Discovery of a novel class of dimeric smac mimetics as potent IAP antagonists resulting in a clinical candidate for the treatment of cancer (AZD5582)

Article

pubs.acs.org/jmc

Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP

Antagonists Resulting in a Clinical Candidate for the Treatment of

Cancer (AZD5582)

Edward J. Hennessy,* Ammar Adam, Brian M. Aquila, Lillian M. Castriotta, Donald Cook,

Maureen Hattersley, Alexander W. Hird, Christopher Huntington, Victor M. Kamhi, Naomi M. Laing,

Danyang Li, Terry MacIntyre, Charles A. Omer, Vibha Oza, Troy Patterson, Galina Repik,

Michael T. Rooney, Jamal C. Saeh, Li Sha, Melissa M. Vasbinder, Haiyun Wang, and David Whitston

Oncology iMed, Innovative Medicines & Early Development, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham,

Massachusetts 02451, United States

S

* Supporting Information

ABSTRACT: A series of dimeric compounds based on the

AVPI motif of Smac were designed and prepared as

antagonists of the inhibitor of apoptosis proteins (IAPs).

Optimization of cellular potency, physical properties, and

pharmacokinetic parameters led to the identiﬁcation of

compound 14 (AZD5582), which binds potently to the

BIR3 domains of cIAP1, cIAP2, and XIAP (IC₅₀= 15, 21, and

15 nM, respectively). This compound causes cIAP1 degrada-

tion and induces apoptosis in the MDA-MB-231 breast cancer

cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14

results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two

weekly doses of 3.0 mg/kg. Antiproliferative eﬀects are observed with 14 in only a small subset of the over 200 cancer cell lines

examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo proﬁle, 14 was nominated as a

candidate for clinical development.

receiving a proapoptotic signal, a variety of intracellular signaling

process occur, eventually culminating in the activation of

cysteine-dependent aspartyl speciﬁc proteases (caspases) that

are responsible for eﬀecting cell death. In recent years, it has

become evident that particular members of a family of

structurally related proteins known as the inhibitor of apoptosis

proteins (IAPs) play a role in the suppression of such

proapoptotic signaling in mammalian cells.^5,6The IAPs are

characterized by the presence of one to three repeating domains

of ∼70−80 amino acids, known as baculovirus IAP repeat (BIR)

domains. Certain members of the IAP family, such as X

chromosome-linked IAP (XIAP), function by directly binding to

and sequestering proapoptotic caspases (such as caspase-3,

caspase-7, and caspase-9) that are critical to the apoptotic

process. In particular, the BIR3 domain of XIAP has been shown

to directly bind caspase-9, preventing the self-dimerization

necessary for caspase-9 catalytic activity.⁷Caspase-3 and caspase-

7 bind to the XIAP BIR2 domain and to a linker region between

the BIR1 and BIR2 domains that blocks the caspase-3 and

caspase-7 catalytic sites.⁸⁻¹⁰Other IAP family members, such as

cellular IAP1 (cIAP1) and cellular IAP2 (cIAP2), have been

shown to play a complementary role in apoptotic signaling owing

INTRODUCTION

■

The process of programmed cell death, known as apoptosis, plays

a critical role in the maintenance of homeostasis and in regulating

the amount of cells in higher organisms.¹Apoptosis plays key

roles during development and aging and is the means by which

damaged or unwanted cells are removed. Aberrations in the

apoptotic process would thus be expected to disrupt this

homeostasis, and, indeed, abnormal apoptosis is implicated in a

variety of diseases, including autoimmune disorders, viral

infections, degenerative diseases of the central nervous system,

and cancer.^2,3

The development of resistance to standard-of-care therapies in

the treatment of cancer is a major cause of disease progression

and represents a critical challenge in the development of novel

cancer therapeutics. Although there can be many causes behind

this acquired drug resistance, a major mechanism of resistance

lies in the dysfunctional survival signaling in cancer cells and the

reluctance of these cells to undergo apoptosis. Indeed, the

propensity of cancerous cells to avoid undergoing apoptosis has

long been recognized as one of the hallmarks of cancer.⁴Thus,

agents that can restore sensitivity of these cells to various

proapoptotic cues might conceivably be employed as cancer

therapies, either alone or in combination with other drugs.

Cells can undergo apoptosis in response to a variety of stimuli,

such as chemotherapy or radiation-induced cellular stresses. On

Received: July 16, 2013

Published: December 9, 2013

9897

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

to the E3 ubiquitin ligase activity of a region of these proteins

called the RING domain.¹¹Speciﬁcally, cIAP1 and cIAP2 can

inhibit apoptosis through ubiquitination of key proteins involved

in canonical and noncanonical NF-κB signaling^12,13and TNF

receptor-mediated activation of caspase-8,¹⁴resulting in their

proteasomal degradation. XIAP, cIAP1, and cIAP2 are frequently

overexpressed in certain types of cancer and are associated with

high-grade disease and poor prognosis.^15,16Moreover, down-

regulation of these IAPs has been shown to restore sensitivity to

apoptotic stimuli.¹⁷Taken together, these features provide a

compelling argument for targeting the IAP family of proteins as a

potential strategy for developing an anticancer agent.

One of the proteins released from mitochondria in response to

apoptotic stimuli, known as Smac (second mitochondrial

activator of caspases), is the endogenous inhibitor of a number

of members of the IAP family. Upon its release into the

cytoplasm, the N-terminal portion of Smac is cleaved, revealing a

region of the peptide that can bind to a groove on the BIR

domains of XIAP. This interaction interferes with the ability of

caspases to bind eﬀectively to XIAP and thus the caspases are

displaced and freed to execute the apoptotic process. Smac also

binds with high aﬃnity toward cIAP1 and cIAP2. The interaction

between Smac and the IAPs has been well-characterized, and it

has been established that the bulk of this binding interaction

involves the four N-terminal amino acid residues of activated

Smac (alanine−valine−proline−isoleucine, AVPI).^18,19In fact,

the tetrapeptide AVPI itself (1; Figure 1) is a moderately potent

can be dosed in vivo to eﬀect antitumor activity. These eﬀorts

have been extensively reviewed,²¹⁻²⁵and a number of

compounds have advanced to human clinical trials. Furthermore,

because Smac protein is known to exist in a dimeric state in

solution and interacts with the IAPs as a dimer,²⁶a number of

examples of bivalent compounds (in which two monomeric

Smac mimetics are covalently linked) have been described in the

literature. These dimers have been shown to interact

concurrently with both the BIR2 and BIR3 domains of

XIAP²⁷⁻²⁹and as a result are typically much more eﬀective

inhibitors than the corresponding monomers in cellular assays

measuring IAP inhibition. Indeed, several dimeric Smac mimetics

are also being evaluated as anticancer agents in clinical trials.²⁴

As part of our drug-discovery program aimed at identifying

novel cancer therapies, we too became interested in the IAP

family of proteins as molecular targets for small-molecule

inhibitors. In this article, we describe our eﬀorts at identifying

novel dimeric Smac mimetics as potential anticancer agents,

culminating in the nomination of a compound as a candidate for

clinical evaluation.

RESULTS AND DISCUSSION

■

At the outset of our studies, we wanted to identify appropriate

positions from which monomeric Smac mimetics could be

eﬀectively dimerized. In a seminal paper surveying the eﬀects of

varying each residue (P1−P4) of the AVPI motif,³⁰Fesik and co-

workers identiﬁed compounds in which the (R)-tetrahydro-

naphthyl and (R)-indanyl groups served as eﬀective replace-

ments for the isoleucine residue at P4 (Figure 1; 2 and 3). Our

analysis of the published structure of (R)-tetrahydronaphthyl

compound 2 bound to the BIR3 domain of XIAP³⁰suggested

that there is room for further substitution at the solvent-exposed

2-position of the tetrahydronaphthyl ring and that this might be a

suitable position to introduce a linkage for dimerization (Figure

2).

In addition to the location of the linker between two

monomeric units, we wanted to choose a linker that would

have minimal steric requirements to prevent disruption of critical

binding interactions between the remainder of the molecule with

the target protein. Given that a 1,3-bisacetylene linkage has been

employed in several previously described eﬀective dimeric Smac

mimetics^27,31and that this linear type of linkage was anticipated

to contribute minimal steric bulk, our initial explorations were

focused on installation of a propargylic ether at the

tetrahydronaphthyl 2-position and subsequent homocoupling

to aﬀord the diyne-containing bivalent compounds.

Figure 1. Structures of AVPI (1) and monomeric Smac mimetics

containing tetrahydronaphthyl or indanyl groups at the P4 position (2

and 3).

To test our initial hypothesis, we ﬁrst assessed the eﬀects of

incorporating a substituent at the 2-position of the tetrahy-

dronaphthyl ring on binding to the IAP proteins of interest. The

four possible diastereomeric matched pairs of 2 were thus

prepared, and the binding aﬃnities of these compounds to the

inhibitor of the interaction between a biotinylated Smac 7-mer

peptide and the BIR3 domain of XIAP (IC₅₀= 0.58 μM).²⁰Given

the promise of antagonists of the IAP family of proteins as

potential anticancer agents, a signiﬁcant amount of work has

been invested in the design of nonpeptide Smac mimetics that

Figure 2. Dimerization strategy based on incorporating a linker at the 2-position of the tetrahydronaphthyl ring.

9898

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

Table 1. cIAP1 BIR3 and XIAP BIR3 Binding Data for the Four Diastereomeric Monomers That Contain a Propargylic Ether at the

Tetrahydronaphthyl 2-Position

Table 2. Cellular Potency Data for the Diastereomeric Monomers and Dimers Containing a 2-Substituted Tetrahydronaphthyl P4

Moiety

tetrahydronaphthyl stereochemistry

(1S,2R)

example

MDA-MB-231 cIAP1 degradation EC₅₀(nM)

MDA-MB-231 antiproliferative GI₅₀(nM)

4

monomer

dimer

4

16

8

0.2

126

4

0.1

192

97

(1R,2S)

(1S,2S)

(1R,2R)

5

monomer

dimer

9

6

monomer

dimer

10

0.6

>370

2

45

10

7

0.3

2190

61

monomer

dimer

11

BIR3 domains of both cIAP1 and XIAP were determined.

Speciﬁcally, the assays used measured the ability of the

compounds to competitively displace a 5-carboxyﬂuorescein-

tagged synthetic peptide from the AVPI binding groove using

ﬂuorescence polarization (see the Supporting Information for

full experimental details). In accord with the previously

published ﬁndings,³⁰we found that the absolute stereochemistry

of the amino-substituted carbon of the tetrahydronaphthyl group

was critical to eﬀective binding to the BIR3 domains of both

cIAP1 and XIAP (Table 1), with the (1S)-amino analogues (4

and 6) being more potent than the (1R) isomers (5 and 7).

Interestingly, the stereochemistry at the ether-substituted carbon

appears to aﬀect the magnitude of the dropoﬀ in potency

between the (1S) and (1R) isomers. For example, (1R,2R)

analogue 7 is nearly 30-fold less potent in the cIAP1 BIR3

binding assay and is over 60-fold less potent in the XIAP BIR3

assay than (1S,2R) compound 4. However, (1R,2S) isomer 5 is

only 3−6-fold less potent than (1S,2S) isomer 6 in these assays.

The impact of stereochemistry is even more pronounced in

assays that assessed the eﬀects of compounds in cells. For

example, it has been demonstrated that the binding of Smac

mimetics to cIAP1 in MDA-MB-231 cells can lead to cIAP1

autoubiquitination followed by proteasomal degradation of the

protein.^12,13We found that monomeric (1S) isomers 4 and 6

induce cIAP1 degradation in MDA-MB-231 cells at lower

concentrations than (1R) isomers 5 and 7 (Table 2). Moreover,

certain cancer cell lines that produce autocrine TNFα (e.g.,

MDA-MB-231 and HCC461) have been shown to be

particularly sensitive to IAP antagonists as single agents,³²and,

indeed, (1S) isomers 4 and 6 are more potent inhibitors of MDA-

MB-231 cell proliferation than are (1R) isomers 5 and 7 (Table

2). These observed diﬀerences in cellular potency between

diastereomers are consistent with the diﬀerences in their ability

to bind to the BIR3 domains of cIAP1 and XIAP, as discussed

earlier. Because we and others have previously shown that

inhibition of both cIAP1 and XIAP is necessary to inhibit

proliferation in such cell lines (including MDA-MB-231),^33,34

the antiproliferative activity observed for compounds 4 and 6

therefore suggests eﬀective binding of these compounds to both

proteins in cells. Thus, inclusion of the propargylic ether moiety

did not seem to have a detrimental eﬀect on either binding

aﬃnity or cellular potency of monomeric Smac mimetics relative

to 2, with the (1S,2R) conﬁguration of the tetrahydronaphthyl

amino alcohol giving the most potent analogue (4).

Because the above data suggested that dimerization of these

monomers through the alkyne should be tolerated with minimal

eﬀect on binding aﬃnity, we next prepared the bivalent analogues

of the compounds in Table 1. A marked increase in

antiproliferative activity was observed with the dimers (Table

2; compounds 8−11), consistent with the postulate that the

dimeric compounds can bind to both the BIR2 and BIR3

domains of XIAP simultaneously. This binding is expected to

9899

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

a

Table 3. Eﬀects of Variation of the P2 Residue on Cellular Potency, Lipophilicity, and Polar Surface Area

a

Values for cLogP and Log D calculated using ACD LogD software (v. 12.01). PSA, polar surface area; NPSA, nonpolar surface area.

disrupt the interactions between XIAP and caspases-3, -7, and -9,

eventually culminating in apoptosis.

mimetics and the well-established paradigm that the likelihood of

a compound being orally bioavailable drops oﬀ signiﬁcantly

above a molecular weight of 500,³⁷it was anticipated that these

compounds would have limited oral exposure. Indeed, the other

dimeric Smac mimetics under evaluation in human clinical trials

are dosed intravenously²⁴and thus we surmised that a compound

based on the scaﬀold described herein would require suﬃcient

aqueous solubility to be a viable candidate drug. Although cell-

potent dimers 8 and 12 did not appear to have limitations in this

respect (measured solubility of >1000 μM at pH 7.4), we wanted

to generate additional compounds with favorable properties as

candidates for in vivo testing.

As a result, we next set out to study the eﬀects of modiﬁcations

at the P2 position of the AVPI motif. The side chain of this

residue is directed away from the AVPI binding groove and as a

result does not contribute to speciﬁc binding interactions with

the protein. Therefore, the P2 residue is tolerant of a range of

diﬀerent substituents of varying polarity,^20,30,38and, in fact, this

position has been utilized as a site of dimerization

previously.^27,39,40Our expectation was that variation of this

residue would provide an opportunity to modulate the physical

properties (and possibly the pharmacokinetic proﬁles) of the

resulting dimers. However, we found that the cellular potency

was greatly aﬀected by the nature of this side chain, with

compounds containing only more lipophilic groups demonstrat-

ing eﬀects on cIAP1 degradation or cell proliferation at the

concentrations evaluated (Table 3).

A signiﬁcant increase in potency in the cIAP1 degradation cell

assay was also observed for all of the dimers relative to the

respective monomers (Table 2). In contrast to the binding of

dimeric compounds to both the BIR2 and BIR3 domains of

XIAP, previously described bivalent Smac mimetics have been

demonstrated to bind only to the BIR3 domain of cIAP1 (and

not the BIR2 domain).^12,35As a result, dimers can concurrently

bind to the BIR3 domains of two cIAP1 molecules. The E3 ligase

activity of cIAP1 that results in autoubiquitination and protein

degradation has been shown to be dependent on dimerization of

the RING domain of the protein;³⁶therefore, it is likely that by

binding to the BIR3 domains of two cIAP1 molecules

simultaneously, these bivalent compounds can facilitate RING

domain dimerization at lower concentrations than the

monomeric compounds.

Having demonstrated that functionalization of the tetrahy-

dronaphthyl moiety at the 2-position is tolerated and that

dimerization from this position aﬀords cell-potent inhibitors of

cIAP1 and XIAP, we next prepared the corresponding

compounds containing an indanyl group in the P4 position.

Our interest in these analogues was not only the expectation that

binding to the target proteins would not be drastically aﬀected

but also that the requisite chiral aminoindanol starting material is

readily available from commercial sources, facilitating the

preparation of multigram quantities of intermediates for further

SAR development. Thus, synthesis of matched pair of 8

(compound 12; Table 3) conﬁrmed that dimerization through

the indanyl 2-position is tolerated with no apparent loss in

cellular potency relative to the tetrahydronaphthyl analogue.

A key goal of our chemistry strategy was to identify

compounds with physical properties that would be compatible

with intravenous administration. Given the very high molecular

weight (typically greater than 1000) of these dimeric Smac

The impact of lipophilicity on cellular potency has been

discussed previously within the context of Smac mimetics,²⁰as

compounds containing less lipophilic groups are often less

potent in cells. This phenomenon is likely the result of poor

membrane permeability, a well-documented characteristic of

peptides and peptidic compounds attributable to a combination

of low lipophilicity and high polar surface area (PSA).⁴¹The

diﬀerences between the cellular potencies of the compounds in

9900

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

Table 4. Modiﬁcations to the P3 Residue and Eﬀects on Cellular Potency and Physical Properties

Table 5. Modiﬁcations to the Linker Region of Dimeric Smac Mimetics

Table 3 can thus be rationalized by comparing parameters for

lipophilicity⁴²and polar surface area (corrected for the positive

contribution to membrane permeability from nonpolar surface

area, according to the method of Stenberg et al.).⁴³Although cell-

potent compounds 12 and 14 have low measured Caco-2

permeability (apparent apical-to-basolateral permeability <0.1 ×

9901

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

Table 6. Dimeric Smac Mimetics Containing Bisamide Linkers

10⁻⁶cm/sec), the compounds containing more polar side chains

at P2 are even more poorly permeable (below the level of

detection), eﬀectively preventing intracellular exposure.

although we found that this compound proved to be somewhat

less eﬀective at inducing cIAP1 degradation and inhibiting cell

proliferation than the corresponding 1,2-disubstituted com-

pounds.

Having demonstrated the necessity for relatively lipophilic

groups to attain cell-potent compounds, we were mindful of the

impact on physical properties that this restriction imposed. To

assess the room for improving upon these compounds, we next

considered modiﬁcations to the proline ring of the AVPI motif

(P3) to see what impact these changes would have. Dimeric

compounds containing a variety of proline mimetics were

prepared and assessed for their IAP antagonistic activity (Table

4). By and large, the changes made to the proline ring were well-

tolerated in terms of cell potency. However, many of the

compounds resulting from these changes had poor aqueous

solubility coupled with high plasma protein binding, limiting the

attractiveness of this approach to identify an agent suitable for

intravenous dosing.

In addition to the compounds described above, we also

investigated alternative means of connecting two monomeric

Smac mimetics through the same region of the scaﬀold (Table 5).

In line with our observations on the binding aﬃnities for the

diﬀerent diastereomers of the tetrahydronaphthyl ring system,

trans-aminoindanol dimer 24 demonstrated comparable levels of

cellular potency to cis-aminoindanol analogue 14. Similarly, we

found that saturation of the diyne moiety of the linker (example

25) does not result in any appreciable change in the cellular

potencies relative to the unsaturated matched pair. Replacing this

n-alkyl chain with a polyether linker (26) likewise aﬀorded a cell-

potent Smac mimetic. Dimerization through a tetrahydroquino-

line P4 group (compound 27) also resulted in potent cell activity.

In an attempt to remove the additional chiral centers imparted by

the substituted tetrahydronaphthyl or indanyl systems described

above, we designed and prepared spiro-aminoamide analogue 28,

In addition to the dimerization linkage through an oxygen

atom at the 2-position of the tetrahydronaphthyl or indanyl ring

system, we also investigated the feasibility of linking two

monomeric Smac mimetics through nitrogen atoms on the

indane ring (as a bisamide); the results of these eﬀorts are

detailed in Table 6. The bisamide linker was expected to be more

conformationally rigid than the corresponding bisether linker,

and, indeed, there does appear to be a dependence on the linker

length with respect to the cellular potencies of these amide

analogues. For example, terephthalamide analogue 29 is less

potent in the MDA-MB-231 proliferation assay than biphenyl

compound 30 or the more conformationally ﬂexible 31,

suggesting that the shorter linker is suboptimal for simultaneous

binding to both the BIR2 and BIR3 domains of XIAP. However,

the length or ﬂexibility of the linker would not be expected to

aﬀect binding to cIAP1 BIR3 drastically, and, indeed, compounds

29−31 have roughly equivalent binding aﬃnities to cIAP1 BIR3

(data not shown) despite the marked diﬀerences in cellular

cIAP1 degradation. Therefore, a more plausible explanation for

the diﬀerences in cellular potency is that the shorter, less

hydrophobic linker of 29 renders this compound less cell-

permeable and thus apparently less potent in cell-based assays.

This eﬀect has been observed previously in bivalent Smac

mimetics with linkers of diﬀerent lengths³⁵and would explain the

diﬀerences in cIAP1 degradation observed despite the similar

binding aﬃnities.

To investigate the cellular mechanism of action of this series

further, studies were conducted to elucidate the biochemical

eﬀects of compound binding to the diﬀerent IAP proteins in

9902

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

MDA-MB-231 cells. Consistent with the data presented in Table

3, compound 14 induces the degradation of cIAP1 in a dose-

dependent manner following 1 h of treatment; the degradation of

cIAP2 protein is likewise observed at similar concentrations

(Supporting Information, Figure S2). No changes in XIAP levels

are observed, however, even after 4 h of exposure to considerably

higher concentrations of 14 (Supporting Information, Figure

S3). These observations are consistent with the eﬀects of

previously described Smac mimetics in this cell line following

similar compound exposure times.¹²

In addition to studying the eﬀects of these Smac mimetics on

the IAP proteins themselves, we wanted to assess the ability of

this series to disrupt the binding of caspases by XIAP. In

particular, the binding of a Smac mimetic such as 14 to the BIR3

domain of XIAP would be expected to interfere with the

interaction between XIAP and caspase-9.⁷Thus, treatment of

MDA-MB-231 cells with 14 for 4 h followed by immunopreci-

pitation of caspase-9 from the cell lysates showed that the

interaction between XIAP and caspase-9 is indeed disrupted,

whereas untreated cells show intact caspase-9−XIAP complexes

(Figure 3).

the basis of these pharmacokinetic data (Table 7). In general,

compounds from this class of dimeric Smac mimetic are quickly

cleared in rat, with many compounds having rates of clearance

approaching or exceeding hepatic blood ﬂow (Q_Hfor rat = 72

mL/min/kg). These values imply an extrahepatic mechanism of

elimination from the plasma. Indeed, for each of the examples

shown in Table 7, the parent compound was detected unchanged

in the urine of the test animals, suggesting renal clearance as a

contributing secondary mechanism of elimination; the extent of

renal clearance varied from compound to compound (<1−14%

of total clearance). In addition, a bile duct-cannulated rat

pharmacokinetic study with 14 demonstrated that biliary

clearance contributed signiﬁcantly (∼30%) to the total clearance

of the compound. Although the other examples in Table 7 were

not subjected to the same experiment, it is feasible that biliary

secretion could play some role in the systemic elimination of

these compounds as well.

In spite of the apparent suboptimal pharmacokinetic proﬁles

for this class of dimers, we were optimistic about the ability of

these compounds to demonstrate in vivo antitumor activity

because of our observation of apoptosis induction (measured by

the extent of caspase-3 cleavage) and cell death in the MDA-MB-

231 cell line following short exposure times. In particular,

cultured MDA-MB-231 cells were treated with increasing

concentrations of compound for brief periods of time (1 and 3

h), at which point the cells were rinsed and exposed to fresh

media to remove the compound. The level of caspase-3 cleavage

was assessed after 24 h, whereas the amount of surviving cells

remaining was determined after a total of 72 h. In the case of

compound 14, substantial apoptosis induction was observed at

subnanomolar concentrations (Figure 4A) despite the cells being

exposed to compound for only the ﬁrst 1 or 3 h of the

experiment. The extent of caspase-3 cleavage correlated well with

the fraction of cells surviving after 72 h (Figure 4B). In contrast,

various monomeric Smac mimetics failed to show any

antiproliferative eﬀects under similar conditions (data not

shown). Although we have not ﬁrmly established the cause of

this prolonged activity, it is possibly a function of the mode of

inhibition of the dimers and the resulting eﬀects on the rate of

dissociation from the protein (k_off). For instance, dimeric Smac

protein has been demonstrated to bind with higher aﬃnity to an

XIAP construct containing both the BIR2 and BIR3 domains

relative to one containing the BIR3 domain alone, with a

resulting 100-fold decrease in k_off.⁴⁴Dimeric Smac mimetics are

similarly known to be much more potent IAP antagonists than

the corresponding monomers, likewise owing to avidity resulting

from simultaneous binding to the BIR2 and BIR3 domains of

XIAP.^12,28This would be expected to result in a much smaller net

k_offthat is a function of the dissociation rates from both the BIR2

and BIR3 domains. In fact, the binding of bivalent Smac mimetics

Figure 3. Eﬀect of compound 14 on caspase-9−XIAP interaction.

MDA-MB-231 cells were either untreated or exposed to 1 μM 14 for 4 h,

and then caspase-9 was immunoprecipitated from the protein lysates.

The levels of caspase-9−XIAP complexes were determined by blotting.

While working to establish what eﬀects structural modiﬁca-

tions to these compounds had on cellular potency and physical

properties, we also examined the eﬀects of these changes on the

rat pharmacokinetic proﬁles of the compounds. Being that a

variety of substitution patterns aﬀord cell-potent IAP antago-

nists, our aim was to prioritize compounds for further study on

a

Table 7. Pharmacokinetic Parameters for Representative Compounds Following Intravenous Dosing in Rat

example

dose (mg/kg)

CL (mL/min/kg)

V_ss(L/kg)

t_1/2(h)

AUC (ng h/mL)

8

0.2

0.5

0.8

1.0

0.2

0.5

111

44

9

3

1.9 0.2

2.3 0.1

0.8 0.1

1.4 0.5

1.9 0.4

1.2 0.4

1.4 0.5

0.5 0.1

1.6 0.8

0.5 0.1

3.6 0.3

1.2 0.4

0.6 0.1

6.6 2.3

30

76

3

5

3

13

14

25

26

27

29

50 0.8

166

45

80

5

1

262 35

206

40

1

9

68 15

37

8

230 46

a

CL, total plasma clearance; V_ss, volume of distribution at steady state; t_1/2, elimination half-life; and AUC, area under the curve.

9903

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

Figure 5. Pharmacodynamic data following a single intravenous dose of

compound 14 to mice bearing MDA-MB-231 xenografts. (A) Measured

tumor levels of cIAP1 protein degradation. (B) Levels of intratumoral

cleaved caspase-3.

the higher initial plasma concentrations following a bolus

intravenous dose. This explains the signiﬁcant activity seen

even at very low doses of 14 (Figure 5A). The cIAP1 levels would

only recover after protein resynthesis, thus resulting in the

prolonged duration of action we observe, similar to what has

been seen with previous Smac mimetics.⁴⁶Although cIAP1

degradation happens very quickly upon exposure to 14 in vivo,

apoptosis induction (as measured by the amount of cleaved

caspase-3) takes longer to reach a maximal eﬀect.^27,46Therefore,

the extent of caspase-3 cleavage in vivo is expected to be more

dependent on the duration of compound exposure above a

threshold concentration, and, as a result, we observe increased

levels of this activity at higher doses (Figure 5B). The levels of

cleaved caspase-3 then diminish over time, returning to near-

baseline levels at 18 h.

Having demonstrated eﬀects of IAP inhibition in vivo, the

ability of compound 14 to exert antitumor activity in the MDA-

MB-231 xenograft model was assessed (Figure 6). Orthotopi-

cally implanted tumors were allowed to grow to ∼120 mm³

before administering a single intravenous dose of 14. After 1

week, an additional dose of 14 was given to the mice, and the

tumors were monitored for regrowth. At the lowest dose tested

(0.1 mg/kg), very modest antitumor activity was observed.

However, at the higher doses (0.5 and 3.0 mg/kg) tumor

regressions occurred, with nearly complete disappearance of the

tumors at the 3.0 mg/kg dose. Regrowth occurred slowly and was

dependent on the initial dosage amount. For example, although

the tumors in the 0.5 mg/kg group returned to their original size

after approximately 10 days following the second dose, the

tumors in the 3.0 mg/kg group took roughly 6 weeks to return to

the size they were before the ﬁrst dose and even then they grew at

a slower rate until the end of the study.

Figure 4. Treatment of cells with compound 14 promotes caspase-3

cleavage and results in cell death. MDA-MB-231 cells were exposed to

14 for 1 or 3 h, and then the culture media was removed and replaced

with drug-free media. (A) Apoptosis was assessed by determining the

level of active caspase-3 in each well after 24 h total. (B) Cell survival was

determined by MTS assay after 72 h total. A reading of 0% survival

represents complete death of the cells.

to XIAP protein constructs containing both the BIR2 and BIR3

domains has been shown to result in conformational changes in

the protein,^28,29,40a phenomenon typically associated with slow

oﬀ kinetics.⁴⁵Therefore, it is feasible that dimeric Smac mimetics

such as 14 will also have a much smaller k_offthan the respective

monomers and will remain bound to the target protein even after

the cells are washed. Thus, these data suggest that apoptosis

induction and cell death should be observed in vivo despite the

relatively short plasma half-lives of this series of compounds.

To explore the potential of compound 14 to demonstrate

antitumor activity in vivo, we examined the pharmacokinetics of

the compound in mice to verify that suﬃcient plasma

concentrations could be achieved. Following a modest 0.5 mg/

kg intravenous bolus dose of 14, unbound plasma levels

remained above the concentrations at which apoptosis induction

and cell death were observed in MDA-MB-231 cells over the

course of several hours. Given the data presented in Figure 4, we

believed this would be a suﬃcient duration of exposure to show

eﬀects using in vivo models.

Compound 14 was administered intravenously to mice

bearing MDA-MB-231 xenografts, and measures of pharmaco-

dynamic activity (intratumoral levels of cIAP1 degradation and

cleaved caspase-3) were measured at 4, 6, and 18 h postdose

(Figure 5). Considerable degradation of cIAP1 was observed at

all doses examined, with no restoration in protein levels observed

over the course of 18 h. Because cIAP1 degradation is known to

occur rapidly following the binding of a Smac mimetic (2−5 min

in vitro),^12,13this eﬀect in vivo would likely be dependent upon

Compound 14 was proﬁled for its antiproliferative activity

across a number of cancer cell lines representing a variety of

tumor types (Figure 7). Of the 200 cell lines examined, only a

small subset of these are sensitive to 14 alone (for example, GI₅₀

≤ 1 μM in only 34 of the 200 cell lines, comparable to the activity

of other Smac mimetics⁴⁶in large cell panels). The most sensitive

9904

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

binds with high aﬃnity to the BIR3 domains of both cIAP1 and

cIAP2 as well as both the isolated BIR2 and BIR3 domains of

XIAP, with measured IC₅₀values eﬀectively at the lower limit of

detection under the assay conditions used (Table 8). In spite of

Table 8. Summary of Measured IC₅₀Values for Compound 14

IAP Protein BIR Domain

IC₅₀(nM)

a

cIAP1 BIR3

cIAP2 BIR3

XIAP BIR2

XIAP BIR3

15 10

b

21

c

21 13

d

15 12

a

b

c

Average of 12 measurements. Average of 2 measurements. Average

d

of 12 measurements. Average of 10 measurements.

its relatively high lipophilicity, the dihydrochloride salt of

compound 14 has suﬃcient aqueous solubility (>7 mg/mL at

pH 4−6) to enable formulation for intravenous administration at

the projected eﬃcacious doses. With respect to chemical stability,

14 was found to be photostable and hydrolytically stable between

pH 4−6, although some amide hydrolysis was observed under

strongly acidic (pH 1) and basic (pH > 8) conditions. In

addition, the compound is stable in the plasma of multiple

species, with no compound degradation observed after several

hours under physiological conditions. In light of the overall in

vitro proﬁle of compound 14 as well as its promising in vivo

activity at readily achievable and tolerable plasma concentrations,

this compound was nominated for clinical development as an

anticancer agent (AZD5582).

Figure 6. Antitumor activity of 14 in the MDA-MB-231 xenograft

eﬃcacy model. Tumors were allowed to grow to an average size of 120

mm³, and the mice were then treated with either vehicle or 14

intravenously once a week for 2 weeks (as indicated by the two black

arrows). Tumor volumes were monitored twice a week for 78 days and

are plotted as the geometric mean volumes standard error (SE).

CHEMISTRY

■

The Smac mimetics described herein were prepared by utilizing

standard peptide coupling reagents and reaction conditions, with

commercially available or readily accessible amino acids. The

preparation of the requisite tetrahydronaphthyl-based amino

alcohols followed a modiﬁcation of published procedures⁵⁰and is

outlined in Scheme 1. Treatment of 1,2-dihydronapthalene with

N-bromosuccinimide in aqueous THF aﬀorded racemic

bromohydrin 32, which, when exposed to ammonium hydroxide,

was transformed to the racemic trans-1,2-aminoalcohol 33 (via

the intermediate epoxide). Benzoylation of the amino group

aﬀorded racemic benzamide 34, which could be separated into

the individual enantiomers by chiral chromatography. The cis-

aminoalcohols were prepared from benzamides 34 by activation

of the hydroxyl group with thionyl chloride, resulting in

intramolecular S_N2 displacement of the intermediate chlor-

osulﬁte esters to aﬀord cis-oxazolines 35.⁵¹Acidic hydrolysis

revealed cis-aminoalcohols 33; comparison of the speciﬁc

rotations to literature values established the absolute stereo-

chemical conﬁguration of each enantiomer.^50,52,53Attempted

hydrolysis of 34 under acidic conditions (6 N HCl, 100 °C) to

recover the enantiopure trans-aminoalcohols instead resulted in a

mixture of the trans- and cis-isomers of 33, presumably through

the intermediacy of oxazoline 35. We instead found that

hydrolysis of the amide group under basic conditions (NaOH,

EtOH/H₂O) gave cleanly the pure enantiomers of trans-1,2-

aminoalcohol 33, with the assignment of absolute stereo-

chemistry of each made by correlation of the assignments for

the cis-aminoalcohols to the common precursor, 34. Comparison

of the speciﬁc rotations of each trans-33 to the previously

published value for the (1S,2S) enantiomer conﬁrmed these

assignments.⁵⁴

Figure 7. Antiproliferative activity of 14 across a panel of 200 cancer cell

lines. The cell lines are ranked in order of decreasing sensitivity to 14.

The horizontal line at pGI₅₀= 6 reﬂects an antiproliferative GI₅₀of 1

μM; those cell lines in which 14 is more potent (i.e., sensitive cell lines)

are colored green, whereas those in which 14 is less potent (i.e., resistant

cell lines) are colored red.

cell lines span a variety of tumor types, such as 97-7 (bladder),

647V (bladder), BT-549 (breast), and MiaPaCa2 (pancreatic).

Previous work has shown that several factors can aﬀect the

underlying senstivity of cancer cell lines to Smac mimetic

treatment, including expression levels of cIAP2^47,48or the cell

surface protein LRIG1.⁴⁹Although the reasons for the sensitivity

proﬁle we observe have not been determined for all cell lines in

this panel, the data in Figure 7 indicate that as a single agent

compound 14 does not exhibit broad-based cytotoxicity but

instead should be employed in selected tumor settings expected

to be sensitive to IAP inhibitors or in rational combinations with

other targeted therapies.

Consistent with the in vitro and in vivo data presented above,

ﬂuoresence polarization assays conﬁrmed that compound 14

9905

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

a

Scheme 1. Synthesis of Chiral Tetrahydronaphthyl Aminoalcohols

a

Reagents and conditions: (a) N-bromosuccinimide, THF/H₂O, rt; (b) NH₄OH, MeOH/H₂O, rt; (c) benzoyl chloride, NEt₃, CH₂Cl₂, 0 °C; (d)

NaOH, EtOH/H₂O, 80 °C; (e) SOCl₂, CH₂Cl₂, rt; (f) HCl, dioxane/H₂O, 100 °C.

a

Scheme 2. Representative Preparation of Monomeric and Dimeric Smac Mimetics

a

Reagents and conditions: (a) Boc-Pro-OH, EDCI, HOBt hydrate, DMF, 0 °C to rt; (b) propargyl bromide, KOH, DMF, 0 °C; (c) 4 N HCl/

dioxane, rt; (d) Boc-Tle-OH, EDCI, HOBt hydrate, 4-methylmorpholine, DMF, 0 °C to rt; (e) Boc-N-Me-Ala-OH, EDCI, HOBt hydrate, 4-

methylmorpholine, DMF, 0 °C to rt; (f) Cu(OAc)₂, pyridine, CH₃CN, 80 °C.

A general preparation of the Smac mimetics described in this

article is shown in Scheme 2, which details the synthesis of

compounds 4 and 8. Coupling of (1S,2R)-cis-33 to Boc-proline

under standard peptide-bond-formation conditions (EDCI,

HOBt, DMF) gave amide 36, which could be selectively

propargylated on the free hydroxyl group using KOH in DMF

to give 37. This was then transformed to Boc-protected

monomeric Smac mimetic 39 by an iterative sequence of

deprotection followed by amide bond formation (ﬁrst with Boc-

tert-leucine and then with Boc-N-methylalanine). Final depro-

tection gave monomer 4.

Monomeric Boc-protected terminal alkyne-containing Smac

mimetic 39 was homocoupled to aﬀord the diyne by heating in

the presence of copper(II) acetate and a stoichiometric base

(such as pyridine or triethylamine). The reaction progress was

monitored carefully, as prolonged reaction times were found to

result in unidentiﬁed side products that were inseparable from

the desired diyne. Removal of the Boc groups under standard

conditions yielded desired dimer 8. The synthetic sequence

outlined in Scheme 2 was also applicable to the preparation of

monomers 5−7 and dimers 9−11.

Smac mimetics containing an indanyl moiety were prepared

analogously to the procedures described above for the

9906

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

a

Scheme 3. Preparation of Aminoindanol-Containing Dimeric Smac Mimetics

a

Reagents and conditions: (a) Boc-Pro-OH, ethyl chloroformate, 4-methylmorpholine, EtOAc, 0 °C to rt; (b) propargyl bromide, KOH, DMF, 0

°C; (c) 4 N HCl/dioxane, rt, or TFA, CH₂Cl₂, rt; (d) NaH, propargyl bromide, THF, 0 °C to reﬂux; (e) EDCI, HOBt hydrate, 4-methylmorpholine,

DMF, 0 °C to rt; (f) isobutyl chloroformate, 4-methylmorpholine, THF, 0 °C; (g) LiOH·H₂O, THF/H₂O, rt; (h) DMTMM, 4-methylmorpholine,

EtOAc, 0 °C to rt; (i) Cu(OAc)₂, pyridine, CH₃CN, 80 °C.

tetrahydronaphthyl-based compounds, only commencing with

commercially available (1S,2R)-cis-1-amino-2-indanol instead

(Scheme 3). Coupling to Boc-proline followed by O-

propargylation yielded cleanly intermediate 42. Alternatively,

we found that the propargyl group could be introduced ﬁrst (via

the alkoxide) to give intermediate 44, which could then be

coupled to Boc-proline. Deprotection of the Boc group from 42

then gave intermediate 43. Building oﬀ of compound 43 with one

protected amino acid at a time as for the tetrahydronaphthyl-

containing compounds described above (Method A) aﬀorded

the Boc-protected monomeric compounds, which could be

subjected under the aforementioned dimerization conditions

followed by deprotection. In some cases, particularly when the

side chain of the P2 residue contained a heteroatom that required

a protecting group, it was advantageous to treat intermediate 43

directly with a dipeptide acid containing the P1 and P2 residues

of the AVPI motif preassembled (Method B); this avoided the

need for orthogonal protecting groups and enabled global

9907

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

a

deprotection of the compound following the dimerization event.

The reagent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-

methyl-morpholinium chloride), previously demonstrated to be

superior to other amide bond-forming reagents for couplings of

sterically hindered peptidomimetics,⁵⁵was employed in these

reactions to minimize epimerization and side-product formation.

The chemistry outlined in Scheme 3 is likewise applicable to

the preparation of compounds in which the proline residue of

AVPI has been replaced with a proline mimetic (19−23, Table

4). Thus, intermediate 44 was coupled to the requisite proline-

mimetic building blocks, which were either commercially

available or were readily prepared following previously published

procedures (e.g., cyclopropyl-fused proline for compound 23⁵⁶).

Elaboration of the resulting products as described above aﬀorded

the target dimers. In the case of spiro-cyclopropylthiaproline

intermediate 46 utilized in the preparation of compound 21, the

reagent was prepared from known 45⁵⁷as a racemate (Scheme

4). As a result, a mixture of diastereomers was formed in the

Scheme 5. Synthesis of Compound 26

Scheme 4. Preparation of Spiro-Cyclopropylthiaproline Used

a

in the Synthesis of Compound 21

a

Reagents and conditions: (a) isobutyl chloroformate, 4-methylmor-

pholine, Chg-OMe·HCl, THF/CH₂Cl₂, 0 °C to rt; (b) LiOH·H₂O,

acetone/H₂O, 0 °C; (c) DMTMM, 4-methylmorpholine, Pro-OMe·

HCl; (d) p-TsCl, triethylamine, CH₂Cl₂, 0 °C to rt; (e) NaH, (1S,2R)-

cis-1-amino-2-indanol, THF, 0 °C to reﬂux; (f) DMTMM, 4-

methylmorpholine, THF/DMF, rt; (g) HCl, dioxane, rt.

a

Reagents and conditions: (a) 37% aqueous formaldehyde, water, rt;

Scheme 6. Synthesis of Compound 27

(b) di-tert-butyldicarbonate, NaHCO₃, water, rt

coupling of 46 with 44. These diastereomers were separated

chromatographically, and each was carried on through the

subsequent sequence of steps to aﬀord diastereomeric Smac

mimetics. The assignment of the absolute stereochemistry at P3

was then made by comparing the binding aﬃnity and cellular

potency measured for each diastereomer (data not shown), with

only one diastereomer demonstrating measurable binding,

cIAP1 degradation, or antiproliferative eﬀects.

Several compounds presented in Table 5 can likewise be

readily prepared following the general protocols described above.

For example, the trans-isomer of 14 (compound 24) was

prepared utilizing the chemistry shown in Scheme 3, only starting

instead with commercially available (1S,2S)-trans-1-amino-2-

indanol. Compound 25, in which the diyne linker in 14 has been

fully saturated, was prepared from di-Boc-protected 14 by

hydrogenation over Pd/C followed by removal of the protecting

groups under standard conditions.

The synthesis of polyether linker compound 26 commenced

by preparation of N-protected tripeptide 48 (Scheme 5) using

chemistry similar to that described earlier. Treatment of this

intermediate with diamine 50 (obtained by O-alkylation of

(1S,2R)-cis-1-amino-2-indanol with the ditosylate 49) aﬀorded

the target compound. Intermediate 48 was also employed in the

synthesis of compound 27, which utilized the tetrahydroquino-

line-derived amine 53 prepared in four steps from commercially

available quinoline 4-carboxylic acid (Scheme 6). The individual

enantiomers of Boc-protected intermediate 52 were obtained by

chiral chromatography, and these were carried forward

independently to give both diastereomers of the dimeric Smac

mimetic. As in the case of compound 21, the absolute

stereochemistry at the chiral center on the tetrahydroquinoline

a

Reagents and conditions: (a) H₂, Raney Ni, HCl, MeOH, rt; (b)

propargyl bromide, K₂CO₃, CH₃CN/MeOH, rt; (c) DPPA, triethyl-

amine, tert-BuOH, 80 °C; (d) chiral chromatography; (e) HCl,

dioxane, rt; (f) 48, EDCI, HOBt hydrate, DIPEA, CH₂Cl₂, 0 °C to rt;

(g) Cu(OAc)₂, pyridine, CH₃CN, 80 °C.

ring was made on the basis of the comparison of the binding

aﬃnities and cellular potencies for each diastereomer.

Compound 28 was also prepared using intermediate 48 by

incorporating the commerically available methyl 2-amino-2,3-

dihydro-1H-indene-2-carboxylate group at the P4 position. Ester

hydrolysis followed by amide bond formation using propargyl-

amine gave Boc-protected monomer 56. Copper-promoted

dimerization followed by removal of the Boc groups yielded the

target compound (Scheme 7).

The synthesis of compounds 29−31 in which a bisamide linker

connects the two monomeric Smac mimetics required the

preparation of protected cis-diaminoindane intermediate 60

(Scheme 8). Regiospeciﬁc radical addition of tert-butyl

dichlorocarbamate (57) across the double bond of indene⁵⁸

9908

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

a

indane ring was made on the basis of the data obtained with the

ﬁnal dimeric compounds as descibed previously. Thus, addition

of the P2 and P1 amino acid residues to 61 followed by Boc-

deprotection of the indane amino group gave intermediate 63.

The linker group between the two monomeric units was installed

by means of a diacid dichloride, and ﬁnal Fmoc-deprotection

revealed target dimers 29−31.

Scheme 7. Synthesis of Compound 28

CONCLUSIONS

■

Given the well-documented role of the IAP family of proteins in

the suppression of apoptosis in cancer cells as well as the

potential for a Smac mimetic to be an eﬀective treatment for

human cancers, we conducted extensive studies to discover and

develop novel inhibitors of the IAPs. As a result, we have

discovered a series of dimeric Smac mimetics based on the AVPI

motif by identifying a site of eﬀective dimerization in monomers

containing a tetrahydronaphthyl P4 substituent. Initial work to

establish the preferred stereochemistry at this residue led to

alkyne-containing monomers 4 and 6 that, when dimerized, gave

very potent inhibitors 8 and 10, respectively, validating our initial

design hypothesis. Further SAR development established the

feasibility of replacing the tetrahydronaphthyl ring with an

indanyl ring and led to an understanding of the drivers for cellular

potency and physical properties. This class of dimers exhibits

very high levels of cellular potency (cIAP1 degradation and

inhibition of cell proliferation) in the MDA-MB-231 cancer cell

line. Importantly, apoptosis induction and cell death are

observed even following transient treatment of these cells with

compound, suggesting that sustained exposure in vivo would not

be necessary to exert pharmacodynamic eﬀects. Thus, upon

intravenous administration of compound 14 to tumor-bearing

mice, reductions in cIAP1 protein levels and increases in cleaved

a

Reagents and conditions: (a) 48, EDCI, HOBt hydrate, 4-

methylmorpholine, DMF, 0 °C to rt; (b) NaOH, MeOH/water, rt;

(c) EDCI, HOBt hydrate, 4-methylmorpholine, propargylamine,

DMF, 0 °C to rt; (d) Cu(OAc)₂, triethylamine, CH₃CN, 70 °C; (e)

HCl, MeOH, rt.

followed by reduction of the N-chloro intermediate gave racemic

trans-58, which could be converted to cis-60 by azide

displacement of the benzylic chloride followed by reduction to

the primary amine. Coupling of this racemic material to Fmoc-

Pro-OH aﬀorded the expected mixture of diastereomers, which

were separated chromatographically following removal of the

Fmoc protecting group (61). Both diastereomers were carried

forward independently through the sequence of steps depicted in

Scheme 8, and the assignment of absolute stereochemistry at the

a

Scheme 8. Synthesis of Dimeric Smac Mimetics Containing a 1,2-Diaminoindane Moiety

a

Reagents and conditions: (a) Ca(OCl)₂, HCl, CH₂Cl₂/H₂O, 0 °C; (b) indene, toluene, 35−40 °C, then NaHSO₃, water, 5−10 °C; (c) NaN₃,

DMF, rt; (d) Pd/C, H₂, EtOH, rt; (e) Fmoc-Pro-OH, HATU, DIPEA, CH₂Cl₂, rt; (f) EtNH₂, THF, rt; (g) Fmoc-Chg-OH, HATU, DIPEA,

CH₂Cl₂, rt; (h) Fmoc-N-Me-Ala-OH, HATU, DIPEA, CH₂Cl₂, rt; (i) 4 N HCl/dioxane, rt; (j) diacid dichloride (for 29, terephthaloyl chloride; for

30, biphenyl-4,4′-dicarbonyl dichloride; for 31, decanedioyl dichloride), DIPEA, CH₂Cl₂, 0 °C.

9909

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

ammonia; 50 mL, 359 mmol) was added, and the mixture was allowed to

stir at room temperature for 1 week (reaction time unoptimized). The

bulk of the unreacted NH₃was evaporated under reduced pressure, and

the resulting mixture was extracted with CH₂Cl₂(3 × 100 mL). The

combined organic extracts were washed with brine, dried (Na₂SO₄),

ﬁltered, and concentrated to give the product as a colorless to pale

caspase-3 were observed, consistent with inhibition of both

cIAP1 and XIAP in vivo. In an MDA-MB-231 xenograft eﬃcacy

study, measurable tumor growth inhibition was observed at lower

doses (0.1 and 0.5 mg/kg) of 14 as a single agent, whereas nearly

complete tumor regression was observed at a higher dose (3.0

mg/kg). Consistent with previously described Smac mimetics,

compound 14 inhibits proliferation in a relatively small subset of

cancer cell lines. Given the high binding aﬃnity to IAP family

members and the cellular potency of 14 coupled with the physical

properties and pharmacokinetics to allow for the in vivo activity

observed following intravenous dosing, this compound was

nominated for further development in human clinical trials.

Further details of the clinical development of this compound will

be reported in the future.

1

yellow solid (5.98 g, 88%). H NMR (400 MHz, DMSO-d₆) δ ppm

1.57−1.70 (m, 1H), 1.79−2.08 (m, 3H), 2.66−2.85 (m, 2H), 3.42−3.58

(m, 2H), 4.83 (br s, 1H), 6.97−7.05 (m, 1H), 7.05−7.18 (m, 2H), 7.47−

7.54 (m, 1H). LC−MS (M + H) 164.

trans-N-(2-Hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)-

benzamide (34). A 250 mL round-bottomed ﬂask containing racemic

trans-33 (3.11 g, 19.1 mmol) was treated with CH₂Cl₂(60 mL) and

triethylamine (3.50 mL, 25.1 mmol). The resulting solution was cooled

to 0 °C, and then benzoyl chloride (2.40 mL, 20.7 mmol) was added

dropwise. After 1 h, the reaction was partitioned between CH₂Cl₂and

water. The aqueous layer was extracted with CH₂Cl₂, and the combined

organics were washed with water and concentrated under reduced

pressure to give a solid residue. This was recrystallized from EtOAc/

EXPERIMENTAL SECTION

■

All reagents and solvents used were purchased from commercial sources

and used without further puriﬁcation. ¹H NMR spectra were obtained

using a Bruker 300 or 400 MHz spectrometer at temperatures ranging

from 23 to 100 °C; chemical shifts are expressed in parts per million

(ppm, δ units) and are referenced to the residual protons in the

deuterated solvent used. Most compounds described in this article

appear as mixtures of amide rotamers by NMR at room temperature,

and the resonances reported in this section are given as ranges of

chemical shifts, reﬂective of these rotameric mixtures. Alternatively,

heating the NMR sample (dissolved in DMSO-d₆) at 100 °C often

causes the resonances to coalesce; spectral data obtained at 100 °C are

denoted as such. Coupling constants are given in units of hertz (Hz).

Splitting patterns describe apparent multiplicities and are designated as s

(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br s

(broad singlet). Optical rotations were measured using a PerkinElmer

Model 341 polarimeter. Mass spectrometry analyses were performed

with an Agilent 1100 equipped with Waters columns (Atlantis T3, 2.1 ×

50 mm, 3 μm or Atlantis dC18, 2.1 × 50 mm, 5 μm) eluted with a

gradient mixture of water and acetonitrile with either formic acid or

ammonium acetate added as a modiﬁer. Reverse-phase chromatography

was performed on a Gilson system using an Atlantis Prep T3 OBD

reverse-phase HPLC column (19 mm × 100 mm) in water/MeCN with

0.1% TFA as mobile phase. Thin-layer chromatography was performed

using EMD silica gel 60 F₂₅₄plates, which were visualized using either

UV light or a stain prepared by dissolving 2 g of KMnO₄and 12 g of

Na₂CO₃in 200 mL of H₂O. Column chromatography was performed

using SiliCycle SiliaSep preloaded silica gel cartridges on Teledyne

ISCO CombiFlash Companion automated puriﬁcation systems. Unless

otherwise indicated, all ﬁnal compounds were puriﬁed to ≥95% purity,

as assessed by analytical HPLC using an Agilent 1100 equipped with

Waters columns (Atlantis T3, 2.1 × 50 mm, 3 μm or Atlantis dC18, 2.1 ×

50 mm, 5 μm) eluted for >10 min with a gradient mixture of water and

acetonitrile with either formic acid or ammonium acetate added as a

modiﬁer, monitored at wavelengths of 220, 254, and 280 nm.

1

hexanes to give the product as a tan colored solid (4.18 g, 82%). H

NMR (400 MHz, DMSO-d₆) δ ppm 1.72−1.84 (m, 1H), 2.03−2.15 (m,

1H), 2.72−2.94 (m, 2H), 3.85−3.95 (m, 1H), 4.96−5.00 (m, 1H),

5.01−5.08 (m, 1H), 7.07−7.19 (m, 4H), 7.42−7.49 (m, 2H), 7.49−7.56

(m, 1H), 7.88−7.97 (m, 2H), 8.58−8.67 (m, 1H). LC−MS (M + H)

268. The racemic material was subjected to supercritical ﬂuid

chromatography with a chiral column (Chiralpak AS column, mobile

phase =80:20 CO₂/EtOH, ﬂow rate = 60 mL/min, temperature = 40

°C) to aﬀord the separate enantiomers each in >98% ee. The absolute

stereochemistry of each was determined by converting to cis- and trans-

aminoalcohols 33 as described below. (1S,2S)-34 [α]²_D⁰+98 (c 0.25,

MeOH), (1R,2R)-34 [α]²_D⁰−99 (c 0.21, MeOH).

(3aR,9bS)-2-Phenyl-3a,4,5,9b-tetrahydronaphtho[1,2-d]-

[1,3]oxazole ((−)-35). A 100 mL round-bottomed ﬂask was charged

with (1S,2S)-34 (803 mg, 3.00 mmol) and CH₂Cl₂(12 mL). The

heterogeneous mixture was cooled to 0 °C, and then thionyl chloride

(0.44 mL, 6.0 mmol) was added dropwise, resulting in a homogeneous

solution. This was allowed to stir at 0 °C with slow warming to room

temperature. After stirring at room temperature overnight, the solution

was concentrated under reduced pressure. The residue was partitioned

between CH₂Cl₂and saturated NaHCO₃, and the aqueous layer was

further extracted with CH₂Cl₂. The combined organics were washed

with water and concentrated under reduced pressure. The crude

material was puriﬁed by silica gel chromatography (gradient elution; R_f

in 80:20 hexanes/EtOAc = 0.42) to give the product as a colorless solid

(717 mg, 96%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.16−1.24 (m,

2H), 1.93−2.05 (m, 1H), 2.29−2.39 (m, 1H), 2.64−2.76 (m, 2H),

3.00−3.09 (m, 1H), 5.66−5.81 (m, 2H), 7.18−7.25 (m, 1H), 7.26−7.35

(m, 2H), 7.57−7.65 (m, 3H), 7.72−7.79 (m, 1H), 8.11−8.19 (m, 2H).

LC−MS (M + H) 250. (3aR,9bS)-35 [α]²_D⁰−309 (c 0.25, MeOH). The

same procedure was followed using (1R,2R)-34 to aﬀord the opposite

enantiomer, (3aS,9bR)-35 [α]²_D⁰+298 (c 0.19, MeOH).

(1S,2R)-cis-1-Amino-1,2,3,4-tetrahydronaphthalen-2-ol (cis-

(+)-33). A 250 mL round-bottomed ﬂask containing (3aR,9bS)-35

(648 mg, 2.60 mmol) was treated with dioxane (2 mL) and 6 N HCl (4

mL). The resulting solution was heated at 100 °C. After heating

overnight, the reaction was allowed to cool and partitioned between

CH₂Cl₂and water. The aqueous layer was further extracted with CH₂Cl₂

and was then treated with NaOH until pH ∼13. The aqueous layer was

then extracted with CH₂Cl₂(3×), and the combined organics were dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure to give the

trans-2-Bromo-1,2,3,4-tetrahydronaphthalen-1-ol (racemic,

32). A 250 mL round-bottomed ﬂask was charged with 1,2-

dihydronaphthalene (7.03 g, 54.0 mmol), THF (50 mL), and water

(25 mL). The resulting biphasic mixture was placed in a room

temperature water bath, and then N-bromosuccinimide (10.66 g, 59.9

mmol) was added. The mixture was allowed to stir at room temperature

overnight and was then partitioned between EtOAc and water. The

aqueous layer was further extracted with EtOAc, and the combined

organics were washed with brine, dried (MgSO₄), ﬁltered, and

concentrated to give a magenta colored solid. This was recrystallized

from EtOAc/hexanes to give the title compound as a colorless,

crystalline solid (9.59 g, 78%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

2.06−2.18 (m, 1H), 2.40−2.51 (m, 1H), 2.78−2.91 (m, 2H), 4.43−4.50

(m, 1H), 4.63−4.71 (m, 1H), 5.90 (br s, 1H), 7.07−7.14 (m, 1H), 7.16−

7.24 (m, 2H), 7.34−7.40 (m, 1H). LC−MS (M + H) 227, 229.

1

product as a colorless solid (404 mg, 95%). H NMR (400 MHz,

DMSO-d₆) δ ppm 1.54−1.79 (m, 3H), 1.82−1.96 (m, 1H), 2.58−2.75

(m, 1H), 2.77−2.90 (m, 1H), 3.61−3.88 (m, 2H), 4.68 (br s, 1H), 6.97−

7.06 (m, 1H), 7.06−7.17 (m, 2H), 7.34−7.44 (m, 1H). LC−MS (M +

H) 164. [α]²_D⁰+72 (c 0.1, MeOH). This is in agreement with the

literature value of [α]²_D⁰+68 (c 0.92, MeOH).⁵⁰The above procedure

was also used to generate the opposite enantiomer starting from

(3aS,9bR)-35. (1R,2S)-cis-33 [α]²_D⁰−64 (c 0.2, MeOH). This is in line

with the literature values of [α]²_D⁰−68 (c 0.88, MeOH),⁵⁰[α]²_D⁰−75.0 (c

1.0, 0.1 M HCl in MeOH),⁵²and [α]_D²⁰−64.2 (c 8.7, MeOH).⁵³

trans-1-Amino-1,2,3,4-tetrahydronaphthalen-2-ol (racemic,

33). A 500 mL round-bottomed ﬂask was charged with 32 (9.47 g,

41.7 mmol) and MeOH (10 mL). Aqueous ammonium hydroxide (28%

9910

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

(1S,2S)-trans-1-Amino-1,2,3,4-tetrahydronaphthalen-2-ol

(trans-(−)-33). A 50 mL round-bottomed ﬂask was charged with

(1S,2S)-34 (270 mg, 1.01 mmol) and NaOH (418 mg, 10.45 mmol).

EtOH (2.5 mL) and water (2.5 mL) were added, and the resulting

mixture was heated in an 80 °C oil bath. After 5 days of heating, the

reaction was allowed to cool and was concentrated under reduced

pressure. The residue was partitioned between CH₂Cl₂and water, and

the aqueous layer was further extracted with CH₂Cl₂. The combined

organic layers were then extracted with aqueous 1 N HCl (2×), and the

combined acidic aqueous extracts were washed with CH₂Cl₂before

being carefully neutralized with solid NaOH to pH ∼13. Finally, this

mixture was extracted with CH₂Cl₂(3×). The combined organics were

dried (Na₂SO₄), ﬁltered, and concentrated under reduced pressure to

give the product as a tan colored solid (93 mg, 56%). [α]²_D⁰−72 (c 0.1,

CHCl₃). This is in accord with the literature value of [α]²_D⁰−85.4 (c

0.634, CHCl₃).⁵⁴The above procedure was also used to generate the

opposite enantiomer starting from (1R,2R)-36. (1R,2R)-trans-33 [α]_D²⁰

+72 (c 0.1, CHCl₃).

reaction was partitioned between EtOAc and 1 N HCl. The aqueous

layer was extracted with EtOAc, and the combined organics were

washed with saturated NaHCO₃and then water and were concentrated

under reduced pressure. The crude material was puriﬁed by silica gel

chromatography (gradient elution; R_fin 60:40 hexanes/EtOAc = 0.23)

to give the product as a colorless, viscous oil (516, 85%). ¹H NMR (400

MHz, DMSO-d₆) δ ppm 0.85−1.03 (m, 9H), 1.33−1.41 (m, 9H), 1.77−

1.93 (m, 3H), 1.93−2.16 (m, 3H), 2.66−2.77 (m, 1H), 2.80−2.91 (m,

1H), 3.37−3.42 (m, 1H), 3.57−3.66 (m, 1H), 3.67−3.76 (m, 1H),

3.84−3.92 (m, 1H), 4.13−4.19 (m, 1H), 4.20−4.27 (m, 2H), 4.45−4.52

(m, 1H), 5.14−5.21 (m, 1H), 6.46−6.54 (m, 1H), 7.05−7.11 (m, 2H),

7.11−7.18 (m, 1H), 7.25−7.31 (m, 1H), 8.08−8.15 (m, 1H). LC−MS

(M + H) 512.

tert-Butyl ((S)-1-(((S)-3,3-Dimethyl-1-oxo-1-((S)-2-(((1S,2R)-2-

(prop-2-yn-1-yloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-

carbamoyl)pyrrolidin-1-yl)butan-2-yl)amino)-1-oxopropan-2-

yl)(methyl)carbamate (39). A 250 mL round-bottomed ﬂask

containing 38 (510 mg, 1.00 mmol) was treated with 4 N HCl in

dioxane (5.0 mL, 20 mmol), and the resulting solution was allowed to

stir at room temperature. After 1 h, the solution was concentrated under

reduced pressure. The residue was dissolved in MeOH and was

reconcentrated to give the intermediate hydrochloride salt as a solid

residue. The ﬂask was charged with Boc-N-Me-Ala-OH (232 mg, 1.14

mmol) and HOBt hydrate (199 mg, 1.30 mmol). Anhydrous DMF (6

mL) was added, and the solution was cooled to 0 °C. 4-Methylmorpho-

line (125 μL, 1.14 mmol) was added followed by EDCI (233 mg, 1.22

mmol), and the resulting mixture was allowed to stir at 0 °C with slow

warming to room temperature. After stirring at room temperature

overnight, the reaction was partitioned between EtOAc and aqueous 1 N

HCl. The aqueous layer was extracted with EtOAc, and the combined

organics were washed sequentially with saturated NaHCO₃and then

water and were concentrated under reduced pressure. The crude

material was puriﬁed by silica gel chromatography (gradient elution; R_f

in 20:80 hexanes/EtOAc = 0.41) to give the title compound as a

colorless, viscous oil (544 mg, 91%). ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 0.83−1.04 (m, 9H), 1.14−1.26 (m, 3H), 1.32−1.47 (m, 9H),

1.76−1.92 (m, 3H), 1.93−2.18 (m, 3H), 2.66−2.80 (m, 4H), 2.80−2.93

(m, 1H), 3.37−3.42 (m, 1H), 3.59−3.74 (m, 2H), 3.84−3.92 (m, 1H),

4.16−4.29 (m, 2H), 4.44−4.55 (m, 2H), 4.55−4.65 (m, 1H), 5.14−5.22

(m, 1H), 7.05−7.12 (m, 2H), 7.12−7.19 (m, 1H), 7.25−7.32 (m, 1H),

7.38−7.64 (m, 1H), 8.09−8.17 (m, 1H). LC−MS (M + H) 597.

(S)-1-((S)-3,3-Dimethyl-2-((S)-2-(methylamino)-

propanamido)butanoyl)-N-((1S,2R)-2-(prop-2-ynyloxy)-1,2,3,4-

tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide hydro-

chloride (4). A 100 mL round-bottomed ﬂask containing 39 (161 mg,

0.27 mmol) was treated with 4 N HCl in dioxane (4.0 mL, 16 mmol),

and the resulting solution was allowed to stir at room temperature for 1

h. The volatile components were then evaporated under reduced

pressure, and the residue was dissolved in MeOH and was

reconcentrated to give the title compound as a solid ﬁlm (138 mg,

(S)-tert-Butyl 2-(((1S,2R)-2-Hydroxy-1,2,3,4-tetrahydronaph-

thalen-1-yl)carbamoyl)pyrrolidine-1-carboxylate (36). A 50 mL

round-bottomed ﬂask was charged with (1S,2R)-cis-33 (283 mg, 1.73

mmol), Boc-Pro-OH (408 mg, 1.90 mmol), and HOBt hydrate (325

mg, 2.12 mmol). Anhydrous DMF (6 mL) was added, and the solution

was cooled to 0 °C. To this was added EDCI (393 mg, 2.05 mmol), and

the resulting mixture was allowed to stir at 0 °C with slow warming to

room temperature. After stirring overnight, the reaction was partitioned

between EtOAc and aqueous 1 N HCl. The aqueous layer was extracted

with EtOAc, and the combined organics were washed with saturated

NaHCO₃and then twice with water before being concentrated under

reduced pressure to give the product as a colorless foam (635 mg,

1

quantitative). H NMR (400 MHz, DMSO-d₆) δ ppm 1.27−1.45 (m,

9H), 1.71−2.00 (m, 6H), 2.02−2.20 (m, 1H), 2.59−2.70 (m, 1H),

2.89−3.00 (m, 1H), 3.23−3.35 (m, 1H), 3.35−3.42 (m, 1H), 3.91−3.99

(m, 1H), 4.24−4.30 (m, 1H), 4.71−4.88 (m, 1H), 4.92−5.04 (m, 1H),

7.01−7.15 (m, 3H), 7.17−7.24 (m, 1H), 7.70−7.87 (m, 1H). LC−MS

(M + H) 361.

(S)-tert-Butyl 2-(((1S,2R)-2-(Prop-2-yn-1-yloxy)-1,2,3,4-tetra-

hydronaphthalen-1-yl)carbamoyl)pyrrolidine-1-carboxylate

(37). A 250 mL round-bottomed ﬂask containing 36 (624 mg, 1.73

mmol) was treated with anhydrous DMF (6 mL). The solution was

cooled to 0 °C, and then propargyl bromide (80% solution in toluene;

300 μL, 2.70 mmol) was added. Powdered potassium hydroxide (287

mg, 5.12 mmol) was added to the solution, and the resulting mixture was

allowed to stir at 0 °C. After 2 h, additional powdered KOH (99 mg) and

propargyl bromide (100 μL) were added, and the mixture was allowed to

stir at 0 °C for 1 h further. The mixture was then partitioned between

EtOAc and water, and the aqueous layer was further extracted with

EtOAc. The combined organics were washed with water and were

concentrated under reduced pressure. The crude material was puriﬁed

by silica gel chromatography (graduent elution; R_fin 60:40 hexanes/

EtOAc = 0.30) to give the product as a colorless to pale yellow solid (475

mg, 69%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.25−1.47 (m, 9H),

1.67−1.78 (m, 1H), 1.78−1.96 (m, 3H), 2.00−2.18 (m, 2H), 2.65−2.79

(m, 1H), 2.81−2.91 (m, 1H), 3.21−3.35 (m, 1H), 3.35−3.43 (m, 2H),

3.84−3.94 (m, 1H), 4.15−4.31 (m, 3H), 5.11−5.26 (m, 1H), 7.06−7.24

(m, 4H), 7.93−8.05 (m, 1H). LC−MS (M + H) 399.

1

96%). H NMR (400 MHz, DMSO-d₆) δ ppm 0.92−1.08 (m, 9H),

1.28−1.35 (m, 3H), 1.74−1.93 (m, 3H), 1.95−2.17 (m, 3H), 2.43−2.49

(m, 3H), 2.65−2.77 (m, 1H), 2.80−2.91 (m, 1H), 3.37−3.43 (m, 1H),

3.63−3.75 (m, 2H), 3.86−3.99 (m, 2H), 4.16−4.28 (m, 2H), 4.44−4.50

(m, 1H), 4.52−4.58 (m, 1H), 5.15−5.22 (m, 1H), 7.04−7.22 (m, 3H),

7.25−7.30 (m, 1H), 8.11−8.18 (m, 1H), 8.57−8.66 (m, 1H), 8.75−8.88

(m, 1H), 9.09−9.22 (m, 1H). LC−MS (M + H) 497.

tert-Butyl ((S)-3,3-Dimethyl-1-oxo-1-((S)-2-(((1S,2R)-2-(prop-

2-yn-1-yloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)-

pyrrolidin-1-yl)butan-2-yl)carbamate (38). A 250 mL round-

bottomed ﬂask containing 37 (469 mg, 1.18 mmol) was treated with

4 N HCl in dioxane (5.0 mL, 20 mmol), and the resulting solution was

allowed to stir at room temperature. After 1 h, the reaction was

concentrated under reduced pressure. The residue was dissolved in

MeOH and was reconcentrated to give the intermediate hydrochloride

as a solid. The ﬂask was then charged with Boc-Tle-OH (297 mg, 1.28

mmol) and HOBt hydrate (226 mg, 1.48 mmol). Anhydrous DMF (5

mL) was added followed by 4-methylmorpholine (155 μL, 1.41 mmol),

and the resulting solution was cooled to 0 °C. EDCI (268 mg, 1.40

mmol) was added, and the resulting mixture was allowed to stir at 0 °C

with slow warming to room temperature. After stirring overnight, the

tert-Butyl (2S,2′S)-1,1′-(2S,2′S)-1,1′-((2S,2′S)-2,2′-

(1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-diylbis(oxy))bis-

(1,2,3,4-tetrahydronaphthalene-2,1-diyl)bis(azanediyl)bis-

(oxomethylene)bis(pyrrolidine-2,1-diyl))bis(3,3-dimethyl-1-ox-

obutane-2,1-diyl)bis(azanediyl)bis(1-oxopropane-2,1-diyl)bis-

(methylcarbamate) (40). A 250 mL round-bottomed ﬂask containing

39 (382 mg, 0.64 mmol) was charged with copper(II) acetate (134 mg,

0.74 mmol). Acetonitrile (6 mL) and pyridine (0.31 mL, 3.8 mmol)

were added, and the resulting heterogeneous mixture was placed in an

oil bath preheated to 80 °C. After 1 h, the reaction was allowed to cool to

room temperature and was concentrated under reduced pressure. The

residue was partitioned between EtOAc and dilute aqueous NH₄OH,

and the aqueous layer was further extracted with EtOAc. The combined

organics were washed with water and were concentrated under reduced

9911

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

pressure. The crude material was puriﬁed by silica gel chromatography

(gradient elution; R_fin EtOAc = 0.36) to give the product as a colorless

solid (312 mg, 82%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.84−1.05

(m, 18H), 1.13−1.28 (m, 6H), 1.33−1.49 (m, 18H), 1.73−1.92 (m,

6H), 1.92−2.18 (m, 6H), 2.65−2.79 (m, 8H), 2.79−2.90 (m, 2H),

3.57−3.71 (m, 4H), 3.80−3.89 (m, 2H), 4.30−4.43 (m, 4H), 4.43−4.49

(m, 2H), 4.49−4.54 (m, 2H), 4.54−4.66 (m, 2H), 5.13−5.22 (m, 2H),

7.04−7.12 (m, 4H), 7.12−7.18 (m, 2H), 7.19−7.64 (m, 4H), 8.11−8.20

(m, 2H). LC−MS (M + H) 1192.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(1,2,3,4-tetrahydronaphthalene-2,1-diyl))bis(1-

((S)-3,3-dimethyl-2-((S)-2-(methylamino)propanamido)-

butanoyl)pyrrolidine-2-carboxamide) (8). A 250 mL round-

bottomed ﬂask containing 40 (210 mg, 0.18 mmol) was treated with

4 N HCl in dioxane (4.0 mL, 16 mmol), and the resulting mixture was

allowed to stir at room temperature for 1 h. The volatile components

were then evaporated under reduced pressure, and the residue was

partitioned between EtOAc and saturated NaHCO₃. The aqueous layer

was further extracted with EtOAc, and the combined organics were

washed with brine, dried (Na₂SO₄), ﬁltered, and concentrated under

reduced pressure to give a viscous oil. This material was lyophilized from

a MeCN/H₂O solution to give the title compound as a colorless,

amorphous solid (172 mg, 99%). ¹H NMR (400 MHz, DMSO-d₆, 100

°C) δ ppm 0.98 (br s, 18H), 1.17 (d, 6H), 1.81−1.94 (m, 4H), 1.97−

2.18 (m, 8H), 2.25 (s, 6H), 2.67−2.77 (m, 2H), 2.84−2.95 (m, 2H),

2.98−3.06 (m, 2H), 3.60−3.70 (m, 2H), 3.71−3.80 (m, 2H), 3.87−3.93

(m, 2H), 4.33−4.43 (m, 4H), 4.48−4.56 (m, 4H), 5.16−5.23 (m, 2H),

7.02−7.17 (m, 6H), 7.25−7.31 (m, 2H), 7.57−7.68 (m, 4H). LC−MS

(M + H) 992.

2H), 7.05−7.18 (m, 6H), 7.25−7.31 (m, 2H), 7.62 (br s, 2H), 7.90 (br s,

2H). LC−MS (M + H) 992.

(S)-1-((S)-3,3-Dimethyl-2-((S)-2-(methylamino)-

propanamido)butanoyl)-N-((1R,2R)-2-(prop-2-ynyloxy)-1,2,3,4-

tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide Hydro-

chloride (7). ¹H NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm 1.03 (s,

9H), 1.38 (d, 3H), 1.83−1.99 (m, 3H), 2.00−2.14 (m, 3H), 2.50 (s,

3H), 2.62−2.77 (m, 1H), 2.77−2.92 (m, 1H), 3.08−3.10 (m, 1H),

3.60−3.79 (m, 2H), 3.83−3.88 (m, 1H), 3.97−4.02 (m, 1H), 4.26 (d,

2H), 4.36−4.42 (m, 1H), 4.51−4.56 (m, 1H), 4.84−4.87 (m, 1H),

7.06−7.12 (m, 1H), 7.12−7.19 (m, 3H), 7.74 (br s, 1H), 8.23 (br s, 1H),

9.03 (br s, 2H). LC−MS (M + H) 497.

(S,S,2S,2′S)-N,N′-((1R,1′R,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(1,2,3,4-tetrahydronaphthalene-2,1-diyl))bis(1-

((S)-3,3-dimethyl-2-((S)-2-(methylamino)propanamido)-

1

butanoyl)pyrrolidine-2-carboxamide) (11). H NMR (400 MHz,

DMSO-d₆, 100 °C) δ ppm 0.97 (s, 18H), 1.16 (d, 6H), 1.80−2.00 (m,

6H), 2.25 (s, 6H), 2.64−2.76 (m, 2H), 2.79−2.87 (m, 2H), 2.98−3.06

(m, 2H), 3.63−3.70 (m, 2H), 3.73−3.77 (m, 2H), 3.81−3.86 (m, 2H),

4.35−4.41 (m, 6H), 4.48−4.53 (m, 2H), 4.83−4.86 (m, 2H), 7.05−7.11

(m, 2H), 7.11−7.18 (m, 6H), 7.62 (br s, 2H), 7.74 (br s, 2H). LC−MS

(M + H) 992.

(S)-tert-Butyl 2-((1S,2R)-2-Hydroxy-2,3-dihydro-1H-inden-1-

ylcarbamoyl)pyrrolidine-1-carboxylate (41). A 1000 mL round-

bottomed ﬂask was charged with Boc-Pro-OH (38.85 g, 180.5 mmol)

and EtOAc (400 mL). The mixture was cooled to 0 °C, and then 4-

methylmorpholine (20.0 mL, 182 mmol) was added, resulting in a

colorless solution. Ethyl chloroformate (17.2 mL, 180 mmol) was added

dropwise, resulting in a heterogeneous, colorless suspension. This was

allowed to stir at 0 °C for 30 min, and then (1S,2R)-1-amino-2,3-

dihydro-1H-inden-2-ol (26.50 g, 177.6 mmol) was added. The resulting

mixture was allowed to stir at 0 °C with slow warming to room

temperature. After stirring at room temperature overnight, the reaction

was transferred to a 1000 mL separatory funnel and was washed with

water (300 mL). The aqueous layer was extracted with EtOAc (150

mL), and the combined organics were washed sequentially with 1 N HCl

(225 mL), half-saturated NaHCO₃(250 mL), and brine (100 mL) and

were then dried (Na₂SO₄), ﬁltered, and concentrated under reduced

pressure to give a solid residue. This was recrystallized from EtOAc/

hexanes to give the title compound as a colorless solid (55.76 g, 91%).

¹H NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm 1.41 (s, 9H), 1.75−1.96

The following compounds were prepared in an analogous manner to

compounds 4 and 8 using the appropriate diastereomer of aminoalcohol

33:

(S)-1-((S)-3,3-Dimethyl-2-((S)-2-(methylamino)-

propanamido)butanoyl)-N-((1R,2S)-2-(prop-2-ynyloxy)-1,2,3,4-

tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide hydro-

chloride (5). ¹H NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm 1.02 (s,

9H), 1.38 (d, 3H), 1.87−1.95 (m, 2H), 2.00−2.19 (m, 4H), 2.52 (s,

3H), 2.68−2.75 (m, 1H), 2.87−2.97 (m, 1H), 3.10−3.23 (m, 3H),

3.73−3.80 (m, 1H), 3.93−4.01 (m, 2H), 4.27 (br s, 2H), 4.46−4.52 (m,

1H), 4.53−4.57 (m, 1H), 5.15−5.19 (m, 1H), 7.06−7.10 (m, 2H),

7.12−7.20 (m, 2H), 7.34−7.42 (m, 1H), 8.19−8.27 (m, 1H), 8.85 (br s,

2H). LC−MS (M + H) 497.

(m, 2H), 2.00−2.09 (m, 1H), 2.09−2.19 (m, 1H), 2.81−2.95 (m, 1H),

3.04−3.13 (m, 1H), 3.36−3.41 (m, 2H), 4.25−4.31 (m, 1H), 4.43−4.49

(m, 1H), 4.59−4.64 (m, 1H), 5.15−5.21 (m, 1H), 7.11−7.27 (m, 4H),

7.29−7.39 (m, 1H). LC−MS (M + H) 346.

(S,S,2S,2′S)-N,N′-((1R,1′R,2S,2′S)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(1,2,3,4-tetrahydronaphthalene-2,1-diyl))bis(1-

((S)-3,3-dimethyl-2-((S)-2-(methylamino)propanamido)-

(S)-tert-Butyl 2-((1S,2R)-2-(Prop-2-ynyloxy)-2,3-dihydro-1H-

inden-1-ylcarbamoyl)pyrrolidine-1-carboxylate (42). A 1000

mL round-bottomed ﬂask was charged with 41 (48.38 g, 139.7

mmol). Anhydrous DMF (300 mL) was added, and the resulting

solution was cooled to 0 °C. Propargyl bromide (80 wt % in toluene;

18.0 mL, 167 mmol) was added, and the cold solution was then treated

in portions with powdered KOH (16.13 g, 287.5 mmol) over 5 min. The

resulting heterogeneous mixture was allowed to stir at 0 °C for 60 min

and was then diluted with EtOAc (300 mL) and water (300 mL). The

mixture was transferred to a separatory funnel, and the layers were

separated. The aqueous layer was diluted with additional water (200

mL) and was extracted with EtOAc (3 × 100 mL). The combined

organics were washed with water (3 × 100 mL) and then brine (∼100

mL) and were dried (Na₂SO₄), ﬁltered, and concentrated under reduced

pressure to give a tan/brown oil. The product was crystallized from

1

butanoyl)pyrrolidine-2-carboxamide) (9). H NMR (400 MHz,

DMSO-d₆, 100 °C) δ ppm 0.96 (s, 18H), 1.16 (d, 6H), 1.84−1.95 (m,

4H), 1.97−2.15 (m, 8H), 2.25 (s, 6H), 2.63−2.80 (m, 2H), 2.86−2.95

(m, 2H), 2.98−3.05 (m, 2H), 3.63−3.72 (m, 2H), 3.75−3.83 (m, 2H),

3.90−3.95 (m, 2H), 4.39−4.44 (m, 4H), 4.46−4.55 (m, 4H), 5.13−5.20

(m, 2H), 7.04−7.10 (m, 2H), 7.11−7.20 (m, 6H), 7.36−7.42 (m, 2H),

7.58−7.67 (m, 2H). LC−MS (M + H) 992.

(S)-1-((S)-3,3-Dimethyl-2-((S)-2-(methylamino)-

propanamido)butanoyl)-N-((1S,2S)-2-(prop-2-ynyloxy)-1,2,3,4-

tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide Hydro-

chloride (6). ¹H NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm 1.05 (s,

9H), 1.38 (d, 3H), 1.83−1.97 (m, 3H), 2.04−2.11 (m, 3H), 2.52 (s,

3H), 2.72−2.77 (m., 1H), 2.79−2.93 (m, 1H), 3.12−3.13 (m, 1H),

3.58−3.72 (m, 2H), 3.72−3.81 (m, 2H), 3.96−4.03 (m, 1H), 4.18−4.31

(m, 2H), 4.38−4.44 (m, 1H), 4.49−4.59 (m, 1H), 4.86−4.90 (m, 1H),

7.03−7.11 (m, 2H), 7.13−7.16 (m, 1H), 7.26−7.28 (m, 1H), 7.86 (br s,

1H), 8.23 (br s, 1H), 8.97 (br s, 2H). LC−MS (M + H) 497.

1

EtOAc/hexanes to give a tan colored solid (49.59 g, 92%). H NMR

(400 MHz, DMSO-d₆, 100 °C) δ ppm 1.40 (s, 9H), 1.73−1.85 (m, 1H),

1.85−1.94 (m, 1H), 1.94−2.06 (m, 1H), 2.06−2.20 (m, 1H), 3.03−3.08

(m, 2H), 3.14−3.20 (m, 1H), 3.30−3.46 (m, 2H), 4.12−4.25 (m, 2H),

4.27−4.30 (m, 1H), 4.35−4.45 (m, 1H), 5.30−5.38 (m, 1H), 7.16−7.29

(m, 4H), 7.46−7.48 (m, 1H). LC−MS (M + H) 385.

(S)-N-((1S,2R)-2-(Prop-2-ynyloxy)-2,3-dihydro-1H-inden-1-

yl)pyrrolidine-2-carboxamide Triﬂuoroacetate (43). A 1000 mL

round-bottomed ﬂask was charged with 42 (30.09 g, 78.26 mmol) and

CH₂Cl₂(100 mL). The resulting solution was placed in a room

temperature water bath, and then triﬂuoroacetic acid (50 mL, 673

(S,S,2S,2′S)-N,N′-((1S,1′S,2S,2′S)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(1,2,3,4-tetrahydronaphthalene-2,1-diyl))bis(1-

((S)-3,3-dimethyl-2-((S)-2-(methylamino)propanamido)-

1

butanoyl)pyrrolidine-2-carboxamide) (10). H NMR (400 MHz,

DMSO-d₆, 100 °C) δ ppm 1.00 (s, 18H), 1.17 (d, 6H), 1.82−1.90 (m,

4H), 1.91−2.00 (m, 2H), 2.00−2.11 (m, 6H), 2.25 (s, 6H), 2.66−2.79

(m, 2H), 2.81−2.88 (m, 2H), 3.00−3.06 (m, 2H), 3.57−3.69 (m, 2H),

3.69−3.82 (m, 4H), 4.39 (s, 6H), 4.50−4.54 (m, 2H), 4.85−4.90 (m,

9912

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

mmol) was added. The resulting solution was allowed to stir at room

temperature for 3 h and was then concentrated under reduced pressure.

The residue was dissolved in diethyl ether (300 mL), and a solid material

soon crystallized from the solution. The solid was isolated by suction

ﬁltration and was washed with ether and dried in air to give the title

5.9 mmol). The resulting suspension was allowed to stir overnight at

room temperature and was then partitioned between EtOAc and water.

The aqueous layer was further extracted with EtOAc, and the combined

organic extracts were washed sequentially with water, 5% aqueous

KHSO₄, saturated aqueous NaHCO₃, and brine before being dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure. The crude

material was puriﬁed by silica gel chromatography (CH₂Cl₂/MeOH =

100:1→ 50:1) to give the Boc-protected monomer as a colorless solid

(450 mg, 75%). LC−MS (M + H) 569.

Preparation of Indanyl-Based Dimeric Smac Mimetics. The

Boc-protected monomers (prepared by either Method A or B) could be

dimerized by the procedure described above for compound 40. Removal

of the Boc groups was then carried out at room temperature under

standard conditions (4 N HCl in dioxane, or 1:1 CH₂Cl₂/triﬂuoroacetic

acid) to reveal the target Smac mimetic as its hydrochloride or

triﬂuoroacetate salt. Select examples were then converted to their

unprotonated form through an aqueous workup with saturated

NaHCO₃.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-(Hexa-2,4-diyne-1,6-diylbis-

(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-3,3-di-

methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-

pyrrolidine-2-carboxamide) (12). Prepared following Method A. ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 0.88−1.04 (m, 18H), 1.07−1.16

(m, 6H), 1.76−1.94 (m, 4H), 1.94−2.13 (m, 6H), 2.13−2.21 (m, 6H),

2.90−3.09 (m, 6H), 3.58−3.79 (m, 4H), 4.24−4.39 (m, 6H), 4.45−4.57

(m, 4H), 5.27−5.36 (m, 2H), 7.13−7.29 (m, 8H), 7.78−7.88 (m, 2H),

8.04−8.11 (m, 2H). LC−MS (M + H) 964.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-(Hexa-2,4-diyne-1,6-diylbis-

(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-3-methyl-

2-((S)-2-(methylamino)propanamido)butanoyl)pyrrolidine-2-

carboxamide) Bistriﬂuoroacetate (13). Prepared following Method

B. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 0.85−1.00 (m, 12H), 1.25−

1.41 (m, 6H), 1.80−2.10 (m, 10H), 2.91−3.10 (m, 4H), 3.50−3.80 (m,

4H), 3.1−4.02 (m, 2H), 4.22−4.35 (m, 6H), 4.35−4.52 (m, 4H), 5.25−

5.32 (m, 2H), 7.15−7.30 (m, 14H), 8.06−8.21 (m, 2H), 8.70−8.92 (m,

4H), 8.92−9.13 (m, 2H). LC−MS (M + H) 936.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-

cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-

pyrrolidine-2-carboxamide) (14). Prepared following Method A. ¹H

NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm 1.00−1.30 (m, 10H),

1.35−1.44 (m, 6H), 1.54−1.94 (m, 14H), 1.97−2.12 (m, 6H), 2.48−

2.52 (m, 6H), 2.96−3.11 (m, 4H), 3.57−3.68 (m, 2H), 3.71−3.81 (m,

2H), 3.85−3.98 (m, 2H), 4.30−4.42 (m, 6H), 4.42−4.50 (m, 2H),

4.52−4.61 (m, 2H), 5.28−5.39 (m, 2H), 7.13−7.30 (m, 8H), 7.57−7.70

(m, 2H), 8.31−8.48 (m, 2H), 8.90−9.33 (m, 4H). LC−MS (M + H)

1015.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-

((S)-2-(methylamino)propanamido)-2-(piperidin-4-yl)acetyl)-

pyrrolidine-2-carboxamide) Tetratriﬂuoroacetate (15). Prepared

following Method B. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 1.20−1.60

(m, 12H), 1.60−2.10 (m, 12H), 2.68−2.95 (m, 4H), 2.95−3.12 (m,

4H), 3.20−3.43 (m, 4H), 3.60−3.90 (m, 6H), 4.20−4.43 (m, 6H),

4.44−4.58 (m, 2H), 4.60−4.80 (m, 2H), 5.19−5.39 (m, 2H), 5.57 (br s,

4H), 7.08−7.39 (m, 8H), 8.12−8.30 (m, 2H), 8.52−8.80 (m, 2H),

8.80−9.07 (m, 6H), 9.07−9.30 (m, 2H). LC−MS (M + H) 1018.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-(1-

acetylpiperidin-4-yl)-2-((S)-2-(methylamino)propanamido)-

acetyl)pyrrolidine-2-carboxamide) Bistriﬂuoroacetate (16). Pre-

pared following Method B. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.05−

1.21 (m, 4H), 1.25−1.34 (m, 8H), 1.60−1.72 (m, 4H), 1.80−2.10 (m,

16H), 2.10−2.33 (m, 2H), 2.35−2.47 (m, 10H), 2.70−2.88 (m, 2H),

2.98−3.15 (m, 6H), 3.60−3.75 (m, 2H), 3.75−3.95 (m, 2H), 4.31−4.89

(m, 4H), 4.80−4.73 (m, 8H), 5.39−5.52 (m, 2H), 7.08−7.33 (m, 10H),

7.65−7.82 (m, 2H). LC−MS (M + H) 1102.

1

compound (28.15 g, 90%). H NMR (400 MHz, DMSO-d₆) δ ppm

1.85−1.94 (m, 2H), 1.94−2.03 (m, 1H), 2.23−2.36 (m, 1H), 2.95−3.13

(m, 2H), 3.17−3.27 (m, 1H), 3.27−3.36 (m, 1H), 3.41−3.47 (m, 1H),

4.15−4.21 (m, 2H), 4.26−4.29 (m, 1H), 4.36−4.40 (m, 1H), 5.34−5.41

(m, 1H), 7.21−7.29 (m, 4H), 8.61 (br s, 1H), 8.71−8.78 (m, 1H), 9.30

(br s, 1H). LC−MS (M + H) 285.

(1S,2R)-2-(Prop-2-ynyloxy)-2,3-dihydro-1H-inden-1-amine

(44). NaH (60% dispersion in mineral oil; 0.89 g, 22 mmol) was added

slowly to a solution of (1S,2R)-1-amino-2,3-dihydro-1H-inden-2-ol

(3.00 g, 20.1 mmol) in anhydrous THF (50 mL) at 0 °C with stirring.

The resulting mixture was allowed to warm to room temperature over 30

min before being heated to reﬂux. A solution of propargyl bromide (80%

in toluene; 3.30 g, 22.2 mmol) in anhydrous THF (10 mL) was added

dropwise to this suspension. The resulting mixture was heated at reﬂux

for 1 h and was then allowed to cool to room temperature. EtOAc (200

mL) and water (200 mL) were added. The layers were separated, and

the aqueous phase was extracted with EtOAc (100 mL × 3). The

combined organic phases were washed with water (300 mL) and then

brine (300 mL) and were dried (Na₂SO₄), ﬁltered, and concentrated

under reduced pressure. The crude material was puriﬁed by silica gel

chromatography (CH₂Cl₂/MeOH = 20:1 →10:1) to give the title

compound as an oil (1.90 g, 50%). ¹H NMR (300 MHz, CDCl₃) δ ppm

1.70 (s, 2H), 2.43 (t, J = 1.8 Hz, 1H), 3.02 (s, 2H), 4.32−4.25 (m, 4H),

7.20−7.25 (m, 3H), 7.38 (m, 1H). LC−MS (M + H) 188.

Method A for the Preparation of Boc-Protected Indanyl-

Based Monomers. The general procedures described for the

preparation of the tetrahydronaphthyl-based compound 4 were

followed utilizing compound 43 as a reagent.

Method B for the Preparation of Boc-Protected Indanyl-

Based Monomers. Amide bond formation between Boc-N-Me-Ala-

OH and a suitably protected amino acid methyl ester followed by ester

hydrolysis gave a dipeptide acid fragment, which could then be coupled

to compound 43. A representative procedure is described here for the

Boc-protected monomer used in the preparation of compound 13.

tert-Butyl Methyl((S)-1-((S)-3-methyl-1-oxo-1-((S)-2-((1S,2R)-

2-(prop-2-ynyloxy)-2,3-dihydro-1H-inden-1-ylcarbamoyl)-

pyrrolidin-1-yl)butan-2-ylamino)-1-oxopropan-2-yl)-

carbamate. Isobutyl chloroformate (0.74 g, 5.4 mmol) was added

dropwise to a solution of Boc-N-Me-Ala-OH (1.00 g, 4.92 mmol) and 4-

methylmorpholine (0.80 g, 7.9 mmol) in anhydrous THF (20 mL) at 0

°C. After stirring at this temperature for 20 min, a solution prepared

from valine methyl ester hydrochloride (820 mg, 4.89 mmol) and 4-

methylmorpholine (1.0 g, 9.9 mmol) in anhydrous THF (10 mL) was

added to this suspension. The mixture was allowed to stir at 0 °C for 1 h

and was then partitioned between EtOAc and water. The aqueous phase

was extracted with EtOAc, and the combined organic extracts were

washed sequentially with water, 5% aqueous KHSO₄, saturated aqueous

NaHCO₃, and brine before being dried (Na₂SO₄), ﬁltered, and

concentrated under reduced pressure. The crude material was puriﬁed

by silica gel chromatography (EtOAc/hexane = 10:1 → 5:1) to aﬀord

the dipeptide ester as a colorless solid (1.30 g, 84%). A solution of this

compound (380 mg, 1.20 mmol) in acetone (10 mL) was cooled to 0 °C

and was treated with a solution of LiOH monohydrate (0.10 g, 2.4

mmol) in water (5 mL). After stirring at 0 °C for 3 h, the reaction was

acidiﬁed with 2 N aqueous HCl to pH ∼3 and was then concentrated

under reduced pressure. The residue was partitioned between EtOAc

and water, and the aqueous layer was further extracted with EtOAc. The

combined organic phases were washed sequentially with water, 5%

aqueous KHSO₄, saturated aqueous NaHCO₃, and brine before being

dried (Na₂SO₄), ﬁltered, and concentrated under reduced pressure to

give the dipeptide acid as a solid (360 mg, 99%). DMTMM (4-(4,6-

dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride, 0.58 g,

2.1 mmol) was added to a cold (0 °C) solution of this dipeptide acid

(320 mg, 1.06 mmol) and compound 43 (500 mg, 1.26 mmol) in THF

(20 mL) and DMF (10 mL) followed by 4-methylmorpholine (0.60 g,

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-(1-

(2-hydroxyethyl)piperidin-4-yl)-2-((S)-2-(methylamino)-

propanamido)acetyl)pyrrolidine-2-carboxamide) Tetratriﬂuor-

1

oacetate (17). Prepared following Method B. H NMR (300 MHz,

9913

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

DMSO-d₆) δ ppm 1.02−1.48 (m, 10H), 1.50−2.30 (m, 20H), 2.75−

3.25 (m, 12H), 3.42−3.65 (m, 6H), 3.65−3.83 (m, 6H), 3.85−4.00 (m,

2H), 4.19−4.41 (m, 6H), 4.43−4.60 (m, 2H), 4.60−4.80 (m, 2H),

5.21−5.43 (m, 2H), 7.05−7.36 (m, 10H), 8.21−8.25 (m, 2H), 8.78−

9.23 (m, 4H), 9.29 (br s, 2H), 9.73 (br s, 2H). LC−MS (M + H) 1106.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-6-

amino-2-((S)-2-(methylamino)propanamido)hexanoyl)-

pyrrolidine-2-carboxamide) Tetratriﬂuoroacetate (18). Prepared

hydro-1H-pyrrole-2-carboxamide) Bishydrochloride (22). ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 0.92−1.26 (m, 10H), 1.26−1.41

(m, 6H), 1.55−1.79 (m, 8H), 1.80−1.89 (m, 2H), 1.93−2.04 (m, 2H),

2.45−2.48 (m, 6H), 2.97−3.07 (m, 4H), 3.78−3.90 (m, 2H), 4.22−4.37

(m, 8H), 4.39−4.48 (m, 2H), 4.49−4.61 (m, 2H), 5.18−5.24 (m, 2H),

5.25−5.40 (m, 2H), 5.72−5.85 (m, 2H), 5.97−6.09 (m, 2H), 7.16−7.30

(m, 8H), 8.27−8.35 (m, 2H), 8.71−8.94 (m, 4H), 9.11−9.36 (m, 2H).

LC−MS (M + H) 1012.

(S,S,1S,1′S,3S,3′S,5S,5′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-

diyne-1,6-diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis-

(2-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)-

acetyl)-2-azabicyclo[3.1.0]hexane-3-carboxamide) Bishydro-

chloride (23). ¹H NMR (400 MHz, DMSO-d₆, 100 °C) δ ppm

0.76−0.85 (m, 2H), 0.94−1.25 (m, 12H), 1.29−1.41 (m, 6H), 1.55−

1.74 (m, 8H), 1.74−1.83 (m, 2H), 1.84−1.99 (m, 6H), 2.37−2.45 (m,

2H), 2.40−2.62 (m, 6H), 2.95−3.07 (m, 4H), 3.70−3.79 (m, 2H),

3.81−3.92 (m, 2H), 4.26−4.38 (m, 6H), 4.67−4.75 (m, 2H), 4.76−4.86

(m, 2H), 5.24−5.32 (m, 2H), 7.15−7.27 (m, 8H), 8.06−8.14 (m, 2H),

8.71−8.90 (m, 4H), 9.09 (br s, 2H). LC−MS (M + H) 1040.

(S,S,2S,2′S)-N,N′-((1S,1′S,2S,2′S)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-

cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-

pyrrolidine-2-carboxamide) (24). ¹H NMR (400 MHz, DMSO-d₆,

100 °C) δ ppm 0.98−1.30 (m, 16H), 1.58−1.81 (m, 12H), 1.84−2.15

(m, 8H), 2.25 (s, 6H), 2.78−2.87 (m, 2H), 2.97−3.05 (m, 2H), 3.25−

3.35 (m, 2H), 3.59−3.65 (m, 2H), 3.66−3.74 (m, 2H), 3.74−3.81 (m,

2H), 4.23−4.33 (m, 2H), 4.38−4.50 (m, 8H), 5.12−5.20 (m, 2H),

7.13−7.25 (m, 8H), 7.60 (br s, 2H), 7.96 (br s, 2H). LC−MS (M + H)

1016.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexane-1,6-diylbis-

(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-cyclo-

hexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidine-

2-carboxamide) Bishydrochloride (25). Prepared by hydrogenation

(10% Pd/C, 1 atm H₂, MeOH, rt) of di-Boc-protected 14 followed by

Boc deprotection (4 N HCl/dioxane, rt). ¹H NMR (400 MHz, DMSO-

d₆, 100 °C) δ ppm 1.04−1.23 (m, 10H), 1.28−1.33 (m, 4H), 1.38−1.40

(m, 6H), 1.47−1.55 (m, 4H), 1.58−1.83 (m, 12H), 1.84−1.93 (m, 2H),

1.96−2.09 (m, 6H), 2.50−2.54 (m, 6H), 2.90−3.07 (m, 4H), 3.43−3.47

(m, 4H), 3.59−3.64 (m, 2H), 3.70−3.79 (m, 2H), 3.85−3.94 (m, 2H),

4.17−4.21 (m, 2H), 4.45−4.49 (m, 2H), 4.54−4.60 (m, 2H), 5.28−5.32

(m, 2H), 7.14−7.26 (m, 8H), 7.50−7.55 (m, 2H), 8.33−8.38 (m, 2H),

8.82−9.15 (m, 4H). LC−MS (M + H) 1024.

(S)-2-((S)-2-((tert-Butoxycarbonyl)(methyl)amino)-

propanamido)-2-cyclohexylacetic Acid (47). Isobutyl chlorofor-

mate (4.04 g, 29.6 mmol) was added dropwise to a solution of Boc-N-

Me-Ala-OH (5.00 g, 24.6 mmol) and 4-methylmorpholine (5.10 g, 50.4

mmol) in anhydrous THF (100 mL) at 0 °C. The mixture was allowed

to stir at this temperature for 20 min and was then treated with a

suspension of cyclohexylglycine methyl ester hydrochloride (5.11 g, 24.6

mmol) and 4-methylmorpholine (5.10 g, 50.4 mmol) in THF (50 mL)

and CH₂Cl₂(50 mL). The mixture was allowed to stir at 0 °C for 1 h and

was then partitioned between EtOAc and water. The layers were

separated, and the aqueous phase was extracted twice more with EtOAc.

The combined organic layers were washed sequentially with water, 5%

aqueous KHSO₄, saturated aqueous NaHCO₃, and then brine and were

dried (Na₂SO₄), ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by silica gel chromatography (EtOAc/hexane =

10:1→ 5:1) to aﬀord Boc-N-Me-Ala-Chg-OMe as a colorless solid (8.10

g, 22.7 mmol). This material was dissolved in acetone (50 mL), and the

solution was cooled to 0 °C. A solution of LiOH·H₂O (5.16 g, 123

mmol) in water (50 mL) was added, and the reaction mixture was

allowed to stir at this temperature for 3 h. The mixture was then acidiﬁed

with 2 N HCl to pH ∼3, and the volatile components were evaporated

under reduced pressure. The residual material was partitioned between

EtOAc and water, and the aqueous layer was further extracted with

EtOAc (2×). The combined organic layers were washed sequentially

with water, 5% aqueous KHSO₄, and then brine and were dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure to give the

title compound as a solid (7.80 g, 93% over 2 steps). LC−MS (M + H)

343.

1

following Method B. H NMR (300 MHz, DMSO-d₆): δ ppm 1.15−

1.80 (m, 20H), 1.80−2.15 (m, 8H), 2.65−2.88 (m, 4H), 2.90−3.10 (m,

4H), 3.47−3.73 (m, 4H), 3.75−3.92 (m, 2H), 4.20−4.41 (m, 6H),

4.41−4.66 (m, 4H), 4.95−5.70 (m, 10H), 7.02−7.51 (m, 10H), 8.08−

8.25 (m, 2H), 8.75−9.03 (m, 4H), 9.05−9.28 (m, 2H). LC−MS (M +

H) 994.

For the compounds presented in Table 4, the chemistry described for

the above compounds was used along with the requisite Boc-protected

proline mimetic (either commercially available or prepared by known

routes).

(S,S,4R,4′R)-N,N′-((1S,1′S,2R,2′R)-(Hexa-2,4-diyne-1,6-diylbis-

(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(3-((S)-3,3-di-

methyl-2-((S)-2-(methylamino)propanamido)butanoyl)-

1

thiazolidine-4-carboxamide) Bistriﬂuoroacetate (19). H NMR

(300 MHz, DMSO-d₆): δ ppm 0.90−1.15 (m, 18H), 1.20−1.45 (m,

8H), 2.92−3.16 (m, 6H), 3.26−3.40 (m, 2H), 3.89−4.07 (m, 2H),

4.23−4.44 (m, 6H), 4.54−4.99 (m, 6H), 5.00−5.19 (m, 2H), 5.28−5.43

(m, 2H), 7.11−7.38 (m, 8H), 8.21−8.37 (m, 2H), 8.66−8.80 (m, 2H),

8.81−9.02 (m, 2H), 9.02−9.22 (m, 2H). LC−MS (M + H) 1000.

(S,S,4R,4′R)-N,N′-((1S,1′S,2R,2′R)-(Hexa-2,4-diyne-1,6-diylbis-

(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(3-((S)-2-cyclo-

hexyl-2-((S)-2-(methylamino)propanamido)acetyl)-

1

thiazolidine-4-carboxamide) Bistriﬂuoroacetate (20). H NMR

(300 MHz, DMSO-d₆): δ ppm 0.92- 1.25 (m, 12H), 1.30−1.40 (m, 6H),

1.55−1.95 (m, 12H), 2.91−3.12 (m, 6H), 3.23−3.40 (m, 2H), 3.80−

3.96 (m, 2H), 4.23−4.40 (m, 6H), 4.45−4.68 (m, 4H), 4.81−4.96 (m,

2H), 5.02−5.16 (m, 2H), 5.15−5.40 (m, 2H), 7.15−7.34 (m, 8H),

8.21−8.36 (m, 2H), 8.74−8.99 (m, 4H), 9.00−9.20 (m, 2H). LC−MS

(M + H) 1052.

6-(tert-Butoxycarbonyl)-4-thia-6-azaspiro[2.4]heptane-7-

carboxylic Acid (46). Compound 45⁵⁷(12.9 g, 70.3 mmol) was

dissolved in water (100 mL), and 37% aqueous formaldehyde (20 mL,

270 mmol) was added. The reaction mixture was allowed to stir

overnight at room temperature and was then cooled in an ice bath.

Absolute EtOH (100 mL) was added followed by pyridine (14.0 mL,

173 mmol), bringing the mixture to pH ∼6. The mixture was cooled to 0

°C and was allowed to stir at this temperature for 1 h. The precipitated

solid was isolated by suction ﬁltration and was washed with cold EtOH

to aﬀord 4-thia-6-aza-spiro[2.4]heptane-7-carboxylic acid as a colorless

solid (4.31 g, 27.1 mmol). This material was suspended in water (20

mL), and NaHCO₃(6.80 g, 81.0 mmol) was added, resulting in a

homogeneous solution. Di-tert-butyldicarbonate (8.76 g, 40.7 mmol)

was then added, and the mixture was allowed to stir overnight at room

temperature. CHCl₃(100 mL) was added, and the mixture was cooled

to 0 °C before being acidiﬁed with 6 N aqueous HCl to pH ∼3. The

layers were separated, and the organic layer was washed with brine, dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure to give to a

colorless foam. Trituration with hexanes provided the target compound

as a colorless solid (6.18 g, 34% over two steps). ¹H NMR (300 MHz,

DMSO-d₆): δ ppm 0.80−1.01 (m, 2H), 1.15−1.31 (m, 2H), 1.41 (s,

9H), 3.98 (s, 1H), 4.61 (m, 2H). LC−MS (M − H) 258.

(7R,7′R)-N,N′-{Hexa-2,4-diyne-1,6-diylbis[oxy(1S,2R)-2,3-di-

hydro-1H-indene-2,1-diyl]}bis{6-[(2S)-2-cyclohexyl-2-{[(2S)-2-

(methylamino)propanoyl]amino}acetyl]-4-thia-6-azaspiro[2.4]-

1

heptane-7-carboxamide} (21). H NMR (300 MHz, DMSO-d₆) δ

ppm 0.80−0.90 (m, 4H), 0.90−1.30 (m, 22H), 1.51−1.91 (m, 12H),

2.10−2.29 (m, 6H), 2.94−3.15 (m, 6H), 4.24−4.41 (m, 6H), 4.49−4.64

(m, 4H), 4.76−4.94 (m, 2H), 5.05−5.16 (m, 2H), 5.29−5.41 (m, 2H),

7.12−7.32 (m, 8H), 7.95−8.14 (m, 4H). LC−MS (M + H) 1104.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-2,2′-(Hexa-2,4-diyne-1,6-

diylbis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-

cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-2,5-di-

9914

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

(S)-1-((S)-2-((S)-2-((tert-Butoxycarbonyl)(methyl)amino)-

propanamido)-2-cyclohexylacetyl)pyrrolidine-2-carboxylic

Acid (48). To a suspension of proline methyl ester hydrochloride (2.40

g, 14.6 mmol) and compound 47 (5.00 g, 14.6 mmol) in THF (100 mL)

and DMF (10 mL) at 0 °C, DMTMM (8.08 g, 29.2 mmol) was added

followed by 4-methylmorpholine (6.1 g, 60 mmol). The suspension was

allowed to stir at room temperature overnight. The reaction mixture was

then partitioned between EtOAc and water, and the aqueous layer was

further extracted with EtOAc (2×). The combined organic layers were

washed sequentially with water, 5% aqueous KHSO₄, saturated aqueous

NaHCO₃, and then brine and were dried (Na₂SO₄), ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by silica

gel chromatography (EtOAc/hexane = 4:1 → 1:1) to aﬀord Boc-N-Me-

Ala-Chg-Pro-OMe as an oil (3.80 g, 8.38 mmol). A portion of this

material (0.59 g, 1.30 mmol) was dissolved in acetone (20 mL), and the

solution was cooled to 0 °C. A solution of LiOH·H₂O (220 mg, 5.24

mmol) in water (20 mL) was added, and the reaction mixture was

allowed to stir at 0 °C. After 3 h, the mixture was acidiﬁed with 2 N HCl

to pH ∼3, and the volatile components were evaporated under reduced

pressure. The residue was partitioned between EtOAc and water, and

the aqueous layer was further extracted with EtOAc (2×). The

combined organic layers were washed sequentially with water, 5%

aqueous KHSO₄, and then brine and were dried (Na₂SO₄), ﬁltered, and

concentrated under reduced pressure to give the title compound as a

solid (0.40 g, 70%). LC−MS (M + H) 440.

mixture was allowed to stir at this temperature for 4 h and was then

concentrated under reduced pressure. The residue was dissolved in

MeOH and was reconcentrated (3×) to give the title compound a a

1

colorless solid (312 mg, 36% over two steps). H NMR (300 MHz,

DMSO-d₆): δ ppm 0.75−1.45 (m, 16H), 1.50−2.12 (m, 14H), 2.32−

2.57 (m, 6H), 2.83−3.11 (m, 4H), 3.26−3.68 (m, 8H), 3.68−4.32 (m,

14H), 4.32−4.70 (m, 4H), 5.16−5.39 (m, 2H), 7.03−7.32 (m, 8H),

7.88−8.10 (m, 2H), 8.67−9.09 (m, 4H), 9.52−9.84 (m, 2H). LC−MS

(M + H) 1012.

1-(Prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinoline-4-carboxylic

Acid (51). A Parr shaker bottle was charged with quinoline-4-carboxylic

acid (5.00 g, 28.9 mmol), MeOH (120 mL), and concentrated HCl (4.0

mL). Raney Nickel (purchased as a slurry in water; approximately 10 g)

was added, and the vessel was sealed on a Parr shaker. The bottle was

degassed and ﬂushed with H₂and was then pressurized with H₂to 60 psi.

The mixture was shaken at room temperature overnight. Additional

Raney Nickel (∼10 g) was added, and the reaction was continued for an

additional 24 h. The vessel was put under an N₂atmosphere, and the

mixture was then suction ﬁltered through a pad of Celite. The ﬁltrate was

concentrated under reduced pressure to give crude 1,2,3,4-tetrahy-

droquinoline-4-carboxylic acid. A portion of this material (2.56 g, 14.4

mmol) was dissolved in acetonitrile (100 mL) and MeOH (40 mL).

Potassium carbonate (5.99 g, 43.3 mmol) was added followed by

propargyl bromide (80% solution in toluene; 2.1 mL, 19.0 mmol). The

reaction mixture was allowed to stir at room temperature for 3 days and

was then concentrated under reduced pressure. The residue was diluted

with water and was acidiﬁed with 1 N HCl until pH 4 to 5. The mixture

was extracted with EtOAc (3×). The combined organic extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The crude material was puriﬁed by silica gel

chromatography (0−100% EtOAc in hexanes) to give the product as

Oxybis(ethane-2,1-diyl) Bis(4-methylbenzenesulfonate) (49).

A solution of diethylene glycol (10.0 g, 94.2 mmol) and triethylamine

(39.0 mL, 280 mmol) in CH₂Cl₂(200 mL) was cooled to 0 °C. A

solution of p-toluenesulfonyl chloride (36.0 g, 189 mmol) in CH₂Cl₂

(100 mL) was added dropwise over 20 min, and the resulting suspension

was allowed to stir at room temperature overnight. Water (200 mL) was

added to quench the reaction, and the organic layer was separated and

was washed with 5% aqueous KHSO₄and then brine, dried over

Na₂SO₄, and concentrated to dryness. The residue was puriﬁed by silica

gel chromatography (CH₂Cl₂/MeOH = 20:1) to aﬀord the title

1

a yellow solid (1.87 g, 60% over two steps). H NMR (400 MHz,

DMSO-d₆) δ ppm 1.91−2.02 (m, 1H), 2.10−2.21 (m, 1H), 3.02−3.05

(m, 1H), 3.13−3.20 (m, 1H), 3.26−3.36 (m, 1H), 3.64−3.69 (m, 1H),

3.96−4.04 (m, 1H), 4.12−4.20 (m, 1H), 6.59−6.66 (m, 1H), 6.74−6.79

(m, 1H), 7.03−7.11 (m, 2H), 12.39 (br s, 1H). LC−MS (M + H) 216.

tert-Butyl (1-(Prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-4-

yl)carbamate (52). A mixture of 51 (1.43 g, 6.66 mmol), diphenyl

phosphorazidate (2.88 mL, 13.3 mmol), and triethylamine (2.32 mL,

16.6 mmol) in anhydrous tert-butanol (32 mL) was heated at reﬂux

overnight. The reaction was allowed to cool and was concentrated under

reduced pressure. The residue was puriﬁed by silica gel chromatography

(0−25% EtOAc in hexanes) to give the racemic product (1.16 g, 61%).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.42 (s, 9H), 1.82−1.93 (m,

1H), 1.93−2.01 (m, 1H), 3.05−3.08 (m, 1H), 3.20−3.26 (m, 2H),

3.95−4.03 (m, 1H), 4.08−4.16 (m, 1H), 4.56−4.66 (m, 1H), 6.60−6.67

(m, 1H), 6.69−6.74 (m, 1H), 7.00−7.09 (m, 2H), 7.13−7.21 (m, 1H).

LC−MS (M + H) 287. This racemic material was subjected to

supercritical ﬂuid chromatography with a chiral column to give the

separate enantiomers (0.52 g each, >98% ee) that were carried on

separately to the respective ﬁnal products.

1-(Prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-4-amine Dihy-

drochloride (53). Hydrochloric acid (4 N in dioxane; 20 mL, 80

mmol) was added to 52 (0.52 g, 1.80 mmol). The resulting solution was

allowed to stir at room temperature for 2 h and was then concentrated

under reduced pressure to aﬀord the title compound as colorless solid

(0.40 g, 86%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.04−2.22 (m,

2H), 3.11−3.15 (m, 1H), 3.22−3.36 (m, 2H), 4.10−4.16 (m, 2H),

4.35−4.44 (m, 1H), 6.64 (br s, 1H), 6.69−6.76 (m, 1H) 6.81−6.87 (m,

1H) 7.17−7.24 (m, 1H), 7.33−7.39 (m 1H), 8.45 (br s, 3H). LC−MS

(M + H) 187.

(S,S,2S,2′S)-N,N′-(1,1′-(Hexa-2,4-diyne-1,6-diyl)bis(1,2,3,4-

tetrahydroquinoline-4,1-diyl))bis(1-((S)-2-cyclohexyl-2-((S)-2-

(methylamino)propanamido)acetyl)pyrrolidine-2-carboxa-

mide) (27). HOBt hydrate (165 mg, 1.08 mmol) and DIPEA (0.47 mL,

2.70 mmol) were added to a mixture of compounds 48 (396 mg, 0.90

mmol) and 53 (200 mg, 0.90 mmol) in CH₂Cl₂(8.5 mL), and the

mixture was cooled to 0 °C. EDCI (207 mg, 1.08 mmol) was added, and

the reaction mixture was then allowed to warm to room temperature and

was allowed to stir overnight. The mixture was diluted with CH₂Cl₂and

1

compound as a colorless solid (25.3 g, 65%). H NMR (300 MHz,

CDCl₃) δ ppm 2.47 (s, 6H), 3.61−3.65 (m, 4H), 4.09−4.18 (m, 4H),

7.36 (d, J = 8.4 Hz, 4H), 7.80 (d, J = 8.4 Hz, 4H).

(1S,1′S,2R,2′R)-2,2′-((Oxybis(ethane-2,1-diyl))bis(oxy))bis-

(2,3-dihydro-1H-inden-1-amine) (50). A solution of (1S,2R)-cis-1-

amino-2-indanol (1.49 g, 10.0 mmol) in anhydrous THF (50 mL) was

added slowly to a suspension of NaH (60% dispersion in mineral oil;

0.40 g, 10 mmol) in THF (20 mL) at 0 °C. The reaction mixture was

allowed to warm to room temperature over 30 min and was then heated

to reﬂux. A solution of compound 49 (1.88 g, 4.54 mmol) in anhydrous

THF (20 mL) was added dropwise to this suspension. The mixture was

heated for 5 h before being allowed to cool. The mixture was carefully

quenched with water (∼300 mL) and was extracted with CH₂Cl₂(3×).

The combined organic extracts were washed with brine, dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure. The

crude material was puriﬁed by silica gel chromatography (CH₂Cl₂/

MeOH/NH₄OH = 50:1:0.1 → 20:1:0.1) to aﬀord the product as an oil

(1.10 g, 66%). ¹H NMR (300 MHz, CDCl₃) δ ppm 2.99−3.06 (m, 4H),

3.70−3.90 (m, 8H), 4.17 (m, 2H), 4.33 (m, 2H), 7.11−7.26 (m, 6H),

7.47 (m, 2H). LC−MS (M + H) 369.

(S,S,2S,2′S)-N,N′-((1S,1′S,2R,2′R)-((Oxybis(ethane-2,1-diyl))-

bis(oxy))bis(2,3-dihydro-1H-indene-2,1-diyl))bis(1-((S)-2-cyclo-

hexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidine-

2-carboxamide) Dihydrochloride (26). A suspension of 48 (0.77 g,

1.75 mmol) and 50 (0.29 g, 0.79 mmol) in THF (30 mL) and DMF (50

mL) was cooled to 0 °C and was then treated with DMTMM (0.97 g, 3.5

mmol). 4-Methylmorpholine (0.70 g, 6.9 mmol) was added, and the

mixture was allowed to stir at room temperature overnight before being

partitioned between EtOAc and water. The aqueous layer was further

extracted with EtOAc, and the combined organic layers were washed

sequentially with water, 5% aqueous KHSO₄, saturated NaHCO₃, and

then brine and were dried (Na₂SO₄), ﬁltered, and concentrated under

reduced pressure. The residue was puriﬁed by preparative HPLC to

aﬀord the di-Boc-protected intermediate as a solid (360 mg). This was

treated with a 4 N solution of HCl in dioxane (10 mL) at 0 °C. The

9915

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

was washed sequentially with 10% aqueous citric acid, saturated

NaHCO₃, and then brine and was dried (MgSO₄), ﬁltered, and

concentrated under reduced pressure. The crude material was puriﬁed

by silica gel chromatography (0−100% EtOAc/hexane) to give the

intermediate Boc-protected monomer as a colorless solid (341 mg, 0.56

mmol). To this material were added copper(II) acetate (153 mg, 0.84

mmol), pyridine (0.18 mL, 2.24 mmol), and acetonitrile (5 mL), and the

mixture was heated at 80 °C for 3 h. On cooling, the mixture was

partitioned between EtOAc and water. The aqueous layer was extracted

with EtOAc, and the combined organic layers were concentrated under

reduced pressure. The crude Boc-protected dimer was thus obtained as a

yellow solid, which was directly treated with hydrochloric acid (4 N in

dioxane; 6.0 mL, 24 mmol). The solution was allowed to stir at room

temperature for 2 h and was then concentrated under reduced pressure.

The crude product was puriﬁed by reverse-phase HPLC to aﬀord the

title compound a colorless solid (110 mg). ¹H NMR (400 MHz,

DMSO-d₆, 100 °C) δ ppm 0.96−1.26 (m, 16H), 1.52−1.82 (m, 14H),

1.82−2.12 (m, 12H), 2.25 (s, 6H), 2.94−3.04 (m, 2H), 3.16−3.34 (m,

4H), 3.52−3.66 (m, 2H), 3.68−3.83 (m, 2H), 4.11−4.28 (m, 4H),

4.34−4.56 (m, 4H), 4.82−4.97 (m, 2H), 6.59−6.68 (m, 2H), 6.68−6.75

(m, 2H), 7.03−7.22 (m, 4H), 7.49−7.64 (m, 2H), 7.64−7.78 (m, 2H).

LC−MS (M + H) 1014.

propanamido)acetyl)pyrrolidine-2-carboxamide) Bistriﬂuoroa-

cetate (28). Boc-protected monomer 56 (90 mg, 0.14 mmol) was

dissolved in acetonitrile (4 mL), and the solution was heated at 70 °C.

To this were added copper(II) acetate (129 mg, 0.71 mmol) and

triethylamine (200 μL, 1.43 mmol). The mixture was heated for 1 h and

was then allowed to cool. The mixture was ﬁltered through a short plug

of silica gel, eluting with EtOAc. Concentration under reduced pressure

aﬀorded the di-Boc-protected dimer (75 mg). This material was

dissolved in methanol (5 mL), and the solution was treated with gaseous

HCl and was allowed to stir at room temperature overnight. The mixture

was concentrated under reduced pressure, and the crude material was

puriﬁed by reverse-phase HPLC (gradient of acetonitrile in H₂O

containing 0.1% triﬂuoroacetic acid) to give the title compound

1

(bistriﬂuoroacetate salt) as a colorless solid (19 mg). H NMR (400

MHz, CD₃OD) δ ppm 0.94−1.39 (m, 10H), 1.39−1.58 (m, 6H), 1.64−

1.94 (m, 16H), 1.96−2.26 (m, 6H), 2.52−2.79 (m, 6H), 3.12−3.54 (m,

6H), 3.56−4.05 (m, 10H), 4.08−4.35 (m, 4H), 4.38−4.61 (m, 2H),

5.41−5.57 (m, 2H), 7.05−7.35 (m, 8H) 8.22−8.40 (m, 2H), 8.71−8.89

(m, 2H). LC−MS (M + H) 1070. Purity: 90%.

tert-Butyl Dichlorocarbamate (57). To a mixture of tert-butyl

carbamate (2.00 g, 17.1 mmol) and calcium hypochlorite (4.89 g, 34.2

mmol) in CH₂Cl₂(40 mL) cooled to 0 °C was added 4 M aqueous HCl

(22.5 mL) over 45 min. The biphasic mixture was allowed to stir at 0 °C

for 1 h, and then the layers were separated. The organic layer was washed

with water, dried over Na₂SO₄, and ﬁltered. Evaporation of the solvent

under reduced pressure provided the product as a liquid (2.67 g, 84%)

that was used without further puriﬁcation.

trans-tert-Butyl (1-Chloro-2,3-dihydro-1H-inden-2-yl)-

carbamate (58). Under a N₂atmosphere, indene (1.23 g, 10.6

mmol) was added dropwise to a solution of 57 (2.00 g, 10.8 mmol) in

anhydrous toluene (20 mL) at a rate such that the reaction temperature

was maintained at 35−40 °C. The resulting solution was heated at 40 °C

for an additional 2 h before being cooled to 5−10 °C. The reaction was

quenched with a 20% aqueous solution of NaHSO₃(11 mL) and was

allowed to stir for 1.5 h. The mixture was partitioned between EtOAc

and water, and the organic layer was washed with brine and dried over

Na₂SO₄. The mixture was ﬁltered and concentrated under reduced

pressure to give the product as a solid (2.5 g, 88%). ¹H NMR (300 MHz,

CDCl₃) δ 1.57 (s, 9H), 3.22−3.25 (m, 2H), 5.28−5.35 (m, 1H), 5.55−

5.59 (m, 1H), 7.20−7.37 (m, 4H), 7.40−7.45 (m, 1H). LC−MS (M +

H) 255.

cis-tert-Butyl (1-Azido-2,3-dihydro-1H-inden-2-yl)carbamate

(59). Compound 58 (18.0 g, 67.4 mmol) was dissolved in anhydrous

DMF (130 mL), and the solution was treated with NaN₃(21.9 g, 337

mmol). The suspension was allowed to stir at room temperature under a

N₂atmosphere overnight. The mixture was then partitioned between

ethyl acetate and water, and the organic layer was washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The crude product was puriﬁed by silica gel chromatography (petroleum

ether/ethyl acetate = 20:1) to give the title compound as a solid (14.8 g,

80%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.41 (s, 9H), 2.85−3.03 (m,

2H), 4.16−4.25 (m, 1H), 4.92−5.02 (m, 1H), 7.20−7.35 (m, 4H),

7.38−7.47 (m, 1H). LC−MS (M + H) 275.

Methyl 2-((S)-1-((S)-2-((S)-2-(tert-Butoxycarbonyl(methyl)-

amino)propanamido)-2-cyclohexylacetyl)pyrrolidine-2-car-

boxamido)-2,3-dihydro-1H-indene-2-carboxylate (54). Com-

pound 48 (310 mg, 0.71 mmol) was dissolved in DMF (4 mL), and

the solution was cooled to 0 °C. EDCI (203 mg, 1.06 mmol), HOBt

hydrate (162 mg, 1.06 mmol), and 4-methylmorpholine (233 μL, 2.12

mmol) were added. The mixture was allowed to stir at 0 °C for 10 min,

and then methyl 2-amino-2,3-dihydro-1H-indene-2-carboxylate (135

mg, 0.71 mmol) was added. The reaction was allowed to stir at 0 °C with

slow warming to room temperature overnight. The mixture was then

partitioned between EtOAc and water. The organic layer was washed

with water and was then dried (Na₂SO₄), ﬁltered, and concentrated

under reduced pressure. The crude material was puriﬁed by silica gel

chromatography (EtOAc in hexanes gradient) to aﬀord the title

1

compound as a colorless solid (298 mg, 69%). H NMR (400 MHz,

CD₃OD) δ ppm 0.92−1.43 (m, 10H), 1.48−1.58 (m, 9H), 1.66−1.96

(m, 6H), 1.96−2.24 (m, 4H), 2.87−2.94 (m, 3H), 3.24−3.43 (m, 2H),

3.51−3.62 (m, 1H), 3.69−3.81 (m, 5H), 3.87−3.96 (m, 1H), 4.41−4.47

(m, 1H), 4.49−4.56 (m, 1H), 4.58−4.70 (m, 1H), 7.18−7.32 (m, 4H).

LC−MS (M + H) 613.

2-((S)-1-((S)-2-((S)-2-((tert-Butoxycarbonyl)(methyl)amino)-

propanamido)-2-cyclohexylacetyl)pyrrolidine-2-carboxami-

do)-2,3-dihydro-1H-indene-2-carboxylic acid (55). Compound

54 (298 mg, 0.49 mmol) was dissolved in methanol (2.5 mL) and water

(2.5 mL), and the solution was treated with NaOH (80 mg, 2.00 mmol).

The mixture was allowed to stir at room temperature for 2 h and was

then concentrated under reduced pressure. The residue was partitioned

between EtOAc and 1 N aqueous HCl, and the organic layer was dried

(Na₂SO₄), ﬁltered, and concentrated to give the product as a solid (290

mg, 98%). LC−MS (M +H) 599. This material was used without further

puriﬁcation.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-oxo-2-((S)-2-((2-(prop-2-

yn-1-ylcarbamoyl)-2,3-dihydro-1H-inden-2-yl)carbamoyl)-

pyrrolidin-1-yl)ethyl)amino)-1-oxopropan-2-yl)(methyl)-

carbamate (56). Compound 55 (290 mg, 0.48 mmol) was dissolved in

DMF (5 mL), and the solution was cooled to 0 °C. EDCI (139 mg, 0.73

mmol), HOBt hydrate (111 mg, 0.73 mmol), and 4-methylmorpholine

(213 μL, 1.94 mmol) were added, and the mixture was allowed to stir at

0 °C for 15 min before propargylamine (32 mg, 0.58 mmol) was added.

The mixture was allowed to warm to room temperature overnight and

was then partitioned between EtOAc and water. The organic layer was

dried (Na₂SO₄), ﬁltered, and concentrated under reduced pressure. The

crude material was puriﬁed by silica gel chromatography (EtOAc in

hexanes gradient) to give the title compound as a colorless solid (100

mg, 33%). LC−MS (M + H) 636. This material was used without further

puriﬁcation.

cis-tert-Butyl (1-Amino-2,3-dihydro-1H-inden-2-yl)-

carbamate (60). To a solution of 59 (15.9 g, 57.8 mmol) in ethanol

(1.0 L) was added 5% Pd/C (4.0 g), and the mixture was allowed to stir

under 1 atm H₂at room temperature for 22 h. The catalyst was removed

by ﬁltration, and the ﬁltrate was concentrated under reduced pressure to

give the title compound as a solid (11.0 g, 77%). ¹H NMR (300 MHz,

DMSO-d₆) δ 1.37 (s, 9H), 2.70−2.82 (m, 1H), 2.86−3.00 (m, 1H),

3.96−4.09 (m, 1H), 4.10−4.17 (m, 1H), 7.20−7.63 (m, 5H), −NH₂

resonances were not observed. LC−MS (M + H) 249.

tert-Butyl (1S,2R)-1-((S)-Pyrrolidine-2-carboxamido)-2,3-di-

hydro-1H-inden-2-ylcarbamate (61). A round-bottomed ﬂask was

charged with Fmoc-Pro-OH (4.12 g, 12.2 mmol) and CH₂Cl₂(15 mL),

and then HATU (5.50 g, 14.5 mmol) was added followed by DIPEA

(6.33 mL, 36.2 mmol). The solution was allowed to stir at room

temperature for 15 min, and then a solution of 60 (3.00 g, 12.1 mmol) in

CH₂Cl₂(15 mL) was added dropwise. The mixture was allowed to stir at

room temperature overnight and was then partitioned between EtOAc

(S,S,2S,2′S)-N,N′-(2,2′-(Hexa-2,4-diyne-1,6-diylbis-

(azanediyl))bis(oxomethylene)bis(2,3-dihydro-1H-indene-2,2-

diyl))bis(1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)-

9916

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

and water. The organic layer was washed with brine, dried (Na₂SO₄),

ﬁltered, and concentrated under reduced pressure. The crude material

was puriﬁed by silica gel chromatography (30% THF in hexanes) to give

the Fmoc-protected intermediate (mixture of diastereomers) as a

colorless solid (6.12 g, 10.8 mmol). This material was treated with

ethylamine (2 M solution in THF; 160 mL, 320 mmol), and the solution

was allowed to stir at room temperature. After 3 h, the mixture was

concentrated under reduced pressure, the residue was treated with

CH₂Cl₂, and the insoluble portion was removed by ﬁltration. The ﬁltrate

was concentrated, and the crude material was puriﬁed by silica gel

chromatography (0 to 100% THF in hexanes gradient) to give the

separated diastereomers: 1.17 g of the undesired diastereomer with the

(1R,2S) conﬁguration at the indane ring was obtained along with 1.31 g

of the diastereomer with the desired (1S,2R) conﬁguration at the indane

ring (61). ¹H NMR (300 MHz, DMSO-d₆) δ ppm 1.37 (s, 9H), 1.53−

1.65 (m, 2H), 1.72−1.84 (m, 1H), 1.89−2.03 (m, 1H), 2.65−2.90 (m,

4H), 3.05−3.16 (m, 1H), 3.52−3.60 (m, 1H), 4.31 (br s, 1H), 5.25−

5.35 (m, 1H), 6.85−6.94 (m, 1H), 7.06−7.14 (m, 1H), 7.14−7.26 (m,

3H), 8.03−8.12 (m, 1H). LC−MS (M + H) 346.

with brine, dried (Na₂SO₄), ﬁltered, and concentrated under reduced

pressure to give the crude di-Fmoc-protected dimer as a solid (611 mg).

This was treated directly with ethylamine (2 M in THF, 15 mL, 30

mmol), and the resulting solution was allowed to stir at room

temperature. After 1 h, the reaction was concentrated under reduced

pressure, and the crude material was puriﬁed by reverse-phase HPLC.

Lyophilization from a CH₃CN/H₂O solution aﬀorded the title

compound as a colorless solid (80 mg, 18% over two steps). ¹H NMR

(300 MHz, DMSO-d₆) δ ppm 0.84−1.02 (m, 4H), 1.02−1.24 (m, 12H),

1.45−1.87 (m, 20H), 2.10−2.21 (m, 6H), 2.86−2.97 (m, 2H), 2.98−

3.10 (m, 2H), 3.12−3.26 (m, 4H), 3.45−3.58 (m, 2H), 3.59−3.71 (m,

2H), 4.25−4.33 (m, 2H), 4.34−4.42 (m, 2H), 4.77−4.88 (m, 2H),

5.37−5.50 (m, 2H), 7.19−7.35 (m, 8H), 7.79−7.86 (m, 2H), 7.87−7.92

(m, 4H), 7.97−8.05 (m, 2H), 8.12−8.21 (m, 2H). LC−MS (M + H)

1070.

N4,N4′-Bis((1S,2R)-1-((S)-1-((S)-2-cyclohexyl-2-((S)-2-

(methylamino)propanamido)acetyl)pyrrolidine-2-carboxami-

do)-2,3-dihydro-1H-inden-2-yl)biphenyl-4,4′-dicarboxamide

(30). Prepared from 63 as described for compound 29 using biphenyl-

4,4′-dicarbonyl dichloride. ¹H NMR (300 MHz, CD₃OD) δ ppm 0.98−

1.22 (m, 6H), 1.22−1.41 (m, 10H), 1.62−1.94 (m, 14H), 1.94−2.15 (m,

6H), 2.35−2.50 (m, 6H), 3.11−3.24 (m, 2H), 3.24−3.33 (m, 2H),

3.37−3.47 (m, 2H), 3.63−3.75 (m, 2H), 3.83−3.96 (m, 2H), 4.36−4.60

(m, 4H), 4.98−5.12 (m, 2H), 5.54−5.68 (m, 2H), 7.25−7.40 (m, 6H),

7.42−7.50 (m, 2H), 7.74−7.86 (m, 4H), 7.94−8.03 (m, 4H), −NH

resonances were not observed. LC−MS (M + H) 1146. Purity: 93%.

N1,N10-Bis((1S,2R)-1-((S)-1-((S)-2-cyclohexyl-2-((S)-2-

(methylamino)propanamido)acetyl)pyrrolidine-2-carboxami-

do)-2,3-dihydro-1H-inden-2-yl)decanediamide (31). Prepared

from 63 as described for compound 29 using decanedioyl dichloride.

¹H NMR (300 MHz, CD₃OD) δ ppm 0.96−1.15 (m, 4H), 1.15−1.38

tert-Butyl (1S,2R)-1-((S)-1-((S)-2-Amino-2-cyclohexylacetyl)-

pyrrolidine-2-carboxamido)-2,3-dihydro-1H-inden-2-ylcarba-

mate (62). A round-bottomed ﬂask was charged with Fmoc-Chg−OH

(1.29 g, 3.41 mmol) and CH₂Cl₂(4 mL). HATU (1.29 g, 3.41 mmol)

was added followed by DIPEA (1.6 mL, 9.3 mmol), and the resulting

mixture was allowed to stir at room temperature for 15 min. A solution

of 61 (1.07 g, 3.10 mmol) in CH₂Cl₂(10 mL) was then added, and the

reaction was allowed to stir at room temperature. After 90 min, the

reaction mixture was concentrated under reduced pressure, and the

crude material was puriﬁed by silica gel chromatography (0 to 100%

THF in hexanes gradient) to give the Fmoc-protected product as a solid

(2.0 g). A portion of this material (1.40 g, 1.98 mmol) was dissolved in

ethylamine (2 M solution in THF; 15 mL, 30 mmol), and the solution

was allowed to stir at room temperature. After 1 h, the mixture was

concentrated under reduced pressure, the residue was treated with

CH₂Cl₂, and the insoluble portion was removed by ﬁltration. The ﬁltrate

was concentrated, and the crude material was puriﬁed by silica gel

chromatography (0 to 100% THF in hexanes gradient) to give the title

compound as a colorless solid (966 mg). LC−MS (M + H) 485.

(S)-((S)-2-((S)-2-((1S,2R)-2-Amino-2,3-dihydro-1H-inden-1-

ylcarbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl) 2-((((9H-

ﬂuoren-9-yl)methoxy)carbonyl)(methyl)amino)propanoate

Hydrochloride (63). A round-bottomed ﬂask was charged with Fmoc-

N-Me-Ala-OH (470 mg, 1.44 mmol) and HATU (603 mg, 1.59 mmol).

CH₂Cl₂(4 mL) and DIPEA (0.76 mL, 4.3 mmol) were added, and the

resulting solution was allowed to stir at room temperature for 15 min. A

solution of 62 (0.70 g, 1.44 mmol) in CH₂Cl₂(4 mL) was then added

dropwise, and the reaction was allowed to stir at room temperature.

After 1 h, the reaction mixture was concentrated under reduced

pressure, and the crude material was puriﬁed by silica gel

chromatography (0 to 100% THF in hexanes gradient) to give the

fully protected monomer as a solid (1.1 g). This material was dissolved

in 4 N HCl/dioxane (5 mL, 20 mmol), and the solution was allowed to

stir at room temperature for 1 h. The mixture was then concentrated

under reduced pressure to give the title compound as a colorless solid

(m, 20H), 1.52−1.89 (m, 16H), 1.90−2.24 (m, 12H), 2.26−2.33 (m,

6H), 2.85−2.96 (m, 2H), 3.06−3.16 (m, 2H), 3.16−3.27 (m, 2H),

3.63−3.74 (m, 2H), 3.85−3.96 (m, 2H), 4.39−4.53 (m, 4H), 4.74−4.81

(m, 2H), 5.37−5.46 (m, 2H), 7.18−7.30 (m, 6H), 7.32−7.40 (m, 2H),

−NH resonances were not observed. LC−MS (M + H) 1106.

ASSOCIATED CONTENT

* Supporting Information

■

S

Procedures for cIAP1, cIAP2, and XIAP binding assays, MDA-

MB-231 cell assays, in vivo experiments, and pharmacokinetic

experiments; representative ﬂuorescence polarization titration

curves for compound 14; western blots of MDA-MB-231 cells

treated with diﬀerent concentrations of 14. This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: (781) 839-4153. E-mail: edward.hennessy@

astrazeneca.com.

■

Notes

The authors declare no competing ﬁnancial interest.

1

(1.0 g, 99% over two steps). H NMR (300 MHz, CD₃OD) δ ppm

0.98−1.36 (m, 9H), 1.63−1.93 (m, 7H), 1.94−2.08 (m, 1H), 2.09−2.32

(m, 3H), 2.79−2.94 (m, 4H), 3.12−3.24 (m, 1H), 3.36−3.47 (m, 1H),

3.61−3.74 (m, 1H), 3.90−4.04 (m, 1H), 4.15−4.26 (m, 1H), 4.27−4.36

(m, 1H), 4.36−4.44 (m, 1H), 4.44−4.53 (m, 3H), 4.55−4.70 (m, 1H),

5.47−5.56 (m, 1H), 7.28−7.40 (m, 5H), 7.41−7.49 (m, 4H), 7.60−7.70

(m, 2H), 7.81−7.91 (m, 3H). LC−MS (M + H) 693.

N1,N4-Bis((1S,2R)-1-((S)-1-((S)-2-cyclohexyl-2-((S)-2-

(methylamino)propanamido)acetyl)pyrrolidine-2-carboxami-

do)-2,3-dihydro-1H-inden-2-yl)terephthalamide (29). A round-

bottomed ﬂask was charged with 63 (615 mg, 0.84 mmol) and

anhydrous THF (10 mL), and the mixture was cooled to 0 °C. To this

was added DIPEA (0.29 mL, 1.68 mmol) and ﬁnally a solution of

terephthaloyl dichloride (85 mg, 0.42 mmol) in anhydrous THF (1

mL). The mixture was allowed to stir at 0 °C for 1 h and was then

partitioned between EtOAc and water. The organic layer was washed

ACKNOWLEDGMENTS

■

We are grateful to Kanayo Azogu, Natascha Bezdenejnih-Snyder,

Nancy DeGrace, Ziling Lu, and Bridget Reaney for analytical and

puriﬁcation support.

ABBREVIATIONS USED

■

IAP, inhibitor of apoptosis proteins; BIR, baculovirus IAP repeat;

XIAP, X chromosome-linked inhibitor of apoptosis protein;

cIAP1, cellular inhibitor of apoptosis protein 1; cIAP2, cellular

inhibitor of apoptosis protein 2; AVPI, alanine−valine−proline−

isoleucine; PSA, polar surface area; NPSA, nonpolar surface area;

PPB, plasma protein binding; Q_H, hepatic blood ﬂow rate; CL,

clearance; V_ss, volume of distribution at steady state; t_1/2, plasma

9917

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation

of caspase-8. J. Cell Sci. 2002, 115, 2757−2770.

(15) Tamm, I.; Kornblau, S. M.; Segall, H.; Krajewski, S.; Welsh, K.;

Kitada, S.; Scudiero, D. A.; Tudor, G.; Qui, Y. H.; Monks, A.; Andreeff,

M.; Reed, J. C. Expression and prognostic significance of IAP-family

genes in human cancers and myeloid leukemias. Clin. Cancer Res. 2000,

6, 1796−1803.

(16) Yang, L.; Cao, Z.; Yan, H.; Wood, W. C. Coexistence of high levels

of apoptotic signaling and inhibitor of apoptosis proteins in human

tumor cells: Implication for cancer specific therapy. Cancer Res. 2003,

63, 6815−6824.

(17) Vucic, D.; Fairbrother, W. J. The inhibitor of apoptosis proteins as

therapeutic targets in cancer. Clin. Cancer Res. 2007, 13, 5995−6000.

(18) Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.;

Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. W. Structural basis for

binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 2000, 408,

1004−1008.

(19) Wu, G.; Chai, J.; Suber, T. L.; Wu, J.-W.; Du, C.; Wang, X.; Shi, Y.

Structural basis of IAP recognition by Smac/DIABLO. Nature 2000,

408, 1008−1012.

(20) Sharma, S. K.; Straub, C.; Zawel, L. Development of

peptidomimetics targeting IAPs. Int. J. Pept. Res. Ther. 2006, 12, 21−32.

(21) Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Qian, D.; Lu, J.;

Qiu, S.; Bai, L.; Peng, Y.; Cai, Q.; Wang, S. Design of small-molecule

peptidic and nonpeptidic Smac mimetics. Acc. Chem. Res. 2008, 41,

1264−1277.

(22) Mannhold, R.; Fulda, S.; Carosati, E. IAP antagonists: Promising

candidates for cancer therapy. Drug Discovery Today 2010, 15, 210−219.

(23) Flygare, J. A.; Fairbrother, W. J. Small-molecule pan-IAP

antagonists: A patent review. Expert Opin. Ther. Patents 2010, 20,

251−267.

(24) Condon, S. M. The discovery and development of Smac-mimetics

− small-molecule antagonists of the inhibitor of apoptosis proteins.

Annu. Rep. Med. Chem. 2011, 46, 211−226.

(25) Fulda, S.; Vucic, D. Targeting IAP proteins for therapeutic

intervention in cancer. Nat. Rev. Drug Discovery 2012, 11, 109−124.

(26) Chai, J.; Du, C.; Wu, J.-W.; Kyin, S.; Wang, X.; Shi, Y. Structural

and biochemical basis of apoptotic activation by Smac/DIABLO. Nature

2000, 406, 855−862.

(27) Li, L.; Thomas, R. M.; Suzuki, H.; De Brabander, J. K.; Wang, X.;

Harran, P. G. A small molecule Smac mimic potentiates TRAIL- and

TNFα-mediated cell death. Science 2004, 305, 1471−1474.

(28) Sun, H.; Nikolovska-Coleska, Z.; Lu, J.; Meagher, J. L.; Yang, C.-

Y.; Qiu, S.; Tomita, Y.; Ueda, Y.; Jiang, S.; Krajewski, K.; Roller, P. P.;

Stuckey, J. A.; Wang, S. Design, synthesis, and characterization of a

potent, nonpeptide, cell-permeable, bivalent Smac mimetic that

concurrently targets both the BIR2 and BIR3 domains in XIAP. J. Am.

Chem. Soc. 2007, 129, 15279−15294.

(29) Cossu, F.; Milani, M.; Mastrangelo, E.; Vachette, P.; Servida, F.;

Lecis, D.; Canevari, G.; Delia, D.; Drago, C.; Rizzo, V.; Manzoni, L.;

Seneci, P.; Scolastico, C.; Bolognesi, M. Structural basis for bivalent

Smac-mimetics recognition in the IAP protein family. J. Mol. Biol. 2009,

392, 630−644.

(30) Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A.-S.; Betz, S.

F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.;

Olejniczak, E. T.; Oleksijew, A.; Oltersdorf, T.; Rosenberg, S. H.;

Shoemaker, A. R.; Tomaselli, K. J.; Zou, H.; Fesik, S. W. Discovery of

potent antagonists of the antiapoptotic protein XIAP for the treatment

of cancer. J. Med. Chem. 2004, 47, 4417−4426.

(31) Laurent, A. IAP BIR domain binding compounds. Patent

US20070093428, 2007.

(32) Petersen, S. L.; Wang, L.; Yalcin-Chin, A.; Li, L.; Peyton, M.;

Minna, J.; Harran, P.; Wang, X. Autocrine TNFα signaling renders

human cancer cells susceptible to Smac-mimetic-induced apoptosis.

Cancer Cell 2007, 12, 445−456.

half-life; AUC, area under the curve; k_off, oﬀ rate; DMSO,

dimethyl sulfoxide; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium];

SCID, severe combined immunodeﬁciency; C₀, initial plasma

concentration; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbo-

diimide hydrochloride; HOBt, 1-hydroxybenzotriazole hydrate;

Boc-Pro-OH, (2S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carbox-

ylic acid; Boc-Tle-OH, (2S)-2-[(tert-butoxycarbonyl)amino]-

3,3-dimethylbutanoic acid; Boc-N-Me-Ala-OH, (2S)-2-[(tert-

butoxycarbonyl)(methyl)amino]propanoic acid; DMTMM, 4-

(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpolinium

chloride; Chg-OMe·HCl, methyl (2S)-amino(cyclohexyl)-

ethanoate hydrochloride; Pro-OMe·HCl, methyl (2S)-pyrroli-

dine-2-carboxylate hydrochloride; DPPA, diphenylphophoryl

azide; DIPEA, N,N-diisopropylethylamine; Fmoc-Pro-OH,

(2S)-1-[(9H-ﬂuoren-9-ylmethoxy)carbonyl]pyrrolidine-2-car-

boxylic acid; HATU, (dimethylamino)-N,N-dimethyl(3H-

[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium hexa-

ﬂuorophosphate; Fmoc-Chg-OH, (2S)-2-{[(9H-ﬂuoren-9-

ylmethoxy)carbonyl](methyl)amino}-2-cyclohexylacetic acid;

Fmoc-N-Me-Ala-OH, (2S)-2-{[(9H-ﬂuoren-9-ylmethoxy)-

carbonyl](methyl)amino}propanoic acid

REFERENCES

■

(1) Kerr, J. F. R.; Wyllie, A. H.; Currie, A. R. Apoptosis: A basic

biological phenomenon with wide-ranging implications in tissue

kinetics. Br. J. Cancer 1972, 26, 239−257.

(2) Carson, D. A.; Ribiero, J. M. Apoptosis and disease. Lancet 1993,

341, 1251−1254.

(3) Solary, E.; Dubrez, L.; Eymin, B. The role of apoptosis in the

pathogenesis and treatment of disease. Eur. Respir. J. 1996, 9, 1293−

1305.

(4) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: The next

generation. Cell 2011, 144, 646−674.

(5) Salvesen, G. S.; Duckett, C. S. IAP proteins: Blocking the road to

death’s door. Nat. Rev. Mol. Cell Biol. 2002, 3, 401−410.

(6) Gyrd-Hansen, M.; Meier, P. IAPs: From caspase inhibitors to

modulators of NF-κB, inflammation, and cancer. Nat. Rev. Cancer 2010,

10, 561−574.

(7) Shiozaki, E. N.; Chai, J.; Rigotti, D. J.; Riedl, S. J.; Li, P.; Srinivasula,

S. M.; Alnemri, E. S.; Fairman, R.; Shi, Y. Mechanism of XIAP-mediated

inhibition of caspase-9. Mol. Cell 2003, 11, 519−527.

(8) Chai, J.; Shiozaki, E.; Srinivasula, S. M.; Wu, Q.; Dataa, P.; Alnemri,

E. S.; Shi, Y. Structural basis of caspase-7 inhibition by XIAP. Cell 2001,

104, 769−780.

(9) Huang, Y.; Park, Y. C.; Rich, R. L.; Segal, D.; Myszka, D. G.; Wu, H.

Structural basis of caspase inhibition by XIAP: Differential roles of the

linker versus the BIR domain. Cell 2001, 104, 781−790.

(10) Riedl, S. J.; Renatus, M.; Schwarzenbacher, R.; Zhou, Q.; Sun, C.;

Fesik, S. W.; Liddington, R. C.; Salvesen, G. S. Structural basis for the

inhibition of caspase-3 by XIAP. Cell 2001, 104, 791−800.

(11) Vaux, D. L.; Silke, J. IAPs, RINGs, and ubiquitylation. Nat. Rev.

Mol. Cell Biol. 2005, 6, 287−297.

(12) Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Federova, A.

V.; Kayagaki, N.; Garg, P.; Zobel, K.; Dynek, J. N.; Elliott, L. O.;

Wallweber, H. J. A.; Flygare, J. A.; Fairbrother, W. J.; Deshayes, K.; Dixit,

V. M.; Vucic, D. IAP antagonists induce autoubiquitination of c-IAPs,

NF-κB activation, and TNFα-dependent apoptosis. Cell 2007, 131,

669−681.

(13) Vince, J. E.; Wong, W. W.-L.; Khan, N.; Feltham, R.; Chau, D.;

Ahmed, A. U.; Benetatos, C. A.; Chunduru, S. K.; Condon, S. M.;

McKinlay, M.; Brink, R.; Leverkus, M.; Tergaonkar, V.; Schneider, P.;

Callus, B. A.; Koentgen, F.; Vaux, D. L.; Silke, J. IAP antagonists target

cIAP1 to induce TNFα-dependent apoptosis. Cell 2007, 131, 682−693.

(14) Fotin-Mleczek, M.; Henkler, F.; Samel, D.; Reichwein, M.;

Hausser, A.; Parmryd, I.; Scheurich, P.; Schmid, J. A.; Wajant, H.

Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of

(33) Ndubaku, C.; Varfolomeev, E.; Wang, L.; Zobel, K.; Lau, K.;

Elliott, L. O.; Maurer, B.; Federova, A. V.; Dynek, J. N.; Kohler, M.;

Hymowitz, S. G.; Tsui, V.; Deshayes, K.; Fairbrother, W. J.; Flygare, J. A.;

Vucic, D. Antagonism of c-IAP and XIAP proteins is required for

9918

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Journal of Medicinal Chemistry

Article

efficient induction of cell death by small-molecule IAP antagonists. ACS

Chem. Biol. 2009, 4, 557−566.

(34) Hennessy, E. J.; Saeh, J. C.; Sha, L.; MacIntyre, T.; Wang, H.;

Larsen, N. A.; Aquila, B. M.; Ferguson, A. D.; Laing, N. M.; Omer, C. A.

Discovery of aminopiperidine-based Smac mimetics as IAP antagonists.

Bioorg. Med. Chem. Lett. 2012, 22, 1690−1694.

(35) Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.; Nikolovska-Coleska, Z.;

McEachern, D.; Yang, C.-Y.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. Potent

bivalent Smac mimetics: Effect of the linker on binding to inhibitor of

apoptosis proteins (IAPs) and anticancer activity. J. Med. Chem. 2011,

54, 3306−3318.

(36) Feltham, R.; Bettjeman, B.; Budhidarmo, R.; Mace, P. D.; Shirley,

S.; Condon, S. M.; Chunduru, S. K.; McKinlay, M. A.; Vaux, D. L.; Silke,

J.; Day, C. L. Smac mimetics activate the E3 ligase activity of cIAP1

protein by promoting RING domain dimerization. J. Biol. Chem. 2011,

286, 17015−17028.

alcohols using chiral cis-1-amino-2-hydroxy-1,2,3,4-tetrahydronaptha-

lene as chiral ligand. Tetrahedron: Asymmetry 1997, 8, 895−902.

(51) Winstein, S.; Boschan, R. The role of neighboring groups in

replacement reactions. XVII. Complex neighboring groups. The

benzamido group. J. Am. Chem. Soc. 1950, 72, 4669−4677.

(52) Higashijima, S.; Itoh, H.; Senda, Y.; Nakano, S. cis-1-Amino-

1,2,3,4-tetrahydro-2-napthalenol: Resolution and application to the

catalytic enantioselective reduction of ketones. Tetrahedron: Asymmetry

1997, 8, 3107−3110.

(53) Lakshman, M. K.; Chaturvedi, S.; Zajc, B.; Gibson, D. T.; Resnick,

S. M. A general chemoenzymatic synthesis of enantiopure cis β-amino

alcohols from microbially derived cis-glycols. Synthesis 1998, 30, 1352−

1356.

(54) Orsini, F.; Sello, G.; Bestetti, G. Enantiopure vic-amino alcohols

and vic-diamines from (1R,2S)-1,2-dihydroxy-1,2-dihydronapthalene.

Tetrahedron: Asymmetry 2001, 12, 2961−2969.

(55) Shieh, W.-C.; Chen, Z.; Xue, S.; McKenna, J.; Wang, R.-M.;

Prasad, K.; Repic, O. Synthesis of sterically-hindered peptidomimetics

using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpolinium

chloride. Tetrahedron Lett. 2008, 49, 5359−5362.

(37) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.

Experimental and computational approaches to estimate solubility and

permeability in drug discovery and development settings. Adv. Drug

Delivery Rev. 2001, 46, 3−26.

(56) Vu, T. C.; Brzozowksi, D. B.; Fox, R.; Godfrey, J. D.; Hanson, R.

L.; Kolotuchin, S. V.; Mazzullo, J. A.; Patel, R. N.; Wang, J.; Wong, K.;

Yu, J.; Zhu, J.; Magnin, D. R.; Augeri, D. J.; Hamann, L. G. Methods and

compounds producing dipeptidyl peptidase IV inhibitors and

intermediates thereof. Patent WO2004052850, 2004.

(38) Kipp, R. A.; Case, M. A.; Wist, A. D.; Cresson, C. M.; Carrell, M.;

Griner, E.; Wiita, A.; Albiniak, P. A.; Chai, J.; Shi, Y.; Semmelhack, M. F.;

McLendon, G. L. Molecular targeting of inhibitor of apoptosis proteins

based on small molecule mimics of natural binding partners.

Biochemistry 2002, 41, 7344−7349.

(57) Notzel, M. W.; Labahn, T.; Es-Sayed, M.; de Meijere, A. A new

̈

(39) Condon, S. M.; LaPorte, M. G.; Deng, Y.; Rippin, S. R. Dimeric

IAP inhibitors. Patent WO2006091972, 2006.

and efficient access to thiazoline-4-carboxylates and cysteine derivatives

incorporating cyclopropyl groups. Eur. J. Org. Chem. 2001, 3025−3030.

(58) Walker, B. J.; Wrobel, P. J. Stereospecific cis radical addition.

Convincing evidence for a 1,3-radical transfer reaction. J. Chem. Soc.,

Chem. Commun. 1980, 462−463.

(40) Nikolovska-Coleska, Z.; Meagher, J. L.; Jiang, S.; Yang, C.-Y.; Qiu,

S.; Roller, P. P.; Stuckey, J. A.; Wang, S. Interaction of a cyclic, bivalent

Smac mimetic with the X-linked inhibitor of apoptosis protein.

Biochemistry 2008, 47, 9811−9824.

(41) Burton, P. S.; Conradi, R. A.; Hilgers, A. R. Transcellular

mechanism of peptide and protein absorption: Passive aspects. Adv.

Drug Delivery Rev. 1991, 7, 365−386.

(42) Because of technical diﬃculties in obtaining accurate

experimental Log D measurements for these compounds, values

Advanced Chemistry Development, Inc.) are presented in Table 3

instead.

(43) Stenberg, P.; Luthman, K.; Artursson, P. Prediction of membrane

permeability to peptides from calculated dynamic molecular surface

dynamics. Pharm. Res. 1999, 16, 205−212.

(44) Huang, Y.; Rich, R. L.; Myszka, D. G.; Wu, H. Requirement of

both the second and third BIR domains for the relief of X-linked

inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by

Smac. J. Biol. Chem. 2003, 278, 49517−49522.

(45) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target

residence time and its implications for lead optimization. Nat. Rev. Drug

Discovery 2006, 5, 730−739.

(46) 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. A potent and

orally active antagonist (SM-406/AT-406) of multiple inhibitor of

apoptosis proteins (IAPs) in clinical development for cancer treatment.

J. Med. Chem. 2011, 54, 2714−2726.

(47) Petersen, S. L.; Peyton, M.; Minna, J. D.; Wang, X. Overcoming

cancer cell resistance to Smac mimetic induced apoptosis by modulating

cIAP-2 expression. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 11936−11941.

(48) Darding, M.; Feltham, R.; Tenev, T.; Bianchi, K.; Benetatos, C.;

Silke, J.; Meier, P. Molecular determinants of Smac mimetic induced

degradation of cIAP1 and cIAP2. Cell Death Differ. 2011, 18, 1376−

1386.

(49) Bai, L.; McEachern, D.; Yang, C.-Y.; Lu, J.; Sun, H.; Wang, S.

LRIG1 modulates cancer cell sensitivity to Smac mimetics by regulating

TNFα expression and receptor tyrosine kinase signaling. Cancer Res.

2012, 72, 1229−1238.

(50) Bellucci, C. M.; Bergamini, A.; Cozzi, P. G.; Papa, A.; Tagliavini,

E.; Umani-Ronchi, A. Catalytic asymmetric synthesis of secondary

9919

dx.doi.org/10.1021/jm401075x | J. Med. Chem. 2013, 56, 9897−9919

Article Doi

Discovery of a novel class of dimeric smac mimetics as potent IAP antagonists resulting in a clinical candidate for the treatment of cancer (AZD5582)

DOI: 10.1021/jm401075x

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm401075x

Full text of DOI:10.1021/jm401075x

Products guided by the article

R&D Labs maybe for 109470-12-4

Relevant to this article

Hot Product