The synthesis and structure-activity relationship of pyridazinones as glucan synthase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.058

A structure-activity relationship study of the lead 5-[4-(benzylsulfonyl) piperazin-1-yl]-4-morpholino-2-phenyl-pyridazin-3(2H)-one 1 has resulted in the identification of 2-(3,5-difluorophenyl)-4-(3-fluorocyclopentyloxy)-5-[4- (isopropylsulfonyl)piperazin-1-yl]-pyridazin-3(2H)-one 11c as a β-1,3-glucan synthase inhibitor. Compound 11c exhibited significant efficacy in an in vivo mouse model of Candida glabrata infection.

Full text of DOI:10.1016/j.bmcl.2011.01.058

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1819–1822
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and structure–activity relationship of pyridazinones
as glucan synthase inhibitors
Pauline C. Ting a, , Rongze Kuang a, Heping Wu a, Robert G. Aslanian a, Jianhua Cao a, David W. Kim a,
⇑
Joe F. Lee a, John Schwerdt a, Gang Zhou a, Samuel Wainhaus b, Todd A. Black c, Anthony Cacciapuoti c,
Paul M. McNicholas c, Yiming Xu c, Scott S. Walker c
a Department of Chemical Research, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Drug Metabolism, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
c Department of Biological Research, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A structure–activity relationship study of the lead 5-[4-(benzylsulfonyl)piperazin-1-yl]-4-morpholino-
Received 10 December 2010
Revised 11 January 2011
Accepted 13 January 2011
Available online 21 January 2011
2-phenyl-pyridazin-3(2H)-one
1 has resulted in the identification of 2-(3,5-difluorophenyl)-4-(3-
fluorocyclopentyloxy)-5-[4-(isopropylsulfonyl)piperazin-1-yl]-pyridazin-3(2H)-one 11c as a b-1,3-glucan
synthase inhibitor. Compound 11c exhibited significant efficacy in an in vivo mouse model of Candida
glabrata infection.
Ó 2011 Elsevier Ltd. All rights reserved.
Keyword:
Glucan synthase inhibitor
O
N
O
Fungal infections are a major risk associated with organ trans-
plant, hematopoietic stem cell transplant, cancer chemotherapy,
HIV infection, venous catheterization, or intensive care hospitaliza-
tion. The standard of care for invasive fungal infections is either the
azoles or amphotericin B, both of which disrupt the fungal cell
membrane function. A relatively new class of antifungal agents
are the echinocandins, such as caspofungin, which disrupt the fun-
gal cell wall by inhibiting b-1,3-glucan synthase (GS). The potent
candicidal activity of the echinocandins offers a treatment option
for azole resistant Candida spp., and their safety allows the clini-
cian to avoid the significant side effects of amphotericin B. How-
ever, since the echinocandins are lipopeptides available only by
IV dosing, an oral GS inhibitor will be an important contribution
to the arsenal of antifungal treatment.1–5 The successful identifica-
tion and development of caspofungin as a marketed antifungal
agent is a prime example of using natural products as a source
for the discovery of new drugs.6,7
O
S
N
O
N
N
1
Analogs were synthesized according to the route depicted in
Scheme 1.10 Condensation of a substituted hydrazine with muco-
chloric acid gave the core pyridazinone 3. N-BOC piperazine added
selectively to the 5-position. A number of different moieties were
then introduced at the 4-position. Removal of the BOC protecting
group and sulfonylation produced the target compounds 6.
Initial SAR studies of the 4-position are summarized in Table 1.
While replacement of the morpholine ring with a cyclohexyl ring
in 7a removed all antifungal activity, the cyclohexyloxy 7b and
cyclopentyloxy 7c showed significantly improved antifungal
activity. The 4-position also tolerated a long acyclic chain (analogs
7d–j) where the preferred linker was oxygen (7f–g) > sulfur
(7h) > nitrogen (7d) or carbon (7e).
In contrast, the piperazine ring was not open to modification
(Table 2). Introduction of a methyl group in 8a–c reduced both
GS and antifungal activity while the constrained diaza-bicy-
clo[2.2.1]heptane was completely inactive. Replacement of the
piperazine ring with an acyclic chain was also not tolerated (data
not shown).
High throughput screening of the legacy Schering–Plough com-
pound collection identified compound 1 as an in vitro inhibitor of
Candida albicans GS (strain BWP17) with an IC50 of 4.6 l
g/mL.8
Although compound 1 exhibited reasonable in vitro antifungal
activity against the yeast Candida glabrata (strain C624), it was
inactive against the yeast C. albicans (strain C693) and produced
no measurable blood levels in the rat upon oral dosing.9 Therefore,
significant optimization was required.
The sulfonamide moiety is critical for antifungal activity. All
attempts to replace the sulfonamide with a variety of groups
⇑
Corresponding author. Tel.: +1 908 740 3534; fax: +1 908 740 7152.
E-mail address: pauline.ting@merck.com (P.C. Ting).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.058

1820
P. C. Ting et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1819–1822
Table 2
tBOC
Cl
Cl
N
Antifungal profile of piperazine-substituted pyridazinones 8a–d
Cl
O
Cl
b
a
O
N
O
Cl
O
N
O
N
N
HO
O
N
O
R1
H
R1
3
4
S
O
N
2
c
R
O
N
O
R2
tBOC
N
d, e
S
R3
R2
N
O
N
O
N
N
a
N
Compd
R
GS IC50
g/mL)
C. glabrata
C. albicans
R1
b
b
N
(
l
MIC100
(
l
g/mL)
MIC100
(lg/mL)
6
N
5
R1
7b
8a
0.53
2.2
0.05
0.78
N
N
N
Scheme 1. Reagents and conditions: (a) R1NHNH2–HCl, NaOH, 100 °C; (b) N-BOC–
piperazine, Et3N, EtOH, , 93%; (c) ROH, NaN(TMS)2, THF or RSH, NaOMe, MeOH or
D
N
Me
N
amine, toluene, or amine, K2CO3, Pd(OAc)2, BINAP, toluene or RB(OH)2, Na2CO3,
D
0.39
0.78
6.3
19c
PdCl2(PPh3)2, CH3CN, H2O,
CH2Cl2.
D
;(d) 4 N HCl in dioxane, CH2Cl2; (e) R3SO2Cl, Et3N,
N
8b
2.8
Me
Table 1
Antifungal profile of 4-substituted pyridazinones 7a–j
N
N
N
8c
4.3
0.39
>50
2.0c
>50
Me
O
O
8d
>10
N
S
R
N
N
a
O
N
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
at least two independent determinations.
N
b
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
average of at least two independent determinations.
c
In general, 100% inhibition of fungal growth is observed for MIC100 determi-
nation. Compounds 8b and 8c exhibit 50% inhibition of fungal growth resulting in a
MIC50 for C. albicans (strain C693) as the average of at least two independent
determinations.
a
Compd
R
GS IC50
g/mL)
C. glabrata
C. albicans
b
b
(
l
MIC100
(lg/mL) MIC100 (lg/mL)
O
N
1
4.6
2.2
0.39
>50
>20
Table 3
Antifungal profile of sulfonamide-substituted pyridazinones 9a–f
7a
>20
O
O
7b
7c
0.53
0.18
0.05
0.03
0.78
0.26
S
O
O
N
N
R
O
N
O
N
EtO
EtO
N
H
7d
7e
2.3
1.5
0.78
0.78
>50
>50
a
Compd
R
GS IC50
g/mL)
C. glabrata
C. albicans
b
b
(l
MIC100
(
l
g/mL)
MIC100
(lg/mL)
7f
0.12
0.90
1.1
<0.10
0.10
0.78
0.78
3.1
7c
9a
0.18
0.59
0.03
0.26
O
S
N
N
7g
7h
O
0.06
0.13
8.4
8.4
3.1c
N
9b
9c
0.20
>10
0.15
0.78c
7i
7j
3.0
0.59
1.6
25
S
O
9d
1.3
0.63
5.0
>10
>50
S
9e
9f
Me
iPr
2.2
1.7
0.63
0.20
>20
25c
O
O
a
a
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
at least two independent determinations.
at least two independent determinations.
b
b
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
average of at least two independent determinations.
average of at least two independent determinations.
c
c
In general, 100% inhibition of fungal growth is observed for MIC100 determi-
In general, 100% inhibition of fungal growth is observed for MIC100 determi-
nation. Compound 7h exhibits 50% inhibition of fungal growth resulting in a MIC50
for C. albicans (strain C693) as the average of at least two independent
determinations.
nation. Compounds 9c and 9f exhibit 50% inhibition of fungal growth resulting in a
MIC50 for C. albicans (strain C693) as the average of at least two independent
determinations.

P. C. Ting et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1819–1822
1821
Table 4
Table 5
Antifungal profile of 2-substituted pyridazinones 10a–g
Antifungal profile of cyclopentyl-substituted pyridazinones 11a–d
O
O
O
O
R'
O
S
S
O
N
N
O
N
N
O
N
N
N
F
N
R
a
R
Compd
R
GS IC50
g/mL)
C. glabrata
C. albicans
b
b
(
l
MIC100
(lg/mL) MIC100 (lg/mL)
Compd
R
R’
GS
C.
C.
A.
Rat AUC(0–6 h)
a
IC50
g/mL) MIC100 MIC100 MECd
g/mL) g/mL) g/mL) (MC)
glabrata albicans fumigatus 10 mg/kg po
b
c
(l
lg h/mL
9f
1.7
8.8
0.20
25c
(
l
(l
(l
10b
11a
H
H
1.0
0.10
0.13
6.3
1.6
4.2
0.67
0.30
10a
0.78
1.6
>50
F
0.42
16.7
10b
6.1
>50
F
F
N
N
11b
11c
11d
H
F
1.1
0.78
0.10
0.05
6.3
3.1
1.6
3.1
0.47
2.1
F
F
10c
10d
10e
>10
1.0
8.4
>20
6.3
3.1
0.62
0.60
6.3
S
F
0.10
0.10
F
12.5
0.44
F
Cl
F
a
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
at least two independent determinations.
C. galbrata (strain C624) MIC100 values are the average of at least two inde-
pendent determinations.
C. albicans (strain C693) MIC100 values are the average of at least two inde-
pendent determinations.
A. fumigatus (strain ND158) MEC values are the average of at least two inde-
pendent determinations.
0.52
b
c
10f
10g
10h
0.62
0.25
0.26
0.40
0.06
0.20
6.3
6.3
3.1
F
d
F
Cl
or heterocycle (pyridine 10b or thiazole 10c) decreased antifungal
activity. Substitution on the phenyl ring was preferred at the meta
position with one electron withdrawing halogen (10d–e) or with
two halogens (10f–h) rather than a hydrogen bonding group
(10i–j). The meta halogen substituted analogs all displayed equiv-
alent rat AUC(0–6 h) at 10 mg/kg po (MC vehicle): 0.67
10d, 0.49 g h/mL for 10e, and 0.49 g h/mL for 10g.
Since the cyclopentyl ring was identified as
metabolism, the mono and disubstituted fluorocyclopentyl analogs
11a–d were synthesized (Table 5). The mono substituted fluoro
compounds 11a and 11c are a mixture of enantiomers. Analogs
11a–d also exhibited activity against the mold Aspergillus fumigatus
(strain ND158), resulting in the morphologic effects on hyphal
growth typical of GS inhibitors.11 Compound 11c possessed the
best rat AUC(0–6 h) of 2.1
addition, compound 11c exhibited MICs to other clinical isolates
of C. albicans (strain C43) and C. glabrata (strain C454) of 12.5 g/
mL and 1.0 g/mL, respectively, and showed no inhibition of the
P450 liver enzymes 2D6, 3A4, and 2C9 at 30 M; low activity in
the herg voltage clamp assay with 22% inhibition at 1 M; reason-
able dog AUC(0–8 h) of 3.5 g h/mL at 2 mg/kg po (MC/HPBCD vehi-
cle); and low hepatocyte clearance of 2.4 L/min/M cells in rat,
1.7 L/min/M cells in dog, and 1.0 L/min/M cells in human.
As proof of concept, compound 11c was tested in a mouse mod-
Cl
HO
Cl
l
g h/mL for
10i
10j
0.90
1.9
2.5
25
>20
>50
l
l
a
site for
H N
2
a
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
at least two independent determinations.
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
average of at least two independent determinations.
b
c
In general, 100% inhibition of fungal growth is observed for MIC100 determi-
lg h/mL at 10 mg/kg po (MC vehicle). In
nation. Compound 9f exhibits 50% inhibition of fungal growth resulting in a MIC50
for C. albicans (strain C693) as the average of at least two independent
determinations.
l
l
l
including amides, ureas, carbamates, and phosphoamides were not
successful (data not shown). In general, the benzylsulfonamides
such as 7c exhibited the best antifungal activity, but zero blood
levels in the rat (10 mg/kg po) due to both metabolism and absorp-
tion issues. Conversion of the benzyl sulfonamide to the pyridyl-
methyl (9a–b), phenyl (9c), or pyridyl (9d) all resulted in a
decrease in antifungal activity (Table 3). Although the alkyl sulfon-
amides (9e–f) also exhibited reduced antifungal activity, the iso-
propyl sulfonamide 9f was the first compound to show moderate
rat AUC(0–6 h) of 1.6
vehicle).
Since compound 9f possessed a moderate PK profile, it was the
basis for further SAR optimization of the 2-phenyl substituent
(Table 4). Replacement of the phenyl ring with a cyclohexyl (10a)
l
l
l
l
l
el of systemic C. glabrata infection. Immunocompromised mice
were infected with C. glabrata strain C624 (107 colony forming
units, CFU/mouse) and treated with compound 11c at 10 and
30 mg/kg po QD for seven days. Following treatment, fungal kidney
burdens were significantly (p <0.001) reduced at the 30 mg/kg dose
(101.30 CFU/kidneys), but not at the 10 mg/kg dose (104.16 CFU/kid-
neys) relative to vehicle treated mice (104.28 CFU/kidneys). Analysis
of plasma levels suggested that at 10 mg/kg, the concentration of
lg h/mL at 10 mg/kg po (methylcellulose, MC

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P. C. Ting et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1819–1822
5. Ma, C.-M.; Abe, T.; Komiyama, T.; Wang, W.; Hattori, M.; Daneshtalab, M.
Bioorg. Med. Chem. 2010, 18, 7009.
6. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
compound 11c was not above the MIC for sufficient time (4 h) ver-
sus the 30 mg/kg dose (10 h).
In conclusion, a new series of GS inhibitors has been discovered.
Optimization of the original lead compound 1 has led to the iden-
tification of compound 11c which exhibits oral efficacy against C.
glabrata in the mouse. Further SAR optimization of compound
11c in terms of C. albicans activity and PK profile as well as addi-
tional details of the biological properties for this GS inhibitor series
will be reported in due course.12
7. Newman, D. J. J. Med. Chem. 2008, 51, 2589.
8. Taft, C. S.; Enderlin, C. S.; Selitrennikoff, C. P. J. Antibiot. 1994, 47, 1001.
9. Antimicrobial susceptibility testing was carried out in accordance with the
Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts; Approved Standard—Third Edition (M27-A3). published by The
Clinical and Laboratory Standards Institute. 2008. All fungi used in these
studies are from the Schering–Plough strain collection.
10. For detailed preparation procedures, refer to Ting, P. C.; Aslanian, R. G.; Cao, J.;
Kim, D. W.; Kuang, R.; Zhou, G.; Herr, R. J.; Zych, A. J.; Yang, J.; Wu, H.; Zorn, N.
World Patent 2008115381, 2008.
11. Kurtz, M. B.; Heath, I. B.; Marrinan, J.; Dreikorn, S.; Onishi, J.; Douglas, C.
Antimicrob. Agents Chemother. 1994, 38, 1480.
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Cacciapuoti, A.; McNicholas, P. M.; Wainhaus, S.; Herr, R. J.; Kuang, R.; Aslanian,
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