Discovery and SAR of orally efficacious tetrahydropyridopyridazinone PARP inhibitors for the treatment of cancer

Article abstract of DOI:10.1016/j.bmc.2012.06.021

PARP-1, the most abundant member of the PARP superfamily of nuclear enzymes, has emerged as a promising molecular target in the past decade particularly for the treatment of cancer. A number of PARP-1 inhibitors, including veliparab discovered at Abbott, have advanced into different stages of clinical trials. Herein we describe the development of a new tetrahydropyridopyridazinone series of PARP-1 inhibitors. Many compounds in this class, such as 20w, displayed excellent potency against the PARP-1 enzyme with a K_i value of <1 nM and an EC₅₀ value of 1 nM in a C41 whole cell assay. The presence of the NH in the tetrahydropyridyl ring of the tetrahydropyridopyridazinone scaffold improved the pharmacokinetic properties over similar carbon based analogs. Compounds 8c and 20u are orally available, and have demonstrated significant efficacy in a B16 murine xenograft model, potentiating the efficacy of temozolomide (TMZ).

Full text of DOI:10.1016/j.bmc.2012.06.021

Bioorganic & Medicinal Chemistry 20 (2012) 4635–4645
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and SAR of orally efficacious tetrahydropyridopyridazinone PARP
inhibitors for the treatment of cancer
Gui-Dong Zhu a, , Jianchun Gong a, Viraj B. Gandhi a, Xuesong Liu a, Yan Shi a, Eric F. Johnson a,
⇑
Cherrie K. Donawho a, Paul A Ellis a, Jennifer J. Bouska a, Donald J. Osterling a, Amanda M. Olson a,
Chang Park b, Yan Luo a, Alexander Shoemaker a, Vincent L. Giranda a, Thomas D. Penning a
a Cancer Research, GPRD, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
b Advanced Technology, GPRD, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
PARP-1, the most abundant member of the PARP superfamily of nuclear enzymes, has emerged as a
promising molecular target in the past decade particularly for the treatment of cancer. A number of
PARP-1 inhibitors, including veliparab discovered at Abbott, have advanced into different stages of clin-
ical trials. Herein we describe the development of a new tetrahydropyridopyridazinone series of PARP-1
inhibitors. Many compounds in this class, such as 20w, displayed excellent potency against the PARP-1
enzyme with a Ki value of <1 nM and an EC50 value of 1 nM in a C41 whole cell assay. The presence of
the NH in the tetrahydropyridyl ring of the tetrahydropyridopyridazinone scaffold improved the pharma-
cokinetic properties over similar carbon based analogs. Compounds 8c and 20u are orally available, and
have demonstrated significant efficacy in a B16 murine xenograft model, potentiating the efficacy of tem-
ozolomide (TMZ).
Received 11 May 2012
Revised 4 June 2012
Accepted 11 June 2012
Available online 19 June 2012
Keywords:
PARP
Cancer therapy
PARP inhibitor
Poly(ADP)ribose polymerase
BRCA
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
in the PARP super family share a common and highly homologous
catalytic domain that catalyzes the transfer of ADP-ribose units
DNA damage occurs constantly due to environmental factors
and normal metabolic processes inside the cell. A DNA repair pro-
cess is rapidly initiated in response to the damage, and is a critical
cellular event to maintain genome integrity. DNA repair is a very
complex process, with up to 150 genes involved. More than 5 prin-
ciple pathways have been identified, including base excision repair
(BER), homology directed repair (HDR), nucleotide excision repair
(NER), mismatch repair (MMR), and non-homologous endjoining
(NHEJ).1 Repair of DNA damage, the double-strand DNA breaks
(DSBs) in particular, have become an increasingly attractive anti-
cancer target since DSBs are the death-provoking effectors of ther-
apeutic radiation and some cytotoxic chemotherapy. Homologous
recombination (HR), a BRCA-dependent and high-fidelity process,
is considered to be the primary repair mechanism for the repair
of DSBs. Therefore, tumors with inherited BRCA1 or BRCA2 muta-
tions are typically defective in DNA repair of double-strand
breaks.2
from intracellular nicotinamide adenine dinucleotide (NAD+) to
the acceptor proteins, leading to the formation of mostly branched
ADP-ribose polymers (PARs). This cellular event is a key process
during base excision repair (BER) of single-strand DNA breaks
caused by ionizing radiation or DNA-damaging chemotherapeutic
treatments, and contributes to the resistance mechanism that of-
ten develops after these cancer therapies. PARP1 is the most abun-
dant member of the PARP family and responsible for >90% of the
poly(ADP-ribosyl)ation activity in cells of all higher eukaryotes.
Evidences have also been reported that PARP1 contributes to
DSB, NHEJ and HR repair pathways.3 PARP2, which is the closest
relative but less active than PARP1, also participates in BER, con-
tributing only 5 to 10% of the total PARP activity in response to
DNA damage.4 Both PARP1, and to a lesser extent PARP2, function
as DNA damage sensor by binding with high affinity to the site of
single- and double-stranded DNA breaks. The binding to the dam-
aged DNA triggers poly(ADP-ribosyl)ation of various proteins that
is believed to be essential in recruiting other repair proteins to
the site of molecular lesions.5
Poly(ADP-ribose)polymerases (PARPs) are a family of nuclear
enzymes that polymerize poly(adenosine diphosphate–ribose) on
substrate proteins critical for cellular regulations including DNA
repair, gene transcription, and chromatin architecture. Members
Abrogation of the PARP-mediated DNA repair would enhance
the anticancer activity of traditional cancer therapies. In addition,
since tumor cells are frequently defective in DNA repair pathways
like homologous recombination, inhibition of PARP-1 leads to the
persistence of single-strand DNA lesions that degenerates
⇑
Corresponding author.
E-mail address: gui-dong.zhu@abbott.com (G.-D. Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.021

4636
G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
O
H
N
NH
N
O
NH2
O
Gly863
2.7
O
F
“Northern Pocket”
N
HN
N
N
H
N
H
N
O
3.1
N
H
F
Ser904
1a
1c
Veliparib
(Abbott)
1b
W2
3.0
Tyr896 3.4
Olaparib
PF-1367338
(AstraZeneca)
(Pfizer)
O
Tyr907
F
O
H
N
NH
N
O
NH2
NH
N
O
2.9
HN
N
N
H
NH
N
O
O
N
N
N
F
W1
N
2.6
O
N
P*
Glu988
1d
1f
1e
8c
F
MK-4827
(Merck)
CEP-9722
BMN-673
(Cephalon)
(BioMarin)
Fig. 2. X-Ray Co-crystal structure of veliparib overlaid with the proposed binding
mode of compound 8c.
*P = Solubilizing prodrug moiety
Fig. 1. Current PARP inhibitors in clinical trials.
veliparib with Ser-904 and Gly-863 in the PARP-1 catalytic domain
were observed. The carboxamide group was restricted into an opti-
mal orientation for the hydrogen-bond interaction through forma-
tion of an intramolecular hydrogen bond with the 3-nitrogen of the
double-strand breaks during DNA replication. Inhibition of homol-
ogous recombination or PARP may be well tolerated in isolation,
but combined inactivation of these distinct DNA-repair pathways
results in cell death — a process called ‘‘synthetic lethality’’. There-
fore PARP inhibition can be toxic to cells deficient in tumor-sup-
pressor genes BRCA1 or BRCA2 that underlie high-penetrance,
hereditary breast and ovarian carcinomas. Normally, homologous
recombination repairs these breaks, but should this mechanism
be unavailable, as is the case when BRCA1 or BRCA2 is absent,
the cell dies. Thus, small-molecule inhibitors of PARP1 standalone
can be an efficient targeted therapy for these cancer patients. Be-
cause of the high homology between PARP-1 and PARP-2, many
of the reported PARP-1 inhibitors actually inhibit PARP-2 to a sim-
ilar extent. Inhibitors of PARP-1 are most likely also inhibitors of
PARP2-4, so the amount of polyADP(ribosylation) is completely
shut down.7 In fact, some clinical evidences have shown that drugs
targeting PARP show promise as treatments for some of the most
aggressive and difficult-to-treat forms of breast cancer.8
As shown in Figure 1, at least six major series of small molecule
PARP inhibitors have been developed in the past decade, and have
advanced into different stages of clinic trials.6 While earlier PARP-
1 inhibitors were investigated primarily in non-oncological indica-
tions, the more recent efforts have been focused on the use both as
mono-anticancer therapy in specific patient populations (e.g. BRCA-
deficient) and in combination with various chemotherapeutics.
We have previously reported our efforts in identifying a series
of potent benzimidazole-containing PARP inhibitors, including
the leading candidate 1c (ABT-888, veliparib), now in Phase II clin-
ical trials.9 This compound demonstrated significant oral efficacy
in a number of preclinical rodent tumor models, potentiating the
efficacy of cytotoxic agents such as temozolomide (TMZ), cisplatin,
carboplatin, and cyclophosphamide, as well as radiation. In this re-
port, we describe the discovery and SAR of a series of novel and or-
ally efficacious tetrahydropyridopyridazinone PARP inhibitors. A
number of these analogs displayed excellent in vitro potency in
both intrinsic and cellular assays, adequate pharmacokinetic prop-
erties as well as oral efficacy in mouse xenograft models.
imidazole moiety. In addition to a characteristic
p-stacking inter-
action for PARP inhibitors between the benzimidazole ring and
Tyr-907, the 1-NH of the benzimidazole ring appears to be in-
volved in a water-mediated hydrogen bond (W1) with Glu-988
of the protein. While olaparib (1b) has demonstrated certain suc-
cess in clinical trials, an overlay (not shown in the figure but sim-
ilar to 8c) with veliparib shows that this compound lacks a
hydrogen bond donor as for the ‘‘NH’’ of veliparib benzimidazole
that forms a hydrogen bond interaction with W1. As modeling sug-
gests in Figure 2, the proposed tetrahydropyridopyridazinone, in
which a 5-NH is incorporated into the ring structure, would pro-
vide additional molecular interaction as compared to olaparib.
There is also a water molecule in the vicinity of the pyrrolidine-
NH near the ‘‘northern pocket’’, displacement of this W2 with a
pyrrolidinone carbonyl would increase the potency through poten-
tial interaction with Tyr896. We have chosen a fluorine atom at the
para-position of the 4-benzyl group as for the case of olaparib due
to a limited space available at this position in the X-ray structure.
3. Chemistry
The tetrahydropyridopyridazinone pharmacophore was con-
structed through a selective Grignard addition of a properly func-
tionalized benzylmagnesium bromide to the 2-carboxylate of a
commercially available dimethyl pyridine-2,3-dicarboxylate (2),
followed by a pyridazinone ring closure and pyridine saturation
(Scheme 1). There is no literature precedence for the high selectiv-
ity observed, and it appears that the presence and position of the
ring nitrogen of pyridine 2,3-dicarboxylate 2 is critical for the
mono-Grignard addition. Lack of or shift of the ring nitrogen failed
to provide reasonable selectivity. Heating the ketoester 4 or 5 with
hydrazine as the mono HCl salt provided pyridopyridazinone 6 or 7
in good yields. Hydrogenation of the pyridopyridazinone ring in
the presence of 5% platinum on charcoal under acidic conditions
furnished desired product 8a. A direct Buchwald amidation of the
bromide 7 failed to provide the coupled product 8c or 8d. However,
after protecting the free lactam NH as benzyloxymethyl ether to
give 9, the Buchwald coupling reaction proceeded smoothly by
using Xantphos as ligand under microwave conditions. Hydrogena-
tion of 10 with a mixture of platinum and palladium hydroxide on
carbon removed the benzyloxymethyl group and saturated the
pyridine ring simultaneously.
2. X-ray Co-crystal structure of veliparib and computer
modeling of a tetrahydropyridopyridazinone PARP inhibitor
Figure 2 displays an X-ray co-crystal structure of veliparib (1c)
overlaid with a tetrahydropyridopyridazinone PARP inhibitor (8c).
Consistent with our previous reports,9 as well as others, three key
hydrogen-bond interactions between the carboxamide group of

G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
4637
O
O
O
R
F
MgBr
O
O
O
OMe
O
3
OMe
OMe
NH
N
n
N
O
CDI
THF
N
N
R
13
N
O
n
H
N
MeCN
DMF
65 o
C
OH
F
2
4 R= H
5 R= Br
O
F
14: n= 1
15: n=2
O
O
NH
N
NH
N
H2
5% Pt/C
O
N
H
R
F
R
F
NH
N
H2
O
8e
8f
N
5% Pt/C
N
F
8
n
6 R= H
7 R= Br
8a: R= H
O
BnOCH2Br
NaH
16: n= 1
17: n=2
O
O
Scheme 3.
N
N
OBn
N
N
OBn
Pd2(dba)3/Xantphos
N
N
of 8b with appropriate acids in the presence of HATU and Hünig’s
base smoothly afforded 8i-l.
Br
F
R
F
As shown in Scheme 3, treating the amine 13 with succinic
anhydride (n = 1) or dihydro-2H-pyran-2,6(3H)-dione (n = 2) in
acetonitrile provided either 14 or 15 that upon exposure to carbon-
yldiimidazole (CDI) furnished compounds 16 or 17. A pyridine ring
saturation under acidic conditions afforded compounds 8e or 8f in
the presence of 5% platinum on carbon.
9
10
O
8c: R=
N
H2
8
O
5% Pt/C & Pd(OH)2
8d: R=
N
Scheme 4 illustrates an alternative protocol in which the pyri-
dine ring of the tetrahydropyridopyridazinone core structure was
first saturated, followed by an amide elaboration. Thus, hydrogena-
tion of 11, in the presence of platinum on carbon and one equiva-
lent of HCl, afforded 18. Saponification of the ester 18, followed by
amide coupling with appropriate amines under the standard EDC/
HOBt conditions, provided 20a–k, 20m,n, and 20r-w. For syntheses
of piperazine derivatives 20l and 20o-q, acid 19 was coupled to N-
Boc-piperazine to give 21. An acid-mediated deprotection of 21 led
to compound 20f which upon further elaboration to give 20l and
20o-q.
Scheme 1.
As shown in Schemes 2 and 3, the carboxamides 8b, 8e through
8l were prepared in a straightforward fashion. Carboxylation of
compound 7 in the presence of Pd(dppf)Cl2 smoothly afforded 11
that, upon treatment with an ammonia solution in methanol, fur-
nished 12. A Hoffman rearrangement of amide 12 provided a key
intermediate 13 that was hydrogenated on platinum on charcoal
under acidic conditions to give 8b. The imidazolidinedione analog
8g and dihydropyrimidinedione 8h were prepared through heating
equal molar equivalent of 8b with methyl 2- or 3-isocyanotopro-
panoate at 65 °C in DMF overnight (Scheme 2). Amide coupling
O
NH
N
LiOH
H2
N
H
O
F
11
5% Pt/C
OMe
O
O
NH
N
NH
N
18
CO
NH3/MeOH
7
N
O
F
N
O
F
O
O
Pd(dppf)Cl2
MeOH
OMe
NH2
NH
N
NH
N
EDC/HOBt
N
H
O
F
N
H
O
F
11
12
OH
R
O
19
20a-k, m,n, r-w
NH
N
H2
Br2/NaOH
8b
N
EDC/HOBt
5% Pt/C
NH2
HO
O
F
O
O
O
O
13
HO
HO
NH
N
O
NH
TFA
N
O
O
O
N
N
H
O
H
N
H
NCO
O
DMF
N
N
O
O
O
NBoc
NH
F
F
O
NCO
HO
21
20f
O
8g, h
8i-l
20l, o-q
Scheme 2.
Scheme 4.

4638
G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
Carboxamides with diverse structures have also been synthe-
4. Results and discussion
sized and evaluated for PARP inhibitory and pharmaceutical prop-
erties (Table 2). While primary and secondary amides displayed
modest to good enzyme activity, as exemplified by 20a–20g, their
potency in cellular settings varied from poor to modest, and rela-
tively poor correlations were observed between their intrinsic
and cellular activities. We were not clear whether the relatively
poor cellular activity observed for some compounds (e.g. 20e–g,
8g) is due to low cellular permeability and/or because they are
MDR substrate. In an attempt to improve cellular potency, the
same strategy as for the benzimidazole series of PARP inhibitors,13
for which introduction of basic amines was beneficial, was utilized.
As shown in Table 2, a blend of cellular data, however, was ob-
tained for these modifications. It appeared that incorporation of a
tertiary amine (e.g. 20d, 20i and 20j) or sterically hindered second-
ary amine (e.g. 20h) is beneficial to the cellular activity, while
those primary amines (e.g. 20f, 20g) or diamines (20e) were less
active. Nevertheless, all compounds displayed excellent intrinsic
activity against PARP1, presumably due to a large binding pocket
at this area of the protein. To explore an impact of the basicity of
the molecule, we synthesized a number of amides (e.g. 20k–
20o), urea (20p) and sulfonamide (e.g. 20q) analogs. Overall these
modifications led to increased potency in cellular assays. Lastly,
weakly basic amine analogs 20r–20w with an aryl- or heteroaryl
group at the para-nitrogen of 20f were also evaluated and resulted
in a series of the most active PARP inhibitors to date with 20w
being the most potent (PARP1 Ki = 0.4 nM, cellular EC50 = 1 nM).
Pharmacokinetic evaluation of tetrahydropyridopyridazinone
series of PARP inhibitors started from a quick screening protocol,
due to the large number of potent PARP inhibitors we identified.
All compounds for the mouse screening PK were dosed orally at
10 mg/kg and the plasma concentrations were determined up to
3 h, seeking orally efficacious PARP inhibitors. Compounds 8f,
We initiated the SAR exploration of the novel tetrahydropyrid-
opyridazinone pharmacophore with a more optimized 4-fluorob-
enzyl substituent at the C4 position of the pyridazinone, based
on previous reports for olaparib and related series.10–12 Consistent
with the SAR in the phthalazinone series, both unsubstituted (8a)
and amino analog 8b displayed a low submicromolar potency in
a PARP1 assay (Table 1). In an attempt to exploit potential binding
interactions in the ‘Northern Pocket’ (Fig. 2) for the tetrahydropy-
ridopyridazinone series, we have incorporated a lactam-like car-
bonyl as illustrated by compounds 8c through 8h. These
modifications provided a series of low single-digit nanomolar PARP
inhibitors with 8f displaying the highest potency both in intrinsic
and cellular assays. Among these cyclic carbonyl substituents, the
imide analogs (8e, 8f) appeared to be more potent than the lactams
(8c and 8d) in the enzyme but not in the cellular assay, and imidaz-
olidine-2,4-dione (8g and 8h) were least active. Good activity was
also observed for acyclic amide substituents (8i–8l), particularly
for those with another carbonyl group at the remote end of the car-
boxylates, rendering a potential hydrogen bond interaction deeper
into the adenosine-ribose binding region.
Table 1
Enzyme and cellular assays of compound 8
O
NH
N
N
H
R
F
8
Compd
R
PARP-1 (Ki, nM)
Cellulara (EC50, nM)
20m, and 20n had AUCs in range of 0.07–0.19 lg-h/mL in this
screening PK protocol, while a subset analogs 20r, 20u, and 20w
showed a slight improvement in oral exposure with AUCs of 2.9–
a
b
H
NH2
O
118
112
ND
ND
6.82 lg-h/mL. Presence of an extra aryl group appeared to improve
pharmacokinetic property within the series.
c
6
3
11
22
N
O
Mouse pharmacokinetic parameters from a standard protocol
for selected compounds were obtained and shown in Table 3.
These compounds were dosed orally at 10 mg/kg and intrave-
nously at 3 mg/kg, and the plasma concentrations were
determined up to 8 h. A head-to-head comparison of the tetrahy-
dropyridyl series (8c or 20u and 20w highlighted) with the tetra-
hydrophenyl series (23)14 indicates that the presence of a NH
functionality is beneficial for an improved pharmacokinetic prop-
erty in this series of PARP inhibitors. All compounds with a
ring-nitrogen (e.g. 8c, 20u, and 20w) displayed significantly higher
plasma exposure in a mouse pharmacokinetic evaluation with an
d
N
O
N
e
f
1.4
0.7
9
56
6
O
O
N
O
O
NH
NH
N
g
h
271
ND
oral AUC0–8h of 3.11, 5.95, and 9.06
lg-h/mL respectively, versus
O
an oral AUC0–8h of 0.33, 2.70, and 2.38
lg-h/mL for the correspond-
O
N
O
ing compounds, (23c, 23u, and 23w) lacking this nitrogen.
Considering a balanced in vitro potency, pharmacokinetic and
pharmaceutical properties and structural diversity, compounds
8c and 20u were evaluated in a murine B16F10 syngeneic mela-
noma model (Figs. 3A and 3B). This B16F10 model, while relatively
resistant to most chemotherapeutics, is moderately sensitive to
TMZ and the sensitivity can be enhanced with PARP inhibitors. It
also closely corresponds with the actual clinical setting of TMZ
used for melanoma. Compound 8c was administered orally on days
5–10 at doses of 10, 30 and 60 mg/kg/day, bid, while TMZ was
administered orally at 50 mg/kg/day, qd, from day 5 to day 10.
As displayed in Figure 3A, 8c significantly potentiated the efficacy
of TMZ in a dose-dependent manner. Significant potentiation was
observed as early as day 12, with TGI (tumor growth inhibition)
values (vs. vehicle control) of 57, 59, and 69 for the 10, 30, and
17
H
N
O
i
12
4
134
27
O
O
O
H
N
j
O
O
H
N
H
N
k
1.4
16
H
N
l
1.3
16
O
O
a
Average of at least two determinations.

G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
4639
Table 2
Table 3
Enzyme and cellular assays of compound 20
Standard mouse pharmacokinetic parameters for 8c, 20u, 20w, and 23c, 23u, 23w
O
O
O
O
NH
N
N
O
R=
N
N
N
N
N
N
N
H
O
F
NH
N
N
R
X
23: X= CH2
23c
8c
23u
20u
23w
20w
R
20
X= NH
F
a
Compd
R
PARP-1 (Ki, nM)
Cellular (EC50, nM)
% Fa (po)
T1/2 (iv)
AUCc
Cmax
Vss
CLf
7.43
11.72
2.04
5.92
0.89
4.55
b
d
e
Compd
a
NH2
17
6
122
45
8c
106
13
67
74
36
48
0.49
0.28
2.6
1.10
3.86
0.36
3.11
0.33
5.95
2.70
9.06
2.38
1.50
0.14
2.01
1.09
5.60
1.03
5.25
4.78
7.66
9.48
4.99
2.33
b
NHEt
23c
20u
23u
20w
23w
c
14
82
HN
N
d
9
111
N
H
N
a
b
c
d
e
f
N
10 mg/kg PO dose, 3 mg/pk IV dose.
h.
e
3.5
290
N
H
po,
po,
L/kg.
L/h/kg.
l
l
g-h/mL.
g/mL.
N
N
NH
f
1.8
1.6
4400
2400
NH2
g
h
7
22
N
N
NH
Max Weight
Loss (%)
3000
N
i
j
6
32
7
N
N
O
2.5
Vehicle
2500
2000
1500
1000
500
TMZ 50
-1
8c/TMZ (60/50)
k
l
0.9
1.3
190
22
N
NH
-12
-3
8c/TMZ (60/50) N=9
8c/TMZ (30/50)
8c/TMZ (10/50)
N
N
N
N
O
m
n
1.3
0.9
180
6
O
CF3
N
N
N
O
o
0.9
11
N
N
O
N
O
0
p
q
1.2
0.7
50
0
5
10
15
20
25
N
N
TMZ PO QD d5-9
8c PO BID d5-10
Days
O
N
S
150
O
Fig. 3A. B16F10 model: 8c in combination with TMZ.
N
N
N
N
N
N
r
s
1.2
1.2
1.7
39
N
t
0.9
0.9
9
N
N
Max Wt
Loss (%)
Vehicle
3500
N
N
N
N
N
N
TMZ 50
-2
-21
-3
u
1.4
20u/TMZ (5/50)
20u/TMZ (2.5/50)
20u/TMZ (1.25/50)
N
N
3000
2500
2000
1500
1000
500
-3
v
0.4
0.4
3.3
1
N
N
w
a
Average of at least two determinations.
60 mg/kg/day 8c combination groups, respectively, compared to
30% for TMZ alone. All three dosing groups continued to differenti-
ate from the TMZ alone group out to end of the trial at day 21. The
8c-TMZ combination was well tolerated for the 10 and 30 mg/kg
dose groups with minimal loss of body weight. For the 60 mg/kg
0
0
5
10
15
20
25
30
TMZ PO QD d5-9
Days
20u/PO BID d5-9
Fig. 3B. B16F10 model: 20u in combination with TMZ.

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G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
group, however, an average of body weight loss of 12% was ob-
served. One animal was euthanized and the data showed reflected
the rest of animals.
by the addition of 150 L of 1.5 mM benzamide (ꢁ1000-fold over its
IC50). 170 L of the stopped reaction mixtures were transferred to
streptavidin-coated Flash Plates, incubated for 1 h, and counted
using a TopCount microplate scintillation counter. Ki data was deter-
mined from inhibition curves at various substrate concentrations.
Similar to 8c, compound 20u demonstrated significant potenti-
ation of the TMZ efficacy but at much lower doses. Compound 20u
was administered orally on days 5–9 at doses of 1.25, 2.5 and 5 mg/
kg/day, bid, while TMZ was administered orally at 50 mg/kg/day
once a day during the same period. As displayed in Figure 3B,
20u significantly potentiated the efficacy of TMZ in a dose-depen-
dent manner, and all combination groups continued to differenti-
ate from the TMZ alone group through the entire study period.
Like compound 8c, the 20u-TMZ combination was well tolerated
at lower doses with an average body weight loss similar to the
TMZ monotherapy group. Significant body weight loss was, how-
ever, observed at the 5 mg/kg combination groups.
6.1.2. Cellular PARP assay
C41 cells were treated with test compound for 30 min in a 96-
well plate. PARP was activated by damaging DNA with 1 mM H2O2
for 10 min. Cells were washed with ice-cold PBS once and fixed
with pre-chilled methanol/acetone (7:3) at ꢂ20°C for 10 min. After
air-drying, plates were rehydrated with PBS and blocked using 5%
non-fat dry milk in PBS-tween (0.05%) (blocking solution) for
30 min at room temperature. Cells were incubated with anti-PAR
antibody 10H (1:50) in blocking solution at room temperature
for 60 min followed by washing with PBS-Tween20 5 times, and
incubation with goat anti-mouse fluorescein 5(6)-isothiocyanate
5. Conclusion
(FITC) -coupled antibody (1:50) and 1 l
g/ml 40,6-diamidino-2-
phenylindole (DAPI) in blocking solution at room temperature for
60 min. After washing with PBS-Tween20 5 times, analysis was
performed using an fmax Fluorescence Microplate Reader set at
the excitation and emission wavelength for FITC or the excitation
and emission wavelength for DAPI. PARP activity (FITC signal)
was normalized with cell numbers (DAPI).
In summary, we have discovered and characterized a novel tetr-
ahydropyridopyridazinone series of PARP inhibitors for the treat-
ment of cancer. Some of the compounds described herein are
among the most potent PARP inhibitors to date in both intrinsic
and cellular assays. Compounds 8c and 20u have demonstrated
significant efficacy in a B16 murine xenograft model, significantly
potentiating the efficacy of TMZ. Pharmacokinetic characterization
through a head-to-head comparisons has demonstrated that the
presence of the NH in the tetrahydropyridyl ring is important for
improved oral exposure. Compound 20u represents a promising
drug candidate for further pre-clinical characterization.
6.1.3. B16F10 tumor model
For B16F10 syngeneic studies, 6 ꢀ 104 cells were mixed with
50% matrigel (BD Biosciences, Bedford, MA) and inoculated by s.c.
injection into the flank of 6–8 week old female C57BL/6 mice,
20 g (Charles River Laboratories, Wilmington, MA). Mice were
injection-order allocated to treatment groups and PARP inhibitor
therapy was initiated on day 5 following inoculation, with tem-
ozolomide treatment also starting on day 5.
6. Experimental
6.1. General
6.2. Synthesis of compound 8a–l
NMR spectra were obtained on Varian M-300, Bruker AMX-400,
Varian U-400, or Varian Unity Inova 500 magnetic resonance spec-
trometers with indicated solvent and internal standard. Chemical
shifts are given in delta (d) values and coupling constants (J) in
Hertz (Hz). The following abbreviations are used for peak multi-
plicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broadened. Mass spectra were performed as follows: ESI (elec-
trospray ionization) was performed on a Finnigan SSQ7000 MS run
as a flow injection acquisition; DCI (desorption chemical ioniza-
tion) was performed on a Finnigan SSQ7000 MS using a direct
exposure probe with ammonia gas; APCI (atmospheric pressure
chemical ionization) was performed on a Finnigan Navigator MS
run as flow injection acquisition. Elemental analyses were per-
formed by Quantitative Technologies Inc. Whitehouse, New Jersey.
All manipulations were performed under nitrogen atmosphere un-
less otherwise noted. All solvents and reagents were obtained from
commercial sources and used without further purification. HPLC
purifications were carried out using a Zorbax C-18, 250 ꢀ 2.54 col-
umn and a eluting with a 0-100% gradient of mobile phase A (0.1%
trifluoroacetic acid (TFA) in water) and mobile phase B (0.1% TFA in
CH3CN).
6.2.1. Methyl 2-(2-(4-fluorophenyl)acetyl)nicotinate (4)
To a solution of dimethyl pyridine-2,3-dicarboxylate (1.0 g,
5.1 mmol) in anhydrous THF (50 ml) was added (4-fluoroben-
zyl)magnesium chloride (0.25 M in THF, 20 mL, 5.1 mmol) through
a syringe at ꢂ78 °C. The reaction mixture was stirred at the same
temperature for 30 min and was quenched with addition of water.
After warming up to room temperature, the reaction mixture was
partitioned between ethyl acetate and brine. The organic phase
was washed with brine and concentrated. The residue was purified
by flash chromatography (15% ethyl acetate in hexane) to give
0.45 g of the title compound. Yield: 33%. MS (DCI/NH3) m/z 274
(M+H)+; 1H NMR. (300 MHz, CDCl3): d 3.88 (s, 3H), 4.38 (s, 2H),
6.94–7.12 (m, 2H), 7.25–7.39 (m, 2H), 7.48 (dd, J = 7.80, 4.75 Hz,
1H), 8.04 (dd, J = 7.97, 1.53 Hz, 1H), 8.74 (dd, J = 4.75, 1.70 Hz, 1H).
6.2.2. 8-(4-Fluorobenzyl)pyrido[3,2-d]pyridazin-5(6H)-one (6)
A solution of 4 (0.46 g, 1.68 mmol) in ethanol (20 mL) was trea-
ted with hydrazine (108 mg, 3.37 mmol) at room temperature for 5
hours. The reaction mixture was concentrated to about 5 mL. The
formed solid was collected by filtration, washed with ethanol
and dried to provide 0.39 g of title compound. Yield: 91%. MS
(DCI/NH3) m/z 256 (M+H)+; 1H NMR. (300 MHz, DMSO-d6): d 4.36
(s, 2H), 6.99–7.13 (m, 2H), 7.29–7.39 (m, 2H), 7.85 (dd, J = 8.13,
4.56 Hz, 1H), 8.59 (dd, J = 8.13, 1.78 Hz, 1H), 9.16 (dd, J = 4.56,
1.78 Hz, 1H).
6.1.1. PARP enzyme assay
Enzyme assay was conducted in buffer containing 50 mM Tris pH
8.0, 1 mM DTT, and 4 mM MgCl2. PARP reactions contained 1.5
lM
[3H]-NAD+ (1.6
lCi/mmol), 200 nM biotinylated histone H1,
200 nM slDNA, and 1 nM PARP-1 or 4 nM PARP-2 enzyme. Auto
reactions utilizing SPA bead-based detection were carried out in
6.2.3. 8-(4-Fluorobenzyl)-1,2,3,4-tetrahydropyrido[3,2-
d]pyridazin-5(6H)-one (8a)
A mixture of compound 6 (150 mg, 0.6 mmol), 5% platinum on
carbon (30 mg), concentrated aqueous HCl (50 lL) and DMF
100 lL volumes in white 96 well plates. Reactions were initiated
by adding 50 L of 2ꢀ NAD+ substrate mixture to 50 L of 2ꢀ enzyme
mixture containing PARP and DNA. These reactions were terminated

G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
4641
(5 mL) in a pressure vessel was stirred at room temperature under
50 psi of hydrogen for 16 h. The reaction mixture was filtered, and
the filtrate was concentrated. The residual solid was purified by
HPLC (Zorbax C-18, 0.1% TFA/CH3CN/H2O) to provide 8 as TFA salt.
Yield: 126 mg (83%). MS (ESI) m/z 260 (M+H)+; 1H NMR. (300 MHz,
DMSO-d6): d 1.55–1.80 (m, 2H), 2.35 (t, J = 6.27 Hz, 2H), 3.10–3.25
(m, 2H), 3.81 (s, 2H), 7.06–7.18 (m, 2H), 7.20–7.31 (m, 2H).
6.2.8. 8-(3-Amino-4-fluorobenzyl)pyrido[2,3-d]pyridazin-5(6H)-
one (13)
A mixture of 1.5 N aqueous KOH solution (2 mL) and 3 g of ice
was treated with bromine (80 mg, 0.5 mmol) at ꢂ10 °C for
10 min. Compound 12 (100 mg, 0.3 mmol) was then added. The
reaction mixture was stirred at ꢂ10 °C for an additional 10 min,
and was then allowed to warm up to 65 °C for 1 h. After cooling,
the mixture was partitioned between ethyl acetate and brine.
The organic phase was washed with brine, and concentrated to
about 10 mL. The formed solid was collected by filtration, washed
with methanol, and dried to provide13. Yield: 50 mg (77%). MS
(DCI/NH3) m/z 271 (M+H)+. 1H NMR. (300 MHz, DMSO-d6): d 4.20
(s, 2H), 4.99 (s, 2H), 6.31–6.54 (m, 1H), 6.67 (dd, J = 8.82, 2.03 Hz,
1H), 6.83 (dd, J = 11.53, 8.14 Hz, 1H), 7.84 (dd, J = 7.97, 4.58 Hz,
1H), 8.59 (dd, J = 8.14, 1.70 Hz, 1H), 9.14 (dd, J = 4.58, 1.86 Hz, 1H).
6.2.4. Methyl 2-(2-(3-bromo-4-fluorophenyl)acetyl)nicotinate
(5)
A mixture of magnesium turnings (880 mg, 37 mmol) and 2-
bromo-4-(bromomethyl)-1-fluorobenzene (1.0 g, 3.7 mmol) in
anhydrous diethyl ether (15 ml) was treated with a piece of iodine.
The mixture was then heated to gentle reflux until the color of the
mixture disappeared, after which the heating continued for addi-
tional hour. The suspension was cooled to room temperature,
and cannulated into a solution of dimethyl pyridine-2,3-dicarbox-
ylate (1.0 g, 5.1 mmol) in tetrahydrofuran (50 ml) at ꢂ78 °C. The
reaction mixture was maintained at the same temperature for
30 min, and was quenched with addition of water. After warming
up to room temperature, the reaction mixture was partitioned be-
tween ethyl acetate and brine. The organic phase was washed with
brine and concentrated. The residue was purified by flash chroma-
tography (15% ethyl acetate in hexane) to provide the title com-
pound. Yield: 500 mg (38%). MS (DCI/NH3) m/z 353 (M+H)+; 1H
NMR. (300 MHz, METHANOL-D4) d 3.88 (s, 3H), 4.38 (s, 2H),
7.09–7.20 (m, 1H), 7.24–7.34 (m, 1H), 7.57 (dd, J = 6.61, 2.20 Hz,
1H), 7.63 (dd, J = 7.97, 4.92 Hz, 1H), 8.18 (dd, J = 7.80, 1.70 Hz,
1H), 8.77 (dd, J = 4.92, 1.53 Hz, 1H).
6.2.9. 8-(3-Amino-4-fluorobenzyl)-2,3,4,6-tetrahydropyrido[2,3-
d]pyridazin-5(1H)-one (8b)
Compound 8b was prepared as TFA salt according to procedure
for compound 8, substituting compound 13 for 6. Yield: 56 mg
(55%). MS (ESI) m/z 275 (M+H)+; 1H NMR (300 MHz, DMSO-d6):
1.62–1.74 (m, 2H), 2.35 (t, J = 6.27 Hz, 2H), 3.10–3.23 (m, 2H),
3.69 (s, 2H), 4.91 (s, 2H), 6.25 (s, 1H), 6.45–6.54 (m, 1H), 6.64
(dd, J = 8.82, 2.03 Hz, 1H), 6.92 (dd, J = 11.53, 8.48 Hz, 1H), 11.93
(s, 1H).
6.2.10. 6-(Benzyloxymethyl)-8-(3-bromo-4-
fluorobenzyl)pyrido[3,2-d]pyridazin-5(6H)-one (9)
A solution of compound 7 (100 mg, 0.3 mmol) in anhydrous
dimethylformamide (10 mL) was treated with potassium t-butox-
ide (1 N solution in tetrahydrofuran, 0.3 mL, 3 mmol) at room tem-
perature for 30 min. Benzyloxychloromethane (52 mg, 0.3 mmol)
was then added, and the mixture was stirred at room temperature
overnight. After quenching with water, the reaction mixture was
partitioned between ethyl acetate and brine. The organic layer
was washed with brine, and concentrated. The residue was puri-
fied by flash chromatography (85% ethyl acetate in hexane) to pro-
vide 9. Yield: 100 mg (74%). MS (DCI/NH3) m/z 454 (M+H)+; 1H
NMR. (300 MHz, CD3OD): d 4.42 (s, 2H), 4.69 (s, 2H), 5.63 (s, 2H),
7.02–7.28 (m, 6H), 7.34–7.47 (m, 1H), 7.69 (dd, J = 6.74, 1.98 Hz,
1H), 7.79 (dd, J = 8.13, 4.56 Hz, 1H), 8.60–8.70 (m, 1H), 9.11 (dd,
J = 4.56, 1.78 Hz, 1H).
6.2.5. 8-(3-Bromo-4-fluorobenzyl)pyrido[3,2-d]pyridazin-5(6H)-
one (7)
Compound 7 was prepared according to the procedure for com-
pound 4 and 6 substituting (3-bromo-4-fluorobenzyl)magnesium
chloride for (4-fluorobenzyl)magnesium chloride. Yield: 180 mg
(38%). MS (DCI/NH3) m/z 335 (M+H)+. 1H NMR. (300 MHz, DMSO-
d6): d 3.31 (s, 2H), 7.27 (t, J = 8.65 Hz, 1H), 7.30–7.41 (m, 1H),
7.66 (dd, J = 6.78, 2.37 Hz, 1H), 7.86 (dd, J = 7.97, 4.58 Hz, 1H),
8.60 (dd, J = 7.97, 1.86 Hz, 1H), 9.17 (dd, J = 4.58, 1.86 Hz, 1H).
6.2.6. Methyl 4-[(4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-
yl)methyl]pyridine-2-carboxylate (11)
A 250 mL high pressure vessel was charged with 7 (4.5 g,
13.47 mmol), a mixture of MeOH (50 mL) and DMF (50 mL),
Pd(dppf)2Cl2ꢃCH2Cl2 (0.099 g, 0.135 mmol) and NEt3 (3.75 ml,
26.9 mmol). The mixture was purged and pressurized with carbon
monoxide (60 psi), and stirred at 100 °C for 5 h. Solid material was
filtered off, and the filtrate was concentrated. The formed solid was
collected by filtration, washed with MeOH, and dried to give the ti-
tle product. Yield: 3.8 g (90%). MS (DCI/NH3) m/z 314 (M+H)+; 1H
NMR. (300 MHz, DMSO-d6): d 3.88 (s, 3H), 3.98 (s, 2H), 7.21 (t,
J = 8.65 Hz, 1H), 7.31–7.47 (m, 1H), 7.61 (dd, J = 6.98, 2.37 Hz,
1H), 7.87 (dd, J = 8.07, 4.58 Hz, 1H), 8.60 (dd, J = 8.07, 1.86 Hz,
1H), 9.17 (dd, J = 4.58, 1.86 Hz, 1H).
6.2.11. 8-[4-Fluoro-3-(2-oxopyrrolidin-1-yl)benzyl]-2,3,4,6-
tetrahydropyrido[2,3-d]pyridazin-5(1H)-one (8c)
A microwave tube was charged with tris(dibenzylideneace-
tone)dipalladium(0) (50 mg, 0.05 mmol), 4,5-bis(diphenylphos-
phino)-9,9-dimethylxanthene (Xantphos) (50 mg, 0.085 mmol), 9
(250 mg, 0.55 mmol), pyrrolidin-2-one (95 mg, 1.1 mmol) and
Cs2CO3 (270 mg, 0.85 mmol). Anhydrous dioxane (10 mL) was
added, and the suspension was heated in a microwave reactor
(CEM Discover) at 200 °C for 60 min. After concentration, the resi-
due was partitioned between ethyl acetate and brine. The organic
phase was concentrated. The residual solid was separated by flash
chromatography on silica gel (100% ethyl acetate) to provide a cou-
pled product. This product was mixed with 5% platinum on carbon
(25 mg), 5% Pd(OH)2 on carbon (25 mg), concentrated aqueous HCl
6.2.7. 2-Fluoro-5-[(5-oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-
yl)methyl]benzamide (12)
A solution of compound 11 (1 g, 3.2 mmol) in 7 N ammonia in
methanol (5 ml) was heated at 70 °C overnight, and cooled to room
temperature. The formed solid was collected by filtration, washed
with methanol and dried to provide the title compound. Yield:
20 mg (28%). MS (DCI/NH3) m/z 299 (M+H)+; 1H NMR. (300 MHz,
DMSO-d6): d 4.39 (s, 2H), 7.17 (dd, J = 10.51, 8.53 Hz, 1H), 7.31–
7.52 (m, 1H), 7.59 (dd, J = 7.14, 2.38 Hz, 1H), 7.85 (dd, J = 8.13,
4.56 Hz, 1H), 8.59 (dd, J = 7.93, 1.98 Hz, 1H), 9.16 (dd, J = 4.56,
1.78 Hz, 1H).
(66 lL) and dimethylformamide (10 ml), and pressurized under
40 psi of hydrogen at rt for 48 h. The volatiles were removed, and
the residue was separated by HPLC (Zorbax C-18, 0.1% TFA/
CH3CN/H2O) to provide compound 8c as TFA salt. Yield: 58 mg.
MS (ESI) m/z 343 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.57–
1.81 (m, 2H), 2.01–2.18 (m, 2H), 2.26–2.46 (m, 4H), 3.17 (m, 2H),
3.72 (t, J = 6.94 Hz, 2H), 3.84 (s, 2H), 6.39 (s, 1H), 7.16–7.19 (m,
1H), 7.18–7.25 (m, 1H), 7.29 (dd, J = 7.54, 1.98 Hz, 1H), 11.89 (s,
1H).

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G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
6.2.12. 8-(4-Fluoro-3-(2-oxoazetidin-1-yl)benzyl)-1,2,3,4-
tetrahydropyrido[3,2-d]pyridazin-5(6H)-one (8d)
ing, methyl 2-isocyanatopropanoate (23 mg, 0.18 mmol) was
added, and the mixture was stirred at 65 °C overnight. The reaction
mixture was concentrated, and the residue was dissolved in DMF
(10 mL). 2 N NaOH solution (1 mL) was added, and the mixture
was stirred at room temperature overnight. Solvent was removed,
and the residue was purified by HPLC (Zorbax C-8, 0.1% TFA/
CH3CN/H2O) to provide 8g as the TFA salt. Yield: 14 mg (23%).
MS (DCI/NH3) m/z 372 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.29 (d, J = 7.46 Hz, 3H), 1.59–1.79 (m, 2H), 2.27–2.36 (m, 2H),
3.17 (d, J = 4.41Hz, 2H), 3.74 (s, 2H), 4.04–4.23 (m, 1H), 6.28 (s,
1H), 6.74–6.88 (m, 1H), 6.97 (d, J = 7.12 Hz, 1H), 7.08 (dd,
J = 11.53, 8.48 Hz, 1H), 8.01 (dd, J = 7.97, 2.20 Hz, 1H), 8.39 (d,
J = 2.37 Hz, 1H).
Compound 8d was prepared according to procedure for8c,
substituting azitidinone for pyrrolidin-2-one. MS (ESI) m/z 329
(M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.61–1.76 (m, 2H), 2.33
(t, J = 6.35 Hz, 2H), 3.12 (t, J = 4.56 Hz, 2H), 3.17 (m, 2H), 3.77 (s,
2H), 3.81 (q, J = 4.36 Hz, 2H), 6.32 (s, 1H), 6.87–7.01 (m, 1H), 7.17
(dd, J = 11.90, 8.33 Hz, 1H), 7.78 (dd, J = 7.54, 2.38 Hz, 1H), 11.80
(s, 1H).
6.2.13. 1-(2-Fluoro-5-((5-oxo-5,6-dihydropyrido[3,2-
d]pyridazin-8-yl)methyl)phenyl)pyrrolidine-2,5-dione 16)
To a solution of 13 (300 mg, 1.1 mmol)) in acetonitrile (20 mL)
was added succinic anhydride (167 mg, 1.7 mmol) and stirred at
80 °C for 18 h. The reaction mixture was heated at 90 °C for an
additional three days. After cooling, the mixture was concentrated.
The crude solid was dissolved in DMF (100 mL) and treated with
CDI (180 mg, 1.1 mmol) and stirred at 80 °C for 16 h. The reaction
mixture was partitioned between ethyl acetate and brine. The or-
ganic layer was washed with brine and concentrated. The residue
was purified by HPLC (Zorbax C-8, 0.1% TFA/CH3CN/H2O) to pro-
vide the title product. Yield: 260 mg (67%). MS (ESI) m/z 353
(M+H)+; 1H NMR (300 MHz, DMSO-d6) d 2.34–2.53 (m, 4H), 4.40
(s, 2H), 7.21–7.34 (m, 2H), 7.41–7.51 (m, 1H), 7.86 (dd, J = 8.13,
4.56 Hz, 1H), 8.57–8.63 (m, 1H), 9.15 (dd, J = 4.56, 1.78 Hz, 1H).
6.2.18. 3-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}dihydropyrimidine-
2,4(1H,3H)-dione (8h)
The title compound was prepared as the TFA salt according to
the procedure for compound 8g, substituting methyl 3-isocyanato-
propanoate for methyl 2-isocyanatopropanoate. Yield: 9 mg (9%).
MS (DCI/NH3) m/z 372 (M+H)+; 1H NMR (DMSO-d6) d 1.68 (m,
2H), 2.27–2.37 (m, 2H), 2.40 (t, J = 6.27 Hz, 2H), 3.17 (m, 2H),
3.33 (m, 2H), 3.73 (s, 2H), 6.24 (s, 1H), 6.66–6.82 (m, 2H), 7.05
(dd, J = 11.36, 8.31Hz, 1H), 8.31 (s, 1H), 11.79 (s, 1H).
6.2.19. N-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}-3-methoxypropanamide (8i)
A mixture of 3-methoxypropanoic acid (19 mg, 0.18 mmol),
HATU (69 mg, 0.18 mmol) and Hunnig’s base (24 mg, 0.18 mmol)
in DMF (0.4 ml) was stirred at room temperature for 10 min. Then
8-(3-Amino-4-fluorobenzyl)-1,2,3,4-tetrahydropyrido[3,2-d]pyri-
dazin-5(6H)-one (50 mg, 0.18 mmol) was added into the reaction.
The mixture was stirred at the same temperature for overnight.
After filtration, the mixture was dissolved in a 1:1 mixture of
dimethylsulfoxide/methanol, and separated by HPLC (Waters Sun-
fire C-8, 0.1% trifluoroacetic acid/water/CH3CN) to provide 8i.
Yield: 10 mg (15%). MS (ESI) m/z 361 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.64–1.73 (m, 2H), 2.33 (t, J = 6.15 Hz, 2H), 2.60 (t,
J = 6.15 Hz, 2H), 3.17 (t, J = 5.16 Hz, 2H), 3.24 (s, 3H), 3.59 (t,
J = 6.15 Hz, 2H), 3.77 (s, 2H), 6.33 (s, 1H), 6.90–7.05 (m, 1H), 7.14
(dd, J = 10.91, 8.53 Hz, 1H), 7.68–7.81 (m, 1H), 9.63 (s, 1H), 11.85
(s, 1H).
6.2.14. 1-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}pyrrolidine-2,5-dione (8e)
Compound 8e was prepared as TFA salt according to the proce-
dure for compound 8, substituting compound 16 for 6. Yield: 38%.
MS (ESI) m/z 357 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.58–
1.78 (m, 2H), 2.33 (t, J = 6.27 Hz, 2H), 2.72–2.90 (m, 4H), 3.07–
3.23 (m, 2H), 3.84 (s, 2H), 6.34 (s, 1H), 7.13 (dd, J = 6.95, 2.20 Hz,
1H), 7.27–7.37 (m, 1H), 7.37–7.43 (m, 1H), 11.83 (s, 1H).
6.2.15. 1-(2-Fluoro-5-((5-oxo-5,6-dihydropyrido[3,2-
d]pyridazin-8-yl)methyl)phenyl)piperidine-2,6-dione (17)
Compound 17 was prepared according to the procedure for 16,
substituting dihydro-2H-pyran-2,6(3H)-dione for succinic anhy-
dride. MS (ESI) m/z 367 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.72–2.15 (m, 2H), 2.74 (t, J = 6.54 Hz, 4H), 4.37 (s, 2H), 7.10–
7.28 (m, 2H), 7.33–7.47 (m, 1H), 7.86 (dd, J = 8.13, 4.56 Hz, 1H),
8.60 (dd, J = 8.13, 1.78 Hz, 1H), 9.15 (dd, J = 4.56, 1.78 Hz, 1H),
12.83 (s, 1H).
6.2.20. N-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}-5-oxohexanamide (8j)
The title compound was prepared as TFA salt according to the
procedure for compound 8i, substituting 5-oxohexanoic acid for
3-methoxypropanoic acid. MS (ESI) m/z 387 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): 1.64–1.80 (m, 4H), 2.08 (s, 3H), 2.27–2.39
(m, 4H), 2.42–2.50 (m, 2H), 3.10–3.23 (m, 2H), 3.77 (s, 2H), 6.34
(s, 1H), 6.94–7.04 (m, 1H), 7.13 (dd, J = 10.85, 8.48 Hz, 1H), 7.66–
7.71 (m, 1H), 9.58 (s, 1H), 11.86 (s, 1H).
6.2.16. 1-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}piperidine-2,6-dione (8f)
To a solution of compound 17 (200 mg, 0.546 mmol) in DMF
(20 mL) in a 100 mL pressure bottle was added 5% Pt/C (60.0 mg,
0.308 mmol) and concentrated HCl (55
mixture was purged and pressurized with hydrogen (30 psi), and
stirred at rt for 3 days. Additional 60 mg of 5% Pt/C and 40 L of
ll, 1.2 equiv). The reaction
l
concentrated HCl were then added, and the mixture was stirred
at rt for 3 days. Solid material was filtered off, and the filtrate
was concentrated. The residue was separated by HPLC (Zorbax C-
18, 0.1% TFA/CH3CN/H2O) to provide 8f as a TFA salt. Yield:
80 mg (40%). MS (DCI/NH3) m/z 371 (M+H)+; 1H NMR (DMSO-d6):
d 1.58–1.78 (m, 2H), 1.82–2.10 (m, 2H), 2.34 (t, J = 6.15 Hz, 2H),
2.76 (t, J = 6.35 Hz, 4H), 3.81 (s, 2H), 3.79–4.01 (m, 2H), 6.37 (s,
1H), 7.07 (dd, J = 7.14, 2.38 Hz, 1H), 7.18–7.28 (m, 1H), 7.28–7.38
(m, 1H), 11.88 (s, 1H).
6.2.21. N-{2-fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}-N0-phenylpentanediamide
(8k)
The title compound was prepared as TFA salt according to the
procedure for compound 8i, substituting 5-oxo-5-(phenylami-
no)pentanoic acid for 3-methoxypropanoic acid. Yield: 13 mg. MS
(ESI) m/z 464 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.62–1.75
(m, 2H), 1.81–1.96 (m, 2H), 2.34 (t, J = 7.12 Hz, 4H), 2.42 (t,
J = 8.14 Hz, 2H), 3.09–3.22 (m, 2H), 3.77 (s, 2H), 6.30 (s, 1H),
6.93–7.07 (m, 1H), 7.14 (dd, J = 10.85, 8.48 Hz, 1H), 7.22–7.34 (m,
3H), 7.59 (d, J = 7.80 Hz, 2H), 7.68–7.77 (m, 1H), 9.62 (s, 1H), 9.87
(s, 1H), 11.82 (s, 1H).
6.2.17. 3-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}-5-methylimidazolidine-2,4-
dione (8g)
A suspension of compound 8b (50 mg, 0.18 mmol) in CH3CN
(15 mL) was heated until a transparent solution formed. After cool-

G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
4643
6.2.22. N-{2-fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]phenyl}-4-oxo-4-phenylbutanamide
(8l)
3.20 (m, 2H), 3.20–3.29 (m, 2H), 3.82 (s, 2H), 6.39 (s, 1H), 7.19
(dd, J = 10.31, 8.33 Hz, 1H), 7.30–7.38 (m, 1H), 7.47 (dd, J = 6.74,
2.38 Hz, 1H), 8.17–8.29 (m, 1H), 11.88 (s, 1H).
The title compound was prepared according to the procedure
for compound 8i, substituting 4-oxo-4-phenylbutanoic acid for 3-
methoxypropanoic acid. MS (ESI) m/z 435 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 1.63–1.73 (m, 2H), 2.32 (t, J = 5.95 Hz,
2H), 2.75–2.79 (m, 2H), 3.08–3.19 (m, 2H), 3.27–3.36 (m, 2H),
3.75 (s, 2H), 6.27 (s, 1H), 6.91–7.04 (m, 1H), 7.14 (dd, J = 10.91,
8.53 Hz, 1H), 7.54 (t, J = 7.54 Hz, 2H), 7.59–7.69 (m, 1H), 7.70–
7.77 (m, 1H), 7.94–8.03 (m, 2H), 9.74 (s, 1H), 11.78 (s, 1H).
6.3.6. N-Cyclopropyl-2-fluoro-5-[(5-oxo-1,2,3,4,5,6-
hexahydropyrido[2,3-d]pyridazin-8-yl)methyl]benzamide (20c)
MS (ESI) m/z 343 (M+H)+; 1H NMR (300 MHz, DMSO-d6): 0.44–
0.59 (m, 2H), 0.63–0.76 (m, 2H), 1.60–1.78 (m, 2H), 2.34 (t,
J = 6.35 Hz, 2H), 2.74–2.90 (m, 1H), 3.09–3.22 (m, 2H), 3.81 (s,
2H), 6.39 (s, 1H), 7.03–7.25 (m, 1H), 7.25–7.37 (m, 1H), 7.42 (dd,
J = 6.74, 2.38 Hz, 1H), 8.33 (d, J = 3.97 Hz, 1H), 11.89 (s, 1H).
6.3. Synthesis of compound 20
6.3.7. 2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]-N-(2-pyrrolidin-1-ylethyl)benzamide
(20d)
6.3.1. Methyl 2-fluoro-5-[(5-oxo-1,2,3,4,5,6-
hexahydropyrido[2,3-d]pyridazin-8-yl)methyl]benzoate (18)
MS (ESI) m/z 400 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.63–
1.76 (m, 2H), 1.76–1.93 (m, 2H), 1.93–2.10 (m, 2H), 2.34 (t,
J = 6.10 Hz, 2H), 2.61–2.76 (m, 2H), 2.96–3.12 (m, 2H), 3.12–3.22
(m, 2H), 3.25–3.40 (m, 2H), 3.52–3.68 (m, 2H), 3.84 (s, 2H), 6.35
(s, 1H,) 7.25 (dd, J = 10.85, 8.48 Hz, 1H), 7.33–7.49 (m, 1H), 7.57
(dd, J = 7.12, 2.37 Hz, 1H), 8.31–8.50 (m, 1H), 11.84 (s, 1H).
In
a 250 mL high pressure vessel, compound 11 (3.3 g,
10.53 mmol) and 5% Pt/C (0.330 g, 1.692 mmol) were suspended
in 30 mL of AcOH. This suspension was purged and pressurized
with 30 psi of hydrogen, and stirred at ambient temperature for
32 h. Solid material was filtered off, and the filtrate was concen-
trated. The formed solid was collected by filtration, washed with
MeOH and dried to give 18. Yield: 2.8 g (84%). MS (ESI) m/z 318
(M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.61–1.75 (m, 2H), 2.34
(t, J = 6.15 Hz, 2H), 3.17 (m, 2H), 3.44 (s, 3H), 3.84 (s, 2H), 6.39 (s,
1H), 7.27 (dd, J = 10.91, 8.53 Hz, 1H), 7.46–7.56 (m, 1H), 7.76 (dd,
J = 7.14, 2.38 Hz, 1H), 11.84 (s, 1H).
6.3.8. 2-Fluoro-N-[2-(4-methylpiperazin-1-yl)ethyl]-5-[(5-oxo-
1,2,3,4,5,6-hexahydropyrido[2,3-d]pyridazin-8-
yl)methyl]benzamide (20e)
MS (DCI/NH3) m/z 429 (M+H); 1H NMR (300 MHz, DMSO-d6): d
1.66–1.72 (m, 2H), 2.34 (t, J = 6.27 Hz, 2H), 2.80 (s, 3H), 2.83–2.96
(m, 2H), 3.05–3.31 (m, 6H), 3.47 (q, J = 6.22 Hz, 6H), 3.83 (s, 2H),
6.39 (s, 1H), 7.23 (dd, J = 10.68, 8.65 Hz, 1H), 7.38–7.45 (m, 1H),
7.51 (dd, J = 7.12, 2.37 Hz, 1H), 8.28 (d, J = 3.39 Hz, 1H), 11.88 (s,
1H).
6.3.2. 2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]benzoic acid (19)
Compound 18 (2.8 g, 8.8 mmol) was dissolved in hot THF
(150 mL), and was treated with LiOH (253 mg, 10.6 mmol) in water
(20 mL) at 50 °C overnight. After cooling to room temperature, the
reaction mixture was acidified with diluted HCl to a pH4, and con-
centrated to about 10 mL. The formed solid material was collected
by filtration, washed with water and dried to provide 19. Yield:
2.4 g (85%). MS (ESI) m/z 304 (M+H)+; 1H NMR (300 MHz, DMSO-
d6): d 1.61–1.77 (m, 2H), 2.34 (t, J = 6.10 Hz, 2H), 3.06–3.25 (m,
2H), 3.84 (s, 2H), 6.36 (s, 1H), 7.22 (dd, J = 10.85, 8.48 Hz, 1H),
7.39–7.52 (m, 1H), 7.73 (dd, J = 7.12, 2.37 Hz, 1H), 11.82 (s, 1H)
13.19 (s, 1H).
6.3.9. 8-[4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl]-2,3,4,6-
tetrahydropyrido[2,3-d]pyridazin-5(1H)-one (20f)
MS (DCI/NH3) m/z 372 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.60–1.77 (m, 2H), 2.35 (t, J = 6.27 Hz, 2H), 3.08 (s, 2H), 3.12–3.31
(m, 4H), 3.44 (s, 2H), 3.77–3.82 (m, 2H), 3.83 (s, 2H), 7.15–7.33
(m, 2H), 7.33–7.44 (m, 1H).
6.3.10. 8-{3-[(4-Aminopiperidin-1-yl)carbonyl]-4-
fluorobenzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-5(1H)-
one (20g)
6.3.3. General procedure for the syntheses of compounds 20
To a solution of compound 19 (100 mg, 0.33 mmol) in anhydrous
DMF (3 mL) was added appropriate amine (0.43 mmol), EDC
(82 mg, 0.43 mmol), 1-hydroxybenzotriazole monohydrate
(66 mg, 0.43 mmol), and triethylamine (43 mg, 0.43 mmol). The
reaction mixture was stirred at rt overnight, and partitioned be-
tween methylene chloride and brine. The organic phase was washed
with brine, water, and concentrated. The residue was separated by
HPLC to provide compounds 20. For compounds 20f, 20g, and 20h
whose syntheses involved the use of BOC-protected amine, the cou-
pling product was then treated with TFA (0.5 ml) in CH2Cl2 (2 mL) at
rt for 1 h. Removal of the volatiles provided the crude 20 that can be
further purified by HPLC to give the title products.
MS (DCI/NH3) m/z 386 (M+H)+; 1H NMR (300 MHz, CD3OD): d
1.44–1.70 (m, 2H), 1.71–1.88 (m, 2H), 1.91–2.04 (m, 1H), 2.12 (d,
J = 11.53 Hz, 1H), 2.51 (t, J = 6.27 Hz, 2H), 2.80–3.00 (m, 1H),
3.14–3.34 (m, 2H), 3.32–3.51 (m, 2H), 3.58–3.73 (m, 1H), 3.91 (s,
2H), 4.74 (d, J = 13.90 Hz, 1H), 7.08–7.26 (m, 2H), 7.31–7.47 (m,
1H).
6.3.11. 8-(4-Fluoro-3-{[(2R),-2-phenylpiperazin-1-
yl]carbonyl}benzyl),-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H),-one (20h)
MS (DCI/NH3) m/z 448 (M+H)+; 1H NMR (DMSO-d6): d 1.48–1.86
(m, 2H), 2.14–2.42 (m, 2H), 3.02–3.37 (m, 2H), 3.40–3.73 (m, 2H),
3.87 (m, 4H), 3.84 (s, 2H), 4.63 (m, 1H), 6.37 (s, 1H), 7.20–7.37 (m,
3H), 7.37–7.47 (m, 2H), 7.48–7.63 (m, 3H), 9.56 (s, 1H), 11.86 (s,
1H).
6.3.4. 2-Fluoro-5-((5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl)benzamide (20a)
MS (ESI) m/z 303 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.58–1.81 (m, 2H), 2.34 (t, J = 6.15 Hz, 2H), 3.12–3.23 (m, 2H),
3.84 (s, 2H), 7.27 (dd, J = 10.91, 8.53 Hz, 1H), 7.40–7.64 (m, 1H),
7.76 (dd, J = 7.14, 2.38 Hz, 1H).
6.3.12. 8-{4-Fluoro-3-[(4-methylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20i)
MS (DCI/NH3) m/z 386 (M+H)+; 1H NMR (DMSO-d6): d 1.52–1.78
(m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 2.84 (s, 3H), 2.92–3.25 (m, 6H),
3.39 (m, 2H), 3.57 (m, 2H), 3.83 (s, 2H), 6.37 (s, 1H), 7.06–7.36
(m, 2H), 7.33–7.51 (m, 1H), 11.86 (s, 1H).
6.3.5. N-Ethyl-2-fluoro-5-[(5-oxo-1,2,3,4,5,6-
hexahydropyrido[2,3-d]pyridazin-8-yl)methyl]benzamide (20b)
MS (ESI) m/z 331 (M+H)+; 1H NMR (300 MHz, DMSO-d6): 1.09 (t,
J = 7.14 Hz, 3H), 1.58–1.74 (m, 2H), 2.34 (t, J = 6.15 Hz, 2H), 3.12–

4644
G.-D. Zhu et al. / Bioorg. Med. Chem. 20 (2012) 4635–4645
6.3.13. 8-{3-[(4-Cyclopentylpiperazin-1-yl)carbonyl]-4-
fluorobenzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-5(1H)-
one (20j)
6.3.21. 8-{4-Fluoro-3-[(4-phenylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20r)
MS (DCI/NH3) m/z 440 (M+H)+; 1H NMR (DMSO-d6): d 1.29–1.86
(m, 8H), 2.01 (m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 2.98 (m, 1H), 3.03–
3.29 (m, 4H), 3.43 (m, 2H), 3.56 (m, 4H), 3.83 (s, 2H), 6.38 (s, 1H),
7.04–7.34 (m, 2H), 7.34–7.54 (m, 1H), 11.86 (s, 1H).
MS (DCI/NH3) m/z 448 (M+H)+; 1H NMR (DMSO-d6): d 1.52–1.78
(m, 2H), 2.34 (t, J = 6.10 Hz, 2H), 3.09 (m, 2H), 3.13–3.26 (m, 4H),
3.35 (m, 2H), 3.68–3.85 (m, 2H), 3.83 (s, 2H), 6.36 (s, 1H), 6.82 (t,
J = 7.29 Hz, 1H), 6.96 (d, J = 7.80 Hz, 2H), 7.22 (d, J = 8.48 Hz, 2H),
7.24–7.30 (m, 2H), 7.30–7.39 (m, 1H), 11.85 (s, 1H).
6.3.14. 8-{4-Fluoro-3-[(3-oxopiperazin-1-yl)carbonyl]benzyl}-
2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-5(1H)-one (20k)
MS (DCI/NH3) m/z 386 (M+H)+; 1H NMR (DMSO-d6): d 1.47–1.78
(m, 2H), 2.34 (t, J = 6.10 Hz, 2H), 3.07–3.24 (m, 2H), 3.39 (m, 2H),
3.69–3.81 (m, 2H), 3.83 (s, 2H), 4.11 (s, 2H), 6.38 (s, 1H), 7.04–
7.30 (m, 2H), 7.31–7.41 (m, 1H), 8.13 (s, 1H), 11.89 (s, 1H).
6.3.22. 8-{4-Fluoro-3-[(4-pyridin-4-ylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20s)
MS (DCI/NH3) m/z 449 (M+H)+; 1H NMR (DMSO-d6 DMSO-d6) d
1.60–1.82 (m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 3.09–3.25 (m, 2H), 3.43
(m, 2H), 3.68 (m, 2H), 3.74–3.88 (m, 4H), 3.84 (s, 2H), 6.38 (s, 1H),
7.17 (d, J = 7.12 Hz, 2H), 7.21–7.29 (m, 1H), 7.28–7.33 (m, 1H),
7.34–7.42 (m, 1H), 8.30 (d, J = 7.46 Hz, 2H), 11.86 (s, 1H).
6.3.15. 8-(3-{[4-(Cyclopropylcarbonyl)piperazin-1-yl]carbonyl}-
4-fluorobenzyl)-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20l)
6.3.23. 8-{4-Fluoro-3-[(4-pyridin-3-ylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20t)
MS (DCI/NH3) m/z 440 (M+H)+; 1H NMR (DMSO-d6) d 0.60–0.82
(m, 4H), 1.59–1.79 (m, 2H), 1.94 (m, 1H), 2.34 (t, J = 6.27 Hz, 2H),
3.00–3.35 (m, 4H), 3.38–3.79 (m, 2H), 3.82 (s, 2H), 3.83–4.11 (m,
4H), 6.37 (s, 1H), 7.15–7.27 (m, 1H), 7.24–7.30 (m, 1H), 7.30–7.40
(m, 1H), 11.87 (s, 1H).
MS (DCI/NH3) m/z 449 (M+H)+; 1H NMR (DMSO-d6): d 1.52–1.80
(m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 3.18 (m, 2H), 3.39 (m, 4H), 3.47–
3.61 (m, 2H), 3.76–3.84 (m, 2H), 3.84 (s, 2H), 6.44 (s, 1H), 7.05–7.33
(m, 2H), 7.30–7.48 (m, 1H), 7.82 (dd, J = 8.98, 5.26 Hz, 1H), 8.02 (dd,
J = 8.81, 2.37 Hz, 1H), 8.24 (d, J = 5.42 Hz, 1H), 8.46 (d, J = 2.71Hz,
1H), 11.92 (s, 1H).
6.3.16. 8-{3-[(4-Acetylpiperazin-1-yl)carbonyl]-4-fluorobenzyl}-
2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-5(1H)-one (20m)
MS (DCI/NH3) m/z 412 (M+H); 1H NMR (300 MHz, DMSO-d6): d
1.66–1.76 (m, 2H), 2.04 (s, 3H), 2.35 (t, J = 6.27 Hz, 2H), 3.12–3.30
(m, 4H), 3.36–3.46 (m, 2H), 3.52 (d, J = 5.09 Hz, 2H), 3.58 (d,
J = 5.09 Hz, 1H), 3.66 (d, J = 5.09 Hz, 1H), 3.83 (s, 2H), 7.14–7.29
(m, 2H), 7.30–7.43 (m, 1H).
6.3.24. 8-{4-Fluoro-3-[(4-pyridin-2-ylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20u)
MS (DCI/NH3) m/z 449 (M+H)+; 1H NMR (DMSO-d6 DMSO-d6): d
1.60–1.80 (m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 3.10–3.24 (m, 2H), 3.39
(m, 2H), 3.55 (m, 2H), 3.68 (m, 2H), 3.73–3.81 (m, 2H), 3.84 (s, 2H),
6.41 (s, 1H), 6.78–6.88 (m, 1H), 7.09 (d, J = 8.81Hz, 1H), 7.20–7.28
(m, 1H), 7.26–7.32 (m, 1H), 7.32–7.42 (m, 1H), 7.79 (t,
J = 7.29 Hz, 1H), 8.00–8.14 (m, 1H), 11.90 (s, 1H).
6.3.17. 8-(4-Fluoro-3-{[4-(trifluoroacetyl)piperazin-1-
yl]carbonyl}benzyl)-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20n)
MS (DCI/NH3) m/z 468 (M+H)+; 1H NMR (DMSO-d6): d 1.57–1.80
(m, 2H), 2.35 (t, J = 6.10 Hz, 2H), 3.09–3.26 (m, 2H), 3.35 (m, 2H),
3.57 (m, 2H), 3.67 (m, 2H), 3.74 (m, 2H), 3.83 (s, 2H), 6.42 (s,
1H), 7.03–7.29 (m, 2H), 7.31–7.48 (m, 1H), 11.94 (s, 1H).
6.3.25. 8-{4-Fluoro-3-[(4-pyrazin-2-ylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20v)
MS (DCI/NH3) m/z 450 (M+H)+; 1H NMR (DMSO-d6): d 1.61–1.76
(m, 2H), 2.34 (t, J = 6.15 Hz, 2H), 3.10–3.24 (m, 2H), 3.34 (m, 2H),
3.54 (m, 2H), 3.67 (m, 2H), 3.74 (m, 2H), 3.83 (s, 2H), 6.43 (s,
1H), 7.20–7.27 (m, 1H), 7.28 (dd, J = 6.54, 2.18 Hz, 1H), 7.31–7.38
(m, 1H), 7.87 (d, J = 2.78 Hz, 1H), 8.10 (d, J = 2.78 Hz, 1H), 8.32 (s,
1H), 11.95 (s, 1H).
6.3.18. 8-{3-[(4-Benzoylpiperazin-1-yl)carbonyl]-4-
fluorobenzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-5(1H)-
one (20o)
MS (DCI/NH3) m/z 476 (M+H)+; 1H NMR (DMSO-d6) d 1.57–1.78
(m, 2H), 2.34 (t, J = 6.10 Hz, 2H), 3.17 (m, 4H), 3.21–3.37 (m, 2H),
3.45 (m, 2H), 3.69 (m, 2H), 3.82 (s, 2H), 6.40 (s, 1H), 7.18–7.30
(m, 2H), 7.29–7.38 (m, 1H), 7.39–7.50 (m, 5H), 11.92 (s, 1H).
6.3.26. 8-{4-Fluoro-3-[(4-pyrimidin-2-ylpiperazin-1-
yl)carbonyl]benzyl}-2,3,4,6-tetrahydropyrido[2,3-d]pyridazin-
5(1H)-one (20w)
6.3.19. 4-{2-Fluoro-5-[(5-oxo-1,2,3,4,5,6-hexahydropyrido[2,3-
d]pyridazin-8-yl)methyl]benzoyl}-N,N-dimethylpiperazine-1-
carboxamide (20p)
MS (DCI/NH3) m/z 450 (M+H)+; 1H NMR (DMSO-d6): d 1.39–1.85
(m, 2H), 2.33 (t, J = 6.10 Hz, 2H), 3.06–3.21 (m, 2H), 3.24–3.35 (m,
2H), 3.55–3.96 (m, 8H), 6.40 (s, 1H), 6.67 (t, J = 4.75 Hz, 1H), 7.16–
7.26 (m, 1H), 7.24–7.30 (m, 1H), 7.30–7.39 (m, 1H), 8.38 (d,
J = 4.75 Hz, 2H), 11.91 (s, 1H).
MS (DCI/NH3) m/z 443 (M+H)+; 1H NMR (DMSO-d6): d 1.37–1.52
(m, 2H), 2.09 (t, J = 6.15 Hz, 2H), 2.17–2.29 (m, 2H), 2.75–2.85 (m,
2H), 2.91 (m, 6H), 2.91–3.01 (m, 4H), 3.38 (m, 2H), 3.56 (s, 2H),
6.17 (s, 1H), 6.91–6.99 (m, 1H), 6.97–7.03 (m, 1H), 7.04–7.10 (m,
1H), 11.68 (s, 1H).
References and notes
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5(1H)-one (20q)
MS (DCI/NH3) m/z 450 (M+H)+; 1H NMR (DMSO-d6): d 1.63–1.81
(m, 2H), 2.35 (t, J = 6.27 Hz, 2H), 2.91 (m, 3H), 3.09 (m, 2H), 3.12–
3.24 (m, 4H), 3.32 (m, 2H), 3.74 (m, 2H), 3.82 (s, 2H), 6.38 (s, 1H),
7.17–7.29 (m, 2H), 7.31–7.39 (m, 1H), 11.89 (s, 1H).

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