The optimization of pyridazinone series of glucan synthase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.06.091

A detailed structure-activity relationship study of a novel series of pyridazine-based small molecule glucan synthase inhibitors is described. The optimization of the PK profile of this series led to the discovery of compound 11g, which demonstrated in vivo potency ip in a lethal fungal infection model.

Full text of DOI:10.1016/j.bmcl.2012.06.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5268–5271
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The optimization of pyridazinone series of glucan synthase inhibitors
Rongze Kuang a, , Heping Wu a, Pauline C. Ting a, Robert G. Aslanian a, Jianhua Cao a, David W. Kim a,
⇑
Joe F. Lee a, John Schwerdt a, Gang Zhou a, R. Jason Herr b, Andrew J. Zych b, Jinhai Yang b, Sang Q. Lam b,
David M. Jenkins b, Samuel A. Sakwa b, Samuel Wainhaus c, Todd A. Black d, Anthony Cacciapuoti d,
Paul M. McNicholas d, Yiming Xu d, Scott S. Walker d
a Department of Chemical Research, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065, USA
b Medicinal Chemistry Department, Albany Molecular Research, Inc. (AMRI), Albany, NY 12203, USA
c Department of Drug Metabolism, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065, USA
d 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 detailed structure–activity relationship study of a novel series of pyridazine-based small molecule
glucan synthase inhibitors is described. The optimization of the PK profile of this series led to the discov-
ery of compound 11g, which demonstrated in vivo potency ip in a lethal fungal infection model.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 4 March 2012
Revised 14 June 2012
Accepted 15 June 2012
Available online 4 July 2012
Keywords:
Glucan synthase inhibitor
Antifungal
Fungal cell wall
Pyridazinone
1,3-b-
D
-Glucan synthase (GS) is a membrane-bound enzyme
class of compounds in general suffers from poor plasma exposure in
the rat and in other animals.
complex that catalyzes the synthesis of 1,3-b-
D
-glucan, a major
component of the fungal cell wall. Inhibition of the glucan synthase
complex, thus disrupting the fungal cell wall, is the mechanism of
action used by a relatively new class of systemic antifungal agents
called echinocandins, such as caspofungin. The potent candicidal
activity and better safety profile of the echinocandins are advanta-
ges over other antifungal agents, such as the azoles (fungistatic
activity) and amphotericin B (significant toxic side effects). 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.1–3
O
N
O
O
O
N
O
N
S
S
preliminary
SAR
O
N
O
N
O
N
Cl
N
N
N
1
2
screening hit
IC50 = 4.6 µg/mL
IC50 = 0.2 µg/mL
We have previously reported the discovery of a novel series of
pyridazine-based glucan synthase inhibitors.4 It was further dem-
onstrated that a compound from this series exhibits oral efficacy
in a non-lethal fungal infection model.5 Our preliminary SAR stud-
ies identified key structural requirements required for good anti-
An examination of the early SAR compounds has shown that the
compounds with high antifungal activity were highly hydrophobic
molecules, with clogP in the range of 3–6. On the other hand, com-
pounds with significant plasma exposure in the rat were found to
have a clogP of 3 or less (Chart 1). This has presented an optimiza-
tion challenge for the development of oral GS inhibitors as anti-
fungal treatment. This paper described our efforts to further
optimize the antifungal activity and pharmacokinetic profiles of
these pyridazine-based glucan synthase inhibitors.
fungal potency, as exemplified by compound 2:
(or fluoro) substituted phenyl group on the left,
a
3-chloro
phen-
a
ylmethanesulfonyl piperazine moiety on the right, and an alkoxyl
group on the C4 position of the pyridazine ring. Unfortunately, this
The general synthesis of the pyridazine-based glucan synthase
inhibitors is shown in Scheme 1.6 Condensation of an aryl hydra-
zine with mucochloric acid 3 provided pyridazine intermediate 4.
⇑
Corresponding author. Tel.: +1 732 594 3742.
E-mail address: rongze.kuang@merck.com (R. Kuang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.091

R. Kuang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5268–5271
5269
Table 1
Antifungal profile of 4-substituted pyridazinones 2, 7, and 8
O
O
R
S
O
N
N
8000
6000
4000
2000
0
O
N
X
N
7:
X = Cl, Y = H
8: X = Y = F
Y
a
Compd
R
GS IC50
Candida glabrata
MIC100
Candida albicans
MIC100 (lg/mL)
b
b
(l
g/mL)
(lg/mL)
2 (7a)
8a
0.2
<0.02
0.4
0.39
0.39
0.78
0.10
0.03
0.8
7b
0.08
<0.02
7c
0.03
F
7d
0.03
<0.02
0.2
7e
7f
0.5
0.9
0.02
0.78
3.13
25
O
HO
HO
N
8g
7h
0.31
4.0
0.78
1.6
25
>50
0
1
2
3
4
5
6
7
N
7i
7j
0.8
0.6
0.78
0.39
12.5
6.25
clogP
N
Chart 1. Rat PK vs clogP AUC (ng h/mL) were obtained at 10 mg/Kg po dose.
a
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
tBOC
at least two independent determinations.5
Cl
N
Cl
b
Cl
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
b
a
O
N
O
Cl
average of at least two independent determinations.7
O
Cl
O
N
N
HO
N
5
N
4
H
R1
R1
3
c
nitroarenes and chloromethanesulfonamides followed by reduc-
tion of the nitro group to an amino moiety (Scheme 2).
O
O
R3
tBOC
R2
N
S
R2
O
N
N
O
N
N
d, e
With oxygen being identified as the optimal linker on the C4
position of the pyridazine ring in our previous reports,4 the SAR
of the alkoxyl moiety was explored (Table 1). A variety of struc-
tures were conveniently introduced from the starting alcohols.
Hydrophobic alkoxyl groups were generally preferred. Aiming to
decrease the overall lipophilicity of the target molecules, the meth-
ylcyclopropymethyl group (7c) and its fluorinated analog (7d)
were found to have the most potent antifungal activity while
maintaining a limited size as a hydrophobic group. Attempts to fur-
ther decrease overall lipophilicity by introducing polar groups,
such as ether (7e), hydroxyl (7f, 8g), and heterocycles (7h–j) re-
sulted in significant reduction in both GS inhibitory activity and
antifungal activity.7
O
N
O
N
N
R1
R1
7
6
Scheme 1. Reagents and condition: (a) R1NHNH2ÁHCl, NaOH, 100 °C; (b) N-Boc-
piperazine, Et3N, EtOH,
D D;(d) 4 N HCl in dioxane,
, 93%; (c) R2OH, NaN(TMS)2
CH2Cl2;)(e) R3SO2Cl, Et3N, CH2Cl2.
N-Boc-piperazine was introduced by selectively displacing the
5-Cl, and the 4-alkoxyl group was then introduced with alcohol
and NaN(TMS)2. After a Boc-deprotection, the piperazine ring
was capped with various sulfonyl chlorides. Another versatile
approach to introduce diversity on the sulfonamide moiety was
to use the vicarious nucleophilic substitution (VNS) reaction with
The right-hand side sulfonamide moiety was found to be critical
for antifungal activity. In general, the benzylsulfonamide exhibited
O
O
O
O
O
O
R2
N
S
R2
N
S
S
R2
N
O
N
N
O
N
N
O
N
N
a, b
Cl
NH2
R'
O
N
O
N
O
N
+
Ar
Ar
R'
Ar
NH2
Scheme 2. Reagents and condition: (a) nitroarene, KOtBu, DMSO, 0 °C to rt; (b) H2, PtO2, EtOAc/MeOH, rt, 2 h.

5270
R. Kuang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5268–5271
Table 2
Table 3
Antifungal profile of sulfonamide-substituted pyridazinones 9 and 10
Antifungal and PK profile of cyclopropyl-substituted pyridazinones 11 and 12
O
O
N
O
O
N
S
S
O
N
R
O
N
R
O
N
O
N
X
N
X
N
9:
X = Cl, Y = H
11:
X = Cl, Y = H
10:
X = Y = F
Y
12:
X = Y = F
Y
a
Compd
R
GS IC50
g/mL)
C. glabrata
C. albicans
Compd R
GS C.
C.
A.
Rat
Cmax
10 mg/
b
b
a
(l
MIC100
mL)
(l
g/
MIC100
mL)
(lg/
IC50 glabrata albicans fumigatus AUC(0–
g/ MIC100 MIC100 MECd
mL) g/mL) ( g/mL) mL)
(
l
g/ 6 h)10 mg/ kg po
kg po
(lg h/mL) (HPBCD)
(HPBCD)
8.1
b
c
(
l
(l
l
(lg/mL)
F
F
9a
0.06
0.04
0.05
1.56
0.1
10b
<0.02
NH2
11a
11b
0.06 0.26
0.04 0.13
1.04
0.52
4.2
1.9
1.4
9c
0.04
0.2
0.39
1.04
2.08
0.52
5.7
NH2
NH2
F
Cl
9d
9e
0.08
0.09
0.31
0.05
0.63
1.56
12c
12d
0.03 0.06
0.04 <0.02
0.26
0.13
0.89
1.1
0.31
0.33
F
NH2
Cl
Cl
NH2
Cl
9f
0.21
0.39
>50
>50
>50
11e
0.39 0.52
>33
>33
0.64
0.44
Cl
CF3
NH2
CF3
11f
0.06 0.06
0.08 0.13
0.52
2.08
2.08
13.1
15.0
3.3
3.0
9g
>150
OMe
NH2
CF3
CO2H
11g
1.04
9h
10i
9j
2.4
25
>50
3.1
Me
NH2
N
0.30
0.05
0.20
0.2
12g
11h
11i
0.07 0.13
0.18 0.13
0.37 16.7
1.04
4.2
4.2
4.2
14.5
9.4
3.5
2.3
0.7
Me
NH2
NH2
NH2
0.78
10k
10l
0.06
0.07
0.05
0.26
0.13
0.26
1.04
0.52
2.1
NH2
> 33
>33
2.1
NH2
OH
NH2
Not
tested
Not
tested
NH2
11j
0.30 8.34
> 33
>33
10m
O
a
NHMe
GS activity (C. albicans membranes, strain BWP17) IC50 values are the average of
11k
11l
0.01 0.78
0.05 0.06
0.05 0.06
0.20 >50
0
0
at least two independent determinations.
b
C. galbrata (strain C624) and C. albicans (strain C693) MIC100 values are the
NMe2
NH2
0.26 16.7
0
0
average of at least two independent determinations.
11m
2.08
2.08
8.4
8.4
3.1
1.0
N
N
the best antifungal activity among unsubstituted alkyl and aryl sul-
fonamides, but led to undetectable blood levels in the rat model
(10 mg/kg po). Therefore, SAR of substitution on the benzyl group
required optimization (Table 2).
11n
11o
0.13 0.06
0.15 1.04
5.2
1.7
NH2
N
Not
tested
Not
tested
As a typical approach to block the potential metabolic sites on
the phenyl ring, substituents such as F, Cl and CF3 were introduced.
Most compounds with F/Cl substituents (9a–e, 10b) retain good
antifungal potency, except for the 3,5-di-Cl (9f) and 3,5-di-CF3
(9g) analogs, which exhibit reduced antifungal activity. However,
these modifications did not result in significant improvement on
the drug plasma exposure in the rat, probably due to the increased
lipophilicity from these modifications(clogP range of 4.5–6.5).
Introducing polar groups to the benzylsulfonamide moiety as an
approach to reduce the overall lipophilicity was then examined.
While a carboxylic group (9h, clogP 4.5) was found to greatly re-
duce the antifungal potency, the pyridylmethyl analog (10i, clogP
2.8) slightly reduces the antifungal activity, but does not improve
16.7
8.4
NH2
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.
b
c
d
antifungal activity. While the hydroxyl group was not beneficial
to improving in vivo rat drug plasma levels (rat AUC for
10m < LOQ at 10 mpk po), the amino group did produce a signifi-
cant increase. Compounds 10k and 10l have AUC of 8.1 and
plasma exposure (rat AUC for 10i = 0.02 lg h/mL at 10 mpk po).
Interestingly, H-bond donor groups, such as hydroxyl (10m,
5.7 lg h/mL at 10 mpk po, respectively.
clogP 3.7) and amino (9j, 10k–l, clogP range of 3.1–3.5), retain

R. Kuang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5268–5271
5271
A series of analogs with the amino group were then explored
(Table 3). The aniline moiety was found to tolerate small substitu-
ents such as F, Cl, Me and OMe. However, larger substituents such as
OCF3, iPr, and CONH2 were found to decrease the antifungal potency
significantly. Although most aniline analogs displayed a much
improved plasma exposure, substitution directly on the aniline
nitrogen (analogs 11k and 11l) led to compounds that retained po-
tency, but provided no measurable exposure after oral dosing. Ami-
nopyridine analogs (11m–o, clogP 2.9) exhibit slightly decreased
activity, and their PK profiles were no better than the aniline
analogs.
with glucan synthase inhibitors such as the echinocandins.5 This
supports the hypothesis that the antifungal activity of this class
of compounds acts through the inhibition of glucan synthase.
In conclusion, a novel series of GS inhibitors has been discov-
ered. Optimization of both the antifungal activity and PK profiles
has led to the identification of compound 11g which exhibits
in vivo efficacy ip in a lethal fungal infection model. Further explo-
ration of this class of compounds as antifungal agents will be
reported in due course.
References and notes
Compound 11g gave high blood levels in both rat and mouse,
1. Moudgal, V.; Sobel, J. Expert Opin. Pharmacother. 2010, 11, 2037.
2. Kitamura, A. Expert Opin. Drug Discov. 2010, 5, 739.
3. (a) Matejuk, A.; Leng, Q.; Begum, M. D.; Woodle, M. C.; Scaria, P.; Chou, S.-T.;
Mixson, A. J. Drugs Future 2010, 35, 197; (b) Hector, R. F.; Bierer, D. E. Expert Opin.
Ther. Pat. 2011, 21, 1597.
and was highly potent against Candida albicans C697 (MIC100
=
0.13 g/mL). This compound was selected for evaluation in a lethal
l
infection model. Normal mice and immune compromised mice
were separately infected with C. albicans C697 (5 Â 107 CFU/
mouse) and subsequently treated with 100 mg/kg (ip) compound
11g, (maleate), 5 mg/kg (ip) caspofungin or vehicle (ip) daily.
Survival was monitored for 4 days. Both 11g and caspofungin pro-
duced 100% survival in normal mice and in immune compromised
mice infected with C. albicans C697 for the duration of the study. In
addition, all treated mice showed significant reductions in fungal
kidney burdens.
For the current series of pyridazine-based GS inhibitors, their
cell-based antifungal activity (MIC) correlates well with their en-
zyme glucan synthase inhibitory activity (IC50). Furthermore, for
filamentous fungi such as Aspergillus fumigatus, 11g caused a dis-
tinct morphological response characterized by substantially trun-
cated and highly branched hyphae, as has been typically seen
4. (a) Ting, P. C.; Kuang, R.; Wu, H.; Aslanian, R. G.; Cao, J.; Kim, D. W.; Lee, J. F.;
Schwerdt, J.; Zhou, G.; Wainhaus, S.; Black, T. A.; Cacciapuoti, A.; McNicholas, P.
M.; Xu, Y.; Walker, S. S. Bioorg. Med. Chem. Lett. 1819, 2011, 21; (b) Zhou, G.; Ting,
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Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts;
Approved Standard—Third Edition (M27-A3). Published by The Clinical and
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Schering-Plough strain collection.