Discovery and SAR of hydrazide antagonists of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor type 1 (PAC1-R)

Article abstract of DOI:10.1016/j.bmcl.2008.01.052

Potent small molecule antagonists for the PAC₁-R have been discovered. Previously known antagonists for the PAC₁-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.

Full text of DOI:10.1016/j.bmcl.2008.01.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2162–2166
Discovery and SAR of hydrazide antagonists of the
pituitary adenylate cyclase-activating polypeptide
(PACAP) receptor type 1 (PAC1-R)
Xenia Beebe,* Daria Darczak, Rachel A. Davis-Taber, Marie E. Uchic, Victoria E. Scott,
Michael F. Jarvis and Andrew O. Stewart
Neuroscience Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-1066, USA
Received 5 December 2007; revised 10 January 2008; accepted 14 January 2008
Available online 18 January 2008
Abstract—Potent small molecule antagonists for the PAC1-R have been discovered. Previously known antagonists for the PAC1-R
were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this
class B GPCR.
Ó 2008 Elsevier Ltd. All rights reserved.
The pituitary adenylate cyclase-activating polypeptide
(PACAP) receptor type 1 (PAC1-R) is a Class B GPCR
closely related to the vasoactive intestinal peptide (VIP)/
integrin/glucagon family of receptors.1,2 PACAP binds
to the PAC1-R selectively over VIP, and also binds to
the VIP receptors, VPAC1 and VPAC2. The truncated
peptide PACAP(6-38) binds selectively to PAC1-R.3
Two other peptides with no significant sequence similar-
ity to PACAP also bind to PAC1-R: maxadilan, a selec-
tive agonist, and a shortened version termed M65, acts
as a specific PAC1-R antagonist.4–6 Recently, the solu-
tion structure and binding mode of PACAP(6-38) to
the extracellular domain of PAC1-Rs have been
reported.7
implicated in neuroprotection,11 and is widely dispersed
in the central nervous system (CNS). It is also upstream
from the MAP-KK signaling system involved in cell cy-
cle regulation. The PAC1-R presents a potential novel
target for small molecule drug discovery in the areas
of neuroscience, oncology, and immunoscience.12,13
Two lead hydrazides shown in Figure 1 were identified
from the Abbott compound library using the 125I-PA-
CAP27 radioligand binding assay to the PAC1-R ex-
pressed in HEK293f membranes.14 Hydrazide
1
efficiently inhibited radioligand binding with a Ki of
56 nM. This compound was subsequently shown to be
a functional antagonist for the PAC1-R in a calcium in-
flux assay with a potent Kb calculated to be 200 nM.15
We assumed that this series is competitive with the pep-
tide ligand in both the radioligand competition and the
calcium flux assay, however because of the affinity of the
PACAP27, equilibrium conditions were difficult to dem-
onstrate. In the radioligand binding assay, hydrazide 2
exhibited similar potency with a Ki of 73 nM. The objec-
Identifying drug-like small molecule antagonists for
class B GPCRs where the natural agonists are large pep-
tides remains a challenge. Only 5 of the 15 known class
B GPCRs have small molecule ligand binders and the
class remains largely unexplored with small molecule
inhibitors. Discovery of small molecule antagonists for
key GPCR protein–protein/peptide interaction remains
a significant challenge in the area of drug discovery.8–10
This is the first report of any small molecule (non-pep-
tide) antagonists for the PAC1-R. The PAC1-R has been
O
O
O
O
N
O
N
N
H
N
N
H
HO
CN
1
HO
2
56nM Ki
Keywords: PACAP; Class B GPCR; PAC1-R; Hydrazides.
*
72nM Ki
Cl
Corresponding author. Tel.: +1 847 937 8291; fax: +1 847 935
0310; e-mail: xenia.b.searle@abbott.com
Figure 1. Structures of lead hydrazides that bind to PAC1-R.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.052

X. Beebe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2162–2166
2163
tive was to develop SAR around these two potent PAC1-
R antagonists. It is interesting to note that these two hits
are potent reference glucagon receptor antagonists.16
However, other compounds in our compound collection
with glucagon activity showed no binding to the PAC1-
R.
inverse
ethers
small changes
tolerated
tolerated
hydrazide
linker
benzyl
ether
R
O
necessary,
tolerates EWG
N
Ar
middle ring
necessary, can be
m- or p- branched
N
distal ring
H
no changes
tolerated
The synthesis of new hydrazides was accomplished by
known methods17 via the coupling of acyl hydrazides
with aldehydes. The aldehydes were synthesized by reac-
tion of the appropriate phenolic aldehyde and aryl bro-
mide. The hydrazides were synthesized by coupling of
HO
EWG
acyl phenol
Figure 2. Structural components and SAR trends of hydrazide leads.
Table 1. Modified phenol and linker hydrazides for PAC1-Ra
O
O
O
O
(LINKER)
O
O
N
HO
R
N
H
O
Cl
Entry
R
Binding Activity
Entry
LINKER
Binding Activity
O
H
)
)
N
3
22%@3lM
7
46%@3lM
(
N
HO
H
)
O
H
N
(
(
(
)
4
5
6
inactive
8
40%@3lM
inactive
N
H
O
)
Cl
)
S
44%@3lM
inactive
9
HO
N
N
O
)
O
)
10
inactive
HN
N
N
N
a All binding activity reported herein was measured with an n = 2 or greater.
Table 2. PAC1-R binding affinity of hydrazide distal ring modifications
O
O
N
R
N
N
H
R
N
H
N
HO
O
HO
Cl
Cl
Entry
R
Ki, nM
152
Entry
R
Ki, nM
85
O
O
CF
3
(
(
11
14
(
15
16
604
O
O
O
(
12
13
167
55%@3lM
(
(
(
N
17
18
49%@3lM
51%@3lM
O
O
O
insoluble18
O
(

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X. Beebe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2162–2166
Table 3. PAC1-R binding activity of middle and distal ring combinations
O
N
R
N
H
HO
Cl
Entry
R
Ki, nM
185
Entry
R
Ki, nM
Entry
R
Ki, nM
522
O
O
O
O
O
O
19
25
inactive/insoluble 31
O
O
(
(
(
(
O
O
O
O
20
21
22
23
204
26
27
28
404
32
33
34
35
537
CF3
CF3
CF3
CF3
CF3
CF3
(
(
O
O
O
O
O
O
O
140
50%@3lM
36%@3lM
34%@3lM
43%@3lM
(
(
(
(
(
(
O
O
O
N
H
CF3
inactive
554
O
O
O
O
CF3
H
N
H
N
H
N
O
43%@3lM 29
inactive
O
O
O
O
O
O
O
(
(
(
(
(
(
O
O
CF3
24
O
41%@3lM 30
274
36
241
CF3
appropriate benzoic acids with Boc-protected hydrazine
followed by deprotection. All new compounds were
purified by flash chromatography and were character-
ing group (EWG) were crucial to high PAC1-R affin-
ity and sequential removal of these substituents
resulted in inactive compounds 3 and 4. Electron
donating groups such as m-OMe reduced binding
activity as shown in entry 5. The 3,4-dioxolane and
20,20-difluoro-3,4-dioxolane analogs were completely
inactive. A p-SO2NH2 in place of the p-OH group
was inactive as was the m-OH phenol. Nitrogen
and oxygen-containing heterocyclic phenol isosteres
similar to compound 6 were inactive. The hydrazide
linker was modified as well. Borohydride-mediated
reduction of the hydrazide gave compound 7 with
the saturated hydrazine linker. This compound
showed weak binding to PAC1-R. Coupling of the
acyl hydrazine with the corresponding acid gave com-
pound 8, which also displayed weak binding to
PAC1-R. Constricting the linker in an oxadiazole or
thiadiazole as shown in entries 9 and 10 resulted in
loss of PAC1-R affinity.
1
ized by H NMR and MS.
For the purposes of systematic SAR studies the lead
compounds were divided into four regions and the over-
all trends are summarized in Figure 2. Both hits were
clearly similar having a p-acyl phenol with a hydrazide
linker to a middle aromatic ring that is further linked
via a 2-atom linkage to a distal aromatic ring. The
SAR studies were undertaken to investigate the effect
of changes in four regions of the hydrazide lead com-
pounds: the phenol portion; the hydrazide linker; the
middle aromatic ring and the distal aromatic ring. We
found that no changes were tolerated for the acyl phe-
nol. Small changes were tolerated in the hydrazide lin-
ker. The middle and distal aromatic rings were
necessary for potency, but many small changes to these
rings and linkers were acceptable. The distal aromatic
ring preferred a lipophilic group appended, but the lipo-
philic group was not crucial for potency.
We next explored small changes to the distal ring of the
hydrazide leads and the results are shown in Table 2.
Our approach was to mix and match the substitutents
on the two lead compounds to determine which changes
were tolerated, if any.
Preliminary SAR is shown in Table 1. We discovered
that both the p-phenol and the m-electron-withdraw-

X. Beebe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2162–2166
2165
Moving the p-isopropyl benzyl ether distal ring to the
meta-position of the middle ring maintains PAC1-R
binding affinity as shown for entry 11. Changing to
the tetramethyl benzyl ether from hydrazide lead com-
pound 2 results in similar binding affinity as shown in
entry 12 or an insoluble18 compound in entry 13.
II GPCR. These acyl hydrazides should be useful in fur-
ther elucidating the biological significance of PAC1-R.
Acknowledgments
The authors thank Steve Pratt of high throughput
screening, Leo Barrett and Paul Richardson for the syn-
thesis of PACAP38, and the structural chemistry group
for the 1H NMR and MS on all compounds.
We also explored different benzyl groups for the inda-
zole lead compound 2 and these results are summarized
in Table 2, entries 14–18. Changes were less tolerated for
these indazole compounds, the best being the m-trifluo-
romethyl (CF3) benzyl group in compound 14 with a
binding affinity similar to that of compound 2. Binding
was also observed with the p-isopropylbenzyl group in
compound 15 and weak binding was observed for com-
pounds simple benzyl (16), 3-pyridyl (17), or extended
benzyloxybenzoyl (18). Lipophilic substituents on the
distal ring are required for binding to PAC1-R for both
the indazole and dimethoxyphenyl middle rings.
References and notes
1. Vaudry, D.; Gonzalez, B. J.; Basille, M.; Yon, L.;
Fournier, A.; Vaudry, H. Pharmacol. Rev. 2000, 52, 269.
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Christophe, J. Eur. J. Biochem. 1992, 207, 239.
Next, SAR studies focused on middle and distal ring
combinations of hydrazide 1 are shown in Table 3.
These examples represent the largest changes to the
hydrazides that were tolerated. The dimethoxy groups
were systematically removed from the middle aryl ring
and these changes were combined with different substit-
uents on the distal benzyl ether. Compounds that retain
the dimethoxy groups of the middle ring with the distal
ring benzyl ether unsubtituted (19), m-CF3 (20), and p-
CF3 (21) are potent binders. Interestingly, we lose all
activity when the m-CF3 benzyl ether is linked through
the m-position (22) instead of the p-position (20) of
the middle ring. The potency drops when the middle
dimethoxy groups are removed, as we compare entries
19 and 31. For compounds containing a m-CF3 in the
benzyl ether, compared to entry 20, lose two-fold po-
tency when one (26) or both (32) methoxy groups are re-
moved from the middle ring. For compounds with a p-
CF3 in the benzyl ether, compared to entry 20, are only
weak binders with one (27) or no (33) methoxy groups in
the middle ring. Remarkably, entry 22 with the m-CF3
benzyl ether linked through the m-position of the middle
ring, regains potency when dimethoxy groups are absent
as in entry 34. Entries 23, 28, and 29 show that weaker
binding is observed upon introduction of an acetamide
group to the distal benzyl ether. In general, the most po-
tent compounds contain both dimethoxy groups in the
middle aromatic and an aliphatic group on the distal
aromatic ring.
4. Moro, O.; Lerner, E. A. J. Biol. Chem. 1997, 272,
966.
5. Moro, O.; Wakita, K.; Ohnuma, M.; Denda, S.; Lerner, E.
A.; Tajima, M. J. Biol. Chem. 1999, 274, 23103.
6. Uchida, D.; Tatsuno, I.; Tanaka, T.; Hirai, A.; Saito, Y.;
Moro, O.; Tajima, M. Ann. N.Y. Acad. Sci. 1998, 865
(VIP, PACAP, and Related Peptides), 253.
7. Sun, C.; Song, D.; Davis-Taber, R. A.; Barrett, L. W.;
Scott, V. E.; Richardson, P. L.; Pereda-Lopez, A.; Uchic,
M. E.; Solomon, L. R.; Lake, M. R.; Walter, K. A.;
Hajduk, P. J.; Olejniczak, E. T. Proc. Natl. Acad. Sci.
U.S.A. 2007, 104, 7875.
8. Hoare, S. R. J. Drug Discov. Today 2005, 10, 417.
9. Klabunde, T.; Evers, A. ChemBioChem 2005, 6, 876.
10. Jacoby, E.; Bouhelal, R.; Gerspacher, M.; Seuwen, K.
ChemMedChem 2006, 1, 760.
11. Shioda, S.; Ohtaki, H.; Nakamachi, T.; Dohi, K.;
Watanabe, J.; Nakajo, S.; Arata, S.; Kitamura, S.; Okuda,
H.; Takenoya, F.; Kitamura, Y. Ann. N.Y. Acad. Sci.
2006, 1070, 550.
12. Pisegna, J. R.; Oh, D. S. Curr. Opin. Endocrinol. Diabetes
Obesity 2007, 14, 58.
13. Dickinson, Tracey; Fleetwood-Walker; Susan, M. Trends
Pharmacol. Sci. 1999, 20, 324.
14. Briefly, membranes prepared from HEK293f cells express-
ing PAC1R (5 lg) were incubated with 0.2 nM 125I-
PACAP27 in 50 mM Tris–HCl, pH7.4, containing 5 mM
MgCl2) for 20 min at 37 °C. Non-specific binding was
defined by 300 nM unlabeled PACAP38. The assay was
terminated by rapid filtration through GF/B filters soaked
overnight in 0.5% PEI at 4 °C and washed three times with
ice cold harvest buffer (50 mM Tris–HCl, pH 7.4,0.5 mM
EDTA and 0.1% BSA). The radioactivity was quantified
by a TopCount (Perkin-Elmer). The Ki values were
determined from the inhibition data determined using
the Cheng–Prusoff equation (Cheng and Prusoff, 1973).
All values were calculated using non-linear regression and
the Prism Analysis package (GraphPad, San Diego).
15. The hydrazides were assessed as functional antagonists
using PACAP-induced Ca2+ mobilization in the rat
pancreatic acinar cell line AR42J that endogenously
expresses PAC1-R. The cells were loaded with the Ca2+
indicator dye Fluo-4AM for 2 h, prior to incubation with
the compounds for 30 min at room temperature offline.
Following, addition of 2 nM PACAP-27 the changes in
calcium concentration were monitored in the FLIPR for
Compounds were also synthesized with the reversed
benzyl ether linkage between the middle and distal aro-
matic rings. Entries 30 and 36 show a two-fold improve-
ment in potency when compared to compound 34. This
trend is not general, however, because when we compare
the unsubstituted benzyl ethers the reversed benzyl ether
for entry 24 is weaker in potency than entry 31.
In conclusion, we have synthesized acyl hydrazides that
show binding to the PAC1-R with nM binding affinity.
We have developed SAR around the linker, middle,
and distal rings of the hydrazides. These compounds
represent the only small molecules that bind to this class

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X. Beebe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2162–2166
3 min. The Kb values were determined from the inhibition Brand, C. L.; Polinsky, A. Bioorg. Med. Chem. Lett. 2002,
curve using the Cheng-Prusoff method (Cheng and Prusoff
1973). All values were calculated using non-linear regres-
sion and the Prism Analysis package (GraphPad, San
Diego).
12, 663.
17. Ling, A.; Hong, Y.; Gonzalez, J.; Gregor, V.; Polinsky, A.;
Kuki, A.; Shi, S.; Teston, K.; Murphy, D.; Porter, J.; Kiel,
D.; Lakis, J.; Anderes, K.; May, John; Knudsen, L. B.;
Lau, J. J. Med. Chem. 2001, 44, 3141.
18. Compound 13 could not be dissolved at 3 lM in DMSO
for the binding assay. Similar compounds in this series
exhibited aqueous solubility at <3 lM.
16. Ling, A.; Plewe, M.; Gonzalez, J.; Madsen, P.; Sams, C.
K.; Lau, J.; Gregor, V.; Murphy, D.; Teston, K.; Kuki, A.;
Shi, S.; Truesdale, L.; Kiel, D.; May, J.; Lakis, J.; Anderes,
K.; Iatsimirskaia, E.; Sidelmann, U. G.; Knudsen, L. B.;