Discovery of a stable macrocyclic o-aminobenzamide Hsp90 inhibitor which significantly decreases tumor volume in a mouse xenograft model

Article abstract of DOI:10.1016/j.bmcl.2011.05.102

An extension of our previously reported series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. Addition of a second methyl group to the tether provided analogs that show increased potency in binding as well as cell-proliferation assays and, more importantly, are stable toward microsomes. We wish to disclose the discovery of a macrocycle which showed impressive biomarker activity 24-h post dosing and which demonstrated prolonged exposure in tumors. When studied in a lung cancer xenograft model, the compound demonstrated significant tumor size reduction.

Full text of DOI:10.1016/j.bmcl.2011.05.102

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4602–4607
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a stable macrocyclic o-aminobenzamide Hsp90 inhibitor
which significantly decreases tumor volume in a mouse xenograft model
a,
⇑
a
a
a
a
a
Christoph W. Zapf b , Jonathan D. Bloom , Zhong Li , Russell G. Dushin , Thomas Nittoli , Mercy Otteng ,
c
c
c
c
d
Antonia Nikitenko , Jennifer M. Golas , Hao Liu , Judy Lucas , Frank Boschelli , Erik Vogan ,
Andrea Olland , Mark Johnson , Jeremy I. Levin
d
d
a
a
Medicinal Chemistry, Pfizer, 401 N. Middletown Road, Pearl River, NY 10965, United States
Discovery Synthetic Chemistry, Pfizer, 401 N. Middletown Road, Pearl River, NY 10965, United States
Oncology Research, Pfizer, 401 N. Middletown Road, Pearl River, NY 10965, United States
b
c
d
Structural Biology & Computational Chemistry, Pfizer, 200 Cambridge Park Drive, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
An extension of our previously reported series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is
reported. Addition of a second methyl group to the tether provided analogs that show increased potency
in binding as well as cell-proliferation assays and, more importantly, are stable toward microsomes. We
wish to disclose the discovery of a macrocycle which showed impressive biomarker activity 24-h post
dosing and which demonstrated prolonged exposure in tumors. When studied in a lung cancer xenograft
model, the compound demonstrated significant tumor size reduction.
Received 18 April 2011
Revised 24 May 2011
Accepted 25 May 2011
Available online 27 June 2011
Keywords:
Hsp90 inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Chaperone inhibitors
ortho-Aminobenzamides
N-terminal ATP-binding site
Macrocycles
Buchwald–Hartwig cyclization
Modern cancer chemotherapy generally suffers from the emer-
tion studies with established chemotherapeutics have been de-
signed and were reported recently as well.
1
,2
16
gence of drug resistance. The selective silencing of a specific cell
signaling pathway by a chemotherapeutic is arguably at the origin
of this problem. By up-regulating alternative pathways, tumors are
able to adapt to the drug treatment and continue their growth. Tar-
geting chaperone proteins offers a promising solution to this tradi-
tional chemotherapy dilemma, since it allows the simultaneous
The first Hsp90 inhibitor reported in the literature was the mac-
rocyclic natural product Geldanamycin 1 (Fig. 1), which belongs to
1
7
the class of the ansamycins. Clinical studies established this com-
pound’s unacceptable toxicology profile, preventing it from further
1
8
development. Optimization studies led to the discovery of 17-
3,4
19
inhibition of multiple pathways with a single agent.
The ATP-dependent 90 kDa molecular chaperone Hsp90 has be-
AAG 2 and 17-DMAG 3 which are currently in clinical trials,
but which are characterized by poor solubility and a narrow ther-
apeutic window, respectively.
come an attractive target for cancer therapy.5
–10
It plays a critical
2
0
role in maintaining the function of a wide range of client proteins,
Vernalis recently disclosed resorcinol 4 which entered clinical
trials in 2007. Isoxazole 4 is one of many small molecules which
have been described in the literature as potential novel Hsp90
inhibitors with clinical application and is part of a recent summary
1
1
many of which are intimately involved in cancer pathology. Inhi-
bition of Hsp90 leads to the destabilization, and ultimately, degra-
dation of the clients, which results in the inhibition of cell growth
1
2
21
and apoptosis.
on this topic.
Different ATP-competitive chemotypes have evolved as potent
N-terminal Hsp90 inhibitors, several of which have transitioned
Serenex recently disclosed their own efforts to discover potent
small-molecule Hsp90 inhibitors that would be devoid of the
drawbacks associated with Geldamamycin and its analogs. Their
1
3–15
22
into clinical trials.
While many of these clinical trials focus
on Hsp90 inhibitors as single agents for cancer therapy, combina-
studies culminated in a series of 2-aminobenzamides which exhib-
ited low-nanomolar potencies in a proliferation assay. Among the
reported compounds, glycine pro-drug SNX-5422 (5) was ad-
⇑
Corresponding author. Tel.: +1 617 665 5602; fax: +1 617 665 5682.
E-mail address: christoph.zapf@pfizer.com (C.W. Zapf).
2
3
vanced to clinical trials.
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.102

C. W. Zapf et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4602–4607
4603
O
O
H N
2
O
R
O
H
N
O
N
O
N
H
NH2
O
HO
N
MeO
N
OH
O
MeO
CF3
OH
O
HN
N
OCONH2
O
1
2
3
Geldanamycin: R = OMe
17-AAG: R = NHallyl
4 NVP-AUY922
5 SNX-5422
17-DMAG: R = NHCH CH NMe
2
2
2
H N
2
O
H N
2
O
H N
2
O
H
N
H
N
H
N
A
B
N
N
N
N
N
NH2
O
O
O
O
A = NH, B = CH : 6a
7
8
2
A = CH , B = NH: 6b
2
Figure 1. Structure of Geldanamycin 1, clinically relevant ansamycins 17-AAG 2, 17-DMAG 3, Vernalis’ clinical candidate NVP-AUY922 4, and Serenex’ clinical candidate SNX-
5
422 5. Previously reported macrocycles 6–8.
A series of potent resorcinol-containing benzisoxazoles as
human microsomes. A critical feature of amide 7 was its prolonged
stability in human microsomes (t1/2 = 26 min), however, the com-
pound was only moderately potent in the enzyme assay
(IC50 = 145 nM). It was generally found to be difficult to predict hu-
man stability for these macrocycles, while enzyme activity could to
some extent be correlated to rigidity in the tether linking the tet-
Hsp90 inhibitors was recently discovered with the aid of struc-
ture-based drug design.24 Guided by X-ray analysis of a structural
analog of 5, we sought to discover potent small molecule Hsp90
inhibitors devoid of the pharmacokinetic liabilities often associ-
ated with resorcinols. Based on this crystallographic information,
2
5,26
27
we were inspired to design structurally unique macrocycles
such as amines 6), which are potent inhibitors of Hsp90 in an en-
rahydroindolone to the benzamide. Consequently the next-gen-
(
eration macrocycles was designed by incorporating a second
methyl group within the tether and installing an exocyclic substi-
tuent as shown in Figure 2. This linker architecture was therefore
designed to provide maximum rigidity for the entire molecule.
The stereochemistry for the second methyl group shown in struc-
tures 9a and 9b was derived from structural considerations based
zyme and HCT116 cell-proliferation assay while retaining excellent
solubility and microsomal stability.2 Not surprisingly, several
amine-based macrocycles were potent at the hERG ion channel.
Subsequently, we discovered macrocyclic amides (such as 7) and
lactams which are potent Hsp90 inhibitors with excellent bio-
marker activity while being devoid of hERG potency.28 When small
heterocycles were incorporated into the macrocyclic linker, Hsp90
inhibitor 8 was obtained which showed in vivo efficacy in a glioma
7
27
on X-ray information of analogs such as 6. Target structures 9a
and 9b vary by the position of the substituted nitrogen within
2
9
xenograft model.
While several of the macrocycles prepared for this program
were stable in rat microsomes, the more potent and attractive
compounds were often characterized by a very short half-life in
H N
2
O
H
N
H
N
H N
2
O
R
N
N
H
N
N
increased rigidity
leads to
enhanced potency?
R
I
II
A
B
O
6
a
+
7
N
X-ray
human
microsomal
stability
R
I
II
R
I
II
H
Me
14a 14b
15a 15b
Ac
Gly
17a 17b
18a 18b
19a 19b
O
9
9
a: A = NR, B = CH2
CH CH OH 16a 16b
COCH OH
2
2
2
b: A = CH , B = NR
2
COCH CH OMe 20a 20b
2
2
Figure 2. X-ray and stability information leads to a proposal for newly designed
analogs.
Figure 3. Newly designed analogs to distinguish architectures I and II.

4
604
C. W. Zapf et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4602–4607
OH
which was mono-Boc protected,30 providing optically pure
a-d
primary amine 11. Macrocycle 13 was obtained following a reduc-
tive amination utilizing the previously reported aldehyde 12 and
amine 11, deprotection and cyclization under standard Buch-
wald–Hartwig conditions. Nitrile 13a was either derivatized prior
to hydrolysis, yielding the desired carboxamides 15a and 16a, or
converted to carboxamide 14a which was subsequently acylated
and deprotected, if required, giving rise to analogs 17a–20a.
Target structures 9b were accessed as outlined in Scheme 2. Iso-
NHBoc
H N
2
OH
1
0
11
CN
R
N
H
N
Br
3
1
xazolidinone 23 could be prepared on a 100-g scale with satisfac-
tory optical purity and was converted to protected methyl-homo-
alanine 24 by means of hydrogenation and Boc-protection. Acid
e-g
NH
N
CHO
2
4 was converted to the primary amide and reduced yielding
amine 25. A reductive amination was carried out followed by
deprotection and cyclization under standard Buchwald–Hartwig
conditions, giving access to nitrile 13b which was prepared on a
O
1
O
2
R = CN: 13a
R = CONH : 14a
h
1
4-g scale. This compound was utilized to access analog 16b, as
2
indicated, or hydrolyzed to carboxamide 14b. Derivatization of
amine 14b involved reductive alkylation or acylation, followed
by deprotection if necessary, yielding analogs 15b and 17b–20b.
Figure 3 highlights the target structures prepared via the routes
described in Schemes 1 and 2. Within this diverse set of analogs, a
direct comparison of the two architectures 9a and 9b is possible.
The analogs incorporating the 1,2-dimethyl-1,3-propanediamine
building block (II) were shown to be more or equally potent in
i,h or
j,k,h
1
1
3a
4a
15a or 16a
17a - 20a
m
(
f or n)
Scheme 1. Reagents: (a) MsCl, Et
PhOCOOtBu, EtOH; (e) 11, NaBH(OAc)
NatOBu, toluene; (h) NaOH,
TBSOCH CHO, NaBH(AcO) , MeOH, DCE; (k) TBAF, THF; (l) Ac
protected) carboxylic acid, HATU, Et N, DCM; (n) NaOH, MeOH.
3
N, CH
, DCE; (f) TFA, CH
EtOH, DMSO; (i) MeI, Et
O; (m) (Boc- or Ac-
2
Cl
2
; (b) NaN
3
, DMF; (c) H
Cl ; (g) Pd
N, CH
2
, Pd/C, EtOH; (d)
dba , BINAP,
Cl (j)
3
2
3
2
2
2
3
the enzyme and the vSrc assays as well as the three cell-prolifer-
ation assays (HCT116, NCI1975, and U87) when compared to the
H
2
O
2
,
3
2
2
;
2
3
2
1
,2-dimethyl-1,2-ethylenediamine building block (I).
3
The unsubstituted macrocycles 14a and 14b are very potent
(Table 1), but the activity drops off to some extent with an increase
in size of the alkyl substituent (15a,b and 16a,b). The potency of
amide 7 led to the investigation of acyl substituents removing
the basic character from the tether. Amides 17a,b–20a,b appear
to be well tolerated in this series as judged by the mostly good
to excellent potencies in the cell-proliferation assays. Further
investigation into the scope of the exocyclic amides appeared to
be warranted.
the tether as both arrangements had led to analogs with promising
potency.
Synthesis of targets corresponding to 9a commenced from com-
mercially available optically pure (2R,3R)-butane-2,3-diol. Conver-
sion to the bis-mesylate, displacement with sodium azide followed
by hydrogenation provided the corresponding diaminobutane
2
8
a,b
c,d
BnN
COOH
BocHN
BocHN
NH2
O
O
2
3, 95% e.e.
24
25
NH
CN
N
R
N
H
N
Br
e - g
CHO
O
O
2
6
R = CN: 13b
h
R = CONH : 14b
2
i,h
1
3b
16b
j, k or l
then m or f)
1
4b
15b, 17b - 20b
(
Scheme 2. Reagents: (a) H
BINAP, NatOBu, toluene; (h) NaOH, H
HATU, Et N, DMF; (m) NaOH, MeOH.
2
, Pd/C, 90% dioxane; (b) Boc
2
O, Et
3
N; (c) (i) iBuOCOCl, NMM, THF; (ii) NH
4
OH; (d) LAH, Et
2
O; (e) 25, NaBH(OAc)
3 2 2 2 3
, DCE; (f) TFA, CH Cl ; (g) Pd dba ,
2
O
2
, EtOH, DMSO; (i) BrCH
2
CH OAc, K CO , DMF; (j) CH O, NaBH
2
2
3
2
3
CN, THF; (k) AcCl, Et
3
N, DMF; (l) (Boc- or Ac-protected) carboxylic acid,
3

C. W. Zapf et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4602–4607
4605
Table 1
in stability. The 2-methylalanine analog 24 is even more potent
than 21, but shows the same stability profile as 22. Cyclopropyl
analog 25 and cyclopentyl analog 26 show an unacceptable drop
in human microsomal stability while mostly maintaining potency.
A comparison of (S)-proline amide 27 and its diastereomer 28
demonstrates the correlation between the chirality of the amino
acid side-chain and the compound’s potency. Proline amide 27
was found to be identical to alanine amide 21 with respect to its
excellent potency, Cyp and stability profile, while simultaneously
showing an improvement in aqueous solubility. Incorporation of
fluoroproline thus providing amide 29 did not maintain the desired
stability characteristics. Lactic acid analog 30 and its corresponding
iso-propyl derivative 31, while very potent, did not quite achieve
the impressive stability that was demonstrated for alanine amide
Potencies to distinguish linker architectures I and II
#
IC50 [lM]
Binding
vSrc
HCT116
NCI1975
U87MG
1
1
1
1
1
1
1
1
1
1
1
1
2
2
4a
0.082
0.064
0.116
0.088
0.142
0.084
0.085
0.058
0.081
0.074
0.077
0.066
0.131
0.083
0.039
0.037
0.070
0.034
0.060
0.030
0.035
0.026
0.810
0.417
0.061
0.043
0.042
0.040
0.029
0.024
0.051
0.027
0.072
0.031
0.027
0.024
0.147
0.216
0.051
0.039
0.055
0.038
0.020
0.027
0.069
0.027
0.056
0.034
0.020
0.015
0.380
0.190
0.054
0.029
0.037
0.031
0.061
0.040
0.133
0.049
0.187
0.055
0.029
0.024
0.481
0.378
0.111
0.047
0.080
0.054
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
0a
0b
2
1 and proline amide 27. When compared to the previously re-
ported analogs 6a, 6b, 7 and 8, macrocycles 21 and 27 represent
an impressive improvement regarding the combination of enzyme
and cell-based potency, with stability, solubility and Cyp inhibition
properties.
H
2
N
O
X-ray crystal structures of macrocycles 21 and 27 were ob-
3
3
H
N
tained. Figure 5 highlights alanine amide 21 bound to the N-ter-
minal ATP-binding site of Hsp90. As seen with analogs previously,
the carbonyl of the tetrahydroindolone interacts through a hydro-
gen bond with Tyr139 whereas the benzamide engages Asp93 in a
direct and a water-mediated hydrogen bond. While no direct H-
bond from the macrocycle side-chain to Asp54 appears to form
due to the distance, a water-mediated interaction of the alanine-
N
N
R
2
NH with the carboxylate of Asp54 seems plausible. The same
O
9
b
water molecule also engages in an interaction with Asn51. Another
water-mediated interaction of the ligand with the protein can be
noticed between the alanine carbonyl attached to the macrocycle
and Gly35. These water-mediated interactions and the usual net-
work of water molecules around Asp93 represents the dense net-
work of conserved water molecules characteristic for the N-
terminal ATP-binding site of Hsp90.
R
#
R
#
R
#
Ac
17b
NH
2
24
28
N
H
COCH NH
18b
2
2
O
O
O
F
Previously we reported on analogs with basic amines within the
heterocyclic tether which were found to be inactive. It was ratio-
2
2
2
1
2
3
25
29
30
31
29
NH2
NH2
N
H
O
nalized that the nitrogen atom of these analogs is directed toward
Lys58 (highlighted in Fig. 5), leading to an unproductive electro-
static interaction and therefore a significant loss in potency.
A biomarker titration experiment using NCI-H1975 cells was
carried out at two time points (5 and 24 h) using macrocycle 21
to determine an effective inhibitor concentration for a subsequent
in vivo study (Fig. 6). When compared to the control experiment in
lane 1, treatment of the cells with the inhibitor at 10 nM concen-
tration (lane 2) leads to a heat-shock response as indicated by
the upregulation of Hsp72 at 5 or 24 h. Increasing the inhibitor
concentration to 30 nM (lane 3) leads to a noticeable increase in
Hsp72 response at both time points. The response remains unaf-
fected when the inhibitor concentration is further elevated to
O
O
26
27
OH
OH
NH2
NH2
O
O
O
O
N
H
NH
2
O
Figure 4. A focused set of exocyclic amides.
Figure 4 highlights a focused set of the amides that were pre-
pared following the synthetic route depicted in Scheme 2. Acetyl
derivative 17b is very potent in the enzyme and cell-based assays.
This macrocycle was more potent than the parent 14b and the al-
kyl substituted analogs (i.e., 15b, Table 1), but it does not show the
desired stability in the microsome assays (Table 2). However, gly-
cine analog 18b is stable in these assays but does not display the
required potency and Cyp profile. A substantial improvement
was achieved with alanine amide 21. This macrocycle was the first
analog prepared in our Hsp90 program that was sufficiently potent
in all assays, showed a promising Cyp profile and demonstrated the
desired half-life in the three microsome assays. The ethyl analog 22
is slightly more potent than 21 but significantly worse with respect
to its Cyp characteristics and also to some extent less stable. Fur-
ther increasing steric bulk (23) has essentially no effect on potency,
and the increase in c log P could possibly contribute to a reduction
1
00, 300 or 1000 nM (lanes 4–6).
However, suppression of Akt activity, a critical protein in cellu-
lar signaling pathways and well-studied biomarker for Hsp90 inhi-
bition, is not evident after 5 h but requires prolonged exposure and
at least 30 nM of the Hsp90 inhibitor 21 (lane 3). Increasing the
inhibitor concentrations to 100 nM (lane 4) leads to a more pro-
nounced effect for the Akt inhibition which remains unchanged
upon further increase of the inhibitor to 300 or 1000 nM (lane 5
and 6, respectively). The inhibition profile of pS473 Akt mirrors
that of Akt. Induction of caspase-mediated cleavage of Parp—a sig-
nal for the induction of apoptosis—is undetectable 5 h post treat-
ment. The effect becomes significant at the 24 h time point when
the cells are exposed to the inhibitor at a concentration of at least
3
0 nM.
With this information available, compound 21 was evaluated in
a biomarker study in a xenograft model using H1975 tumor bear-

4
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C. W. Zapf et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4602–4607
Table 2
Biological activities, profiling data, and selected calculated properties for Geldanamycin and macrocycles
For experimental details about assay conditions see Ref. 24.
Control
6 h
24 h
HSP70
pS6K
p473
pS6
Actin
Figure 7. Biomarker evaluation of 21 in H1975 tumor bearing mice when dosed at
5 mg/kg iv. Each vertical lane with its five markers represents data from an
2
individual animal.
ing mice (Fig. 7). When administered at 25 mg/kg iv, the compound
clearly elicits a heat-shock response characterized by an upregula-
tion of Hsp70, which is pronounced even 24 h post administration.
Treatment with compound 21 also caused prolonged diminution of
phosphorylation of two downstream markers of Hsp90 inhibitory
activity, Akt S473 and ribosomal protein S6 S235/236 (Fig. 7).
Tissue and blood levels of 21 were also measured in two differ-
ent xenograft models. As can be seen in Table 3, rapid clearance of
the compound from the blood was observed in all models within
Figure 5. Macrocycle 21 bound to the N-terminal ATP-binding site of Hsp90.
Hydrogen bonds (black dotted lines) are labeled with distances in Å. A portion of the
protein has been omitted for clarity.
6
h. Compound levels in the tumor remained high in a subcutane-
ous lung cancer tumor model (NCI-H1975) throughout the 24 h
experiment. After iv dosing, the tumor levels were also high in
the subcutaneous glioma tumor model (U87) after 6 h but slightly
lower after 24 h. The compound levels were even lower after oral
dosing at both time points in spite of the increase in dose. This dif-
5
h
24 h
1
2 3 4 5 6 1 2 3 4 5 6
Hsp90
Hsp72
Akt
Table 3
pS473 Akt
Exposure levels of 21 after dosing
6
h
24 h
Actin
Parp
a
2
5 mg/kg iv
NCI1975 tumor
0 mg/kg iv
U87 tumor
0 mg/kg po
U87 tumor
Plasma
Tumorb
Plasma
36
9
910
45
772
6
693
102
967
53
a
5
Cleaved Parp
Tumorb
a
5
Plasma
Tumorb
Figure 6. Biomarker titration in vitro using NCI-H1975 cells. Lane 1: 0; lane 2:
309
242
1
2
0 nM; lane 3: 30 nM; lane 4: 100 nM; lane 5: 300 nM; lane 6: 1000 nM compound
1. Each vertical lane with its seven markers represents data from an individual
a
[
[
ng/mL].
ng/g].
b
animal.

C. W. Zapf et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4602–4607
4607
Acknowledgments
We thank Professor Mukund Sibi, Department of Chemistry,
North Dakota State University and Professor David Macmillan,
Princeton University for stimulating discussions.
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0. Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.; Cansfield, J. E.;
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Finch, H.; Fink, A.; Hayes, A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.;
Lockie, A.; Martins, V.; Massey, A.; Matthews, T. P.; McDonald, E.; Northfield, C.
J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S. Y.;
Surgenor, A.; Walmsley, D. L.; Webb, P.; Wood, M.; Workman, P.; Wright, L. J.
Med. Chem. 2008, 51, 196.
Table 4
PK parameters for 21 in CD-1 mice following a single 2 mg/kg iv or 20 mg/kg oral dose
t
[
1/2
AUC0–inf
[hÁng/mL]
Clp [mL/
min/kg]
V
kg]
ss [l/
C
mL]
max [ng/
t
[h]
max
%F
h]
21. Biamonte, M. A.; Van de Water, R.; Arndt, J. W.; Scannevin, R. H.; Perret, D.; Lee,
W. C. J. Med. Chem. 2010, 53, 3.
Iv
Po
10.7
6.8
542
234
62
30
4
2
2. Barta, T. E.; Veal, J. M.; Rice, J. W.; Partridge, J. M.; Fadden, R. P.; Ma, W.; Jenks,
M.; Geng, L.; Hanson, G. J.; Huang, K. H.; Barabasz, A. F.; Foley, B. E.; Otto, J.;
Hall, S. E. Bioorg. Med. Chem. Lett. 2008, 18, 3517.
22
1
2
3. Huang, K. H.; Veal, J. M.; Fadden, R. P.; Rice, J. W.; Eaves, J.; Strachan, J. P.;
Barabasz, A. F.; Foley, B. E.; Barta, T. E.; Ma, W.; Silinski, M. A.; Hu, M.; Partridge,
J. M.; Scott, A.; DuBois, L. G.; Freed, T.; Steed, P. M.; Ommen, A. J.; Smith, E. D.;
Hughes, P. F.; Woodward, A. R.; Hanson, G. J.; McCall, W. S.; Markworth, C. J.;
Hinkley, L.; Jenks, M.; Geng, L.; Lewis, M.; Otto, J.; Pronk, B.; Verleysen, K.; Hall,
S. E. J. Med. Chem. 2009, 52, 4288.
ference in the two tumor models appears to be consistent with the
cell-proliferation data.
Compound 21 was also characterized in a xenograft model
using NCI-H1975 bearing nude mice (Fig. 8). In this study, the mac-
rocycle significantly suppressed tumor growth when dosed iv once
weekly at 12.5 mg/kg compared to the control group, and even lead
to reduction in tumor size at the 25 mg/kg dose.
2
4. Gopalsamy, A.; Shi, M.; Golas, J.; Vogan, E.; Jacob, J.; Johnson, M.; Lee, F.;
Nilakantan, R.; Petersen, R.; Svenson, K.; Chopra, R.; Tam, M. S.; Wen, Y.;
Ellingboe, J.; Arndt, K.; Boschelli, F. J. Med. Chem. 2008, 51, 373.
2
5. Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. Nat. Rev. Drug Discovery 2008, 7,
608.
The pharmacokinetic parameters for macrocycle 21 were deter-
mined (Table 4). The compound shows a long half-life which corre-
lates to the mouse microsomal stability. It was found to have high
clearance and volume of distribution. The molecule has low bio-
availability and shows low potency at the hERG ion channel
26. Marsault, E.; Peterson, M. L. J. Med. Chem. 2011, 54, 1961.
2
7. Zapf, C. W.; Bloom, J. D.; McBean, J. L.; Dushin, R. G.; Nittoli, T.; Ingalls, C.;
Southerland, A.; Sonye, J. P.; Eid, C. N.; Golas, J. M.; Liu, H.; Boschelli, F.; Vogan,
E. M.; Hu, Y.; Levin, J. I. Bioorg. Med. Chem. Lett. 2011, 21, 2278.
28. Zapf, C. W.; Bloom, J. D.; McBean, J. L.; Dushin, R. G.; Nittoli, T.; Otteng, M.;
Ingalls, C.; Golas, J. M.; Liu, H.; Lucas, J.; Boschelli, F.; Vogan, E. M.; Hu, Y.; Levin,
J. I. Bioorg. Med. Chem. Lett. 2011, 21, 3411.
(
IC50 > 33
lM).
2
9. Zapf, C. W.; Bloom, J. D.; McBean, J. L.; Dushin, R. G.; Golas, J. M.; Liu, H.; Lucas,
J.; Boschelli, F.; Vogan, E.; Levin, J. I. Bioorg. Med. Chem. Lett. 2011, 21, 3627.
0. Pittelkow, M.; Lewinsky, R.; Christensen, J. B. Org. Synth. 2007, 84, 209.
1. Sibi, M. P.; Prabagaran, N.; Ghorpade, S. G.; Jasperse, C. P. J. Am. Chem. Soc. 2003,
In conclusion, compound 21 is a metabolically stable Hsp90
3
3
inhibitor, potent in enzyme and cell-based assays. It demonstrated
excellent biomarker activity and has shown a significant effect on
tumor growth suppression. Furthermore, it demonstrated good tu-
mor exposure 24 h after iv dosing in two different xenograft
models.
1
25, 11796.
32. Boschelli, F.; Golas, J. M.; Petersen, R.; Lau, V.; Chen, L.; Tkach, D.; Zhao, Q.;
Fruhling, D. S.; Liu, H.; Nam, C.; Arndt, K. T. Cell Stress Chaperones 2010, 15, 913.
33. The structure has been assigned PDB ID code 3RKZ.