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Discovery of a Potent Non-peptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-linked Inhibitor of Apoptosis Protein (XIAP) Emiliano Tamanini, Ildiko Maria Buck, Gianni Chessari, Elisabetta Chiarparin, James E. H. Day, Martyn Frederickson, Charlotte M. Griffiths-Jones, Keisha Hearn, Tom D. Heightman, Aman Iqbal, Christopher Norbert Johnson, Edward J. Lewis, Vanessa Martins, Torren Peakman, Michael Reader, Sharna J. Rich, George A. Ward, Pamela A. Williams, and Nicola E. Wilsher J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Discovery of a Potent Non-peptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-linked Inhibitor of Apoptosis Protein (XIAP)

Emiliano Tamanini*, Ildiko M. Buck, Gianni Chessari, Elisabetta Chiarparin, James E. H. Day, Martyn Frederickson, Charlotte M. Griffiths-Jones, Keisha Hearn, Tom D. Heightman, Aman Iqbal, Christopher N. Johnson, Edward J. Lewis, Vanessa Martins, Torren Peakman, Michael Reader,. Sharna J. Rich, George A. Ward, Pamela A. Williams, Nicola E. Wilsher.

Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.

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ABSTRACT: XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in ATIAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.


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1.

INTRODUCTION

Apoptosis, a closely regulated programmed cell death mechanism, is an essential process to maintain tissue homeostasis. Resistance to apoptotic stimuli is one of the hallmarks of cancer1, and can be achieved by overexpression of anti-apoptotic proteins. Inhibitor of apoptosis proteins (IAP), such as cellular IAP1 and 2 (cIAP) and X-linked IAP (XIAP) play a critical role in the anti-apoptotic and pro-survival signaling pathways; XIAP directly inhibits caspases, whilst cIAPs prevent the formation of pro-apoptotic signaling complexes. This leads to suppression of apoptosis through both the extrinsic and intrinsic apoptosis pathways.2 Deregulation of IAPs, through amplification, overexpression or loss of endogenous antagonists, has been observed in various cancers and can contribute to tumor growth, disease progression and poor prognosis. Therefore IAPs are attractive therapeutic targets for anticancer drug discovery.3 In addition, IAPs have been shown to play a role in resistance to treatment; XIAP is upregulated in response to ionizing radiation,4 suggesting a role for IAP antagonists in combination therapy. IAPs are distinguished by their baculovirus IAP repeat (BIR) domains which are mediators of protein-protein interactions; family members such as cIAP and XIAP additionally possesses really interesting new gene (RING) domains with ubiquitin ligase activity.5 The antiapoptotic effect of XIAP are mediated by direct binding of its BIR domains to caspases 3, 7 and 9 resulting in caspase inactivation; caspases 3 and 7 bind to the linker between domains BIR1 and BIR2 whilst caspase 9 binds at the peptide binding groove of the BIR3 domain.6 IAP antagonists such as the endogenous second mitochondria derived activator of caspases (SMAC) bind to the IAP BIR domains and can disrupt interactions such as those between caspase 9 and XIAP.7 On binding to cIAP1, SMAC induces a conformational change resulting in activation of its E3 ligase activity with subsequent rapid autoubiquitination and proteasomal degradation.8 This combined loss of cIAP1 and release of the XIAP-mediated Page 3 of 47 ACS Paragon Plus Environment


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block on caspases results in a sustained pro-apoptotic effect via the extrinsic apoptosis pathway in the presence of TNF-α. Tumors with sufficient levels of TNF-α in the tumor microenvironment may, therefore, be particularly sensitive to IAP antagonism.9 In addition, antagonism of XIAP-mediated caspase inhibition is important for promoting apoptosis via the intrinsic apoptosis pathway, in response to stimulation by agent such as chemotherapeutics and DNA damaging agents.10 This suggests that dual cIAP1/XIAP antagonists can be used to promote apoptosis through both the extrinsic and intrinsic pathways. IAPs have been therapeutically targeted by antisense oligonucleotides and small molecule antagonists.

XIAP

antisense

oligonucleotide

(AEG35156)2b

sensitized

cells

to

chemotherapeutic agents and TRAIL receptor agonists and showed evidence of clinical activity.2b The first generation of IAP antagonists is based on the SMAC N-terminal peptide sequence AVPI. SMAC-mimetics, such as 1 – 411a-d (Figure 1), all contain a terminal alanine group and have shown promising preclinical activity allowing them to progress into clinical trials.2a, 2b


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Figure 1. Examples of SMAC mimetics in clinical trials

We previously reported the application of a fragment-based approach to the IAP family12 where weakly binding fragment 5 was optimized into early lead compound 6 (Figure 2). This is the first published example of a non-peptidic, sub-micromolar affinity dual antagonist of cIAP1 and XIAP, lacking the alanine group present in previously reported SMAC mimetics. When dosed intraperitoneally, compound 6 exhibited in vivo biomarker modulation and concomitant tumor growth inhibition in a mouse xenograft model based on a breast cancer cell line (MDA-MB-231). Here we describe further optimization of 6 into 26, an orally bioavailable and efficacious low nM antagonist of both cIAP1 and XIAP.



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O N

N N

N HN

HN

X Y

O

O

5

X = CH, Y = N 6

XIAP IC50 38% @ 5 mM LE < 0.20 cIAP1 IC50 16% @ 5 mM LE < 0.20

XIAP IC50 = 160 nM LE = 0.30 cIAP1 IC50 = 10 nM LE = 0.35 X = N, Y = CH 7 XIAP IC50 = 220 nM LE = 0.29 cIAP1 IC50 = 14 nM LE = 0.35

Figure 2. Structures of previously published piperazine fragment 5 and early lead compound 612 are shown alongside close analogue 7, the starting point of the work presented in this paper. 2.

OPTIMIZATION OF THE AZAINDOLINE SCAFFOLD

Figure 3A shows the X-ray crystal structure of previously published compound 6 bound to XIAP-BIR3. The piperazine ring occupies the P1 pocket with the protonated nitrogen engaged in a bidentate hydrogen bond interaction with the side chain of Glu314 and the backbone carbonyl of Asp309. The methyl substituent efficiently fills the small lipophilic sub-pocket in P1 forming a van der Waals contact with the side chain of Trp310, strongly contributing to binding affinity for XIAP. The methoxymethyl group lies in the P2 pocket and contributes 4 fold to binding affinity,12 despite making no obvious direct contact with the protein. When we first synthesized compound 6, we lacked a good explanation for this affinity increase but comparison with X-ray crystal structures of bound peptidic IAP antagonists (e.g. as shown in Figure 3B) suggested that variation of the P2 substituent would provide good scope for further optimization. Compound 6 extends into P3 with a 5azaindoline bicycle connected to the piperazine moiety via an amide linker. The carbonyl of


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the central amide forms a hydrogen bond with the backbone NH of Thr308. The saturated ring of the azaindoline stacks against the side chain of Trp323 in the P3 pocket while the electron deficient aromatic portion of the bicycle contacts a negative patch in the electrostatic potential surface of the protein formed by the backbone carbonyl of Gly306 and the phenolic oxygen in the side chain of Tyr 324.12 A benzylic substituent grows from C-6 of the azaindoline into P4, partially filling the lipophilic pocket.

Figure 3. (A) X-ray crystal structures of 6 bound to XIAP-BIR3 (PDB 5C83). Hydrogen bonds between ligand and protein are shown as dashed red lines. The Connolly surface of the protein is colored by electrostatic potential (red = negative, blue = positive). (B) Comparison of X-ray crystal structures of 7 (orange) and the peptide AVPF (light green) bound to XIAPBIR3. The Connolly surface of the protein is colored gray.

We started our SAR exploration by synthesizing a small number of analogues of 6 replacing the methoxy group in the P2 pocket, with the aim of improving interactions in that region of the protein. It became evident that some of these compounds showed different degrees of chemical instability. In a 24 hour NMR-based stability assay test compounds were dissolved



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in pH 1, 4 and 7 aqueous buffers (at a nominal concentration of 0.5 mM) and the compounds showed different levels of cleavage of the central amide bond. This instability was particularly notable when methoxy was replaced by a more nucleophilic substituent such as an amine. We ascribed this observation to the electronic effect of the aromatic nitrogen (N-5) of the indoline scaffold and its ability to stabilize an incipient negative charge on N-1 of the cleavage product. Hence the 5-azaindoline moiety is a relatively good leaving group and the entire structure susceptible to amide bond cleavage. We reasoned that repositioning of the nitrogen to N-4 (as in 7, Figure 2) would lessen the degree of anion stabilization and, gratifyingly, this change allowed us to improve the chemical stability, whilst still maintaining almost identical levels of affinity against the two target proteins as measured by XIAP and cIAP1 fluorescence polarization assay (7 XIAP IC50 220 nM, LE 0.29; cIAP1 IC50 14 nM, LE 0.35). We report herein the results of the lead optimization campaign that started with compound 7 with a particular emphasis on the structure activity relationships (SAR) for two of the subpockets within the protein binding site: P2 and P4 (see Figure 3B). Data presented will primarily focus on the 4-azaindolines, though some 5-azaindolines are shown in order to illustrate key SAR points and learnings that were subsequently applied to the 4-azaindoline series.

3.

OPTIMIZATION OF THE P2 AND P4 SIDE CHAINS

A number of features suggested that the P2 pocket was a promising region to target. Firstly, the crystal structure of 7 bound to XIAP-BIR3 domain (Figure 3B) shows the ether side chain on the piperazine ring occupies the P2 pocket of the protein mimicking the iso-propyl group of the valine amino acid of the AVPF sequence. Previous work on peptidomimetics13 suggests the potency of the ligand can be modulated by altering the size and polarity of the Page 8 of 47 ACS Paragon Plus Environment

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substituent in this pocket. Secondly, the electron density of the ether side chain is not well resolved in the crystal structure which suggests it can assume a number of energetically similar conformations in the crystal structure of 7 and may not be optimal. Finally, we consistently observed a water molecule which mediates an H-bond interaction between the tertiary amine of the piperazine ring and the NH of Trp323 (Figure 4A, labeled W1). Modeling studies suggested that a carefully positioned substituent on the ligand scaffold, such as the amide shown in Figure 4A, could displace the water molecule and engage in a direct interaction with the protein resulting in an increase in binding affinity.



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Table 1. Initial exploration of structure activity relationships (SAR) in the P2 pocket

XIAP LE b XIAP IC50 Compound

X

Y

R (nM)a

cIAP1 LE b cIAP1 IC50

-1

(kcal mol per

(nM)a

non-H atom)

a

(kcal mol-1per non-H atom)

7

N

CH

220

0.29

14

0.35

8

N

CH

250

0.27

47% @ 12

>0.33

9

N

CH

150

0.26

62% @ 12

>0.30

10

CH

N

49% @ 40

>0.29

80% @ 12

>0.31

11

N

CH

110

0.27

64% @ 12

>0.31

12

N

CH

44

0.29

82% @ 12

>0.31

Values were determined by fluorescence polarization assay (see Experimental Section).

Values are the geometrical mean of at least two separate measurements. bLigand efficiency (LE) values calculated according to the Hopkins formula.14


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Figure 4. (A) X-ray crystal structure of compound 9 (light green) bound to XIAP-BIR3 overlaid with modeled morpholine side chain (purple in ball and stick representation). (B) and (C) X-ray crystal structure of XIAP-BIR3 bound to compound 10 (B) and 12 (C). (D) Water clusters (red maps) identified from the analysis of on molecular dynamics simulation performed on a truncated analog of 7 lacking the P2 substituent (light blue ligand). Hydrogen bonds are shown as dashed lines and Connolly surface of the protein is colored gray. PDB Coordinates for Computational Models reported in Figures 4A and 4D are available in the Supporting Information.

The results of the assay (Table 1), however, indicate that the amide linker in P2 (compounds 8 and 9) is tolerated but did not lead to a significant increase in XIAP binding affinity. The Page 11 of 47 ACS Paragon Plus Environment


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crystal structure of 9 (Figure 4A) reveals that the morpholine amide side chain does not bind on the top of Gln319 as predicted by modeling studies. Instead, the morpholine prefers to stack on the top of the main scaffold above the tertiary amide, with the conserved water W1 still bound. A similar binding mode with folded conformation was observed with the 4methyl pyrazole 10 (see Figure 4B) and we were intrigued to discover whether this bound conformation represented a low energy state for the ligand. Interestingly, NMR spectroscopy (see Supporting Information, Figure S1) on 10 dissolved in phosphate buffer indicated that the X-ray pose is consistent with the lowest energy conformation in solution. In order to investigate the impact of the folded conformations on potency, we designed a small array of compounds where the P2 substituents were selected so that their electrostatic potential surfaces (EPS) were complementary to the EPS of the central amide region of the piperazine-indoline section of the ligand (see dashed area in Figure 5A). This approach allowed us to maximize the intramolecular stacking interaction between the P2 substituent and the central amide moiety of the same molecule in order to favor collapsed folded geometries. Figures 5B and 5C show two P2 sidechains (an aromatic and a saturated heterocycle) which satisfy the ESP complementarity and that were selected for synthesis.


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Figure 5. Electrostatic potential surface (EPS) of the piperazine-azaindoline scaffold (A) and a selection of P2 side chains which were selected for synthesis (B and C). Negative areas are colored red and positive areas are colored blue. The key region surrounding the central amide of A is enclosed by the dashed red line.

A 2-fold increase in activity was observed with 4-fluoro pyrazole 11, and the 2-pyrrolidinone 12 was the most ligand efficient group we identified in this first round of SAR exploration, with XIAP IC50 44 nM (LE = 0.29). The crystal structure of 12 confirmed that the pyrrolidinone ring stacked on top of the central linker amide group, resulting in the folded conformation (see Figure 4C). Interestingly, the lactam ring gives a 5-fold increase in potency compared to the simple ether side chain 7 despite no direct involvement in any van der Waals interactions with the protein. In order to understand this effect, molecular dynamic (MD) simulations were performed on the complex between XIAP-BIR3 and a truncated analog of 7 lacking the P2 substituent. An analysis of the MD trajectories revealed a well-defined water molecule (Figure 4D, labeled



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W2) on the top of the key hydrogen bond interaction between the linker carbonyl group of the ligand and the backbone NH of Thr308. We hypothesized that the lactam side chain in compound 12 displaces W2, shielding the hydrogen bond interaction from bulk water. This may have a positive impact on the overall potency of the ligand.15,16 With lactam 12 in hand, we started exploration of the P4 pocket (see Table 2) and quickly found that many compounds reached potency levels close to or below the lower limit of the fluorescence assays, hence we increasingly relied on XIAP and cIAP1 cell-based assays12 for generation of primary SAR. For XIAP, we used a XIAP-caspase 9 immunoprecipitation (IP) assay in HEK293 cells overexpressing XIAP and caspase 9, which directly measures the ability of compounds to disrupt the protein-protein interaction inside the cell. For cIAP1, we measured intracellular cIAP1 degradation in MDA-MB-231 cells as a surrogate for cIAP1 affinity.17 SAR were also generated in a MDA-MB-231 cell proliferation assay (highly sensitive to cIAP1 inhibition)12 and an IAP-insensitive human colon cancer cell line (HCT116) to control for off-target cytotoxicity. XIAP/cIAP1 selectivity was defined as the ratio between the EC50 values in the HEK293 XIAP-caspase 9 IP and cIAP1 degradation assays. The crystal structure of compound 12 bound to XIAP-BIR3 (Figure 4C) showed that the benzyl group is slightly smaller than the pocket suggesting that small substituents at C-4 or C-2 and C-4 of the phenyl ring could maximize shape complementarity between the ligand and the protein.


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Table 2. Exploration of SAR in the P4 pocket

Compound

a

R

XIAP IP

cIAP1 deg.

EC50

EC50

(nM)a

(nM)b

Selectivity XIAP IP/

MDA-MB 231

cIAP1 deg.

EC50 (nM)c

HCT-116 EC50 (µM)d

12

-CH2Ph

120

7.5

16

100

-9% @ 10

13

-CH2-4F-Ph

73

2.3

32

20

2% @ 10

14

-CH2-2,4,diF-Ph

69

2.4

28

19

1% @ 10

15

-CF2Ph

17

20

0.8

140

10% @ 10

16

-CF2Me

130

240

0.5

2000

-10% @ 10

17

-CF2Et

150

150

1.0

1800

1% @ 10

18

-CF2Pr

32

24

1.3

210

-8% @ 10

Values were determined by immunoprecipitation assay. bIntracellular cIAP1 degradation in

MDA-MB-231 cells (see Experimental Section). cMDA-MB-231 cell proliferation assay (sensitive to cIAP1 inhibition). dHuman colon cancer cell line (HCT-116) cell proliferation assay (IAP insensitive).



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Encouragingly, the fluorinated analogues 13 and 14 gave a modest XIAP potency increase in the cellular XIAP IP assay (respectively EC50 73 nM and 69 nM) compared to 12 (EC50 120 nM) and were also 5-fold more active in the MDA-MB-231 cell proliferation assay (EC50 20 nM and 19 nM respectively) compared to 12 (EC50 100 nM). We have previously described how XIAP affinity can be improved by increasing the electron deficient character of the aromatic ring of the bicycle, due to close proximity of this moiety to an electronegative patch on the protein surface.12 Therefore two fluorine atoms were introduced at the benzylic position (compound 15). Gratifyingly, this change elicited a 7-fold increase in XIAP potency. However, a lack of increased cIAP1 potency is reflected in no potency improvement in the MDA-MB231 cell proliferation assay. Encouraged by the XIAP potency increase seen with 15 a series of 1,1-difluoroalkyl substituents (16-18) was also investigated in order to mimic the P4 binding mode of the Ile side chain of the SMAC tetrapeptide AVPI. Of these, 18 had exceptional potency in the IP assay for its size. However, all these compounds had lower activity in the MDA-MB-231 cell assay compared to the fluorobenzyl analogues, likely reflecting only moderate affinity for cIAP1. The contrasting selectivity profiles achieved with larger P4 groups (e.g. fluorobenzyl) versus smaller P4 groups (e.g. 1,1-difluoropropyl) can be explained by differences in P4 pocket size between XIAP and cIAP1. The base of the P4 pocket is formed by the side chain of Leu292 in XIAP, resulting in a smaller pocket than cIAP1 which contains Val292 in this position. Hence for small P4 groups, cIAP1 P4 is improperly filled (Figure 7) and the compounds tend toward weaker cIAP1 affinity. For a large P4 group, the act of filling P4 optimally for cIAP1 results in suboptimal contact in XIAP between the azaindoline bicycle and the protein surface, leading to a tendency for greater cIAP1 selectivity.


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Whilst we were delighted by the potent XIAP activity obtained with small P4 substituents, we were concerned with the relative lack of cIAP1 activity. cIAP1 lies upstream on the apoptotic pathways and there is a risk that insufficient target engagement here would result in poor caspase activation. Given that XIAP lies downstream of caspase activation, high potency XIAP antagonism might be expected to be effective only in the presence of a strong caspase activation signal. We therefore decided to prioritize the 2,4-difluorobenzyl or the 4fluorobenzyl as P4 substituents and focus our attention to further developing SAR in the P2 pocket. In particular, we wanted to continue with the identification of P2 groups capable of adopting the folded conformation that shields the key hydrogen bond interaction with Thr308. As described above, potential chemical instability was observed with 5-azaindolines bearing a nucleophilic P2 substituent, suggesting an interaction between frontier orbitals of the amide carbonyl and P2 substituents. Therefore we investigated orbital overlap between the Lowest Unoccupied Molecular Orbital (LUMO) of the piperazine-indoline core (Figure 6A) and the Highest Occupied Molecular Orbital (HOMO) of different P2 substituents (e.g. Figures 6B and 6C). Although the orbitals have contributions from other atoms, the LUMO corresponds to the π* orbital on the carbonyl of the piperazine-azaindoline core, while the HOMO on the morpholine or fluoro substituted pyrrolidine corresponds to the nitrogen lone pair. Fluoro substituted pyrrolidine and morpholine rings, for example, showed high degree of complementarity with the piperazine-indoline scaffold and were selected for synthesis. Table 3 shows the activity value of P2 nucleophile containing compounds as measured in the XIAP IP cell assay, with lactam 19 as comparison.



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Figure 6. LUMO of the piperazine-azaindoline scaffold (A) and HOMO of examples of P2 side chains (B and C) which were selected for synthesis. Red arrows indicate the positive orbital overlap between the piperazine-azaindoline scaffold and the P2 side chains.

The pyrrolidine 20 (Table 3) had lower activity compared to lactam 19. The result could be explained by considering the ionization state of the P2 amine at physiological pH. The calculated pKa of this side chain was 9.4 suggesting appreciable protonation and a consequent disruption of the favorable HOMO-LUMO interaction.


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Table 3. SAR for tertiary amine P2 substituents

XIAP IPa Compound

R

EC50 (nM)

a

19

67

20

180

21

24

22

17

23

14

Values were determined by immunoprecipitation assay.

Basic side chains with reduced basicity were then targeted, such as fluoropyrrolidines (21 and 22) and morpholine 23. As anticipated, all had much improved potency compared to 20, with Page 19 of 47 ACS Paragon Plus Environment


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23 close to 10 nM in the XIAP cell IP assay. An X-ray crystal structure of 23 bound to XIAPBIR3 (data not shown) also showed the morpholine ring binding in the folded conformation, with the nitrogen lone pair close to linker carbonyl of the ligand. Comparison of the ligand and protein surfaces suggested an opportunity to append methyl groups adjacent to the morpholine nitrogen. These changes were tested in our 4-azaindoline main series (Table 4) in order to avoid the potential issue of chemical instability. We decided to prioritize the 4fluorobenzyl substituent in P4 for further investigation over 2,4-difluorobenzyl based on the identical level of potency measured for compounds 13 and 14 (Table 2) and on grounds of reduced molecular weight and lipophilicity. Compound 26 stood out by virtue of sub-10 nM cell based XIAP activity and exceptional potency in the cIAP1 degradation assay. Addition of the methyl group results in a gain of 2.6-fold in XIAP potency and a significantly larger increase in cIAP1 potency compared to compound 24. The high cIAP1 potency led to very potent antiproliferative effect in the MDA-MB-231 assay. Given the high potency of 26 we wanted to know whether the folded conformation seen in the X-ray crystal structure (Figure 7) was representative of the conformation in aqueous solution as previously shown for 10.


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Table 4. SAR for substituted morpholine P2 substituents

Compd

R

R1

24

XIAP

cIAP1

IP

Deg.

EC50

EC50

(nM)a

(nM)b

13

2.5

5.2

20

10% @ 10

37

2.4

15.4

39

-10% @ 10

5.1

0.32

15.9

4.4

24% @ 3

Selectivity

MDAMB-231

XIAP IP/ EC50 cIAP1 deg.

(nM)c

HCT-116 EC50 (µM)d

O

25

N

26

a

Values were determined by immunoprecipitation assay. bIntracellular cIAP1 degradation in

MDA-MB-231 cells (see Experimental Section). cMDA-MB-231 cell proliferation assay (sensitive to cIAP1 inhibition). dHuman colon cancer cell line (HCT-116) cell proliferation assay (IAP insensitive).



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Figure 7. X-ray crystal structures of compound 26 bound to XIAP-BIR3 (A and B) and to cIAP1-BIR3 (C and D). Water molecules are represented as red spheres. Red dashed lines represent key hydrogen bond interactions between the ligand and the protein residues. Connolly surface are shown for XIAP (green) and cIAP1 (gray)

A full NMR structural assignment was performed on 26 and the solution conformation was assessed in pH 7 phosphate buffer at 20 °C using a ROESY experiment (see Figure 8). Correlations were seen between hydrogens 1 and 3 and between hydrogens 2 and 4, defining the conformation of the linker relative to the azaindoline moiety. Crucially, strong ROESY correlation was observed between hydrogens 5 and 6 (Figure 8) confirming the close proximity of the morpholine to C7 of the azaindoline, consistent with the X-ray bound


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conformation. On acidification of the NMR sample to pH 4, the key correlation between hydrogens 5 and 6 disappeared, consistent with protonation of the morpholine nitrogen and disruption of the folded over conformation. Hence there is a strong degree of structural preorganization of 26 in solution at neutral pH, favoring a conformation consistent with that seen in the X-ray crystal structure of the co-complex. This suggests a low conformational entropic penalty would be incurred on ligand binding to the protein, thus contributing to the high affinity observed.

Figure 8. Chemical structure and solution conformation of 26 as observed by NMR. Red arrows indicate through space ROESY correlations in the NMR spectrum.

When 26 was tested in the XIAP and cIAP1 cellular assays we observed that the compound had a more balanced profile compared to IAP inhibitors in the clinic such as 1-4, which were between 40 and 200 fold selective for cIAP1 over XIAP (see Supporting Information, Table S1). This is likely due to the fundamental difference in the binding interactions of the piperazine moiety in the P1 region compared to those of the alanine terminus of the peptidomimetics, as has previously been discussed.12



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In vivo target engagement of cIAP1 after a single 30 mg/kg dose PO of 26 was demonstrated in a PK/PD study in Balb/c scid mice bearing MDA-MB-231 xenografts (see Figure 9A). There was a clear reduction in tumor cIAP1 levels measured by Western blotting at 24 hours (3/3 mice) and 48 hours (2/3 mice) post dose; before returning to vehicle-treated levels 72 hours post dose. Furthermore, effects on markers of apoptosis were demonstrated in the same study with elevation of both cleaved PARP and cleaved caspase-3. The mean plasma and tumor levels of 26 after the single 30 mg/kg dose from this PK/PD study demonstrated a prolonged exposure in the tumor compared to the plasma (see Figure 9B). To demonstrate efficacy in a mouse xenograft model, 26 was dosed daily for 24 days at 7.5 or 15 or 30 mg/kg in Balb/c scid mice bearing MDA-MB-231 xenografts. Significant dosedependent reduction in tumor growth was measured with daily dosing PO of 26 (see Figure 9C), comparable with compound 2, with no significant effect on body weight. Efficacy was also demonstrated in an A375 melanoma xenograft model (see Supporting Information, Figure S2).

In vivo target engagement of XIAP by 26 was demonstrated in an engineered HEK293 cell line xenograft model in which the HEK293 cell line stably over-expresses both FLAG-tagged XIAP and caspase-9. This cell line, when grown as sub-cutaneous xenografts in Bald/c nude mice, allowed us to demonstrate 26 induced disruption of the interaction between XIAP and caspase-9 by immunoprecipitation of FLAG-tagged XIAP (Figure 9D). Lanes 1-5 in the left panel of Figure 9D shows the results of the immunoprecipitation experiments performed with anti-FLAG antibody on tumor lysates from mice dosed with vehicle control or with 25 mg/kg of compound 26. In the control mice, caspase-9 is pulled down in complex with FLAG-tagged XIAP in the presence of anti-FLAG antibody but the amount of caspase-9 pulled down in animals dosed with compound 26 is considerably reduced compared to the control mice (and also compared to the total pull down control


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experiment shown in the right panel, lanes 6-10, of Figure 9D) indicating that compound 26 efficiently disrupts the XIAP-caspase-9 complex.

Figure 9. (A) Pharmacodynamic (PD) and (B) pharmacokinetic (PK) effect of a single 30mg/kg dose of 26 PO in Balb/c scid mice bearing MDA-MB-231 xenografts. (C) In vivo efficacy in Balb/c scid mice bearing MDA-MB-231 xenografts dosed daily PO with 26 at 30 mg/kg (blue line), 15 mg/kg (red line) and 7.5 mg/kg (green line) or dosed PO with 2 at 30mg/kg (orange line). (D) Immunoprecipitation (using anti-FLAG antibody) of FLAGtagged XIAP from tumor lysates taken 2 h after a single 25 mg/kg dose of 26 to HEK293 xenografted Balb/c nude mice (lanes 1-5) and total pull down control experiments (without anti-FLAG antibody) (lanes 6-10).

Pharmacokinetic parameters of compound 26 in mouse are shown in Table 5. Moderate clearance in vivo (>30% liver blood flow), volume of distribution (Vss), and 3 h half life were measured when compound 26 was dosed IV at 1 mg/kg in mice. Following single oral dosing



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in mice at 5 mg/kg, a rapid rate of absorption was observed and oral bioavailability (F) was 22%.

Table 5. Mouse pharmacokinetics of 26 dose IV at 1 mg/kg, and PO at 5 and 30 mg/kg Dose

Tmax

Cmax

CLp

Vss

Half life

AUC

F

(h)

(µg/mL)

(mL/min/kg)

(L/kg)

(h)

(µg*h/mL)

(%)

40.9

6.4

3.0

0.41

1.8

0.45

Route (mg/kg) 1

IV

5

PO

2

0.12

30

PO

1

1.7

4.

22

12.1

CHEMISTRY

For compounds 7-26 we required carboxylic acid functionalized piperazine (Scheme 1) and azaindoline (Scheme 2) building blocks. Functionalized piperazines 28-29 were prepared from intermediates (27a and 27b) described in a previous publication.12 Hydroxymethyl piperazine 27b was readily oxidized to the corresponding carboxylic acid intermediate followed by amide coupling with dimethylamine or morpholine to generate compounds 28a and 28b. Carboxylic acid functionalized piperazines 30a-c, were prepared by reaction of intermediate 27a with methanesulfonyl chloride, subsequent reaction with a variety of amine or amide nucleophiles to give 29a-e, then reaction with benzyl bromoacetate and final hydrogenolysis in the presence of palladium on carbon. The fluoro-pyrazole and methylpyrazole compounds 31a-b were prepared via displacement of the mesylate formed in situ from the protected hydroxymethyl piperazine and further elaboration as for 29a-c.


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Scheme 1. Reagents and conditions: a) NaIO4, RuCl3, water/dimethyl carbonate/MeCN, RT, 78%; b) Carbonyldiimidazole, then dimethylamine or morpholine, DCM, RT, quant.; c) Pd/C, EtOH, H2, RT, 71% - quant.; d) NaBH(OAc)3, benzaldehyde, dichloroethane, RT, 76%; e) methanesulfonyl chloride, triethylamine, DCM, RT, 77%; f) NaH, R3H, DMF, 80°C; g) R3H, KI, K2CO3, MeCN, 70°C, 59% - quant.; h) Pd/C, H2,



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AcOH/EtOH, RT, 73-95%; i) benzyl bromoacetate, K2CO3, MeCN, RT, 56-98%; j) Pd/C, H2, EtOAc, RT, 80-94%; k) methanesulfonic anhydride, triethylamine, 4-fluoropyrazole or 4-methyl-pyrazole, 0-20°C, 30-59%.

Scheme

2.

Reagents

and

conditions:

a)

ArZnBr,

[1,3-bis(2,6-

diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, LiBr, NMP/THF, RT, 70% - quant.; b) 4M HCl in dioxane, MeOH, RT, 78% - quant.; c) nBuLi, RCON(Me)OMe, -78 °C, 45-61%; d) Bis(2-methoxyethyl)aminosulfur trifluoride, THF, 70-90 °C, 34-47%. All 4-azaindoline intermediates 32a-c and 33a-d were prepared from a common starting material, Boc protected 3,3-dimethyl-6-bromo-4-azaindoline18 as shown in Scheme 2. Differently substituted benzylic groups were introduced at the C-6 position of the indoline via Negishi coupling19 with the appropriate benzylzinc bromide in the presence of [1,3-bis(2,6diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride catalyst. The Page 28 of 47 ACS Paragon Plus Environment

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same procedure was also used to synthesize the 5-azaindoline intermediates 34a-b starting from Boc protected 3,3-dimethyl-6-chloro-5-azaindoline.12,18 Gem-difluoro substituted compounds 33a-d were prepared by fluorination of the corresponding ketones with bis(2methoxyethyl)aminosulfur trifluoride and subsequent deprotection. The required 6-keto intermediates were synthesised in good yields via lithiation at C-6 using nBuLi followed by quench with the appropriate Weinreb amide. Three different routes were employed to assemble compounds 7-26 as shown in Schemes 3, 4 and 5. In the first method (Scheme 3) the carboxylic acid functionalised piperazines were directly coupled to the appropriate indoline in a HATU or PyBroP mediated amide coupling reaction. Compound 7 required late stage Negishi coupling with benzylzinc bromide to elaborate C-6 of the indoline scaffold. Scheme 4 shows a second approach: the “three component coupling”. In this two-step-onepot reaction the indoline was firstly reacted with chloroacetylchloride to establish the amide bond followed by the addition of the appropriately decorated piperazine to produce the Bocprotected final compounds in moderate yields. Finally, Scheme 5 describes the third synthetic strategy. Using methods similar to those described previously, chloromethyl intermediates 35a-b were prepared allowing introduction of the P2 group at a late stage. All final compounds were isolated as hydrochloride salts following deprotection under acidic conditions.



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Scheme 3. Reagents and conditions: a) HATU (or PyBroP), diisopropylethyl amine, DMF (or DCM), RT, 17-91%; b) HCl, EtOAc or MeOH, RT, 91% - quant.; c) benzylzinc

bromide,

[1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-

chloropyridyl)palladium(II) dichloride, LiBr, NMP/THF, RT, 81%.


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Scheme 4. Reagents and conditions: a) chloroacetyl chloride, diisopropylethyl amine, RT, then appropriate piperazine, diisopropylethyl amine, RT, 17-77%; b) HCl in EtOAc or dioxane, RT, 66% - quant.

Scheme 5. Reagents and conditions: a) chloroacetyl chloride, 0 °C – 20 °C, 83%; b) 27a or 29c, K2CO3, KI, MeCN, RT, quant.; c) HCl in dioxane, RT, 90% - quant.; d) Page 31 of 47 ACS Paragon Plus Environment


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MeSO2Cl, triethylamine, 0 °C - RT, 77%; e) appropriate amine, K2CO3, KI, MeCN, 70 °C, 32% - quant. 5.

CONCLUSIONS

We applied structure-based drug design to our previously published fragment-derived early lead 6 to develop a potent, orally bioavailable, dual XIAP and cIAP1 antagonist 26, which we have designated AT-IAP.

A combination of structural information from X-ray

crystallography, computational studies, NMR solution conformational work and docking allowed us to efficiently grow into the P2 and P4 pockets. Compound 26 has demonstrated potent dual target engagement in cells and robust XIAP and cIAP1 pharmacodynamic effects in vivo. Compound 26 represents a structurally novel, potent chemical probe for further investigation of IAP biology and has been synthesised on a multi-gram scale. It also represents an advanced lead for IAP drug discovery, and further work leading to the identification of a clinical candidate will be reported in due course.

6.

EXPERIMENTAL SECTION

General Chemistry. All solvents employed were commercially available anhydrous grade, and reagents were used as received unless otherwise noted. Hereafter, petrol denotes the petroleum ether fraction boiling at 40−60 °C. Flash column chromatography was performed on a Biotage SP1 system (32−63 µm particle size, KP-Sil, 60 Å pore size). NMR spectra were recorded on Bruker AV400 (Avance 400 MHz) and on Bruker AV500 spectrometers. Analytical LC−MS was conducted using an Agilent 1200 series with mass spec detector coupled with an Agilent 6140 single quadrupole mass detector and an Agilent 1200 MWD SLUV detector. LC retention times, molecular ion (m/z), and LC purity (by UV) were based on the method below. Purity of compounds (as measured by peak area ratio) was >95%, as determined by the LC method described in Table 6. Page 32 of 47 ACS Paragon Plus Environment

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Table 6. LC Method (BASIC) eluent A 95:5 10 mM NH4HCO3 + NH4OH:CH3CN (pH = 9.2) eluent B CH3CN gradient 5−95% eluent B over 1.1 min flow 0.9 mL/min column Waters Acquity UPLC BEH C18; 1.7 µ; 2.1 mm × 50 mm column T 50 °C

Preparation of compound 26 (Scheme 5): 1-{6-[(4-fluorophenyl)methyl]-3,3-dimethyl1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}-2-[(2R,5R)-5-methyl-2-{[(3R)-3methylmorpholin-4-yl]methyl}piperazin-1-yl]ethan-1-one Hydrochloride Salt. Step1:

2-Chloro-1-[6-(4-fluoro-benzyl)-3,3-dimethyl-2,3-dihydro-pyrrolo[3,2-b]pyridin-1-

yl]-ethanone hydrochloride salt To a stirred suspension of 6-(4-fluoro-benzyl)-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[3,2b]pyridine (6.7 g, 26.0 mmol, see Supporting Information) in acetonitrile (20 mL) at 20 oC was added, steadily over 0.2 h, a solution of chloroacetyl chloride (3.8 g, 2.7 mL, 33.9 mmol) in acetonitrile (10 mL), maintaining the reaction mixture at or below 20 oC using an external ice-methanol bath. A clear solution resulted then, as the internal temperature reached 0 oC, a solid began to crystallize from the reaction mixture. Stirring at 20 oC was continued for 1 h then toluene (20 mL) and 40 – 60 petroleum ether (20 mL) were added slowly and stirring continued for 0.2 h. The resulting colourless solid was collected by filtration to give the title compound (8.0 g, 83%).

1

H NMR (Me-d3-OD): 8.81 (1H, s), 8.31 (1H, s), 7.39-7.29 (2H,

m), 7.16-7.04 (2H, m), 4.45 (2H, s), 4.19 (4H, s), 1.58 (6H, s); LCMS: [M+H]+ = 333.



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Step 2: (2R,5S)-4-{2-[6-(4-fluoro-benzyl)-3,3-dimethyl-2,3-dihydro-pyrrolo[3,2-b]pyridin-1yl]-2-oxo-ethyl}-2-methyl-5-((R)-3-methyl-morpholin-4-ylmethyl)-piperazine-1-carboxylic acid tert-butyl ester A

mixture

of

2-chloro-1-[6-(4-fluoro-benzyl)-3,3-dimethyl-2,3-dihydro-pyrrolo[3,2-

b]pyridin-1-yl]-ethanone hydrochloride (6.6 g, 17.9 mmol), (2R,5S)-2-methyl-5-((R)-3methyl-morpholin-4-ylmethyl)-piperazine-1-carboxylic acid tert-butyl ester (5.6 g, 17.9 mmol, see Supporting Information), potassium carbonate (9.9 g, 72.0 mmol) and finely ground potassium iodide (5.9 g, 36.0 mmol) in acetonitrile (40 mL) was stirred at 20 oC for 1 h. Water (200 mL) was added and mixture stirred for 0.2 h, then the mixture was extracted with EtOAc (200 mL). The organic phase was dried (Na2SO4) and evaporated in vacuo to give the title compound (11.3 g) as an oil, used without further purification. 1H NMR (Med3-OD): 8.28-8.19 (1H, m), 8.13-8.04 (1H, m), 7.25 (2H, dd), 7.04 (2H, t), 4.23-4.13 (1H, m), 4.13-4.04 (2H, m), 4.04-3.95 (3H, m), 3.81-3.58 (4H, m), 3.54 (1H, d), 3.25 (2H, dd), 3.01-2.66 (4H, m), 2.62-2.42 (2H, m), 2.42-2.17 (2H, m), 1.47 (9H, s), 1.43-1.37 (6H, m), 1.29-1.21 (3H, m), 1.01 (3H, d); LCMS: [M+H]+ = 610. Step 3: 1-{6-[(4-fluorophenyl)methyl]-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}-2[(2R,5R)-5-methyl-2-{[(3R)-3-methylmorpholin-4-yl]methyl}piperazin-1-yl]ethan-1-one hydrochloride salt (26) To a solution of (2R,5S)-4-{2-[6-(4-fluoro-benzyl)-3,3-dimethyl-2,3-dihydro-pyrrolo[3,2b]pyridin-1-yl]-2-oxo-ethyl}-2-methyl-5-((R)-3-methyl-morpholin-4-ylmethyl)-piperazine-1carboxylic acid tert-butyl ester (6.1 g, 10.0 mmol) in EtOAc at room temperature was added a 4M HCl in dioxane and the resulting mixture was stirred under inert atmosphere for 18 h. The solid that formed was collected by filtration and dried in a vacuum oven to give 5.3g of the desired product as a white solid (quantitative yield). [α]20D 1.6 (c 5.0, H2O); 1H NMR (400 MHz, Me-d3-OD): 8.70 (1H, s), 8.45 (1H, s), 7.36 (2H, dd), 7.13 (2H, t), 4.25 (5H, t), 4.16-


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3.90 (5H, m), 3.79 (3H, d), 3.67-3.40 (5H, m), 3.26-2.96 (3H, m), 1.62 (6H, s), 1.39 (6H, d); 13

C NMR (101 MHz, Methanol-d4) δ 175.08, 163.35 (d, J = 244.9 Hz), 155.23, 143.08,

141.91, 135.69, 135.46 (d, J = 3.3 Hz), 133.00, 132.30 (d, J = 8.1 Hz), 116.81 (d, J = 21.4 Hz), 70.70, 65.32, 61.84, 60.90, 57.28, 55.10, 53.73, 53.43, 52.58, 52.06, 48.04, 38.85, 27.06, 26.97, 15.32, 13.28; LCMS: [M+H]+ = 510; HRMS m/z: Calcd for C29H40FN5O2 510.3239; Found 510.3241. Crystallography. XIAP-BIR3 250-354 was crystallized using a 1:1 ratio of 10 mg/ml protein and 0.1 M Hepes-NaOH pH 8.0, 3.0-3.9 M NaCl. Crystals appeared over the course of a few days at 4 ºC. Crystals were soaked in fragments using 2.5 µl of compound in 100% DMSO, 47.5 µl 0.1 M Hepes-NaOH pH 8.0, 4 M NaCl to give a final concentration of fragment in the range of 50-100 mM. The pH of the soaking solution was adjusted if necessary and crystals were left at 4 ºC for 24-72 hours. Crystals were cryo-protected using 0.05 M Hepes-NaOH pH 8.0, 4M NaCl, 15% ethylene glycol. The crystals had cell dimensions of approximately 70 Å, 70 Å, 105 Å, and belong to space group P4122. The diffraction observed ranged from 1.73.0 Å. cIAP1-BIR3 267-363 was crystallized using a 1:1 ratio of 10mg/ml protein and 0.1 M sodium citrate pH 5.6, 0.4-0.7 M lithium sulphate, 0.5-0.7 M ammonium sulphate, 10% glycerol and 5mM TCEP. Crystals were soaked in solutions containing compounds at 5mM concentration in 5% DMSO overnight at room temperature prior to data collection. The crystals diffract in the range of 1.5-2.5Å and belong to space group P212121 with cell dimensions 31 Å, 70 Å, 119 Å. Fluorescence Polarization Binding Assay. The interaction between the SMAC peptide and the BIR3 domains of XIAP and cIAP1 was measured using a fluorescence polarization assay, utilizing a fluorescent peptide tracer (AbuRPFK(5&6FAM)-amide; Peptide Synthetics Ltd, Fareham, Hampshire, UK ) derived from SMAC. Compounds were incubated with XIAP (20



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nM) or cIAP1 (4 nM) proteins in 50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, 1 mM DTT, 2% DMSO and 5 nM or 2 nM fluorescent tracer respectively. After equilibration at room temperature fluorescence polarization was measured (excitation 485 nm / emission 538 nm) using a BMG Pherastar plate reader (BMG Labtech, Orttenburg, Germany). IC50 curves were generated using GraphPad prism version 6 (LaJolla, CA, USA) and fitted using the four parameter logistic curve fit. The data presented are the geometrical mean of at least 2 separate measurements. Compounds were not intrinsically fluorescent. cIAP1 Kd for SMAC tracer- 8nM [Tracer]=2nM ([T]/Kd = 0.25) xIAP Kd for SMAC tracer – 20nM, [Tracer] 5nM ([T]/Kd =0.25) Binding affinity results for the tetrapeptide AVPI: cIAP1 IC50 mean = 0.017µM, SD = 0.003, n = 72; XIAP IC50 mean = 0.32 µM, SD = 0.08, n = 111. Cell Line Proliferation Assay. Inhibition of cell growth was measured using the Alamar Blue assay.22 For each proliferation assay cells were plated onto 96 well plates and allowed to recover for 16 hours prior to the addition of inhibitor compounds (in 0.1% DMSO v/v) for a further 72 hours. At the end of the incubation period 10% (v/v) Alamar Blue (Bio-Rad AbD Serotec, Oxford, UK) was added and incubated for a further 6 hours prior to determination of fluorescent product at 535 nM excitation / 590 nM emission. The anti-proliferative activities of 1-4, 12-18 and 24-26 were determined by measuring the ability of the compounds to inhibit growth in 2 cancer cell lines: (1) MDA-MB-231 (human breast carcinoma) [ECACC, Salisbury, UK], (2) HCT116 (human colon carcinoma) [ECACC, Salisbury, UK] - insensitive cell line used as a control for non-specific cytotoxicity. The data reported in the paper is the result of two duplicates. Compounds 26 and 2 were tested multiple times in independent experiments and statistical limits are calculated for those compounds: Compound 26: XIAP IP EC50 = 4.4 nM, SD = 2.0, n = 21.


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Compound 2: XIAP IP EC50 = 7.8 nM, SD = 3.6, n = 4. XIAP Antagonism Immunoprecipitation Assay. An engineered HEK293 cell line was generated by transfecting the HEK293 cell line (ECACC, Salisbury, UK) with a full-length FLAG-tagged XIAP expression construct [Origene Technologies Inc., Rockville, USA] and a full-length untagged caspase-9 construct [Origene Technologies, Inc., Rockville, USA]. Stable co-transfectants were selected after culture in selection medium containing Geneticin (Life Technologies, Paisley, UK). Stable HEK293-XIAP-Caspase-9 cells were plated out into 96-well plates and left overnight at 37 °C to recover. Compounds were added to duplicate wells in 0.1% DMSO for 2 h at 37 °C. Cells were lysed in 50 µL lysis buffer (1% Triton X-100 in 20 mM Tris.Cl (pH 7.6), 150 mM NaCl, including protease inhibitors (Roche Diagnostics Ltd., Burgess Hill, UK) for 20 min rocking at room temperature. Streptavidin-coated high-bind MSD plates (Meso Scale Discovery, Gaithersburg, USA) were coated with biotinylated anti-FLAG M2 antibody (Sigma, Poole, UK) and then blocked with 3% BSA in TBST (20 mM Tris.Cl (pH 7.6), 150 mM NaCl, 0.1% Tween-20). Cell lysate was added to the 96-well anti-FLAG coated MSD plate and placed on a shaker overnight at 4 °C. After washing 3 times with TBST, rabbit antiCaspase-9 (Cell Signaling Technology Inc., Danvers, USA) was added for 2 h at room temperature, with shaking. After washing 3 times with TBST, anti-rabbit-sulfo tag (Meso Scale Discovery, Gaithersburg, USA was added for 2 hours at RT. Plates were washed 3 times with TBST and then read buffer was added, before reading the plate on a MESO QuickPlex SQ 120 (Meso Scale Discovery, Gaithersburg, USA). The data reported in the paper is the result of two duplicates. Compounds 26 and 2 were tested multiple times in independent experiments and statistical limits are calculated for those compounds: Compound 26: XIAP IP EC50 = 5.1 nM, SD = 2.3, n = 43. Compound 2: XIAP IP EC50 = 35 nM, SD = 11.8, n = 6.



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Cellular cIAP1 Antagonism Assay. MDA-MB-231 cells were plated out into 96-well plates and left overnight at 37 °C to recover. Compounds were added to duplicate wells in 0.1% DMSO for 2 h at 37 °C. Cells were lysed in 50 µL lysis buffer (1% Triton X-100 in 20 mM Tris.Cl (pH 7.6), 150 mM NaCl, including protease inhibitors (Roche Diagnostics Ltd., Burgess Hill, UK) for 20 min rocking at room temperature. Streptavidin-coated high-bind MSD plates (Meso Scale Discovery, Gaithersburg, USA) were coated with biotinylated anticIAP1 antibody (R&D Systems, Abingdon, UK) and then blocked with 3% BSA in TBST (20 mM Tris.Cl (pH 7.6), 150 mM NaCl, 0.1% Tween-20). Cell lysate was added to the 96well anti-cIAP1 coated MSD plate and placed on a shaker for 2 hours at room temperature. After washing 3 times with TBST, anti-cIAP1 antibody (R&D Systems, Abingdon, UK) which had been tagged with sulfo-tag (Meso Scale Discovery, Gaithersburg, USA) was added for 2 h at room temperature, with shaking. Plates were washed 3 times with TBST and then read buffer was added, before reading the plate on a MESO QuickPlex SQ 120 (Meso Scale Discovery, Gaithersburg, USA). The data reported in the paper is the result of two duplicates. Compounds 26 and 2 were tested multiple times in independent experiments and statistical limits are calculated for those compounds: Compound 26: cIAP1 degradation EC50 = 0.32 nM, SD = 0.15, n = 30 Compound 2: cIAP1 degradation EC50 = 0.40 nM, SD = 0.13, n = 7

Western Blot Analysis of MDA-MB-231 Xenografts. MDA-MB-231 xenograft tumor lysates were prepared by grinding the frozen tissue to a fine powder with a mortar/pestle under liquid nitrogen, and then adding ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris.HCl pH 7.5, plus protease inhibitors (Roche Diagnostics Ltd., Burgess Hill, UK), 50 mM NaF and 1 mM Na3V04), to the ground-up tumor powder. Samples were vortexed and


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left on ice for 30 min. Lysates were cleared and sample of the supernatant removed for protein determination (BCA assay – Pierce, Life Technologies, Paisley, UK). Equivalent amounts of protein lysate had SDS sample buffer and a final concentration of 50 mM DTT added, before being boiled. Samples were resolved by SDS PAGE (4-12% Nu-PAGE gels – Novex, Life Technologies, Paisley, UK), blotted onto nitrocellulose filters, blocked with Odyssey Blocking Buffer (LI-COR, Cambridge, UK) and incubated overnight at 4 °C with the following primary antibodies: goat polyclonal anti-cIAP1 antibody (R& D Systems, Abingdon, UK), goat polyclonal anti-XIAP antibody (R& D Systems, Abingdon, UK), rabbit polyclonal anti-cleaved PARP (Cell Signaling Technology Inc., Danvers, USA) and rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling Technology Inc., Danvers, USA). After washing and incubation with the appropriate IR-conjugated secondary antibody (LI-COR, Cambridge, UK), detection was achieved using an Odyssey Infrared Imaging System (LICOR, Cambridge, UK). Dosing. Rodent studies were performed according to the Animal (Scientific Procedures) Act (1986) law. 26 was formulated in 100% saline or 100% water for IV or oral administration respectively. Intravenous dosing was administered via the lateral tail vein at dose volumes of 1-5 mL/kg. 26 was administered orally by nasogastric gavage at dose volumes of 2-10 mL/kg. All doses were calculated as freebase equivalent per kg of bodyweight. Pharmacokinetic studies were performed in male Balb/c mice, obtained from Harlan Laboratories Inc. (Shardlow, UK). Following dosing in mice, blood samples (0.2 mL) were drawn in tubes containing potassium EDTA, via either saphenous vein bleeding or cardiac puncture at various time points over 30 hours using sparse sampling (n=3 per time point), prior to centrifugation (2000 g at 4°C, 10 min). The resultant plasma was separated from the erythrocyte pellets for analysis and stored at -20°C. Non-compartmental pharmacokinetic (PK) analyses were performed using Phoenix 6.3.0.395® (Certara USA, Inc.) software. Page 39 of 47 ACS Paragon Plus Environment


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Xenograft studies. MDA-MB-231 xenografts were prepared by subcutaneously injecting 5x106 cells into the right hind flank of male Balb/c severe combined immunodeficient (SCID) mice. Tumor load was estimated from external digital calliper measurements. For PKPD studies, a single dose of 26 at 25 or 30 mg/kg was administered to groups of nine mice via oral gavage. Following dosing, blood samples (approximately 0.5 mL) were drawn in tubes containing potassium EDTA, via either saphenous vein bleeding or cardiac puncture at various time points over 72 hours, prior to centrifugation (2000 g at 4ºC, 10 min). The resultant plasma was separated from the erythrocyte pellets for analysis and stored at -20ºC. Tumors were immediately excised and flash-frozen in liquid nitrogen. For efficacy studies, eight mice were grouped to achieve a mean tumor volume of approximately 100 mm2. Mice were dosed orally once a day at a dose of 3.75 or 7.5 or 30 mg/kg 26 for 22 days. During the dosing period bodyweights were recorded daily and every 3-4 days after cessation of dosing. Tumor volumes were measured every 3-4 days until the study was complete. A control group, receiving vehicle only, was included. Bioanalysis. Samples were extracted by protein precipitation with acetonitrile containing internal standard (1:3 v/v). Tumor samples were prepared by homogenization in water (5 mL/g tumor tissue) using a Precellys 24® tissue homogenizer. For quantitative studies, calibration standards and quality controls were prepared in blank matrix and extracted under the same conditions. All samples were centrifuged at 3700 rpm at 4°C for 20 min. Computational Protocols. Docking. Docking experiments were used to predict the binding mode of designed piperazines and to select compounds for synthesis. The X-ray crystal structure of XIAP-BIR3 with compound 6 (PDB 5C83) was used to dock ligands. Hydrogens were added to the protein, and a binding pocket was generated with all protein atoms within 6 Å of any non-hydrogen atom in ligand 1. All calculations were run on a Linux cluster using


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Goldscore21 scoring function within the Astex Web-based docking and virtual screening platform.22 Methods and settings have been previously described by Verdonk et al.23 Ab Initio Calculations. EPSs, HOMOs and LUMOs were calculated using the ab initio quantum chemistry package Q-Chem,24 using the 6-31G* basis set and B3LYP method. Molecular dynamics simulation. Molecular dynamics (MD) simulations were conducted by using the AMBER 14 program.25 The starting coordinates of the X-ray crystal structure of the complex between compound 6 and XIAP-BIR3 (PDB 5C83) were used as a starting model. A modified version of the ligand structure was produced where the P2 side chain of 7 was deleted. Topology and parameter files for the protein, ligand, and complex were generated using the LEaP module in AMBER 14. TIP3P water molecules were added in a truncated octahedral periodic box, sized so that the edges of the box were at least 9Å from any protein atom. To ensure overall neutrality of the system, appropriate numbers of Na+ or Cl– were added to the box. For each system, energy minimization, equilibration and production MD simulation were carried out using the GPU version of the PMEMD program in AMBER 14. The production MD simulations were performed for a total of 50 ns. The coordinates of the complexes were saved every 5 ps during the production phase, and then taken for detailed analysis. A harmonic restraint of 1 kcal mol-1 Å-2 was applied to all protein C alpha during the entire production run, targeted on the initial structure. 7.

AUTHOR INFORMATION

Corresponding Author Phone: +44(0)1223226200. E-mail: [email protected] Notes The authors declare no competing financial interest. Compound 26 is available for use as a chemical probe by the broader scientific community.



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8.

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ACKNOWLEDGEMENTS

We warmly acknowledge scientific discussions with many Astex colleagues, including David C. Rees and Christopher W. Murray. The authors would also like to thank Anne Cleasby for critical discussions and crystallographic support. 9.

ABBREVIATIONS USED

cIAP1, cellular inhibitor of apoptosis protein 1; XIAP, X-linked inhibitor of apoptosis protein; BIR, baculovirus IAP repeat domain; RING, Really Interesting New Gene; TNFα, tumor necrosis factor alpha; SMAC, second mitochondria derived activator of caspases; FBDD, fragment-based drug discovery; PPI, protein-protein interaction; LE, ligand efficiency; EPS, electrostatic potential surface; MD, molecular dynamics; ROESY, rotatingframe nuclear Overhauser effect correlation spectroscopy; DMF, dimethyl formamide; NMP, N-methyl

pyrrolidone;

Cbz,

carbobenzyloxy;

EDC,

1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide; DCM, dichloromethane; THF, tetrahydrofuran; RT, room temperature; MeCN, acetonitrile. 10.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at… Protein production for bioassay and crystallography. Synthesis of compounds 7-25, 28a-b, 29a-e, 30a-b, 31a-b, 32a-c, 33a-d, 34a-b and 35. Molecular formula strings Accession Codes Coordinates for XIAP-BIR3 complexes with compounds 7, 9, 10, 12 and 26 and for cIAPBIR3 complex with compound 26 have been deposited in the Protein Data Bank (PDB) under accession codes 5m6f, 5m6h, 5m6e, 5m6m, 5m6l and 5m6n respectively. 11.

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26 XIAP EC50 = 5.1 nM cIAP1 EC50 = 0.32 nM F = 22%


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