Lipophilic isosteres of a π-π Stacking interaction: New inhibitors of the Bcl-2-Bak protein-protein interaction

Article abstract of DOI:10.1021/ml300095a

The discovery of new Bcl-2 protein-protein interaction antagonists is described. We replaced the northern fragment of ABT737 (π-π stacking interactions) with structurally simplified hydrophobic cage structures with much reduced conformational flexibility and rotational freedom. The binding mode of the compounds was elucidated by X-ray crystallography, and the compounds showed excellent oral bioavailability and clearance in rat PK studies.

Full text of DOI:10.1021/ml300095a

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Letter
Lipophilic Isosteres of a pi-pi Stacking Interaction: Design of
New Inhibitors of the Bcl-2-Bak Protein-Protein Interaction.
Naeem Yusuff, Michaël Doré, Carol Joud, Michael Visser, Clayton
Springer, Xiaoling Xie, Kara Herlihy, Dale Porter, and B. Barry Touré
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml300095a • Publication Date (Web): 27 May 2012
Downloaded from http://pubs.acs.org on May 29, 2012
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Lipophilic Isosteres of a π-π Stacking Interaction: New Inhibi-
tors of the Bcl-2-Bak Protein-Protein Interaction.
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Naeem Yusuff*, Michaël Doré, Carol Joud, Michael Visser, Clayton Springer, Xiaoling Xie, Kara
Herlihy, Dale Porter, B. Barry Touré*
Novartis Institutes for BioMedical Research Inc., Global Discovery Chemistry, 250 Massachusetts Avenue, Cambridge, MA
02139.
KEYWORDS Bclꢀ2 inhibitors, proteinꢀprotein interaction, πꢀ π stacking interactions, πꢀ π stacking isosteres, bioisoteres,
cancer therapeutics.
Supporting Information Placeholder
ABSTRACT: The discovery of new Bclꢀ2 proteinꢀprotein interaction antagonists is described. We
replaced the northern fragment of ABT737 (πꢀ π stacking interactions) with structurally simplified
hydrophobic cage structures with much reduced conformational flexibility and rotational freedom.
The binding mode of the compounds was elucidated by xꢀray crystallography, and the compounds
showed excellent oral bioavailability and clearance in rat PK studies.
Although the principles and energetic components of bioꢀ
molecular binding interactions have been well studied, the design
and implementation of these principles remain a challenge. Noꢀ
where is this challenge more pronounced than in the search for
small molecule inhibitors of proteinꢀprotein interactions (PPI’s).1
A notable recent success was achieved with the discovery of
ABT737,2 a dual Bclꢀ2, BclꢀxL inhibitor with potent in vivo acꢀ
tivity against a number of tumors.3 Bclꢀ2 and BclꢀxL are repreꢀ
sentative members of the Bꢀcell lymphomaꢀ2 (Bclꢀ2) family of
antiapoptotic proteins, which also include Bclꢀw, A1, and Mclꢀ1.4
The overexpression of these proteins in cancer cells is a defining
event in chemoꢀ and radiotherapy resistance, and also marks the
progression of the disease. Mechanistically, the antiapoptotic Bclꢀ
2 family members prevent the programmed cell death by bindꢀ
ing/sequestering the proꢀapoptotic Bclꢀ2 family members (e.g.
bid, bim, noxa). This sequestration prevents the oligomerization
of Bax/Bak, which is essential for the permeation of the mitoꢀ
chondrial membrane, subsequent release of cytochrome C, and
initiation of downstream apoptosis events.5
difficult to accurately quantify the energetics of these conformaꢀ
tional changes, but it is generally accepted that they will largely
negatively impact the binding potency.9 Thus, reducing the conꢀ
formational flexibility of ABT737 represents a viable approach to
improving not only its drugꢀlike properties, but perhaps also its
binding potency.10 Efforts along this line have been reported in
the literature.11 Herein, using ABT737 as a starting point, we
describe an orthogonal design principle for developing new Bclꢀ2
inhibitors with significantly reduced conformational flexibility.
Efforts to improve the solubility of this novel series are also disꢀ
cussed.
At the outset, we elected to maintain the chlorophenyl and linkꢀ
er region (piperazine to sulfone) constant as they make several
important interactions with the protein.12 Thus, our efforts started
with compound 3a, wherein the entire northern ABT737 fragment
is replaced with a simple methyl group. Remarkably, measurable
binding interactions remained with an EC50 of 8.62 ꢁM (Table 1,
entry 1).13 Sequential homologation of this methyl group quickly
exposed the promises and challenges of our approach. For inꢀ
stance, while 3b was more potent (5.31 ꢁM, Table 1), the potency
was reduced when the methyl group was replaced with tꢀBu (3c,
Table 1, entry 3), indicative of steric clashes with the protein. In
agreement, 3d was also inactive. Despite these setbacks, we conꢀ
tinued to prepare bulkier analogues such as 3e and 3f; this time
we also inserted a methylene linker between the sulfonamide
functionality and the p4 probe. We were pleased to observe that
3e showed a near 8ꢀfold improvement in potency compared to 3a.
Moreover, enantiomeric cisꢀmyrtanol derived analogues 3g and
3h, lacking any polar atoms, displayed significantly improved
inhibition of the target (0.56 and 0.42 uM respectively). Again,
similar to the camphor derivatives (vide supra) no stereodiscrimiꢀ
nation was observed in the binding.
Inspection of the binding interactions between ABT737 and
Bclꢀ2 reveals a highly optimized ligand (with respect to the proꢀ
teinꢀligand interactions) spanning the p2 to p4 binding pockets.6
Interestingly, these two pockets were independently identified by
alanineꢀscanning mutagenesis as being critical “hot spots” in the
interaction of Bclꢀ2 with Bak.7 Notable among the interactions is
a unique turn in the northern fragment of ABT737 where the thiꢀ
ophenyl is folded underneath the nitroaromatic ring (intramolecuꢀ
lar πꢀ π stacking interaction) with the latter ring forming an addiꢀ
tional intermolecular πꢀπ stacking with Tyr 161 of Bclꢀ2. This
folding of the ligand in the p4 pocket has three negative conseꢀ
quences: First, the electronic demand of the πꢀπ interactions reꢀ
stricts the medicinal chemistry approaches to improving the drugꢀ
like properties of ABT737.8 Secondly, the highly engineered naꢀ
ture of the p4 ligand portion of ABT737 introduces a large
amount of rotational freedom and lipophilicity to the molecule.
High levels of rotatable bonds and lipophilicity has a general
negative impact on solubility and permeability. Lastly, the foldꢀ
ing itself would entail significant conformational changes. It is
With a good understanding of the binding requirements in the
p4 pocket, we revisited the adamantane analogue 3d. Its appeal
stems from its high degree of symmetry and relative synthetic
tractability. The synthesis of 3i, featuring two carbon linker, is
outlined in Scheme 1.14,15 First, the commercially available primaꢀ
ry alcohol 5 was treated with triphenylphosphine and iodine to
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afford iodide 6, which was immediately displaced with potassium
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3
4
5
6
7
8
thioacetate to yield intermediate 7 in quantitative yield. The oxiꢀ
dation of this newly formed thioacetate 7 with sulfuryl chloride
and subsequent treatment with ammonium hydroxide allowed
rapid access to 8 in 71% yield. Further oxidation of 8 into sulfonꢀ
amide 9 using potassium permanganate paved the way for the
final coupling step as shown in Table 1 to afford 3i. 3j was also
prepared in an analogous fashion. Gratifyingly, both compounds
were active against the target, with 3j being a subꢀuM inhibitor of
Bclꢀ2 (Table 1, entry 9). These results further highlight the necesꢀ
sity for a linker with one carbon being the optimal length. The
nature of this spacer was also critical. For instance, the conversion
of the carbon linker into amine (sulfonyl urea, synthesis described
in Scheme 2) negatively impacted the potency (3k, Table 1).
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Having optimized the linker, we now turned our attention to the
p4 binding region of the molecule. Again, sharp SAR trends were
manifest. The contraction of the adamantane ring by one carbon
resulted in 4ꢀfold loss in potency (3l vs 3j, Table 1). Leaving this
region of the molecule intact, we inserted an additional carbon
into the distal position in the adamantane framework (3m, Table
1). Remarkably, this expansion of the ring by a single carbon
atom resulted in a net 6 fold gain in potency. These results
demonstrate how potency may be modulated in the series. We
also tested key compounds against BclꢀxL, a closely related Bclꢀ2
family member. In all cases (compounds 3h, 3j and 3 m, Table 1)
the compounds were marginally less active with 3j providing the
largest window of selectivity (4.3 fold).
At this juncture, we sought to solve the crystal structure of
some of our potent Bclꢀ2 binders to validate our design hypotheꢀ
sis and inform the SAR optimization strategy. This campaign led
to the xꢀray complex of 3j and Bclꢀ2 (Figure 1a). It shows that the
adamantane is occupying the hydrophobic p4 pocket per the iniꢀ
tial design assumptions. The adamantane ring fits within the bindꢀ
ing envelope defined by the northern region of ABT737, although
a close inspection shows movement of Tyr 161, a key residue for
the binding of ABT737 (not shown). This movement creates a
deeper hydrophobic pocket to accommodate the binding of the
ligand. Other notable differences between the two crystal strucꢀ
tures are highlighted in Figure 1b. The acylsulfonamide moiety
(3l) is rotated away from the protein. This repositioning of the
sulfonamide was also observed in the xꢀray coꢀcomplex of 3a, a
less sterically demanding analogue, and Bclꢀ2. From this we infer
that the position of the sulfonamide found in the ABT737 strucꢀ
ture is being repositioned by the πꢀπ stacking motif that forces the
sulfonamide from its lowest energy positioning. Contrast this with
the high symmetric adamantyl group which imposes much less
bias on the sulfonamide position. More importantly, this rotation
results in the loss of a hydrogen bonding interaction between the
sulfone oxygen (3j) and the backbone NH of Gly104 in Bclꢀ2
(this interaction is seen in the xꢀray coꢀcomplex of ABT737 and
Bclꢀ2). The above results clearly validated our design premise,
and demonstrated for the first time that the northern region of
ABT737 can be replaced with simple rigid structural motifs with
much reduced rotational freedom.
aAverage of at least two measurements, the compounds are inꢀ
b
active against Mclꢀ1 (IC50 > 50 µM). Stereochemistry of the
c
starting camphor sulfonamides. Enantiomeric (1S, 2S, 5S) and
(1R, 2R, 5R)ꢀmyrtanol were used respectively to prepare these
analogues. dThis is the bottom limit of the assay.
Guided by this coꢀcrystal structure, we next attempted to access
the region occupied by the dimethyl amine moiety in the crystal
structure of ABT737/Bclꢀ2 complex as well as the sub pocket
utilized by the nitro group (ABT737). An additional goal of these
efforts was to decrease the clogP of our compounds, in order to
improve the solubility of our analogues. The synthesis of these
analogues is detailed in Scheme 3. It started with 3ꢀbromoꢀ
adamantaneꢀ1ꢀcarboxylic acid 12. First, reduction of the carboxꢀ
ylic acid with BH3.THF afforded alcohol 13 in excellent yield
(87%). The latter compound was converted into 14 via 4 straightꢀ
forward and high yielding synthetic operations as described above
in Scheme 1. This versatile intermediate lent itself to many interꢀ
esting chemistries. For instance, the angular Br can be displaced
Table 1. Redesigning the northern fragment of ABT737.
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with aqueous phosphate under microwave conditions to afford
tolerated
at
this
position.
1
2
3
4
5
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8
alcohol 15 in 71 % yield. Further, 16 was obtained in good yield
when the latter compound was reacted with DAST in DCM at
room temperature. Additionally, treatment of 14 with alcohols,
exemplified by 2ꢀmorpholinoethanol, under forcing conditions
afforded alkoxyadamantane analogues in moderate yield. Finally,
bromide 14 can be carried through the Ritter reaction with a
variety of nitrile coupling partners to afford the corresponding
Nꢀadamantylamides in good yield (18, Scheme 3). These inꢀ
termediates, as well other commercially available 3ꢀ
functionalized adamantane methyl sulfonamides were then
successfully coupled to the bottom ABT737 carboxylic acid
(20) to afford the desired final targets 19a-d and 19f-i, (Table
2). The 3ꢀposition of the adamantane could also be probed via
a late stage diversification strategy. For instance, the fluoride
in 19c was replaced with a methyl group by simple treatment
with trimethyl aluminum (19e, Scheme 3).
Scheme 3. Derivatization of adamantane core
d
O O
S
O O
S
H2N
H2N
OH
F
15 (71%)
16 (78%)
c
O O
b
a
HO
S
HO
H2N
Br
Br
e
Br
O
12
14
13 (87%)
9
f
10
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12
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O O
O O
S
S
H2N
H2N
NH
R
O
17 (43%)
18
O
N
R = Me, i-Pr, cyclopropyl, t-Bu
O
13-18
g
or other 3-functionalized
admantane-1-methyl
19
Figure 1. Crystal structures.
sulfonamides
p-ClPh
N
N
HO2C
20
19e
h
19c
a. BH3.THF, THF, 0 oC, then reflux, 4h; b. See Scheme 1; c. Aq. NaH2PO4,
microwave, 180 oC, 10 min; d. DAST, DCM, 24 h; e. 2-morpholinoethanol,
microwave, 200 oC, 90 min; f. RCN, ZnCl2 (1M, in Et2O), DCE, 90 oC, 12 h;
g. 20, EDCI, DMF/DCE; h. Me3Al, DCM, rt, 30 min;
Table 2. Modulation of the drugꢀlike properties and potency in
the adamantane series.
1a. Xꢀray complex of 3j and Bclꢀ2 at 2.0 Å resolutions (left). 1b. Overlay of xꢀray
crystal structure of ABTꢀ737 and compound 3j (right)
.
Scheme 2. Synthesis of the sulfonyl urea
O
O
O
S
a, b
S
Cl
N
10
C O
H2N
N
H
O
11 (87%)
a. t-BuOH, DCM, rt,10 min, then adamantylamine, 0 oC to rt, 45 min;
b. HCl, dioxane.
The potency of these second generation analogues was measꢀ
ured in the SPR assay, and the results are summarized in Table 2.
Interestingly, hydroxylation of the adamantane at the 3ꢀposition
was not only tolerated but also afforded a more potent compound
(19a, Table 2). Furthermore, sterically larger polar substituents
such as the side chain ethyl morpholinyl (19b, Table 2) were also
aAverage of at least two measurements (duplicate EC50 values
were generated in each assay). bNumber of rotatable bonds.
However 19a and 19b were equipotent hinting that the latter
compound binds orienting the 3ꢀ position away from the pocket
(solvent exposed). In agreement with this data, hydrophobic subꢀ
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stituents at this position were not tolerated. For instance a simple
Author Information
1
2
3
4
5
6
7
8
methylation (19e) resulted in a two fold loss in potency. Furtherꢀ
more, the introduction of a phenyl ring (19f) was even less favorꢀ
able. In contrast, halogens (fluoro (19c), bromo (19d)) were tolerꢀ
ated. Finally, while smaller hydrophobic amides substituents were
tolerated, and even improved the potency of our series in some
instances (19gꢀi), bigger hydrophobic amides were less desirable
(19i). Overall, we concluded that this position may be ideally
used for the introduction of solubilizing groups, and this goal can
be combined with minor gains in potency as shown for the hyꢀ
droxyl and smaller amide analogues.
Correponding Author: Tel: 1ꢀ617ꢀ871ꢀ4197. Email: barꢀ
ry.toure@novartis.com or naeem.yusuff@yahoo.com
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The design concept of introducing a simple rigid
structure to replace the northern portion of ABT737 was inꢀ
tended to improve the overall pharmaceutical properties of our
Bclꢀ2 inhibitors by reducing MW, clogP and rotatable bonds
(the clogP and number of rotatable bonds values are shown in
Table 2 for our compounds16). The pharmacokinetic properꢀ
ties obtained for compounds 3j and 19a after a single solution
dose in SpragueꢀDawley rat is summarized in Table 3. The
systemic clearance values were low relative to the hepatic
blood flow (6 and 16 mL/min.Kg respectively). The volume of
distributions were moderate (3j, 0.7 L/Kg and 19a, 1.2 L/Kg)
with the drug being primarily confined to the plasma. Both
compounds showed excellent oral bioavailability (66, and
100%17) indicative of improved permeability and solubility
relative to ABT737. Overall the pharmacokinetic properties of
both compounds were favorable.
3
Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong, R.
C.; Augeri, S. W.; Belli, B. A.; Bruncko, M.; Deckwerth, T. L.;
Dinges, J.; Hajduk, P. J.; Joseph, M. K.; Kitada, S.; Korsmeyer, S.
J.; Kunzer, A. R.; Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D.
J.; Ng, S.; Nimmer, P. M.; O’Connor, J. M.; Oleksijew, A.;
Petros, A. M.; Reed, J. C.; Shen, W.; Tahir, S. K.; Thompson, C.
B.; Tomaselli, K. J.; Wang, B.; Wendt, M. D.; Zhang, H.; Fesik,
S. W.; Rosenberg, S. H. An inhibitor of Bclꢀ2 family proteins
induces regression of solid tumours. Nature, 2005, 435, 677ꢀ681.
(b) Tse, C.; Shoemaker, A. R.; Adickes, J.; Anderson, M. G.;
Chen, J.; Jin, S.; Johnson, E. F.; Marsh, K. C.; Mitten, M. J.;
Nimmer, P.; Roberts, L.; Tahir, S. K.; Xiao, Y.; Yang, X.; Zhang,
H.; Fesik, S.; Rosenberg, S. H.; Elmore, S. W. ABTꢀ263: A Poꢀ
tent and Orally Bioavailable Bclꢀ2 Family Inhibitor Cancer Res.
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Table 3. In vivo Pharmacokinetics in SpraguesDawley rata
Parameters
Cl (mL/min.kg)
Vdss (L/kg)
t1/2 (hours)
%F
3j
19a
16
6
0.7
1.3
66
9.5
1.2
1.3
>100b
6.4
clogP
4 Cory, S., Adams, J. M. The Bcl2 family: regulators of the celꢀ
aSpragueꢀDawley rat was dosed 1mg/Kg IV and 10 mg/Kg p.o. The compound was
administered as a solution in 10% NMP, 30% PEG300, 60 (20%) vitamin ETPGS for
3j, and 10% NMP, 25% PEG300, 5% cremophor EF,WFI for 19a. bSee related comꢀ
lular lifeꢀorꢀdeath switch. Nat. Rev. Cancer 2002, 2, 647–656.
5
(a) Hanahan, D.; Weinberg, R. A. The Hallmarks of Cancer.
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overexpression in cancers see: (c) Berghella, A. M., Pellegrini, P.,
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the Treatment of Cancer: New Strategies of Biotherapy which
should not be Underestimated. Cancer Biother. Radiopharm.
1998, 13, 225–236. For correlation between Bclꢀ2 and resistance
to treatment with radiation and chemotherapy see: (d) Reed, J. C.
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ments in ref. 17
.
In summary, we have demonstrated that simple and rigid cageꢀ
structures can replace the highly engineered northern fragment
in ABT737, albeit with a minimal loss in potency. Herein, the
binding is primarily driven by hydrophobic interactions, but
we have identified areas of the molecules that can be modified
to improve the solubility of the series. More importantly, a
favorable PK profile is obtained in the process as compared to
ABT737. Combining good potency and oral exposure has
been challenging in targeting Bclꢀ2, and the results reported
herein are very promising in this regard. Further efforts to
improve the potency of our compounds while preserving the
favorable PK profile are in progress and will be reported in
due course.
6 Lee, E. F.; Czabotar, P. E.; Smith, B. J.; Deshayes, K.; Zobel,
K.; Colman, P. M.; Fairlie, W. D. Crystal structure of ABTꢀ737
complexed with BclꢀxL: implications for selectivity of antagonists
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lan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.;
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Bak peptide complex: recognition between regulators of apoptoꢀ
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Supporting Information Available: Synthetic procedures and
characterization data (1H NMR, HRMS and HPLC analysis) for
compounds 3aꢀ3m, 19aꢀe, g-j, biochemical assay conditions. This
material is available free of charge via the internet at
http://pubs.acs.org.
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Lipinsky, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility
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See supporting information for a description of this assay
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er, J. H.; Smith, W.W.; Burger, M. T. Design of Macrocyclic
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15 Thea, S.; Cevasco, G. A facile synthesis of sulfinyl chlorides
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In comparaison, ABT737 has a clogP of 10.0 and 17 rotataꢀ
Park, C.ꢀM.; Oie, T.; Petros, A.M.; Zhang, H.; Nimmer, P.
ble bonds.
M.; Henry, R. F.; Elmore, S. W. Design, Synthesis, and Computaꢀ
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17 The oral bioavailability values were respectively 97, 108, and
150% in individual rats. Saturation of clearance at higher po relaꢀ
tive to IV dose may explain the >100% F in third rat.
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clogP
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rotatable bond
PK
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NO2
S
NH
OH
Our work
Flat structure to 3D
N
ABT737
Intra- and Intermolecular
π-π interactions
Hydrophobic interactions
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Lay Summary
The discovery of new Bclꢀ2 proteinꢀprotein interaction antagonists is described. We replaced the northern fragment of ABT737 (πꢀ
π stacking interactions) with structurally simplified hydrophobic cage structures with much reduced conformational flexibility and
rotational freedom. The binding mode of the compounds was elucidated by xꢀray crystallography, and the compounds showed exꢀ
cellent oral bioavailability and clearance in rat PK studies.
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