Discovery of novel selective HER-2 sheddase inhibitors through optimization of P1 moiety

Article abstract of DOI:10.1016/j.bmcl.2009.07.052

A novel series of carbamates was discovered as potent and selective HER-2 sheddase inhibitors. Significant enhancement in potency and selectivity was achieved through attenuating the P1 moiety, which was conventionally believed to be exposed to solvent.

Full text of DOI:10.1016/j.bmcl.2009.07.052

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5037–5042
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel selective HER-2 sheddase inhibitors through optimization
of P1 moiety
*
Yun-Long Li , Eric Shi, David Burns, Yanlong Li, Maryanne B. Covington, Maxwell Pan, Peggy Scherle,
Steve Friedman, Brian Metcalf, Wenqing Yao
Incyte Corporation, Experimental Station—E336, Route 141 and Henry Clay Road, Wilmington, DE 19880, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of carbamates was discovered as potent and selective HER-2 sheddase inhibitors. Signifi-
cant enhancement in potency and selectivity was achieved through attenuating the P1 moiety, which was
conventionally believed to be exposed to solvent.
Received 24 June 2009
Revised 7 July 2009
Accepted 8 July 2009
Available online 25 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HER-2
Sheddase
Metalloprotease
ADAM-10
Selectivity
P1
The ErbB family of tyrosine kinase receptors is composed of four
members, epidermal growth factor (EGF) receptor (EGFR; HER-1 or
ErbB1), HER-2 (neu or ErbB2), HER-3 (ErbB3), and HER-4 (ErbB4).
Following ligand binding, EGF receptor family members form
homo- or heterodimers, autophosphorylate tyrosine residues in
their cytoplasmic tails and thereby trigger intracellular signaling
cascades which lead to cell growth, survival, or differentiation.1
Although HER-2 lacks a functional ligand binding domain, it is
the preferred partner for heterodimerization with other family
members.2 Moreover, when overexpressed, HER-2 homodimers
with signaling activity form spontaneously. Non-mutated HER2 is
overexpressed in a significant fraction of human breast (ꢀ25%) as
well as ovarian, gastric, and non-small cell lung tumors. In fact,
the presence of high HER-2 levels is associated with aggressive
clinical progression of breast and ovarian cancer.3 A humanized
monoclonal antibody, trastuzumab (HerceptinÒ), directed against
the extracellular domain (ECD) of HER-2 has proven to be an effec-
tive therapy for breast cancer patients who overexpress HER-2.4
In HER-2 overexpressing cancer cells, the ECD of HER-2 is
cleaved by the ADAM (a disintegrin and metalloproteinase) family
of zinc-dependent, membrane-associated metalloprotease. The
ectodomain shed of HER-2 renders its remaining transmembrane
portion, p95, a constitutively active, phosphorylated tyrosine ki-
nase.5 In vitro studies indicate that the p95 fragment of HER-2 is
10–100 times more oncogenic than the full length receptor.6 In
the clinic, the presence of the HER-2 ECD in the serum of cancer pa-
tients has been linked to a poor prognosis,7 with decreases in ser-
um ECD levels during treatment being a predictor of response to
trastuzumab therapy.8 Accordingly, inhibition of the protease
responsible for HER-2 ECD shedding and p95 production may have
potential therapeutic benefit in HER-2 positive patients.
Many of the existing broad-spectrum metalloprotease inhibi-
tors, such as marimastat, inhibit not only MMPs (matrix metallo-
proteinases) but also ADAMs in vitro and thereby prevent the
processing of multiple ErbB ligands.9,10 However, their clinical
development has been hampered by side effects, many of which
are likely due to MMP inhibition.11 Recently we have identified
ADAM-10 as a major source of HER-2 ectodomain sheddase
activity in breast cancer cells that overexpress HER-2.12 We have
also shown that selective inhibitors of HER-2 sheddase decrease
tumor size, decrease cleaved HER-2 ECD plasma levels, and
potentiate the effects of trastuzumab in vivo.13,14 The selectivity
profile of these novel azaspiro-hydroxamic acids was optimized
by exploiting the subtle differences between the S1’ pocket of
the MMPs and the HER-2 sheddase by attenuating the P10
substituent.
Herein we report that the potency and selectivity profile of
sheddase inhibitors can also be attenuated by modification of the
P1 substituent. In the following discussion HER-2 potency
describes the cellular inhibition of HER-2 sheddase and ADAM-10
potency denotes the enzymatic inhibition of HER-2 sheddase. The
* Corresponding author.
E-mail addresses: yunli@incyte.com, yunlongster@gmail.com (Y.-L. Li).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.052

5038
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5037–5042
selective against MMP-2 than compound 3. The nitrogen linked
NH2
HN
OH
P1
compounds 6 and 7 also inhibit ADAM-10 with potency similar
to 3, but their selectivity against MMP-2 was found to be
decreased.
HN
O
Zn2+
HN
O
OH
O
O
N
To further characterize the P1 moiety, a series of compounds
with different P1 were prepared in the non-selective,13 4-phenyl-
1,2,3,6-tetrahydropyridin-1-yl P10 series as shown in Table 2.
While all the analogs were found to be generally more potent than
spiro-cyclopropyl compound 8, the oxygen linked compound 10
was outstanding with its 10-fold selectivity against MMP-2. This
result suggested some specific unfavorable interactions between
the oxygen linked carbamate and the MMP-2 S1 cliff. On the other
hand, the selectivity profile of the carbon linked amides (4 vs 9)
apparently depended on the P10 moiety.
N
H
N
P1'
N
H
P2
O
1: HER-2 IC50 358 nM
ADAM-10 IC50 323 nM
MMP-2 IC50 998 nM
2: HER-2 IC50 162 nM
ADAM-10 IC50 161 nM
MMP-2 IC50 1662 nM
Figure 1. Starting point of P1 optimization.
ADAM-10 data is used to evaluate the MMP selectivity, since it is
more comparable with the MMP affinity data.
At this point, the configuration at C-5 position of carbamate 10
was determined to be axial. Its equatorial counterpart 14 (Fig. 2)
was found to be less active for HER-2 and ADAM-10 and not selec-
tive against MMP-2, resembling the profile of the spiro-cyclopropyl
Conventional SAR studies of metalloproteases have focused pri-
marily and often entirely on the S10 pocket to achieve both potency
and selectivity, since it was believed that the P1 substituent was
primarily solvent exposed.15–17 Recently, Condon et al disclosed
the optimization of a series of TACE inhibitors through modifica-
tion of the P1 moiety.18 Our investigation into the SAR of the P1
moiety was triggered by the finding that compound 2 designed
as a protein probe was two fold more potent than parent com-
pound 1 (Fig. 1). When the more drug-like compound 4 was pre-
pared (Table 1), we were delighted to find that it was 16 times
more potent in the HER-2 cellular assay and almost seven times
more potent in the ADAM-10 enzymatic assay than compound 3
(the single diastereoisomer of 1). While the cellular activity is com-
plicated by protein binding and permeability, the ADAM-10 affinity
assay results are experimentally significant. More impressively,
compound 4 was 2.5 times more selective against MMP-2 than
compound 3.
Table 2
P1 SAR in phenyl tetrahydrapyridinyl series21
OH
HN
R1
O
R2
N
N
H
O
Compound
R1 and R2
HER-2 IC50
(nM)
ADAM-10 IC50
(nM)
MMP-2 IC50
(nM)
8
Spiro-cyclopropyl
18
33
<5
Other possible P1 moiety linkages were also investigated, as
shown in Table 1. The oxygen linked compound 5 has comparable
potency to 3 in the ADAM-10 assay, but is more than 2.4 times
O
N
9
4.3
17
<5
O
Table 1
10
5.6
15.5
10
9
23
12
11
102
11
N
O
P1 SAR in phenyl pyrrolidinyl series21
OH
HN
R1
O
N
R2
O
N
11
12
13
N
H
O
O
N
Compound
R1 and R2
HER-2 IC50
(nM)
ADAM-10 IC50
(nM)
MMP-2 IC50
(nM)
12
3
Spiro-cyclopropyl
92
97
960
O
N
O
4
5
5.6
14
365
8.5
<5
N
O
O
Note: The stereochemistry at C-5 position was not determined. Data shown is for
the more active isomer in each case.
28
52
29
83
138
84
>2000
502
N
O
O
N
OH
OH
HN
HN
O
6
7
N
O
N
O
O
N
O
N
O
O
N
H
Ph
N
Ph
H
O
N
O
210
10: HER-2 IC50 5.6 nM
ADAM-10 IC50 9 nM
MMP-2 IC50 102 nM
14: HER-2 IC50 30 nM
ADAM-10 IC50 78 nM
MMP-2 IC50 < 5 nM
Note: The stereochemistry at C-5 position was not determined. Data shown is for
the more active isomer in each case.
Figure 2. Diastereomers 10 and 14.

Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5037–5042
5039
compound 8. Although the diastereomers of 9, 11, 12, and 13 differ
in potency, the selectivity profile was found to be similar (data not
shown). The difference in selectivity observed between 10 and 14
suggested that an appropriately directed P1 moiety may interact
with a S1 pocket, in addition to the possible interactions between
the carbamate oxygen and S1 cliff.
tion of the scaffold piperidine ring.13 Therefore, substitution on
nitrogen was expected to alter the orientation of the P10 moiety
and thus modify the binding profiles. In the carbamate series,
piperidine core nitrogen substitution resulted in a modest loss of
potency for HER-2 and ADAM-10, but a slight increase in selectiv-
ity against MMP-2, as demonstrated in Table 4.
Looking into different carbamates, the inhibition potency for
HER-2 and ADAM-10 were found to increase when the amino
group was changed from azetidinyl to pyrrolidinyl and to piperid-
inyl. Continued increase in the ring size of the amino group to
azepanyl and azocanyl then resulted in decreased potency as
shown in Table 5. The selectivity for MMP-2 was also found to in-
crease as the amino ring size enlarged up to the six-membered
ring. Partial unsaturated pyrrolidinyl compound 24 was less active
for ADAM-10, but more active for HER-2 than saturated analog 20.
Bis-fluoro substituted analog 25 was less potent than 24 in both
HER-2 and ADAM-10 assays. All compounds tested in MMP-1,
MMP-3, and MMP-9 did not show any inhibitory activities when
Comparing different P10 moieties in the carbamate series as
shown in Table 3, saturated compound 15 is less active, but more
selective than 10. The P10 moieties of compound 16 and 10 would
be expected to have similar coplanar conformations (due to conju-
gation between the p-system of the aromatic ring and the lone pair
of electrons on the piperazine ring)19 and possess similar activity.
However, 16 is more selective than 10.
With small P10 inner ring, the pyrrolidinyl analogs 17, 18, and
19 are less active for HER-2 and ADAM-10. When the compounds
with different P10 moieties were evaluated in the inhibition of
other MMPs, the phenyl piperazinyl compound 16 showed better
selectivity profile than 10.
Substitution on piperidine nitrogen of 16 was hypothesized to
alleviate potential hydrogen bonding between the scaffold N–H
and P10 carbonyl and twist the P10 amide C@O to avoid steric and
torsional strain. It could also change the conformational distribu-
tested at 5 lM.
The caco-2 permeability of compound 26 was determined to be
0.9 ꢁ 10ꢂ6 cm/s. Its projected clearance from human microsome
incubation is 0.58 L/h/kg. When dosed 5 mg/kg po as a suspension
Table 3
In vitro profile of different P10 analogs21
OH
HN
N
O
O
O
N
NRR
O
H
Compound
NRR
HER-2 IC50 (nM)
5.6
ADAM-10 IC50 (nM)
9
MMP-1 IC50 (nM)
MMP-2 IC50 (nM)
102
MMP-3 IC50 (nM)
>5000
MMP-9 IC50 (nM)
498
10
>5000
>5000
>5000
>5000
>5000
2500
N
Ph
Ph
Ph
15
16
17
18
19
42
3.8
11
28
70
99
11
>2000
235
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
N
N
N
Ph
60
506
N
Ph
83
>2000
>2000
N
186
N
Table 4
The effects of piperidine core substitution21
OH
HN
O
N
O
O
N
O
N
R
N Ph
Compound
R
HER-2 IC50 (nM)
ADAM-10 IC50 (nM)
MMP-1 IC50 (nM)
MMP-2 IC50 (nM)
MMP-3 IC50 (nM)
MMP-9 IC50 (nM)
16
20
21
H
Me
CO2Me
3.8
11.5
8.1
11
18
20
>5000
>5000
>5000
235
471
613
>5000
>5000
>5000
>5000
>5000
>5000

5040
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5037–5042
Table 5
SAR of P1 moiety in phenyl piperazine series21
OH
HN
RRN
O
O
N
O
N
N Ph
O
Compound
NRR
HER-2 IC50 (nM)
31
ADAM-10 IC50 (nM)
58
MMP-1 IC50 (nM)
>5000
MMP-2 IC50 (nM)
730
MMP-3 IC50 (nM)
>5000
MMP-9 IC50 (nM)
>5000
22
NMe2
23
20
24
44.5
11.5
3.8
20
18
30
>5000
>5000
>5000
432
471
581
>5000
>5000
>5000
>5000
>5000
>5000
N
N
N
F
F
25
26
50.5
7.3
125
9.6
ND
>2000
344
ND
ND
N
>5000
>5000
>5000
N
27
4.4
35
26
>5000
>5000
843
>5000
>5000
>5000
>5000
N
28
27.5
1475
N
ND = not done.
in 10% DMAC in 0.5% methylcellulose in an n-in-1 study, com-
pound 28 exhibited an AUC of 323 nM/L h and T1/2 of 0.88 h in
mice.
protective group. The resulting acid 35 was then subjected to BOP
catalyzed coupling with hydroxylamine hydrochloride salt in the
presence of diisopropylethylamine to afford analog 16. The axial-
and equatorial isomers were separated by reversed phase HPLC
in a ratio of 1:1.
The reduction of ketone 30 with sodium borohydride afforded
an alcohol 31 and 5% of lactone 36, presumably generated by
in situ cyclization. The axial- and equatorial isomers could be sep-
arated by chiral HPLC and the ratio was determined to be 1:1
(shown in Scheme 2). When the reduction was carried out with
L-Selectride in THF, the equatorial isomer 31b was generated
exclusively. With (S)-oxazaborolidine-borane complex as cata-
lyst,20 the reduction of 30 in dichloromethane produced the two
isomers in a ratio of 2:1. When the (R)-oxazaborolidine-borane
The novel HER-2 sheddase inhibitors were synthesized from
known compound 2913 as exemplified by the preparation of com-
pound 16 (Scheme 1). Ozonolysis of 29 gave ketone 30 in 90% yield.
Reduction of 30 with sodium borohydride provided alcohol 31 as
an inseparable mixture of axial and equatorial isomers in 86%
yield. Treatment of 31 with carbonyl diimidazole followed by pyr-
rolidine yielded carbamate intermediate 32 in a yield of 92%. After
hydrogenation, the acid 33 was coupled with phenylpiperazine in
the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phos-
phonium hexafluoro-phosphate (BOP) to generate amide 34. The
later was treated with trifluoroacetic acid to remove the tert-butyl
O
CO2-t-Bu
CO2Bn
CO2-t-Bu
CO2Bn
HO
CO2-t-Bu
CO2Bn
31
N
O
CO2-t-Bu
CO2Bn
N
O
CO2-t-Bu
CO2H
b)
a)
c)
d)
N
N
Cbz
N
Cbz
O
O
N
N
Cbz
30
H
Cbz
33
32
29
N
O
CONHOH
N
O
CO2H
N
O
CO2-t-Bu
f)
g)
e)
O
O
O
N
N
N Ph
N
N
H
35
N Ph
N
H
N
O
N Ph
H
O
O
16
34
Scheme 1. Synthesis of compound 16. Reagents and conditions: (a) O3, CH2Cl2, ꢂ78 °C, 90%; (b) NaBH4, MeOH, 86%; (c) carbonyl diimidazole, CH2Cl2, rt, then pyrrolidine,
92%; (d) H2, 10% Pd/C, MeOH, 98%; (e) 1-phenylpiperazine, BOP, i-Pr2NEt, DMF, rt, 78%; (f) TFA, CH2Cl2, 100%; (g) NH2OHꢃHCl, BOP, i-Pr2NEt, DMF, 60%.

Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5037–5042
5041
O
O
O
CO2-t-Bu
CO2Bn
HO
CO2-t-Bu
CO2Bn
HO
CO2-t-Bu
CO2Bn
+
+
N
N
N
N
CO2Bn
Cbz
Cbz
30
Cbz
Cbz
36
31a
31b
NaBH4
L-Selectride
1
0
:
:
1
1
Ph
Ph
O
BH3.SMe2
2
:
1
N B
Ph
O
Ph
BH3.SMe2
1
:
1.1
N B
Scheme 2. Reduction of ketone 30.
complex was used, alcohols 31a and 31b were afforded in 1:1.1
ratio.
ane. While 2-oxa-5-azabicyclo[2.2.2]octan-3-one was detected,
no 6-oxa-3-azabicyclo[3.2.1]octan-7-one was identified. Since the
C-5 stereochemistry was carried from chiral starting material, the
formation of 2-oxa-5-azabicyclo[2.2.2]octan-3-one but not the 5-
membered lactone suggested C-3 configuration was trans to C-2
and C-5. Reaction of alcohol 41 with carbonyl diimidazole followed
by piperidine afforded carbamate 42. Hydrogenation of the latter
followed by BOP promoted coupling with phenyl piperazine
yielded amide 44. Reductive amination of 44 with formaldehyde
then produced piperidine N-methylated compound 45 in a yield
of 86%. Treatment of 45 with freshly prepared hydroxylamine
methanol solution successfully gave the desired product 26 in
71% yield.
In summary, a novel series of carbamates has been identified as
potent and selective HER-2 sheddase inhibitors. Although it was
believed that the P1 substituent was primarily solvent exposed,
we were able to demonstrate that significant enhancement in po-
tency and selectivity could be achieved by attenuating the P1 moi-
ety. While the increase in binding potency maybe the result of
solvent-repulsion, non-specific lipophilic interaction at the solvent
exposed S1 pocket, the astonishing increase in the selectivity
against MMP-2 clearly indicates a specific interaction between
the P1 moiety and S1 cliff and pocket. Since the dose-limiting
Since the selectivity of ketone reduction was less than satisfac-
tory and the resulting alcohol mixtures were inseparable by flash
chromatography, a novel synthesis was devised to accommodate
the imperative need of biologic evaluation, as shown in Scheme
3. Reaction of secondary amine 37 with commercially available
(S)-(+)-glycidyl tosylate in the presence of trimethylaluminum
gave alkylated product 38 in 91% yield. The use of trimethylalumi-
num as catalyst has been proven to be superior, since other Lewis
acids have been less effective. For instance, when lithium perchlo-
rate was used as catalyst, there was no reaction; while the ytter-
bium(III) triflate catalyzed reaction in acetonitrile takes 4 days to
achieve 80% conversion. When dichloromethane was used as sol-
vent, the ytterbium(III) triflate catalyzed reaction was fast, but
gave a mixture of starting material, the desired alkylated product,
and a cyclized lactone in a ratio of 1:1:1.
The alcohol 38 was then protected as its ethoxy ethyl ether in a
yield of 98%. Subjection of protected 39 to the intra-molecular
alkylation conditions provided piperidine 40 in 74% yield. The C-
2 and C-3 cis-isomer was not detected. The C-3 configuration
was established through chemical correlation. After deprotection,
alcohol 41 was subjected to trimethylaluminum in dichlorometh-
OTs
OTs
O
O
CO2Me
CO2Bn
CO2Me
HO
CO2Me
CO2Bn
O
O
CO2Me
CO2Bn
c)
a)
b)
N
BnHN
d)
CO2Bn
N
N
Bn
Bn
Bn
37
38
39
40
HO
CO2Me
CO2Bn
N
O
CO2Me
CO2Bn
N
O
CO2Me
CO2H
g)
e)
f)
O
N
O
N
N
H
Bn
Bn
42
43
41
N
O
CO2Me
N
O
CONHOH
N
O
O
CO2Me
N
O
44
h)
i)
O
O
N
N
O
N
Ph
N
N
N Ph
N
H
N Ph
O
26
45
Scheme 3. Stereoselective synthesis of compound 26. Reagents and conditions: (a) (S)-(+)-glycidyl tosylate, AlMe3, CH2Cl2, ꢂ78 °C, 91%; (b) ethyl vinyl ether, PPTS, CH2Cl2,
98%; (c) LiHMDS, PhMe–THF (8:1), ꢂ78 °C to 0 °C, 74%; (d) 0.5 N HCl, THF, 97%; (e) carbonyl diimidazole, CH2Cl2, rt, then piperidine, 94%; (f) H2, 10% Pd/C, MeOH, 98%; (g) 1-
phenylpiperazine, BOP, i-Pr2NEt, DMF, rt, 80%; (h) 36% HCHO, NaBH(OAc)3, THF–AcCN, 86%; (i) 1.64 M NH2OH in MeOH, 71%.

5042
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5037–5042
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are attributed to their non-selective inhibition of multiple MMPs,11
it is desirable to design potent HER-2 sheddase inhibitors that are
selective against MMPs to avoid deleterious physiological side ef-
fects. This discovery opened a new area which could be explored
to obtain a desired selectivity profile for improved HER-2 sheddase
inhibitors.
Acknowledgements
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analytical group, Cindy A. Marando and Kamna Katiyar of the dis-
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