Development of substituted 6-[4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl]-4,5-dimethylpyridazin-3(2H)-ones as potent poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors active in BRCA deficient cells

Article abstract of DOI:10.1016/j.bmcl.2009.11.087

We describe an extensive SAR study in the 6-[4-fluoro-3-(substituted)benzyl]-4,5-dimethylpyridazin-3(2H)-one series which led to the identification of potent PARP-1 inhibitors, capable of inhibiting the proliferation of BRCA-1 deficient cancer cells in th

Full text of DOI:10.1016/j.bmcl.2009.11.087

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1100–1105
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of substituted 6-[4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl]-4,5-
dimethylpyridazin-3(2H)-ones as potent poly(ADP–ribose) polymerase-1 (PARP-
1) inhibitors active in BRCA deficient cells
*
Federica Ferrigno , Danila Branca, Olaf Kinzel, Samuele Lillini, Laura Llauger Bufi, Edith Monteagudo,
Ester Muraglia, Michael Rowley, Carsten Schultz-Fademrecht, Carlo Toniatti, Caterina Torrisi, Philip Jones
IRBM, Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia 00040, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe an extensive SAR study in the 6-[4-fluoro-3-(substituted)benzyl]-4,5-dimethylpyridazin-
3(2H)-one series which led to the identification of potent PARP-1 inhibitors, capable of inhibiting the pro-
liferation of BRCA-1 deficient cancer cells in the low nanomolar range, and displaying >100-fold selectiv-
ity over the BRCA wild type counterparts. The series of compounds was devoid of hERG channel activity,
and CYP inhibition and induction liabilities. Several analogs were stable in rat and human liver micro-
somes and displayed moderate rat clearance, with urinary excretion of parent as the major route of
elimination.
Received 27 October 2009
Revised 17 November 2009
Accepted 17 November 2009
Available online 22 November 2009
Keywords:
Anticancer agents
PARP-1 inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
BRCA1 deficient cancer cells
6-[4-Fluoro-3-(piperazin-1-
ylcarbonyl)benzyl]-4,5-dimethylpyridazin-
3(2H)-ones
Coupling reactions
SAR exploration
Pharmacokinetic properties
Poly(ADP–ribose) polymerase (PARP)-1 is the main isoform of
the PARP family, which include some 18 proteins.1 PARP catalyses
the polymerization of NAD+ into long poly(ADP–ribose) chains.
PARP-1 is one of the most abundant nuclear enzymes and has a
well established role in DNA repair processes.2 PARP inhibitors
have been used in cancer therapy as both chemo- and radio-sensi-
tizers,3 and more recently, have been employed alone in treatment
of tumors displaying BRCA mutations.4
Several PARP inhibitors are currently on-going clinical trials
including: AZD-2281,5 ABT-888,6 AG-014699,7 and MK-4827,8 as
well as the undisclosed BS-201 and INO-1001.
We have recently described the identification of a novel series
of pyrrolo[1,2-a]pyrazin-1(2H)-one PARP inhibitors characterized
by A1 and A2 (Fig. 1).9
Both compounds were potent PARP-1 inhibitors (IC50 <2.5 nM)
with good antiproliferative activity against BRCA-1 deficient HeLa
cells (CC50 = 48 and 53 nM, respectively) and displaying >100-fold
selective cytotoxicity over BRCA wild type cells.
Despite these encouraging characteristics this series suffered
from some off-target activities and poor rat pharmacokinetics. In
particular, weak binding to the hERG channel, and micromolar
CYP inhibition were observed. Furthermore, A2 also caused modest
CYP induction at 10 lM. The pharmacokinetic properties were also
suboptimal with moderate–high intrinsic clearance in rat liver
microsomes and in rats clearance in excess of hepatic blood flow
was measured. With the aim to solve these issues while maintain-
ing PARP inhibition activity, we extensively worked to modify the
heterocyclic scaffold, hypothesizing that the electron rich pyrrole
motif could be the cause of metabolic instability. We also made
an extensive SAR study on the right end side part of our scaffold
supposing that the lipophilic substituents caused the unwanted
off-target activities.
In this Letter we describe the identification and optimization of a
new series of dimethylpyridazin-3(2H)-one PARP inhibitors B lead-
ing to very potent analogs with clean off-target profiles and im-
proved pharmacokinetic properties both in vitro and in vivo in rats.
We prepared two common precursors which allowed us to
introduce diversity at the late stage of the synthesis: 5-[(4,5-di-
methyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl]-2-fluorobenzoic
acid (5), and 6-[4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl]-4,5-
dimethylpyridazin-3(2H)-one (6) (Scheme 1).
In brief, the 3,6-dichloro-4,5-dimethylpyridazine (1) was con-
densed with 5-(cyanomethyl)-2-fluorobenzonitrile (3) to afford
* Corresponding author.
E-mail address: federica_ferrigno@libero.it (F. Ferrigno).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.087

F. Ferrigno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1100–1105
1101
scaffold A
scaffold B
O
O
NH
NH
N
Cl
N
O
F
O
F
Cl
R
R
N
N
N
R
N
H
O
HN
A1
Compds
A2
2.0
PARP-1 IC50 (nM)
BRCA1- CC50 (nM)
BRCAwt CC50 (nM)
2.5
48
53
>5000
>5000
Rat liver microsomes
65
122
Clint (µl/min/mg)
Rat Clearance
(mL/min/kg)
130
>200
hERG IC50 (nM)
12000
11000
CYP inhibition
(%inh. at 10µM)
2D6: 35
-
3A4: 25
14% rifampicin
response
CYP induction (at 10µM)
Figure 1. Profile of pyrrolo[1,2-a]pyrazin-1(2H)-one PARP inhibitors A1 and A2 and the modification to novel series of dimethylpyridazin-3(2H)-ones B.
Me
CN
F
2
i) NBS, AIBN,
CCl4, 80°C, o/n
ii) TMS-CN, TBAF,
CH3CN, 2h
40%
CN
i)
HN
NC
N
i) AcOH:HClconc.: H2O =
1:2:1, reflux, o/n
ii) AcOH, NaOAc, reflux, 1h
iii) aq. NaOH, 90°C, 30 min.
iv) 2M HCl, 10 min.
Boc
F
Cl
O
O
O
3
TBTU, DIPEA,
DMF, o/n
acid, HATU,
Cl
NaH (2eq.), THF,
0°C -> RT, 2h
N
N
NH
N
NH
N
HOBT, DIPEA,
DMF, 40°C, o/n
NH
N
ii) TFA, DCM
N
N
O
F
O
F
O
F
51%
80%
CN
95%
OH
NC
N
N
Cl
NH
N
1
F
R
4
5
6
13, 15, 19, 20, 28
i) Boc-protected
amine, TBTU, DIPEA,
DMF, o/n
TFA, DCM
or
Et2NH, THF
or
H2, MeOH
ii) TFA, DCM
O
O
NH
N
NH
N
O
R
F
O
N
N
F
R
7, 10, 11
12, 14, 16, 23-27, 29-37
aldehyde,
NaBH3CN,
MeOH, 2h
aldehyde, AcOH,
NaBH3CN, DCE,
MeOH, 1h
O
O
NH
N
NH
N
O
O
R
F
N
N
F
R
17, 18, 21, 22
8, 9
Scheme 1. Synthesis of compounds 1–37.

1102
F. Ferrigno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1100–1105
intermediate 4 using 2 equiv of NaH.10 Compound 3 was prepared
from 2-fluoro-5-methylbenzonitrile (1) by radical mono-bromina-
tion followed by displacement with TMS-CN in the presence of
TBAF. The chloro-dimethylpyridazine 4 was then submitted to full
hydrolysis and decarboxylation by a three step sequence which
consist of hydrolysis and decarboxylation of the cyano group in
the benzylic position, followed by formation of the pyridazinone
core, and hydrolysis of the fluorobenzonitrile to give the desired
carboxylic acid scaffold 5.11 This intermediate was used to obtain
the compounds listed in Table 1 using a standard coupling proce-
dure followed by Boc-deprotection and reductive amination. Alter-
natively, the carboxylic acid intermediate 5 was coupled with tert-
butyl piperazine-1-carboxylate to install the piperazine moiety
and obtain, after Boc-removal, the intermediate 6. This piperazine
was employed for the synthesis of the compounds described in Ta-
bles 2 and 3 by coupling reactions with the appropriate carboxylic
acids, followed by removal of the N-protecting group and reductive
amination to add the alkyl fragments. Compound 22 was obtained
from the Fmoc protected amino acid, as the cyclobutyl fragment
was discovered to be unstable under acidic conditions required
for N-Boc cleavage. The coupling reaction of 6 with the sterically
hindered carboxylic acids proved to be slow, and was optimized
using a combination of HATU and HOBT with gentle heating for ex-
tended reaction time.12
Table 2
SAR study in the
a
,
a
-disubstituted amino acid series
O
NH
N
O
N
N
F
R
Compds
R
PARP-1
IC50 (nM)
BRCA1-
CC50 (nM)
BRCAwt
CC50 (nM)
a,b
c,b
d,b
N
H
12
13
14
3.7
280
360
240
>5000
> 2500
>5000
O
N
1.8
3.6
5.7
O
O
O
N
H
15
16
N
350
81
7700
The replacement of the pyrrolo[1,2-a]pyrazin-1(2H)-one scaffold
A by the dimethylpyridazin-3(2H)-one B was beneficial and we pre-
paredtheanalogof A1(7)inthenewseries(Table1). Thespiro-deriv-
ative 7 showed comparableactivityto A1 both in thePARP-1enzyme
and in inhibiting the proliferation of BRCA-1 deficient cells
(IC50 = 2.6 nM, CC50 = 49 nM vs IC50 = 2.5 nM, CC50 = 48 nM). Simi-
larly 7 maintained the excellent selectivity seen with A1, displaying
2.7
3.1
2.4
2.7
3.9
>5000
N
H
O
O
O
O
O
17
18
19
20
91
4000
2100
1500
870
N
H
71
N
H
Table 1
Exploration of the spiro-bicycle motif in the amide portion of 5
O
89
N
NH
N
O
130
N
H
R
F
a,b
c,b
Compds
R
PARP-1 IC50
(nM)
BRCA1-CC50
(nM)
BRCAwt
CC50 (nM)
21
22
3.5
3.2
170
24
1900
450
d,b
N
H
O
O
N
N
7
2.6
4.5
49
>5000
>5000
N
H
HN
N
a–d
See footnotes of Table 1.
8
9
100
N
>100-fold selective cytotoxicity for BRCA-1 deficient cells compared
to their wild type counterparts (CC50 >5 M). In contrast, unlike A1
whichdisplayedmultiple off-targetsactivities, 7 was devoid of bind-
ing to the hERG channel and CYP inhibition or induction at 10 M.
Furthermore, 7 was found to be extremely stable in rat and human
liver microsomes (Clint <1 l/min/mg). In spite of this, when profiled
in vivo in rats, 7 showed a high plasma clearance (Cl = 57 mL/min/
kg) with a t1/2 of 3.7 h mainly due to a high volume of distribution
(Vdss = 6.7 L/kg). Routes of elimination studies revealed that excre-
tion of parent to urine was an important route of clearance, with
37% of the dose excreted intact in the first 24 h following iv dosing.
Starting from 7 we tried to modify the spiro-bicycle motif with
the aim to improve first the cellular activity, and eventually the PK
9.7
170
>5000
l
N
l
N
N
l
10
11
4.7
4.6
>5000
>5000
>5000
NH
>5000
HN
a,c,d
b
8
The assays were performed as described in Ref.
Values are means of at least two experiments.
.

F. Ferrigno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1100–1105
1103
Table 3
SAR study in the
Table 3 (continued)
a
-substituted proline derivative series
a,b
c,b
d,b
Compds
R
PARP-1 IC50
(nM)
BRCA1-CC50
(nM)
BRCAwt CC50
(nM)
O
NH
F
N
O
N
N
36
4.2
290
15,000
F
R
(S) N
H
a,b
c,b
d,b
Compds
R
PARP-1 IC50
(nM)
BRCA1-CC50
(nM)
BRCAwt CC50
(nM)
O
F
23
2.8
3.8
1.3
3.1
2.7
2.8
90
>5000
N
H
O
O
O
O
37
2.5
15
3300
(R) N
H
(S) N
H
24
25
26
27
28
780
45
>40,000
>5000
O
a-d
See footnotes of Table 1.
(R) N
H
profile. Compared to the lead 7, the N-methylated derivative 8 was
a less potent PARP-1 inhibitor and displayed reduced antiprolifer-
ation effect on BRCA-1 deficient cells. The introduction of larger
lipophilic substituents, as in 9, further decreased the activity both
in the enzyme and the cell based assays and confirmed the impor-
tant role of the free NH moiety. Shifting the NH to the b position of
the spiro-centre resulted in 10, which although maintained enzyme
activity (IC50 = 4.7 nM), was devoid of activity in cells at 5 lM. The
same result was obtained by changing the five-membered cycle to
a fused azetidine, as in 11.
(S)
3000
220
380
>10,000
>10,000
11,000
N
H
(R)
N
H
O
O
O
Although 7 displayed an encouraging profile, we were unable to
further improve the antiproliferation activity of this series, so we
decided to turn our attention to the amino acid derivative A2,
and accordingly analog 12 was prepared (Table 2).
As seen previously with the pair A1 and 7, 12 displayed compa-
rable enzyme activity to the pyrrolo[1,2-a]pyrazin-1(2H)-one A2
(IC50 = 3.7 and 2.0 nM, respectively), although it was fivefold weak-
er than A2 in inhibiting the proliferation of BRCA-1 deficient cells
(CC50 = 280 nM compared to 53 nM for A2). More importantly the
pyridazinone 12 was also devoid of off-target cardiac ion channel
NH
29
30
3.9
12
>5000
2000
>5000
O
>20,000
N
H
O
O
NH
(R)
activity, and CYP inhibition or induction at 10
the amino acid derivative 12 displayed good stability in rat and hu-
man liver microsomes (Clint = 7 and 10 l/min/mg) but high plas-
lM. Similarly to 7,
31
32
3.1
3.8
78
24
>40,000
16,000
l
ma clearance in rats (Cl = 64 mL/min/kg). Equally to 7, there was
an extensive excretion of the parent in urine, 34% of the iv dose ex-
creted as parent in first 24 h.
N
H
O
O
Driven by the results of 12 an extensive SAR study was under-
taken. Unsurprisingly, the cyclopropyl analog 14 was comparable
to 12 both in enzyme and cell based assays, but the enlargement
to the cyclopentyl 16 resulted in an improvement in activity
(IC50 = 2.7 nM) and encouragingly 16 displayed double digit anti-
proliferation activity against BRCA1 deficient cells (CC50 = 81 nM)
with greater that 60-fold selectivity. The addition of a second
methyl on the nitrogen in 13 and 15 was slightly detrimental for
the activity, demonstrating once again the importance of a second-
ary amine moiety in this region of the molecule. The homologation
of the methyl group of 16 to an ethyl in 17, or to a cyclopropyl in 18
caused only slight changes in enzyme potency and cell based activ-
ity. Unfortunately the introduction of a larger and more lipophilic
substituent in this region of the molecule, as the methylcyclopro-
pyl group, resulted in a loss of selectivity between BRCA-1 deficient
and wild type cells, with 18 displaying only 30-fold selectivity
compared to the 60-fold selectivity of 16. This trend was confirmed
by the pyrrolidine analog 19, which showed only 15-fold selectiv-
33
34
1.8
3.3
31
53
19,000
>5000
N
H
N
H
O
F
35
3.6
9.1
2700
N
H
O

1104
F. Ferrigno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1100–1105
ity, and the N-phenyl derivative 20, which was the least selective
compound of this series, displaying only sixfold selectivity. The
replacement of the cyclopentyl fragment with the corresponding
cyclohexyl in 21 was detrimental for both enzyme and cellular
activity. Reducing the ring size to the corresponding cyclobutyl
22 produced instead a more potent compound. In fact 22 displayed
a threefold improvement in the BRCA-1 deficient cells assay com-
pared to 18, but with only 20-fold selectivity.
The threefold boost in cellular activity seen when we cyclised
the gem-dimethyl group of 12 to the corresponding cyclopentyl
of 16 drove us to explore if a similar result could be obtained by
cyclising the gem-dimethyl group onto the N-methyl amino group,
thereby forming a pyrrolidine or related derivative (Table 3).
31 nM, respectively). The ethyl analog 32 displayed good stability
in presence of rat and human liver microsomes (Clint = 6 and
13 ll/min/mg, respectively), but unfortunately it showed high
plasma clearance in rats (Cl = 63 mL/min/kg). The introduction of
the simple benzyl substituent in 34 caused a modest improvement
in cellular activity compared to the racemic
a-methyl derivative
23, with 34 displaying CC50 = 53 nM and more than 100-fold selec-
tivity. Further exploration of 34 demonstrated that when we in-
stalled the para-fluoro benzyl group in 35 we obtained a further
improvement in cellular activity with antiproliferation activity in
the BRCA-1 deficient cells in the low nanomolar range
(CC50 = 9.1 nM). Again the majority of the activity resided in one
enantiomer and, when the corresponding enantiomers 36 and 37
were prepared, the (R)-enantiomer displayed superior cellular
activity compared to the (S)-enantiomer (CC50 = 10 and 290 nM,
respectively). Unfortunately this class of compounds, bearing an
Indeed, the
a-methyl proline derivative 23 was a more potent
PARP-1 inhibitor than 12 (IC50 = 2.8 and 3.7 nM, respectively),
and, similarly to what we previously observed by cyclising to 16,
12 showed
a
threefold improvement in cell based activity
a
-benzyl proline residue, was metabolically unstable, displaying
high intrinsic clearance when compounds were incubated with
rat and human liver microsomes (34: Clint = 101 and 50 l/min/
mg, respectively). Probably the additional lipophilicity was also
the source of some CYP3A4 inhibition (35: 73% inh. at 10 M),
(CC50 = 90 nM for 23 compared to 280 nM for 12). Moreover, 23
was completely stable in presence of rat and human liver micro-
somes (Clint <1 ll/min/mg), and, when dosed in rats, it showed
l
only a moderate plasma clearance (Cl = 38 mL/min/kg). Further
studies revealed that the compound was essentially cleared intact
with 82% of the iv dose being eliminated in the urine as parent
within 72 h, and a further 3% in the bile. Trace amounts of 6 were
also detected as a minor elimination route. Given that the clear-
ance value is significantly higher than the renal glomerular filtra-
tion rate an active transport mechanism is suspected.
Given the encouraging profile of 23, we first elucidated which
features of the proline residue were essential for the activity. Prep-
aration of the enantiomers of 23 revealed that the (R)-25 was more
active than the enantiomer 24 both in PARP-1 enzyme (IC50 = 1.3
vs 3.8 nM) and antiproliferation (CC50 = 45 and 780 nM, respec-
tively) assays. The (R)-enantiomer 25 was one of the most interest-
ing compounds and displayed more than 100-fold selectivity
between BRCA-1 deficient and wild type cells. The same difference
was observed for other pairs of enantiomers with the (R)-enantio-
mer displaying at least 10-fold improved activity in the BRCA-1
l
although 35 remained devoid of hERG activity and CYP induction
liabilities.
Nevertheless the issue of interesting analogs, as 23, 31 and 32,
was the lack of oral bioavailability (F <3%). This was probably due
to the lack of absorption, as confirmed for compound 23 that re-
mained undetected after sampling of the hepatic portal vein of rats
dosed orally.
In summary, we have developed a new class of PARP-1 inhibi-
tors with good enzyme activity and demonstrating low nanomolar
antiproliferative activity in BRCA-1 deficient cells. Our compounds
showed excellent selectivity between BRCA1 deficient and wild
type cells, displaying no off-targets activities on cardiac ion chan-
nels and no CYP liabilities. Several compounds showed good stabil-
ity in liver microsome incubations and acceptable clearance in rats,
being eliminated unchanged in the urine and bile.
deficient cells assay (26 and 27; 36 and 37). Removal of the
methyl group produced in 27 a fivefold drop in antiproliferation
a-
Acknowledgments
activity compared to 25, demonstrating the importance of the ste-
The authors thank Mauro Cerretani, Claudia Giomini, Monica
Bisbocci, Maria Orsale, Massimiliano Fonsi, Fabrizio Fiore and Anna
Alfieri for their support to this work.
ric crowding in the
a-position of the carbonyl. Other important
features in this series for both the enzyme and the cell based assays
activity included the presence of the free NH, as shown by the
replacement of the NH with an oxygen in tetrahydrofuran 28,
which was fourfold less potent than 23 in cells and only displayed
30-fold selectivity between BRCA deficient and proficient cells.
Similarly, the position of the nitrogen in the cycle was critical for
cell based activity, as pyrrolidin-3-yl derivative 29 was devoid of
References and notes
1. (a) Hassa, P. O.; Hottiger, M. O. Front. Biosci. 2008, 13, 3046; (b) Amé, J. C.;
Spenlehauer, C.; de Murcia, G. Bioessays 2004, 26, 882.
2. (a) Jagtap, P.; Szabó, C. Nat. Rev. Drug Disc. 2005, 4, 421; (b) Schreiber, V.;
Dantzer, F.; Amé, J.-C.; de Murcia, G. Nat. Rev. Mol. Cell Biol. 2006, 7, 517.
3. (a) Tentori, L.; Graziani, G. Pharmacol. Res. 2005, 52, 25; (b) Rodon, J.; Iniesta, M.
D.; Papadopoulos, K. Expert Opin. Investig. Drugs 2009, 18, 31; (c) Ratnam, K.;
Low, J. A. Clin. Cancer Res. 2007, 13, 1383.
4. (a) Bryant, H. E.; Schultz, N.; Thomas, H. D.; Parker, K. M.; Flower, D.; Lopez, E.;
Kyle, S.; Meuth, M.; Curtin, N. J.; Helleday, T. Nature (London, UK) 2005, 434,
913; (b) Farmer, H.; McCabe, N.; Lord, C. J.; Tutt, A. N. J.; Johnson, D. A.;
Richardson, T. B.; Santarosa, M.; Dillon, K. J.; Hickson, I.; Knights, C.; Martin, N.
M. B.; Jackson, S. P.; Smith, G. C. M.; Ashworth, A. Nature (London, UK) 2005,
434, 917.
antiproliferation activity in BRCA-1 deficient cells at 5 lM. As in
the previous series the size of the ring was important for activity,
where the six-membered ring analog 30 was fourfold less potent
in the PARP-1 assay compared to pyrrolidine 23. Interestingly,
the azetidine analog 31 retained most of the activity of pyrrolidine
derivative 25, being twofold less active in cells with CC50 = 78 nM,
but displaying excellent selectivity. Moreover 31 was stable when
incubated with rat and liver microsomes (Clint <1 and 3 ll/min/mg,
5. Menear, K. A.; Adcock, C.; Boulter, R.; Cockcroft, X.; Copsey, L.; Cranston, A.;
Dillon, K. J.; Drzewiecki, J.; Garman, S.; Gomez, S.; Javaid, H.; Kerrigan, F.;
Knights, C.; Lau, A.; Loh, V. M., Jr.; Matthews, I. T. W.; Moore, S.; O’Connor, M. J.;
Smith, G. C. M.; Martin, N. M. B. J. Med. Chem. 2008, 51, 6581.
respectively) and it displayed moderate plasma clearance in rats
(Cl = 23 mL/min/kg), with 11% of the iv dose excreted intact in
the urine as parent in the first 24 h.
6. Penning, T. D.; Zhu, G.-D.; Gandhi, V. B.; Gong, J.; Liu, X.; Shi, Y.; Klinghofer, V.;
Johnson, E. F.; Donawho, C. K.; Frost, D. J.; Bontcheva-Diaz, V.; Bouska, J. J.;
Osterling, D. J.; Olson, A. M.; Marsh, K. C.; Luo, Y.; Giranda, V. L. J. Med. Chem.
2009, 52, 514.
Given the excellent cell based activity and selectivity seen with
the
a-methyl proline derivative 25, a further exploration was con-
ducted for the replacement of the
a-methyl substituent. Here we
7. Thomas, H. D.; Calabrese, C. R.; Batey, M. A.; Canan, S.; Hostomsky, Z.; Kyle, S.;
Maegley, K. A.; Newell, D. R.; Skalitzky, D.; Wang, L. Z.; Webber, S. E.; Curtin, N.
J. Mol. Cancer Ther. 2007, 6, 945–956.
8. Jones, P.; Altamura, S.; Boueres, J.; Ferrigno, F.; Fonsi, M.; Giomini, C.;
Lamartina, S.; Monteagudo, E.; Ontoria, J. M.; Orsale, V.; Pesci, S.; Roscilli, G.;
obtained compounds, such as the ethyl derivative 32 and the allyl
analog 33, which improved cellular activity compared to the race-
mic compound 23, with both 32 and 33 displaying low nanomolar
antiproliferation activity in BRCA-1 deficient cells (CC50 = 24 and

F. Ferrigno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1100–1105
1105
Rowley, M.; Scarpelli, R.; Schultz-Fademrecht, C.; Toniatti, C. J. Med. Chem.
2009, 52, 7170.
9. Pescatore, G.; Branca, D.; Kinzel, O.; Llauger Bufi, L.; Muraglia, E.; Orvieto, F.;
Rowley, M.; Toniatti, C.; Torrisi, C.; Jones, P. Bioorg. Med. Chem. Lett., accepted
for publication.
10. 5-[(6-Chloro-4,5-dimethylpyridazin-3-yl)(cyano)methyl]-2-fluorobenzonitrile (4):
To a ice-cold solution of 1 (13.3 g, 75 mmol) and 3 (10 g, 62 mmol) in dry THF
(200 mL), was added portion wise NaH (5.0 g, 125 mmol). The reaction was
stirred at 0 °C for 15 min and at rt for 2 h, then quenched with a satd aq
NaHCO3 solution. Aqueous work up, followed by crystallization from EtOAc
afforded 4 (9.5 g, 51%) as white solid. MS (ES) C15H10ClFN4 requires 300, 302,
found: 301, 303 (M+H)+.
with EtOAc. The crude product was suspended in 4 M aq NaOH solution
(50 mL), and stirred at 90 °C for 30 min. After cooling to rt, 2 M HCl solution
was added until pH 4, and filtration gave 5 (7.0 g, 80%) as yellow powder. 1H
NMR (300 MHz, DMSO-d6) d: 13.23 (br s, 1H), 12.66 (br s, 1H), 7.71–7.63 (m,
1H), 7.47–7.39 (m, 1H), 7.29–7.19 (m, 1H), 3.98 (s, 2H), 2.01 (s, 3H), 1.99 (s,
3H). MS (ES) C14H13FN2O3 required: 276 found: 277 (M+H)+.
12. 6-(4-Fluoro-3-{[4-(2-methylprolyl)piperazin-1-yl]carbonyl}benzyl)-4,5-dimethyl-
pyridazin-3(2H)-one (23):
a solution of Boc-2-methyl-D,L-proline (50 mg,
0.22 mmol), HOBT (33 mg, 0.22 mmol) and HATU (83 mg, 0.22 mmol) in
DMF (1.4 mL) was stirred at rt for 30 min. then 6 (50 mg, 0.15 mmol) and
DIPEA (38
lL, 0.22 mmol) were added. The mixture was stirred at 40 °C for
12 h, then diluted with EtOAc. Aqueous work up, followed by preparative RP-
HPLC purification (column: Waters X-Terra C18) afforded 23 (38 mg, 51%) as
white powder. 1H NMR (400 MHz, DMSO-d6) d: 12.67 (br s, 1H), 9.19 (br s, 1H),
7.36–7.17 (m, 3H), 3.96 (s, 2H), 3.75–3.42 (m, 6H), 3.33–3.03 (m, 4H), 2.28–
2.09 (m, 2H), 2.06–1.82 (m, 8H), 1.63-1.46 (m, 3H). MS (ES) C24H30FN5O3
requires 455, found: 456 (M+H)+.
11. 5-[(4,5-Dimethyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl]-2-fluorobenzoic acid
(5):
4 (9.5 g, 32 mmol) was dissolved in AcOH, concd HCl, H2O (1:2:1,
180 mL) and the mixture was stirred at 95 °C for 12 h. Volatiles were
removed under reduced pressure and the crude product was dissolved in
AcOH (180 mL). NaOAc (3.7 g, 63 mmol) was added and the mixture was
stirred at 100 °C for 1 h. The reaction mixture was cooled to rt and extracted