Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5022–5029

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Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists Kyoung Soon Kim ⇑, Liping Zhang, David Williams, Heidi L. Perez, Erik Stang, Robert M. Borzilleri, Shana Posy, Ming Lei, Charu Chaudhry, Stuart Emanuel, Randy Talbott Bristol-Myers Squibb Research & Development, PO Box 4000, Princeton, NJ 08543, USA

a r t i c l e

i n f o

Article history: Received 18 July 2014 Revised 4 September 2014 Accepted 8 September 2014 Available online 16 September 2014 Keywords: Terahydroisoquinoline Bivalent IAP A875 melanoma

a b s t r a c t Bivalent heterodimeric IAP antagonists that incorporate (R)-tetrahydroisoquinoline in the P30 subunit show high affinity for the BIR2 domain and demonstrated potent IAP inhibitory activities in biochemical and cellular assays. Potent in vivo efficacy was observed in a variety of human tumor xenograft models. The bivalent heterodimeric molecule 3 with a P3–P30 benzamide linker induced pharmacodynamic markers of apoptosis and was efficacious when administered intravenously at a dose of 1 mg/kg to mice harboring A875 human melanoma tumors. Analog 5, with a polyamine group incorporated at the P20 thiovaline side chain exhibited antiproliferative activity against the P-gp expressing HCT116/VM46 cell line. Ó 2014 Elsevier Ltd. All rights reserved.

Apoptosis plays an important role in maintaining cell homeostasis, and resistance to apoptosis is one of the key hallmarks of cancer.1 Proteolytic protein caspases play a major role in apoptosis, and sequential activation of the various caspases leads to the execution of cell death.2 Inhibitor of apoptosis proteins (IAPs) are key apoptotic regulators that inactivate caspases and are overexpressed in many tumors, making them attractive targets for the treatment of cancer.3 Cellular IAP (cIAP) and human X-linked IAP (XIAP) regulate apoptosis through their involvement in the extrinsic and intrinsic apoptotic pathways.4 The IAP family of proteins contains three baculovirus repeat (BIR) domains (BIR1, 2, 3) and a RING zinc finger domain.5 cIAP inhibits the extrinsic death receptor-mediated apoptotic pathway through association and subsequent ubiquitination of the death receptor signaling complex which enhances prosurvival NF-jB activity.6 XIAP inhibits apoptosis via the intrinsic mitochondrial apoptotic pathway by blocking the proteolytic activation of the caspase-9 zymogen.7 XIAP can also inhibit apoptosis by binding pre-activated effector caspase-3 and -7, thus blocking their hydrolytic potential. The second mitochondria-derived activator of caspases (Smac/ Diablo), an endogenous regulator of IAPs, is a protein capable of binding IAPs in a manner that inhibits their antiapoptotic properties and plays a key role in regulating apoptosis.8 Following mitochondrial depolarization and release to the cytosol, Smac dimerizes ⇑ Corresponding author. Tel.: +1 609 252 5181. E-mail address: [email protected] (K.S. Kim). http://dx.doi.org/10.1016/j.bmcl.2014.09.022 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

to form a structure with exposed N-terminal tetrapeptide AVPI residues which concurrently bind both BIR2 and BIR3 domains of cIAP and XIAP, effecting IAP downstream interactions.9 Based on the ‘AVPI’ binding motif, many tetrapeptide mimetics have been developed as inhibitors of IAPs for the potential treatment of cancers.10 Monovalent inhibitors induce cell death primarily by binding to the BIR3 domain of cIAP (cBIR3) and subsequently inducing proteasome-mediated cIAP degradation via ubiquitinylation by the RING domain.6 While this approach can lead to effective induction of apoptosis in Type I cell lines sensitive to extrinsic pathway induction, XIAP-dependent Type II cell lines can evade cell death by blocking effector caspase-3 and -7 activities, making it desirable to inhibit both cIAP and XIAP simultaneously.11 With this rationale in mind, our efforts were focused on the discovery of bivalent molecules capable of inducing apoptosis in both Type I and Type II cancer cell lines. The P3 proline of the ‘AVPI’ binding motif induces a ‘U-shaped’ binding conformation and is crucial for binding to IAP BIR3 domains through its hydrophobic interaction with Trp323.12 Several fused proline-based replacements have been shown to maintain binding to BIR3.13 In the context of bivalent inhibitors, requirements for binding to the BIR2 domain are less stringent with many different amino acid residues tolerated at P30 .14 Based on this knowledge, two monomeric prototypes (Fig. 1) were selected for further study: 110a which maintains the P3 proline for BIR3 binding, and 2 which contains (R)-tetrahydroisoquinoline amino acid (Tic) as a P3 proline surrogate15 for BIR2 binding. These two molecules were tethered through various linkers to afford

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K. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5022–5029

P1

P2

P3

P1

P4

O

H N

O

O

N H

P2

O

N H

HN

N H

N

O

O HN

NH

O

H N

P1'

O

O N H

O

N

O

N H HN

3

N H

P4

O

O

H N

O

2 P3

P4

N

N H

1 P1

P3

O

H N

N

N H

P2

O

H N

NH

O

4

N

N H

O

O

N

N O

P2'

F O

P3'

N H

P4'

N H H2 N R

N H

N

O

NH

O

NH

O

NH

N

HN

5 R = -(CH2 )3NH-(CH2 )4-NH-(CH2)3 -NH2

O

O

H N

O S

H N

O N H

N O

O

N H

Figure 1. Structures of monovalent and bivalent heterodimers (1–5).

potent heterodimeric bivalent IAP inhibitors, such as 3, 4 and 5 (Fig. 1). Herein, the synthesis, SAR and biological activities of several heterodimeric bivalent analogs containing the P30 Tic moiety are discussed. The bis-benzamide P3–P30 linked heterodimer 3 was prepared starting from (R)-6-nitro tetrahydroisoquinoline acid 616 (Scheme 1). Coupling of 6 with commercially available (R)-tetrahydronaphthyl amine, followed by successive incorporation of P20 tert-leucine and P10 N-methylalanine using EDC–HOAt coupling reagents followed by reduction of the nitro group provided intermediate amine 10. Following a similar procedure, commercially available Fmoc-protected 4-aminoproline 11 was converted to intermediate 12. After introducing the carboxybenzamide linker onto 12 to give 13, subsequent coupling with 10 led to heterodimer 3 after deprotection of the Boc-groups. Heterodimer 4, with a P3–P40 linker was prepared using commercially available (R)-2-fluoro-a-methylbenzylamine 14 (Scheme 2). Alkylation of amine 14 with p-carboxymethylbenzyl bromide17 followed by subsequent couplings with the P3, P2 and P1 amino acid residues provided 18 after hydrolysis of the ester. Intermediate acid 18 was coupled with 12 and deprotection of the Boc-groups afforded 4.

Polyamine substituted thiovaline heterodimer 5 was synthesized using commercially available thiovaline 19. Compound 19 was converted to intermediate 22 following a similar synthetic sequence to that described above (Scheme 3). Coupling of 22 with 13, followed by hydrolysis produced acid intermediate 23. Guanylated polyamine 25 was obtained by reductive alkylation of tri-Boc spermine 2418 followed by guanylation with bis-Boc-thiourea in the presence of mercuric chloride19 and debenzylation using Pd(OH)2 under 1 atm of H2 gas. Coupling of 25 with 23 followed by global deprotection of the Boc groups provided guanylated polyamine substituted heterodimer 5. Monovalent analog 2 with (R)-tetrahydroisoquinoline (Tic) at P3 demonstrated potent inhibition of cIAP1 BIR2-3 (cBIR2-3) binding in an FP binding assay20 (IC50 = 13 nM) and growth inhibition of Type I MDA-MB-231 human breast carcinoma cells (IC50 = 183 nM).21 While the compound demonstrated moderate inhibition of XIAP BIR3 (xBIR3) binding in an HTRF binding assay (IC50 = 290 nM)22 it was inactive in XIAP BIR2-3 (xBIR2-3) HTRF (IC50 >5lM) and was unable to inhibit the proliferation of the Type II A875 human melanoma cell line (IC50 >10 lM). The lack of cellular activity against a Type II cell line is not surprising since monovalent compounds bind preferentially to IAP BIR3 domains with reduced

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NO 2

NO 2

NO 2 NH2

Boc

O

b

a

+

N

HN

OH

O

7

6

Boc O N

c

N

H2 N O

N H

O

9

8

NH 2

N H

N

N H

O

O

N H

10

O OH

O HO

HN

Boc

Boc

N

H N

N

N H

N

O

d

12

NH 2

O

O

H N

O

O

11

Fmoc

N

Boc

O

O

N H

N

O HN

e

O

3

13 O

OH

Scheme 1. Reagents and conditions: (a) EDC, HOAt, Hunig’s base, DMF, rt, 87%; TFA, CH2Cl2, rt, 98%; (b) L-N-Boc-tert-Leu, EDC, HOAt, Hunig’s base, DMF, rt, 98%; TFA, CH2Cl2, rt, 100%; (c) N-Boc-N-Me-Ala, EDC, HOAt, Hunig’s base, DMF, rt, 97%; H2, Pd–C, 92%; (d) HATU, Et3N, DMF, 86%; LiOH–H2O, THF–MeOH, rt, 99%; (e) compound 10, HATU, Hunig’s base, DMF, rt, 84%; HCl, dioxane, rt, 75%.

O

O

O NH2

a

b

F

HN

14

F

O

N

H 2N O

17

O

Cbz

N

F

N O

c N

16

15 F

O

O

d

O

Boc O N

e

N

N H

O

18

O

OH

4

N

F

Scheme 2. Reagents and conditions: (a) p-Br-CH2Ph-CO2CH3, K2CO3, DMF, 78%; (b) N-Cbz-Tic acid, 1-chloro-N,N-2-trimethylpropenamine, CH2Cl2, Hunig’s base, 92%; (c) H2, MeOH, Pd-C, 92%; L-N-Boc-tert-Leu, HOAt, Hunig’s base, DMF, rt; TFA, CH2Cl2, rt, 60%; (d) N-Boc-N-Me-Ala, EDC, HOAt, Hunig’s base, DMF, rt, 82%; aq LiOH, MeOH, 97%; (e) compound 12, HATU, Hunig’s base, DMF, 62%; HCl, dioxane, rt, 100%.

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K. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5022–5029

O SH Boc

N H

O

O

S

a

OH

Boc

O

S

b

OH

N H

Boc

O

N H

N O

20

19

O

S Boc O N

N H

N O

NH

O

NH

d N

N H

O

HO O

Boc N

O

N H

S Boc O N

22

H 2N

N H

O

O

H N

NH 2

O

O

21

N Boc O

c

NO 2

O

N Boc

N H

Boc

O

+

N H

23

N O

H N

O

N H

e

Cbz

24 H Boc N H2 N

N Boc N

25

Boc N

N Boc

N H

Boc

f

5

Scheme 3. Reagents and conditions: (a) BrCH2CO2CH3, Hunig’s base, DMF, rt, 53%; (b) 8, EDC, HOAt, Hunig’s base, DMF, 77%; (c) TFA, CH2Cl2, rt, 100%; N-Boc-Ala, EDC, HOAt, Hunig’s base, DMF, rt, 86%; H2, 20% Pd(OH)2, MeOH, 84%; (d) 13, HATU, Hunig’s base, DMF, 42%; LiOH, MeOH–H2O, 100%; (e) NaBH(OAc)3, EtOH, 54%; 1,3-bis-Boc thiourea, HgCl2, Et3N, DMF, rt, 91%; H2, 20% Pd(OH)2, EtOH–HOAc, 44%; (f) 23, HATU, Hunig’s base, DMF, rt, 26%; 4 N HCl in dioxane, rt, 100%.

ability to inhibit IAP BIR2 activities. It is expected that monovalent compound 2 stimulates MDA-MB-231 cell death through binding the cIAP1 BIR3 domain, inducing the proteins autoubiquitination resulting in extrinsic apoptotic pathway activation. When compound 2 was incorporated into bivalent analogs (Tables 1 and 2), the resulting compounds maintained potent inhibition of cBIR2-3 binding activity, but also inhibited the binding properties of xBIR3, xBIR2-3 as well as the growth of both MDA-MB-231 and A875 cell lines. Compound 3 also displayed potent rescue of xBIR2-3 inhibited caspase 3 activity with an EC50 of 65 nM.23 This is consistent with our previous findings that the BIR2 P3 binding pocket is flexible and accommodates a variety of P30 amino acid residues in the context of a bivalent compound binding to BIR2–BIR3.24 The biochemical SAR of bivalent IAP antagonists constructed using various linkers to tether P3 groups of the two corresponding monomeric molecules was relatively flat except the oxazole linker analog 30 against cBIR2-3 (Table 1), suggesting minimal participation of the linkers in binding to these proteins. The high cBIR2-3 inhibitory activity of compound 30 compared to the other analogs is not well understood. It is remarkable that the activity of analog 27, which contains a very rigid acetylenic benzamide linker, is similar to that of analog

28 with a flexible ethyl benzamide linker. We speculate that this conformational insensitivity to the linker stems from the flexibility of the peptide linker between the XIAP BIR1 and BIR2 domains as described by Sun et al.,25 thus allowing an induced fit of the protein to the bivalent molecule. We hypothesize that the P3 proline subunit of the bivalent antagonist first binds to the BIR3 domain with high affinity followed by intramolecular interaction of the P30 Tic containing subunit with the BIR2 domain. This induced fit is facilitated by the flexibility of the IAP protein and favorable entropic effect stemming from its intramolecular binding. These bivalent heterodimers displayed potent antiproliferative activities when tested in both the Type I and Type II cell lines, indicating they impact both the intrinsic and extrinsic anti-apoptotic pathways. The antiproliferative profile of the analogs differs in that compounds with a more lipophilic linker are generally more cytotoxic (e.g., 3 vs 30), potentially due to increased cell permeability as observed by others.13c Considering the high avidity of the bivalent molecules for the BIR2-3 protein and the reported examples of various linkers at different positions,10b bivalent analogs with a P3–P40 linker containing different P40 capping groups were explored. The BIR2 P4 binding site is shallower than that of BIR3 due to the former containing relatively

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K. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5022–5029

Table 1 SAR of P3–P30 linker (IC50, nM)

Table 2 P40 SAR with P3–P40 linker (IC50, nM)

P1

P2

H N

N H

HN

O

O

NH O

N H

N

P2

P1

P4

P3

P3 O

P4 O

N

N H

O NH

HN O

L

H N

O

H N

O

P1'

O

P2'

Number Linker (L)

N H

P1'

N

N H

O

N H

P3'

P4'

xBIR3 xBIR2-3 cBIR2-3

MDAA875 MB-231

N O

P2'

O

N R

P3'

P4'

Number R

xBIR3 xBIR2-3

cBIR2-3

MDA-MB-231 A875

31

58

57

25

30

101

28

3.3

7

1.0

3.3

60

10

NT

3.4

4.7

10

40

5

6.8

415

7.9

9.5

8

1.5

3.6

4

F

O 3

O

17

3.3

15

1.2

7.1 32

NH

F F

O 26

45

16

76

0.9

6.9

100

10

79

2.0

8.5

40

6.8

25

3.1

20

O 33

O

O

O

27

34

NT: not tested.

O

28

29

HN

N S

O

11

11

0.9

4.8

10

3.6

2

11

136

N

O 30

37

O

N H

O

O

bulky amino acid residues (K206 vs G306, and K208 vs T308).26 With this in mind, the large tetrahydronaphthyl group of 31 was replaced with smaller P40 capping groups. Analogs 4 and 32, with smaller amethyl mono- or di-fluorobenzyl P40 groups, were more potent when tested in the XIAP BIR2-3 binding and A875 antiproliferative assays compared to 31 (Table 2). Interestingly, analog 34 with the opposite stereochemistry of the methoxymethyl side chain compared to that of compound 3 was more potent in BIR2-3 binding assays than analog 33 (IC50 9.5 nM vs 40 nM). Furthermore, 34 is significantly more potent than 33 in the A875 antiproliferation assay (IC50 3.6 nM vs 415 nM). Modeling studies suggest that the bulkier methoxy side chain of 34 flips to occupy the bulk of the P40 binding site instead of the phenyl ring, thus avoiding a steric clash with the back of the P40 pocket.

Several analogs tested during our studies demonstrated variable aqueous solubilities prompting us to explore alternative side chains to improve physicochemical properties of the series. The P2 binding site of BIR2 engages in H-bonding and hydrophobic interactions with the P2 backbone, while the side chain of the P2 amino acid extends out toward solvent.27 Fortunately, the unnatural amino acid thiovaline was found to be a suitable replacement for the P2 valine of the ‘AVPI’ motif. Importantly, the thiovaline provides a handle to incorporate various polar groups onto its solvent-exposed free thiol functionality to improve the physicochemical properties of these molecules. Polyamines were of special interest since it is known that they facilitate cellular uptake via the cationic transport system.28 In addition, a polyarginine-conjugated Smac peptide has been reported to enhance cellular penetration.29 The polyamine-substituted analogs 5 and 35–37, having a total of 5–6 cationic charges per molecule, were proposed to serve as substrates for cationic transporters (Table 3). However, the overall cellular antiproliferative activities of these polycationic molecules were weaker than the non-polyamine analogs (Tables 1 and 2) which presumably enter cells through passive diffusion. Despite the overall high positive charge of these compounds, the polyamine analogs demonstrated meaningful cellular antiproliferative activities. Spermine analog 36 displayed better cellular potency

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K. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5022–5029 Table 3 P20 polyamine analogs (IC50, nM)

N H

O

O

H N

N H

N

O

R

O

O

NH

O

NH

S O

H N

Number

N O

R

3 3

With verapamil

35

H2N

36

H2 N

H N

H2 N

H N

H N

N H

5

N H

N H

N H

N H

N H

N

N H

HN HN

37

N H

HN

H N NH 2

O

N H

xBIR3

xBIR2-3

MDA-MB-231

A875

HCT 116/VM46

17

3.3

1.2

7.1

>2500 8

20

3.4

50

140

1560

10

2.4

30

120

340

9.5

2.2

1.79

40

310

3.6

8.9

101

NT

650

NH 2

NH2 N

N HN

N NH2HN

N H NH2

Figure 2. Pharmacodynamic protein profile from compound 3 treated A875 tumor xenografts.

relative to spermidine analog 35. Many of our monomeric and dimeric IAP inhibitors were found to be P-gp substrates (data not shown). Analog 3 was inactive against the P-gp-expressing HCT116/VM46 cell line, while its antiproliferative activity was potentiated in the presence of the P-gp inhibitor verapamil

(Table 3).30 Interestingly, the polyamine analogs demonstrated good antiproliferative activity in this P-gp-expressing cell line, and guanidine-containing polyamine analog 5 displayed the most potent cellular activities. Too many guanidinium ions in 37 led to lower cellular potency. Further studies are warranted to determine

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K. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5022–5029

(a)

References and notes

A875 Melanoma Xenograft Model 3500

Tumor Volume (mm3 )

3000 Vehicle 2500 1 mg/kg 2000 3 mg/kg 1500 5 mg/kg 1000 500

Dose q3dx6; iv

0 20

25

30

35

40

45

50

55

60

65

Days Post Implant

Tumor Volume (mm 3 )

(b)

PANC1 Pancreatic Ca Xenograft Model

2000 1800 1600 1400 1200 1000 800 600 400 200 0

Vehicle 10 mg/kg

Dose q3dx6; iv 5

10

15

20

25

30

35

40

Days Post Implant Figure 3. In vivo efficacy of compound 3.

the tissue distribution and toxicity profiles of these polyamine containing analogs. Several of the bivalent heterodimeric compounds described herein induced pharmacodynamic markers of apoptosis and demonstrated potent in vivo efficacy in human tumor xenograft models implanted in athymic mice. For example, sustained degradation of cIAP1 protein31 followed by time dependent processing of caspase3 and PARP cleavage was apparent in lysates from staged (200– 300 mm3) A875 human melanoma tumors treated with compound 3 at a dose of 1.0 mg/kg (Fig. 2). In a subsequent A875 in vivo efficacy study using mice staged with large (750 mm3) tumors compound 3 provided significant antitumor activity at all three dose levels (1, 3 and 5 mg/kg) tested (Fig. 3a).32 Tumor regression was observed at both the 3 and 5 mg/ kg dose levels (iv dosing starting on day 20) without noticeable changes in average body weight (4% to +5%). The compound was also efficacious when administered at 10 mg/kg (q3dx6, iv) to mice with human PANC1 pancreatic tumors at 100 mm3 average size, showing comparable activity to gemcitabine administered at a 30 mg/kg dose (q2dx5, iv Fig. 3b). In the A875 xenograft model compound 4 was also efficacious with regression at 5 mg/kg (q3dx6, iv dosing, data not shown) without noticeable changes in average body weight. In conclusion, we have identified potent tetrahydroisoquinoline containing bivalent heterodimeric IAP antagonists with P3–P30 and P3–P40 linkers. These compounds displayed potent inhibitory activities in XIAP BIR3, XIAP BIR2-3 and cIAP1 BIR2-3 assays as well as antiproliferative cellular assays with both Type I and Type II tumor cell lines. Compounds 3 and 4 were efficacious in a human melanoma A875 xenograft model providing tumor regression at doses of 65 mg/kg, and compound 3 was efficacious in a human pancreatic PANC1 xenograft model at 10 mg/kg. Polyamine analogs such as 5 exhibited good antiproliferative cellular activity in the P-gp-expressing HCT 116/VM46 cell line.

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(a) Cai, Q.; Sun, H.; Peng, Y.; Lu, J.; Nikolovska-Coleska, Z.; McEachern, D.; Liu, L.; Qiu, S.; Yang, C.-Y.; Miller, R.; Yi, H.; Zhang, T.; Sun, D.; Kang, S.; Guo, M.; Leopold, L.; Yang, D.; Wang, S. J. Med. Chem. 2011, 54, 2714; (b) Seneci, P.; Bianch, A.; Battaglia, C.; Belvisi, L.; Bolognesi, M.; Caprini, A.; Cossu, F.; de Franco, E.; de Matteo, M.; Delia, M.; Drago, C.; Khaled, A.; Lecis, D.; Manzoni, L.; Marizzoni, M.; Mastrangelo, E.; Milani, M.; Motto, I.; Moroni, E.; Potenza, D.; Rizzo, V.; Servida, F.; Turlizzi, E.; Varrone, M.; Vasile, F.; Scolastico, C. Bioorg. Med. Chem. 2009, 17, 5834; (c) Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.; NikolovskaColeska, Z.; McEachem, D.; Yang, C.-Y.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. J. Med. Chem. 2011, 54, 3306. 14. (a) Sweeney, M. C.; Wang, X.; Park, J.; Liu, Y.; Pei, D. Biochemistry 2006, 45, 14740; (b) Eckelman, B. P.; Drag, M.; Snipas, S. J.; Salvesen, G. S. Cell Death Differ. 2008, 15, 920. 15. Zhang, Y.; Fang, H.; Xu, W. Curr. Protein Pept. Sci. 2010, 11, 752. 16. Anderson, P. C., European Patent Application 1990, EP 401676 A1. 17. Wright, A. T.; Song, J. D.; Cravatt, B. F. J. Am. Chem. Soc. 2009, 131, 10692. 18. Blagbrough, I. S.; Geall, A. J. Tetrahedron Lett. 1998, 39, 439. 19. (a) Poss, M. A.; Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z. Synth. Commun. 1993, 23, 1443; (b) Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677. 20. Peptide fluorescence polarization assays (FPA). Assays were performed in black, flat-bottom, 96-well reduced volume HE microplates. The final assay volume was 30 lL prepared from additions of fluoresceinated modified dimeric SMAC peptide, His-Tb-cBir2-3(154–352) and test compounds in assay buffer consisting of 20 mM sodium phosphate, 1 mM EDTA, 50 mM NaCl, and 0.05% Pluronic F68. The reaction mixture was incubated at room temperature for 60 min and fluorescence polarization of the reaction was detected on the LJL Analyst Plate Reader (LJL Biosystems). Inhibition data were calculated from mP values generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 5.9 nM N-His-Tb-cBir2-3(154– 352), 3 nM fluoresceinated modified dimeric SMAC peptide, and 1% DMSO. Dose response curves were generated to determine the concentration required for inhibiting 50% of polarization activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis. 21. MTS cell proliferation assay: Tumor cells were treated with compounds in the presence of TNFa for 72 h before adding MTS (Promega) reagents. MTS tetrazolium salt is bioreduced by live cells to form a formazan product which is soluble in aqueous solution. The measured absorbance value of formazan at 490 nm is directly proportional to the living cell numbers in the culture. Assays were performed in 96 well plates. Briefly cells were plated on day 0 and drug, dissolved at 10 mM in dimethylsulfoxide, added on day 1 in 8 serial 3 fold dilutions. TNFa at a final concentration of 2 ng/mL was included in all wells. Relative cell numbers were determined by adding MTS on day 4, incubating for 3 h at 37 °C and reading the absorbance at 490 nm wavelength in a Spectra Max 250 plate reader (Molecular Devices). The percent inhibition was determined by the following formula; %Inhibition = (1  ODavg treated/ODavg untreated)  100%. Sensitivity and IC50 values were determined by non-linear regression analysis using in-house software. 22. XIAP-BIR2-3 Dimeric SMAC Peptide Homogeneous Time Resolved (HTRF) Assays were performed in black, flat-bottom, 384-well plates. The initial assay volume was 40 lL prepared from additions of His-BIR2-3 (125-356, C202A/ C213G, XIAP), fluorescein labeled dimeric SMAC peptide, and test compounds in assay buffer consisting of 20 mM sodium phosphate, 1 mM EDTA, 50 mM NaCl, 50 lg/mL BSA, and 0.05% PLURONICÒ F68. The reaction mixture was incubated at room temperature for 60 min followed by a 10 lL addition of

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23.

24.

25. 26. 27.

28. 29. 30. 31.

mouse anti-6xHis-Tb IgG (Medarex, Cisbio). The final reaction mixture (50 lL) was incubated for an additional 30 min at room temperature. The HTRF signal, ratio of fluorescence intensities at emission wavelengths for fluorescein acceptor (520 nm) and terbium donor (615 nm), the 520/615 ratio, generated by the reaction was then measured on the Envision Plate Reader. Inhibition data were calculated from the 520/615 ratio generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 0.5 nM N-His-BIR2-3(125-356, C202A/C213G, XIAP), 20 nM fluorescein labeled dimeric SMAC peptide, 0.25 nM anti-His-Tb-Fab, and 0.1% DMSO. Dose response curves were generated to determine the concentration required for inhibiting 50% of the HTRF signal (IC50). Compounds were dissolved at 3 mM in dimethylsulfoxide (DMSO) and evaluated at eleven serially diluted concentrations. IC50 values were derived by non-linear regression analysis. Further details on HTRF assay development and optimization will be submitted for publication elsewhere. Caspase 3 activation assay: Human monocytic THP-1 cells were used as a source of caspase 3 following the protocol described in Sun, W.; NikolovskaColeska, Z.; Qin, D.; Sun, H.; Yang, C. Y.; Bai, L.; Qiu, S.; Wang, Y.; Ma, D.; Wang, S. J. Med. Chem. 2009, 52, 593. We found that bivalent IAP antagonists release caspase-3 from XIAP-mediated inhibition by binding to both the BIR3 and BIR2 domains despite the poor BIR2binding affinities of each independent monomeric component. This work will be submitted for publication elsewhere. Sun, C.; Cai, M.; Gunasekera, A. H.; Meadows, R. P.; Wang, H.; Chen, J.; Zhang, H.; Wu, W.; Xu, N.; Ng, S. C.; Fesik, S. W. Nature 1999, 401, 818. Wu, G.; Wagner, K. W.; Bursulaya, B.; Schultz, P. G.; Deveraux, Q. L. Chem. Biol. 2003, 10, 759. Lukacs, C.; Belunis, C.; Crowther, R.; Danho, W.; Gao, L.; Goggin, B.; Janson, C. A.; Li, S.; Remiszewski, S.; Schutt, A.; Thakur, M. K.; Singh, S. K.; Swaminathan, S.; Pandey, R.; Tyagi, R.; Gosu, R.; Kamath, A. V.; Kuglstatter, A. Acta Crystallogr. Sect. D 2013, D69, 1717. Xie, S.; Wang, J.; Zhang, Y.; Wang, C. Expert Opin. Drug Deliv. 2010, 7, 1049. Yang, L.; Mashima, T.; Sato, S.; Mochizuki, M.; Sakamoto, H.; Yamori, T.; Ohhara, T.; Tsuruo, T. Cancer Res. 2003, 63, 831. Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Cancer Res. 1981, 41, 1967. Pharmacodynamic (PD) effects following treatment of A875 tumor bearing mice. Female Balb/C athymic (nu+/nu+) mice (n = 2 per time point) were implanted with A875 tumor fragments subcutaneously in the hind flank using

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an 18 g trocar. Approximately two weeks post implant, when tumor sizes reached 200–300 mm3, intravenous dosing was initiated using gavage needles with compound 3 at the indicated concentrations in vehicle (7.5% 1 M NaCl, 12% hydroxypropyl beta-cyclodextrin 80.5% water). Lysates were prepared from excised tumor tissue at various time points following treatment and analyzed by immunoblot for markers of IAP antagonism and apoptosis. 32. In vivo Xenograft Models: Tumor fragments were implanted subcutaneously in the hind flank of 6–8 weeks old female Balb/C athymic (nu+/nu+) mice (n = 8). Intravenous dosing was initiated when A875 tumor sizes reached 750 mm3 (day 20) and Panc1 tumor reached 100 mm3 (day 8) using gavage needles with either compound at the indicated concentrations or vehicle in the control group. Tumor growth was assessed twice weekly by vernier caliper measurement. Antitumor activity was determined by calculating the maximum percent tumor growth inhibition (TGI) of treated animals at the indicated time points using the formula: %TGI = {(Ct  Tt)/(Ct  C0)}  100, where Ct = the median tumor volume (mm3) of vehicle treated control (C) mice at time t. Tt = median tumor volume of treated (T) mice at time t. C0 is the median tumor volume of control mice at time 0. Activity is defined as a continuous %TGI >70% for two tumor volume doubling times after the start of drug treatment.

Further reading 33. Spectroscopic data for 3 as 2 HCl salt: 1H NMR (400 MHz, CD3OD) d 8.06 (d, J = 2.6 Hz, 4H), 7.82 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 7.25 (br s, 1H), 7.18–7.05 (m, 6H), 5.22–5.13 (m, 4H), 5.03 (m, 1H), 4.83–4.73 (m, 3H), 4.66 (s, 1H), 4.60 (m, 1H), 4.18 (m, 1H), 3.98–3.86 (m, 3H), 3.69 (s, 2H), 3.22–3.14 (m, 2H), 2.84 (m, 4H), 2.68 (s, 6H), 2.20–2.13 (m, 1H), 1.97 (br s, 2H), 1.89–1.77 (m, 4H), 1.49 (d, J = 7.0 Hz, 4H), 1.41 (d, J = 7.0 Hz, 2H), 1.20 (s, 6H), 1.11 (s, 12H); MS(ESI+) m/z 1108.0 (M+H)+. 34. Spectroscopic data for 4 as 2 HCl salt: 1H NMR (400 MHz, CD3OD) d 8.57 (d, J = 8.6 Hz, 0.5H), 7.96–7.80 (m, 0.5H), 7.73–7.59 (m, 1.5H), 7.56–7.46 (m, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.39–6.84 (m, 11.5H), 5.98–5.80 (m, 1H), 5.26–4.97 (m, 4H), 4.78–4.49 (m, 6H), 4.22–3.76 (m, 4H), 3.16–3.04 (m, 1H), 2.97–2.71 (m, 3H), 2.70–2.46 (m, 7H), 2.17–1.74 (m, 5H), 1.70 (d, J = 6.8 Hz, 2H), 1.62–1.51 (m, 1H), 1.47 (d, J = 6.8 Hz, 3H), 1.41–1.26 (m, 3H), 1.19–0.97 (m, 18H); MS(ES+) m/z 1070.8 (M+H)+.