Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.04.110

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Full text of DOI:10.1016/j.bmcl.2010.04.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3618–3622
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists
b
b
b
a
Brian S. Lucas a, , Wade Aaron , Songzhu An , Richard J. Austin , Matthew Brown ,
Hon Chan c, Angela Chong b, Randall Hungate a, Tom Huang c, Ben Jiang c,
Michael G. Johnson a, Jacob A. Kaizerman a, Gary Lee b, Dustin L. McMinn a, Jessica Orf b,
Jay P. Powers a, , Minqing Rong b, Maria M. Toteva d, Craig Uyeda c, Dineli Wickramasinghe b,
Guifen Xu c, Qiuping Ye c, Wendy Zhong b
*
a Department of Medicinal Chemistry, AMGEN, 1120 Veterans Blvd., S. San Francisco, CA 94080, USA
b Department of Oncology, AMGEN, 1120 Veterans Blvd., S. San Francisco, CA 94080, USA
c Department of Pharmacokinetics and Drug Metabolism, AMGEN, 1120 Veterans Blvd., S. San Francisco, CA 94080, USA
d Department of Small Molecule Process and Product Development, AMGEN, 1120 Veterans Blvd., S. San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tis-
sues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a trans-
ducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of
unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally
bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant
tumor volume reduction in a mouse medulloblastoma model.
Received 9 March 2010
Revised 22 April 2010
Accepted 26 April 2010
Available online 28 April 2010
Keywords:
Smoothened
SMO
Ó 2010 Elsevier Ltd. All rights reserved.
Smo
Hedgehog
Hh
Patched
Ptch
Cancer
Medulloblastoma
The Hedgehog (Hh) signaling pathway plays a significant role in
the regulation of cell growth and differentiation during embryonic
development.1 Abnormal activation of the Hh pathway has been
implicated in a number of cancer2,3 types, including basal cell car-
cinoma4 and medulloblastoma.5 Members of the Hh family (Sonic
Hedgehog, Indian Hedgehog, and Desert Hedgehog) can bind to
the 12-pass transmembrane protein Patched (Ptch), a repressor
of Smo signaling. Thus, the Hh family of proteins indirectly activate
Smo, often measured downstream as a corresponding increase in
Gli transcription factors. Interruption of Hedgehog signaling via
Smo antagonism was first established with plant alkaloids includ-
ing cyclopamine,6 and has subsequently resulted in considerable
interest in targeting Smo for cancer.7
phthalazine 1 (Fig. 1). The Smo antagonism (IC50) of 22.0 and
7.0 nM in our respective mouse (mSmo)9 and human (hSmo)10 as-
says was confirmed upon resynthesis. Lead compound 1 was fur-
ther evaluated in a number of high-throughput assays, including
stability to microsomes11 and inhibition of CYP enzymes 3A4 and
2D6.12 Compound 1 was found to have relatively high turnover
in rat (RLM) and human (HLM) microsomes, as well as high levels
of CYP 2D6 inhibition. Therefore, we initiated a medicinal chemis-
try program around 1 with the aim of maintaining potency for
hSmo IC50 = 7.0 nM
mSmo IC50 = 22.0 nM
O
Our investigation8 began with a high-throughput screen which
identified a number of potent Smo antagonists, including the
T.O. HLM = 47% a
N
N
T.O. RLM = 31% a
S
N
N
Inhib. CYP 3A4 = 10%b
Inhib. CYP 2D6 = 47%b
1
* Corresponding author. Tel.: +1 650 244 2128.
E-mail address: blucas@amgen.com (B.S. Lucas).
Current address: ChemoCentryx, Inc., 850 Maude Avenue, Mountain View, CA
94043, USA.
Figure 1. Lead compound 1. aPercent of 1 turned over (consumed) after 30 min
incubation with human (HLM) or rat (RLM) liver microsomes.11 bPercent inhibition
of CYP 3A4 or 2D6 at 3 l
M concentration.12
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.110

B. S. Lucas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3618–3622
3619
Table 1
Smoothened antagonism and microsomal stability of amide modifications
O
N
N
R1
N
N
R1
hSMO IC50 (nM)
T.O. HLMb (%)
T.O. RLMb (%)
CYP 3A4 inhib.c
CYP 2D6 inhib.c
a
Compound
1
2
3
4
5
2-Thienyl
2-Furyl
2-Thiazoyl
Methyl
7.0
13.0
9.5
91.0
19.0
48
43
48
10
19
31
32
25
10
12
<10
<10
<10
<10
<10
47
48
89
29
11
Phenyl
a
b
c
Values are the mean of a minimum of two measurements with a standard deviation of 35% of the mean.
Percent turnover after 30 min incubation with human (HLM) or rat (RLM) liver microsomes.
Percent inhibition of CYP 3A4 or 2D6 at 3
l
M concentration.
afforded intermediate 8. Compounds 1–5 were obtained by acyla-
tion of 8 with an appropriate acid chloride.
a
b
While heterocyclic thiophene replacements such as furyl (2)
and thiazoyl (3) did not address microsomal turnover, the simple
bioisosteric phenyl group in compound 5 afforded reasonable po-
tency, reduced turnover in microsomes, and minimal CYP 3A4
and 2D6 inhibition. Thus, the benzoyl piperazine motif was incor-
porated in subsequent compounds as shown in Scheme 2. Starting
once again from 1,4-dichlorophthalazine 6, piperazines 9 and 10
were incorporated to afford piperazinylphthalazines 11 and 12,
respectively. Key intermediates 11 and 12 were subjected to a
combination of deprotection with trifluoroacetic acid (TFA, 11
only), acylation with benzoyl chloride, and a palladium catalyzed
cross-coupling in various order to generate compounds 13–43.
Metabolite ID studies of 5 performed in rat and human hepato-
cytes identified the piperazine ring as an additional site for metab-
olism (Fig. 2).16 Investigation of methylation about the piperazine
ring revealed significant effects on potency and stability as de-
scribed in Table 2. The 2-S-methyl piperazine 13 afforded a nearly
fourfold increase in potency as well as lowered turnover in human
microsomes. Incorporation of the regioisomeric 3-R-methyl piper-
azine (16) led to even more potent Smo antagonism, however
microsomal clearance was increased significantly over the other
piperazine cores.
Cl
Cl
Cl
N
N
N
7
N
6
c
O
N
NH
N
N
R1
N
N
N N
1-5
8
Scheme 1. Synthesis of unsubstituted piperazine compounds 1–5. Reagents and
conditions: (a) PhBr, n-BuLi, ZnCl2, Pd(PPh3)4, À78 °C to rt, 48%; (b) piperazine,
MIBK, reflux, 100%; (c) R1COCl, Et3N, 30–54%.
Smo, improving metabolic stability, and reducing P450 inhibition,
beginning with the replacement of the thiophene ring in 1
(Table 1).
Synthesis of compounds 1–513 was accomplished according to
Scheme 1. Dichlorophthalazine 6 was subjected to Negishi cross-
coupling conditions14 to afford phenylpthalazine 7. Treatment of
7 with piperazine in refluxing methylisobutyl ketone (MIBK)15
Our initial SAR around the pendant aryl group was performed
on the unsubstituted piperazine core. A simple Topliss17 scan (Ta-
ble 3, 5 and 17–20) showed insufficient spread in potency and thus
failed to provide clear direction. Additional analogs (21–25) were
synthesized but likewise showed little dynamic range in terms of
potency. However, the relative microsomal stability and general
Cl
Cl
N N
6
HN
N
Boc
HN
NH
*
*
a
b
9
10 R1
R1= Me or H
O
N
N
Cl
N
N
Boc
26,
30
Cl
N
NH
R1
*
OH and -OSO3H
N
N
11
*
d, g
N
N
12
e, d
O
O
c, d, e
e,c, d
15, 16
d, e
NH HN
N
HN
OH
d, f
27
5
17-19,
29
13,14,
35,36,
38-43
20-25,
OH
28,31-34
O
N
HN
OH
Figure 2. Metabolites of 5 identified in rat and human hepatocytes.
Scheme 2. Synthesis of benzoyl-piperazine bearing compounds 13–43. (a) Neat,
120 °C, 27–50%; (b) MIBK, reflux, 71–89%; (c) TFA, DCM, 100%; (d) Benzoyl chloride,
Et3N, DCM, room temperature, 35–89%; (e) Ar–B(OH)2,, Pd(PPh3)4, Na2CO3, toluene/
water, reflux, 23–95%; (f) 3-pyridylboronic acid, K3PO4, Pd2(dba)3, SPhos 87%; (g)
Aryl-SnBu3, Pd(PPh3)4, 15–73%.

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B. S. Lucas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3618–3622
Table 2
Methylation of the piperazine ring
O
N
3
N
CH32
N
N
T.O. HLMb (%)
T.O. RLMb (%)
CYP 3A4 inhib.c
CYP 2D6 Inhib.c
a
Compound
Piperazine
hSMO IC50 (nM)
5
13
14
15
16
Piperazine
2-S-Me
2-R-Me
3-S-Me
19.0
5.3
13.0
11.0
2.2
19
<10
17
<10
44
12
21
21
23
39
<10
<10
<10
ND
11
11
<10
ND
47
3-R-Me
<10
a
b
c
Values are the mean of a minimum of two measurements with a standard deviation of 35% of the mean.
Percent turnover after 30 min incubation with human (HLM) or rat (RLM) liver microsomes.
Percent inhibition of CYP 3A4 or 2D6 at 3
l
M concentration.
Table 3
Initial SAR around the pendant aryl group
O
N
N
N
N
R
T.O. HLMb (%)
T.O. RLMb (%)
CYP 3A4 inhib.c
CYP 2D6 inhib.c
a
Compound
Phenyl
hSMO IC50 (nM)
5
17
18
19
20
21
22
23
24
25
H
3,4-Cl
4-Cl
4-CH3
4-OCH3
4-F
3-OCH3
3-CN
2-CH3
2-Cl
19.0
17.3
5.2
19
31
20
45
27
17
43
10
90
62
12
10
10
72
22
10
33
10
40
15
<10
<10
16
<10
<10
<10
<10
<10
<10
<10
11
ND
24
<10
19
29
19
18
24
<10
4.4
15.0
10.8
20.0
212.0
15.0
20.3
a
b
c
Values are the mean of a minimum of two measurements with a standard deviation of 35% of the mean.
Percent turnover after 30 min incubation with human (HLM) or rat (RLM) liver microsomes.
Percent inhibition of CYP 3A4 or 2D6 at 3 lM concentration.
Table 4
Aryl group substitutions within the 2-S-methyl piperazine series
O
R
N
N
N
N
T.O. HLMb (%)
T.O. RLMb (%)
CYP 3A4 inhib.c
CYP 2D6 inhib.c
a
Compound
Aryl
hSMO IC50 (nM)
13
26
27
28
29
30
31
32
33
34
Ph
5.3
110
213
96
2340
2700
3.2
7.0
11.8
3.6
10
<10
10
10
<10
10
38
<10
10
21
<10
12
25
<10
23
43
<10
15
<10
<10
12
11
<10
<10
52
15
27
10
43
18
<10
2-Pyridyl
3-Pyridyl
4-Pyridyl
2-Pyridazyl
2-Oxazoyl
4-CH3-Ph
4-Cl-Ph
58
<10
<10
13
40
20
4-F-Ph
4-CF3-Ph
<10
<10
49
a
b
c
Values are the mean of a minimum of two measurements with a standard deviation of 35% of the mean.
Percent turnover after 30 min incubation with human (HLM) or rat (RLM) liver microsomes.
Percent inhibition of CYP 3A4 or 2D6 at 3 lM concentration.

B. S. Lucas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3618–3622
3621
Table 5
Effects of p-substituents on the phenyl ring in the 3-R-methyl piperazine series
O
N
N
N
N
R
T.O. HLMb (%)
T.O. RLMb (%)
CYP 3A4 inhib.c
CYP 2D6 inhib.
a
c
Compound
Phenyl
hSMO IC50 (nM)
16
35
36
37
38
39
40
41
42
43
H
4-Cl
2.2
0.7
0.4
2.3
7.3
10.1
2.7
3.1
5.2
2.8
44
38
56
34
41
89
14
<10
59
13
39
12
55
21
73
94
13
<10
82
11
<10
<10
<10
31
14
25
15
12
90
<10
<10
60
<10
<10
20
4-CH3
4-cPr
4-iPr
4-tBu
4-CH2OH
4-CN
10
<10
<10
26
4-N(CH3)2
4-CF3
15
a
b
c
Values are the mean of a minimum of two measurements with a standard deviation of 35% of the mean.
Percent turnover after 30 min incubation with human (HLM) or rat (RLM) liver microsomes.
Percent inhibition of CYP 3A4 or 2D6 at 3
l
M concentration.
attention to the 3-R methyl piperazine core to evaluate the effects
of phenyl group substitution on stability within this more potent
series. As illustrated in Table 5, introduction of a series of para-sub-
stituents onto the pendant phenyl ring in 16 afforded a number of
promising compounds, including the sub-nanomolar hSMO antag-
onists 35 and 36.
An excellent balance of properties was achieved with 4-CF3
compound 43. Compound 43 was potent in both our mSMO and
hSMO assays with IC50s of 2.0 and 2.8 nM, respectively. Moreover,
an excellent cross-species in vivo pharmacokinetic profile of 43
was established in mouse, rat, dog, and cynomologous monkey
(Table 6), enabling in vivo efficacy studies.
Table 6
In vivo pharmacokinetic properties of 43
c
c
Species
Cl (IV, L/h/kg)
Vdss (L/kg)
t
(h)
F (%)
½
Rata
0.41
0.30
0.20
0.53
5.07
1.40
2.96
2.52
9.5
3.4
10.0
4.3
39
65
60
31
Mouseb
Doga
Cynoa
a
b
c
Nominal dose = 0.5 mg/kg iv, 2.0 mg/kg po.
Nominal dose = 1.0 mg/kg iv, 2.0 mg/kg po.
After iv dosing.
tolerance of para-substituents such as -Cl (18) and -F (19) was
noted. Focusing then on the promise of the 2-S-methyl piperazine
series, we explored modifications to the pendant aryl group in 13.
As shown in Table 4, a number of aryl and heteroaryl groups
were incorporated in compounds 26–34. While heteroaryl replace-
ments 26–30 were generally less potent, the previously noted tol-
We sought to validate the in vivo Smo antagonism of 43 with a
rodent hair follicle study. The role of the Shh pathway in hair fol-
licle morphogenesis is well known, and the activation of the Shh
signaling pathway in the skin of rodents following depilation has
been used as a model of pathway antagonism with known Smo
antagonists such as cyclopamine.18 Adapted from the method of
Paladini et al.,19 the hind flanks of mice were depilated with wax
to activate the hair follicle cycle. Five days after depilation, the
mice were treated with a single oral dose20 of 2, 20, or 100 mg/
kg of compound 43. After 6 h the skin was harvested and evaluated
for induction of Gli1. A dose dependent reduction of Gli1 expres-
erance of
a para-substitution was confirmed in this series.
Especially notable were 4-Cl and 4-CF3 substituted compounds
32 and 34, respectively, that afforded comparable potency without
compromising stability in microsomes. Finally, we turned our
sion was observed reflecting an approximate EC50 = 2.0 lM, along
40
30
20
10
0
6000
4000
2000
0
Gli1/RGS
Plasma (ng/ml)
4000
3000
2000
Vehicle
Rx
1000
0
10 mg/kg 43
7
8
9
10
11
12
13
14
15
16
Figure 3. Reduction of Gli1 expression in skins of mice treated with 43. Four mice
per group, two samples per mouse: r (2 mg/kg) = 0.0016, r (20, 100 mg/kg) <0.0001
(Dunnett’s method). For the no wax treatment, there were two mice per group, two
samples per mouse. Gli1 RNA was measured using a quantitative real time PCR and
was normalized to RGS (Pitx2) RNA.
Days Post Implantation
Figure 4. Tumor volume reduction in mouse Ptch +/À p53 À/À medulloblastoma
model induced by treatment with 10 mg/kg 43, QD oral. Ten mice per group,
< 0.0001 (Dunnett’s method).
q

3622
B. S. Lucas et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3618–3622
2002, 14071). An oligonucleotide cassette with five consensus Gli1 binding
sites was ligated into the luciferase reporter plasmid pGL4.16 (Promega,
Madison, WI, USA). A stable clone of NIH-3T3 cells stably transfected with the
Gli1-binding-site plasmid was used for the reporter assay. To measure
compound activity, compounds were incubated with cells for 15 h in the
presence of Optimem medium (Invitrogen, Carlsbad, CA, USA) supplemented
with 0.5% charcoal–dextran treated fetal bovine serum (HyClone) and 10 mM
myristoylated mouse Shh protein (Williams et al., 1999). Luciferase activity
with a corresponding dose-dependant increase in plasma exposure
of 43 (Fig. 3).
Having established the in vivo modulation of Smo signaling in
the rodent hair follicle model, we sought to evaluate the efficacy
of compound 43 in a rodent tumor model. It has been well estab-
lished that Ptch +/À mice are prone to develop medulloblastomas.
Addition of a second genetic defect such as p53 À/À increases the
incidence and decreases the latency associated with development
of these tumors.21 Following the method of Sasai et al.,22 p53 À/
À and Ptch +/À mice were crossbred. Tumors from p53 À/À Ptch
+/À mice were harvested and transplanted into immunocompro-
mised mice. After 8 days, the tumor-allograft bearing mice were
randomized and separated into a treatment group (10 mg/kg 43
QD, oral)20 and control group (vehicle). As shown in Figure 4, the
treatment group showed inhibition of tumor growth relative to
control, and significant tumor volume reduction on day 15 with re-
spect to onset of treatment on day 8.
was measure by addition of Bright-Glo (Promega) and reading on
a
luminometer. For single IC50 assay, compounds were tested in
a
quadruplicate using a threefold dilution series. Assays were performed in
384-well plates.
10. IC50 assay for human Smo: HEPM cells were used to measure human Smo
activity in vitro using a modified version of the method described in US Patent
6,613,798. In 96-well tissue culture plates compounds were incubated with
HEPM cells in the presence of MEM media supplemented with 0.5% charcoal–
dextran treated fetal bovine serum (HyClone) and 50 mM myristoylated mouse
Shh protein. (Williams, K. P.; Rayhorn, P.; Chi-Rosso, G. Garber, E. A.; Strauch, K.
L.; Horan, G. S. B.; Reilly, J. O.; Baker, D. P.; Taylor, F. R.; Koteliansky V.;
Pepinsky, R. B. J. Cell Sci. 1999, 112, 4405.) Twenty-four hours after the addition
of compound and Shh, GLI expression was measured using a Quantigene assay
(Affymetrix, Santa Clara, CA, USA).
11. Pooled human or rat liver microsomes (0.25 mg/mL) were incubated at 37 °C in
In conclusion, we have described the evolution of a class of 4-
aryl-1-piperazinyl phthalazines as potent and metabolically stable
antagonists of SMO. Replacement of the thiophene ring in 1 with a
phenyl group significantly improved the outcome in our rat and
human microsomal stability assays. The interplay of piperazine
methylation and aryl group substitution was explored, elucidating
the SAR of concomitant changes to these regions. Ultimately the
in vivo efficacy of 43 was demonstrated in a Ptch +/À medulloblas-
toma tumor allograft model.
a phosphate buffer (pH 7.4) with the test compound (1 lM). The reaction was
started with the addition of NADPH (1 mM final concentration). Incubations
were stopped after 0 or 30 min with the addition of organic solvent. Quenched
samples were analyzed for unchanged test compound by reversed phase HPLC
with tandem mass spectrometric detection. Percent turnover is determined by
the ratio of the amount (peak area) of unchanged test compound remaining in
incubated samples to the amount of unchanged test compound in non-
incubated samples (0 min).
12. CYP 3A: Pooled human liver microsomes (0.1 mg/mL) were incubated at 37 °C
in a phosphate buffer (pH 7.4) with the selective 3A substrate midazolam at a
concentration of 2.5 lM in the presence and absence of test compound (3 lM).
The reaction was started with the addition of NADPH (1 mM final
concentration). Incubations were stopped after 5 min with the addition of
organic solvent and 1-hydroxymidazolam metabolite formation is measured
by an HPLC–MS detection method. Inhibition was determined by the ratio of
the amount of metabolite in the presence of test compound to the amount of
metabolite in the absence of test compound. CYP 2D6: Pooled human liver
microsomes (0.25 mg/mL) were incubated at 37 °C in a phosphate buffer (pH
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a
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