equiv.) and azide 61 (0.998 g, 3.26 mmol, 1.00
equiv.) in dry dioxane (17.0 mL) for 105 min was followed by filtration of the mixture. The
filtrate was evaporated under reduced pressure and triturated with cold MeOH to give 22
(0.417 g, 33%) as an amorphous white solid: mp 154-156°C, 152-154 °C; 1H NMR (CDCl3):
 7.75 (s, 1H), 7.52 (m, 2H), 7.38 (m, 3H), 5.45 (s, 2H), 2.76 (s, 3H), 2.56 (s, 3H); HRESIMS
calcd. for C16H1481BrN5NaO2 m/z [M + Na]+ 412.0203, found 412.0201; calc. for
C16H1479BrN5NaO2 m/z [M + Na]+ 410.0223, found 410.0217; TLC Rf = 0.53 (40%
EtOAc:hexanes); HPLC purity: 97.9%.
4.1.40. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-2-benzylpyridazin-3(2H)-one (23).
Similar reaction of 2,4-pentanedione (0.16 mL, 1.56 mmol, 1.01 equiv.), triethylamine (0.21
mL, 1.50 mmol, 0.98 equiv.) and azide 60 (0.348 g, 1.53 mmol, 1.00 equiv.) in dry dioxane
(4.0 mL) for 170 min, followed by evaporation of the mixture and trituration of the residue
with cold MeOH gave 23 (0.131 g, 28%) as an amorphous white solid: mp 147-148 °C; 1H
NMR (CDCl3):  8.24 (d, J = 2.6 Hz, 1H), 7.48 (m, 2H), 7.35 (m, 3H), 6.99 (d, J = 2.6 Hz,
1H), 5.39 (s, 2H), 2.74 (app d, J = 2.6 Hz, 6H); HRESIMS calcd. for C16H15N5NaO2 m/z [M

+ Na]+ 332.1118, found 332.1117; calc. for C16H16N5O2 m/z [M + H]+ 310.1299, found
310.1296; TLC Rf = 0.04 (20% EtOAc:hexanes); HPLC purity: 99.8%.
4.1.41. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(4-iodobenzyl)pyridazin-
3(2H)-one (24). Similar reaction of 2,4-pentanedione (0.30 mL, 2.92 mmol, 0.99 equiv.),
triethylamine (0.41 mL, 2.94 mmol, 0.99 equiv.) and azide 62 (1.281 g, 2.97 mmol, 1.00
equiv.) in dry dioxane (43 mL) for 125 min, followed by filtration through Celite®,
evaporation of the filtrate and purification of the residue by column chromatography on silica
gel, eluting with 0–20% EtOAc:hexanes, followed by re-precipitation from EtOAc:hexanes,
gave 24 (0.677 mg, 44%) as an amorphous white solid: mp 140-141 °C; 1H NMR (CDCl3): 
7.74 (s, 1H), 7.72 (m, 2H), 7.28 (m, 2H), 5.37 (s, 2H), 2.76 (s, 3H), 2.56 (s, 3H); HRESIMS
calcd. for C16H1381BrIN5NaO2 m/z [M + Na]+ 537.9170, found 537.9181, calcd. for
C16H1379BrIN5NaO2 m/z [M + Na]+ 535.9190 found 535.9196; TLC Rf = 0.50 (40%
EtOAc:hexanes); HPLC purity: 99.4%.
4.1.42. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-phenethylpyridazin-
3(2H)-one (25). Similar reaction of 2,4-pentanedione (0.40 mL, 3.90 mmol, 1.01 equiv.),
triethylamine (0.54 mL, 3.87 mmol, 1.00 equiv.) and azide 63 (1.236 g, 3.86 mmol, 1.00
equiv.) in dry dioxane (60.0 mL; added as a suspension) at 20 oC for 150 min, followed by
filtration and evaporation of the filtrate gave a crude product contaminated with the amine
derived from 61A. The mixture was triturated with EtOAc, and the resulting suspension was
filtered and the filtrate was evaporated. The residue was purified by column chromatography
on silica gel, eluting with 0–40% EtOAc:hexanes, followed by re-precipitation from
EtOAc:hexanes, to give 25 (0.187 g, 12%) as a semi-crystalline white solid: mp 150-151 °C;
1H NMR (CDCl3):  7.71 (s, 1H), 7.29 (m, 5H), 4.54 (m, 2H), 3.19 (m, 2H), 2.77 (s, 3H),

2.57 (s, 3H); HRESIMS calcd. for C17H1681BrN5NaO2 m/z [M + Na]+ 426.0360, found
426.0347; calc. for C17H1679BrN5NaO2 m/z [M + Na]+ 424.0380, found 424.0367; TLC Rf =
0.52 (40% EtOAc:hexanes); HPLC purity: 99.7%.
4.1.43. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(3-
phenylpropyl)pyridazin-3(2H)-one (26). Similar reaction of 2,4-pentanedione (0.30 mL,
2.92 mmol, 1.00 equiv.), triethylamine (0.41 mL, 2.94 mmol, 1.01 equiv.) and azide 64
(0.976 g, 2.92 mmol, 1.00 equiv.) in dry dioxane (20.0 mL) at 20 oC for 280 min, followed by
evaporation of the mixture under reduced pressure and purification of the residue by column
chromatography on silica gel, eluting with 0–40% EtOAc:hexanes, gave 26 (0.531 g, 44%) as
a pale yellow gummy resin: 1H NMR (CDCl3):  7.72 (s, 1H), 7.25 (m, 5H), 4.35 (app t, J =
7.3 Hz, 2H), 2.77 (m, 5H), 2.57 (s, 3H), 2.26 (app p, J = 22.2, 14.8, 7.5 Hz, 2H); HRESIMS
calcd. for C18H1881BrN5NaO2 m/z [M + Na]+ 440.0516, found 440.0523; calc. for
C18H1879BrN5NaO2 m/z [M + Na]+ 438.0536, found 438.0540; TLC Rf = 0.41 (40%
EtOAc:hexanes); HPLC purity: 98.4%.
4.1.44. 5-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)-4-bromo-2-(4-((2-
(dimethylamino)ethyl)-amino)benzyl)pyridazin-3(2H)-one (27). Using a general literature
method,38 iodide 24 (150 mg, 0.29 mmol, 1.00 equiv.), CuI (20 mg, 0.17 mmol, 0.34 equiv.),
2-dimethylaminoethylamine (80 μL, 0.74 mmol, 2.55 equiv.) and L-proline (20 mg, 0.17
mmol, 0.60 equiv.) were combined in dry DMSO (1.0 mL), and the resulting royal blue
solution was deoxygenated by three freeze-evacuate-thaw cycles. The reaction mixture was
then heated at 95 °C for 5 min, then K2CO3 (111 mg, 0.80 mmol, 2.75 equiv.) was added, and
the resulting mixture deoxygenated by a second series of three freeze-evacuate-thaw cycles,
and finally stirred at 95 °C for 16 h. The mixture was cooled, diluted with dry DMSO and

filtered through Celite® and the pad washed with a small amount of EtOAc. The combined
filtrate was washed with brine (x 3), dried (Na2SO4), filtered and evaporated under reduced
pressure. The crude product from this and a second, 3-fold larger, reaction were combined
and purified by column chromatography on silica gel, eluting with 0-20% MeOH:CH2Cl2,
containing 0.5% v/v aq. NH3 solution (twice), and then by preparative HPLC. The product
material thus obtained was unstable as the free base, so was dissolved in saturated aqueous
NaHCO3 (pH~8), filtered, the solids washed with EtOAc, and the aqueous filtrate extracted
with EtOAc (x 4). The combined organic extracts were dried (MgSO4) and evaporated, and
the residue was dissolved in a minimum amount of dry MeOH and solution was diluted with
~3 equiv. of methanolic HCl followed by EtOAc:hexanes, afforded 27 (diHCl salt) as an
amorphous white hygroscopic solid (36 mg, 23%): mp 101-104 °C (powder→glue), 149-153
°C (glue→liquid); 1H NMR (CDCl3):  10.00 (v. br. s, 2H), 7.55 (s, 1H), 7.51 (d, J = 8.3 Hz,
2H), 7.32 (d, J = 8.3 Hz, 2H), 5.29 (s, 2H), 3.72 (br s, 2H), 3.30 (br s, 2H), 2.83 (s, 3H), 2.74
(s, 3H), 2.59 (s, 3H), 2.27 (s, 3H) [–NH– not visible]; HRESIMS calcd. for C20H2581BrN7O2
m/z [M + H]+ 476.1228, found 476.1235, C20H2579BrN7O2 m/z [M + H]+ 474.1248, found
474.1251; TLC Rf = 0.29 (10% MeOH:HCl + 2 drops aqueous NH3); HPLC purity: 94.6%.
4.2. Biology Experimental
4.2.1. Purification of recombinant PFKFB3. GST-tagged human PFKFB3 was expressed in
Escherichia coli BL21(DE3) and purified using glutathione sepharose 4 Fast flow beads (GE
Healthcare). The resulting pure protein was concentrated to 10 mg ml-1 protein at pH 7.4, in a
mixture of 50 mm Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 0.01% TX-100.