Discovery of a potent, selective, and orally active human epidermal growth factor receptor-2 sheddase inhibitor for the treatment of cancer

Article abstract of DOI:10.1021/jm061344o

The design, synthesis, evaluation, and identification of a novel class of (6S,7S)-N-hydroxy-6-carboxamide-5-azaspiro[2.5]octane-7-carboxamides as the first potent and selective inhibitors of human epidermal growth factor receptor-2 (HER-2) sheddase is des

Full text of DOI:10.1021/jm061344o

J. Med. Chem. 2007, 50, 603-606
603
teases are comprised of the a disintegrin and metalloprotease
(ADAM) and matrix metallo-protease (MMP) family members,
which are highly conserved within the active site. Baselga et
al. have demonstrated that HER-2 shedding can be induced by
MMP activators and inhibited by broad spectrum MMP inhibi-
tors, such as (2R,3S)-N(1)-[(1S)-1-benzyl-2-(methylamino)-2-
oxoethyl]-N(4)-hydroxy-2-iso-butyl-3-[(2-thienylthio)methyl] suc-
cinamide (batimastat) and naturally occurring tissue inhibitor
of metalloproteinase 1 (TIMP-1).4 Based on the latter result,
Baselga et al. concluded that ADAM-17, which has been widely
implicated in the shedding of numerous proteins, was not the
protease responsible for HER-2 ECD shedding, because ADAM-
17 is not inhibited by TIMP-1.4d,5 Conversely, ADAM-10 is
inhibited by batimastat6 and TIMP-1,7 and Blobel et al. recently
identified ADAM-10 as a major sheddase for two crucial EGFR
ligands in mouse cells.6 We therefore postulated that ADAM-
10 may play a critical role in HER-2 ECD shedding, which
was recently confirmed by Burn et al. of Incyte.8 Irrespective
of the exact nature of the protease, it is well-known that
metalloproteases and sheddase activity can be inhibited by
hydroxamic acid-derived compounds.1c,9
Discovery of a Potent, Selective, and Orally
Active Human Epidermal Growth Factor
Receptor-2 Sheddase Inhibitor for the Treatment
of Cancer†
Wenqing Yao,* Jincong Zhuo, David M. Burns,
Meizhong Xu, Colin Zhang, Yun-Long Li, Ding-Quan Qian,
Chunhong He, Lingkai Weng, Eric Shi, Qiyan Lin,
Costas Agrios, Timothy C. Burn, Eian Caulder,
Maryanne B. Covington, Jordan S. Fridman,
Steven Friedman, Kamna Katiyar, Gregory Hollis,
Yanlong Li, Changnian Liu, Xiangdong Liu,
Cindy A. Marando, Robert Newton, Max Pan,
Peggy Scherle, Nancy Taylor, Kris Vaddi,
Zelda R. Wasserman, Richard Wynn, Swamy Yeleswaram,
Ravi Jalluri, Michael Bower, Bing-Bing Zhou, and
Brian Metcalf
Incyte Corporation, Experimental Station,
Wilmington, Delaware 19880
ReceiVed NoVember 20, 2006
A number of hydroxamic acid-derived broad spectrum MMP
inhibitors have been described in the literature. However, early
clinical studies indicate that these compounds exhibit dose-
limiting toxicities, such as fibroplasia and tendonitis (frozen
shoulder syndrome).10 Therefore, it is desirable to design a
potent HER-2 sheddase inhibitor that is selective against MMPs
to avoid deleterious physiological side effects. We envisioned
that this could be achieved by exploiting subtle differences
within the S1′ subsite of the otherwise highly conserved active
sites within the metalloprotease family.9
At the inception of the project, Yoshiizumi et al. reported a
series of 5,6,7,8-tetrahydropyrido[3,4-b] pyrazine-based hy-
droxamic acids 1 that were designed to inhibit the shedding of
heparin-binding epidermal growth factor (HB-EGF) by inhibit-
ing an unknown ADAM (Figure 1).11 Our hypothesis was that
a similar enzyme may play a role in HER-2 shedding and lead
to the expedient synthesis and evaluation of pipecolic acid-
derived hydroxamates. Compounds of this class, such as 2,
displayed moderate cellular inhibition of HER-2 shedding
(0.67-1.9 µM), suggesting that the protease responsible for
HER-2 and HB-EGF shedding might be similar. However, these
compounds were found to be nonselective broad spectrum MMP
inhibitors.
Xue et al. reported using cyclic succinates to inhibit ADAM-
17.12 Although, we knew that ADAM-17 was not the sheddase
of interest based on Baselga’s results,4d we believed that the
trans-6-membered succinate might be a viable lead that could
be attenuated to target the HER-2 sheddase by modifying the
P1′ substituent and thus exploiting potential differences within
the S1′ subsite of ADAM-17 and the HER-2 sheddase, as
discussed above.
Thus, a series of cyclic succinates, such as compound 3, were
prepared and were found to have moderate HER-2 sheddase
activity with an IC50 of 560 nM accompanied with a high affinity
for MMPs (Figure 2).
Abstract: The design, synthesis, evaluation, and identification of a
novel class of (6S,7S)-N-hydroxy-6-carboxamide-5-azaspiro[2.5]octane-
7-carboxamides as the first potent and selective inhibitors of human
epidermal growth factor receptor-2 (HER-2) sheddase is described.
Several compounds were identified that possess excellent pharmaco-
dynamic and pharmacokinetic properties and were shown to decrease
tumor size, cleaved HER-2 extracellular domain plasma levels, and
potentiate the effects of the humanized anti-HER-2 monoclonal antibody
(trastuzumab) in vivo in a HER-2 overexpressing cancer murine
xenograft model.
The human epidermal growth factor receptor-2 (HER-2a or
ErbB-2) is a tyrosine kinase receptor that is activated upon
homo-and heterodimerization with another member of the HER
family or by proteolytic cleavage (shedding) of the extracellular
domain (ECD).1 Once activated, intracellular signal transduction
pathways are initiated that mediate a diverse range of essential
cellular activities such as cell proliferation, differentiation,
motility, adhesion, and survival.2 Overexpression of the onco-
gene HER-2/neu has been associated with aggressive patho-
genesis, poor prognosis, and decreased responsiveness to
conventional chemotherapeutic and hormonal treatment regimes
in non-small cell lung cancer, ovarian cancer, and breast cancer
patients.1a In addition, elevated plasma levels of HER-2 ECD
have been associated with an increased metastatic potential and
a decrease in disease-free and overall survival in patients with
breast cancer.1b,3 Therefore, inhibition of the protease responsible
for HER-2 ECD shedding may be therapeutically desirable for
treating cancer patients that overexpress HER-2, particularly
breast cancer patients.
At the onset of the project, the protease that is responsible
for HER-2 shedding had not been identified, although there was
strong evidence suggesting that protein ectodomain shedding
is mediated by zinc-dependent metalloproteases.1c Metallopro-
† Dedicated to Professor Ralph F. Hirschmann on the occasion of his
85th birthday.
In an effort to enhance the binding profile, the conforma-
tionally flexible piperidine scaffold ring was rigidified by the
installation of a cyclopropyl group at the C-5 position. It was
hypothesized that by rigidifying the piperidine core the spatial
arrangement of the P1′ substituent would be more defined, which
* To whom correspondence should be addressed. Tel.: 302-498-6707.
Fax: 302-425-2704. E-mail: wyao@incyte.com.
a Abbreviations: HER-2, human epidermal growth factor receptor-2;
ECD, extracellular domain; ADAM, a disintegrin and metalloprotease;
MMP, matrix metalloprotease; TIMP-1, tissue inhibitor of metalloproteinase-
1.
10.1021/jm061344o CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/26/2007

604 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Letters
the tetrahydropyridine double bond or the lone pair of electrons
on the piperazine nitrogen.14 Conversely, the two ring systems
of the 4-phenyl piperidine analog 12 prefer an orthogonal
conformation with respect to one another. It was speculated that
this difference in the orientation of the phenyl ring within the
narrow and deep loop-3 region of the S1′ pocket may be
exposing a subtle difference at residue 223, which is tyrosine
for MMP-2, -3, and -9 and alanine for ADAM-10.
Figure 1. HB-EGF inhibitor structural leads.
To explore this hypothesis, a series of compounds were
prepared and evaluated that had varying torsion angles with
respect to the P1′ aryl ring and the heterocycle that it is attached.
Twisting the two ring systems out of coplanarity was achieved
by either substituting the 3-position of the heterocycle ring, such
as 13, or the ortho-position of the aryl ring, such as 14. Both
of these designed compounds were found to be inactive in the
HER-2 sheddase assay. However, introduction of a nitro group
at the para-position of compound 14 to provide the 4-(2-methyl-
4-nitrophenyl)piperazine analog 15 resulted in a dramatic
increase in sheddase potency with excellent selectivity against
other MMPs. In comparison to the unsubstituted 4-phenylpip-
erazine analog 10, compound 15 displayed a slight loss in
potency that was offset by the increase in selectivity against
MMP-2 and -9. The corresponding 4-(2-methyl-4-nitrophenyl)-
tetrahydropyridine compound 16 exhibited an increase in
selectivity by more than 55-fold against MMP-2 and 3-fold
against MMP-9 in comparison to the unsubstituted 4-phenyl-
tetrahydropyridine analog 11, albeit with a concomitant loss in
potency. One explanation for this dramatic observed substituent
effect is that the strong electron withdrawing properties of the
nitro group render the aromatic π-system electron deficient and
predisposed to electron donation, thus amplifying the conjugative
effects and mitigating the steric strain of the ortho-methyl group.
The counterbalancing effect of these two groups results in the
aryl ring having an optimal torsion angle to achieve the desired
selectivity.
Figure 2. Design of spiro-pipecolic acid scaffold.
Scheme 1. Synthesis of Spiro-pipecolic Acid Analogsa
a Reagents and conditions: (a) BnBr, DBU, PhMe, 46%; (b) CH2dC(CH2-
Cl)2, K2CO3, NaI, ACN, 70%; (c) NaI, acetone, 96%; (d) LiHMDS, THF,
-78 to -30 °C, 72%; (e) CbzCl, 93%; (f) CH2N2, Pd(OAc)2, 98%; (g) H2,
Pd/BaSO4, MeOH, 98%; (h) (i) BOP, HNR2R3, DIEA, DMF; (ii) R1X,
DIEA, DCM; (i) (i) TFA, DCM; (ii) NH2OH, BOP, DIEA, DMF.
In an effort to mimic this optimal twisted coplanar conforma-
tion exhibited by the 4-(2-methyl-4-nitro-phenyl)piperazine
analog 15, the cyclic compound 17 was prepared (Table 1).
Despite the slight loss in potency and selectivity against MMP-2
in comparison to compound 15, there was a significant gain in
potency in comparison to the unsubstituted 4-(2-methyl-phenyl)-
piperazine analog 14. These results support the hypothesis that
the torsion angle between the heterocycle and appended aromatic
ring is critical in determining the metalloprotease binding profile.
An alternative approach to exploit the amino acid difference at
position 223 was to change the directionality of the aryl ring
by reducing the size of the heterocycle ring to which it was
attached from a six- to a five-membered ring. 3-Phenylpyrro-
lidine 18 exhibited both exceptional potency against HER-2
sheddase and selectivity against MMPs (Table 1). The corre-
sponding 3-phenylpyrrolid-3-ene 19 was 7-fold more potent,
however, the selectivity against MMP-9 was diminished by
5-fold and the selectivity against MMP-2 was completely
abrogated.
would be beneficial in ascertaining information regarding the
bioactive conformation from SAR studies. This hypothesis was
supported by the 5-fold increase in potency observed for the
C-5 cyclopropyl analog 18. It was also speculated that the
decrease in conformational flexibility may also enhance the oral
bioavailability.13
Based on the above rational, a series of compounds with the
general formula 4 were prepared from the designed polyfunc-
tionalized chiral building block 8, which was prepared from
L-aspartic acid â-tert-butyl ester 5 and 3-chloro-2-(chloro-
methyl)-1-propene as depicted in Scheme 1.13
A series of secondary amides such as 9 were prepared and
found to be inactive against the HER-2 sheddase. In contrast,
4-phenyl-piperazine 10 and 4-phenyl tetrahydropyridine 11
analogs exhibited excellent cellular activity against HER-2
sheddase (<60 nM, Table 1). The high degree of selectivity of
these two compounds against MMP-1, which has a shallow S1′
pocket, and overall modest selectivity against MMP-2 and -9,
which have a narrow and deep S1′ pocket, suggests that the S1′
pocket of the HER-2 sheddase might be similar to that of
MMP-2 and -9. Replacing the tetrahydropyridine of compound
11 with a piperidine group resulted in an 8-fold loss in potency
against HER-2 sheddase, but was compensated for by a
substantial gain in selectivity against MMP-2 and -9, implying
that there are subtle differences in the S1′ pocket between MMP-
2/9 and the HER-2 sheddase.
In summary, the above results suggest that the S1′ pocket of
the HER-2 sheddase, like other metalloproteases, is extremely
sensitive to minor spatial modifications of the P1′ substituent.
This is manifested by the corollary that the orientation of the
aryl group in the S1′ pocket can have dramatic effects on the
binding properties, particularly with regard to the selectivity
profile.
4-Phenyl-tetrahydropyridine and 4-phenyl-piperazine groups
prefer to adopt a coplanar conformation due to the π-system of
the aromatic ring being in conjugation with the π-system of
It was hypothesized that the scaffold piperidine NsH and
the P1′ amide CdO could participate in hydrogen bonding and
thus direct the orientation of the P1′ substituent. Substitution of

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 605
Table 1. In Vitro Data for P1′ and P2′ Substituents
a BT-474 cellular proliferation assay, see reference 8. b See reference 15. c See reference 16. d See reference 17. e See reference 18. f NA ) not active.
Table 2. PK Properties of 11 and 22
the scaffold piperidine nitrogen would alleviate this interaction
mouse PK (po)a
and twist the P1′ amide CdO to avoid steric and torsional strain,
resulting in a different orientation for the P1′ substituent. In
addition, substitution of the scaffold piperidine nitrogen may
result in a change in the conformational distribution of the core
piperidine ring. This global conformational change would also
alter the orientation of the P1′ substituent and would modify
the binding properties.
proj. Cl
(L/h/kg)
PB
(% free)
AUC(0-24h)
Cmax
cmpd
(nM‚h)
(nM)
11
22
0.9
0.4
60
30
350
580
3900
7700
a Average of three animals administered at 10 mg/Kg as a suspension in
10% DMAC in 0.5% methylcellulose.
Methylation of the scaffold piperidine nitrogen resulted in a
modest loss in potency and overall selectivity in comparison to
the corresponding NsH compounds (Table 1, compounds 20
and 21). However, there was a 2- to 5-fold gain in selectivity
against MMP-9 for the piperazine 20 and tetrahydropyridine
21 NsMe analogs in comparison to their corresponding free
NsH compounds 10 and 11, respectively.
The introduction of a methyl carbamate to the spiro-piperidine
core of compound 11, which afforded compound 22, resulted
in a 4-fold decrease in HER-2 sheddase activity while maintain-
ing a similar selectivity profile. However, comparison of the
pharmacokinetic properties of compounds 11 and 22 indicated
that compound 22 had a superior murine plasma exposure with
an AUC of 3900 nM‚h and a Cmax of 7700 nM, following oral
administration (Table 2).
In an effort to assess the working hypothesis, the pharma-
codynamic properties of compound 22 were evaluated. In vivo,
22 (INCB3619)8a was shown to decrease HER-2 ECD plasma
levels and tumor size in the HER-2 overexpressing human breast
cancer mouse xenograft model BT-474-SC1 (Figure 3).8 Com-
Figure 3. Combination effect of 22 and trastuzumab in a HER-2
overexpressing human breast cancer mouse xenograft model BT-474-
SC1. Compound 22 was administered subcutaneously using ALZET
osmotic pumps at 30 mg/kg/d for 28 d, as indicated by the black arrow
below the x-axis. Tumor volumes are shown as mean values with error
bars (SEM; n ) 7).8
pound 22 was also shown to potentiate the effects of humanized
anti-HER-2 monoclonal antibody (trastuzumab) with regards to
tumor size reduction in this model. These results support the

606 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
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cell lines that overexpress HER-2 can be therapeutically
beneficial and may potentiate the anti-cancer effects of existing
chemotherapeutics in a clinical setting.
In conclusion, a novel class of conformationally restricted
azaspiro-hydroxamic acids has been developed to inhibit the
protease that is responsible for HER-2 shedding, which has been
implicated in the pathogenesis of various cancers. The potency
and selectivity profile was optimized by exploiting the subtle
differences in the S1′ pocket of the MMPs and the HER-2
sheddase by attenuating the P1′ substituent, in particular the
orientation of the aryl group. The encouraging in vitro and in
vivo data prompted comprehensive SAR, pharmacodynamic, and
pharmacokinetic studies, and the results are impending.
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