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Figure 17 Src SH2 binding IC50 (Fp) for compound 25 (AP21773), which

contains a bone-targeted, 4-diphosphonomethylphenylalanine (Dmp) pTyr

mimic. (From Ref. 16.)

groups, we expected the Dmp to bind with greater affinity than pTyr, and

the Src SH2 binding results for AP21773 (Dmp) and AP21733 (pTyr)

confirm this prediction (Figs. 13 and 17). X-ray and NMR structural

studies involving AP21773 [16] verify these additional Dmp-related

contacts in the pTyr pcket, as well as other key Src SH2 interactions

observed earlier with this benzamide class as already discussed. The Dmp

moiety not only increases Src SH2 binding affinity, but also provides a

mechanism for tissue selectivity by targeting bone [16,35]. This targeting

feature provides a higher local concentration of compound on bone than

Figure 18 Solid phase synthetic scheme and molecular diversity groups for

compound 27. (From Ref. 36.)

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Table 6 Src SH2 Binding (FP), Rabbit Pit, and Rat TPTX Data for Analogs of

Compounds 27 and 35 (AP22209)

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

in solution, which in addition increases the amount of Src inhibitor

delivered to the resorbing osteoclasts associated with bone. Compounds

containing the Dmp group, including AP21773, also bind to hydroxyapatite (data not shown), a major component of bone [16].

Building on the SAR information obtained from the pY+3 study,

we focused on improving binding affinity and cellular potency by means of

structure-based, parallel synthesis. A resin-bound, enantiomerically

enriched benzamide template 26 (Fig. 18) [28] was synthetically elaborated

in a manner similar to that described in Scheme 3 to provide the desired

Dmp-containing products. A total of 22 structurally biased analogues of

27 were generated (not all combinations synthesized) having specific R1

and R2 groups as shown in Figure 18 [36]. Table 6 shows the SAR results

for a selected series of the benzamide analogues. Similar to the earlier

study, increasing hydrophobicity at the pY+3 position leads to increased

binding affinity, as demonstrated by compounds 28 to 30. Interestingly,

the overall effect on Src SH2 binding of the 3-pentyl group of compound

30 appears to be similar to that of the cyclohexylmethyl group of

AP21773, although the latter group contains two more carbon atoms.

All the derivatives show an approximately 5- to 10-fold reduction in Src

SH2 binding affinity with no substitution (R2 = H, compounds 31–34) at

Figure 19 Solid-phase synthetic scheme and molecular diversity groups for

compound 36.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

the R2 position. Finally, in an effort to select compounds for testing in an

in vivo thyroparathyroidectomized (TPTX) animal model [37,38], Src SH2

inhibitors were evaluated in a cell-based resorption assay mediated by

rabbit osteoclasts. A potent compound, 35 (AP22209; Table 6), was discovered and showed significant bone resorption inhibition in test animals

(55% inhibition at 25 mg/kg b.i.d.), thus providing in vivo validation for

an Src SH2 inhibitor (C. A. Metcalf III, unpublished results).

A recent series of proprietary, nonpeptide Src SH2 inhibitors synthesized by our solid phase, parallel synthetic method is outlined in Figure 19.

This inhibitor series was based on a set of compounds disclosed earlier

[35,39,40] and containing a novel, high-affinity bicyclic benzamide template designed to interact favorably with the hydrophobic Tyr205 Src SH2

protein residue. A bone-targeting, 3,4-diphosphonophenylalanine (Dpp)

mimic of pTyr was also incorporated [35,40]. The Dpp moiety can be

correlated to both pTyr and citrate (Fig. 20). The biological data for the

library analogs of 36 will be described elsewhere.

Figure 20 Representation of the design rationale for two novel, bone-targeting

pTyr mimics, Dmp and Dpp, relative to an x-ray structure [16] of citrate

complexed with Src SH2.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.


The increasing number of therapeutic targets available to drug discovery

programs has challenged chemists to devise new and efficient strategies for

the advancement of lead compounds to clinical candidate status. One

evolving approach, as described in this chapter, is the integration of

synergistic technologies (e.g., structure-based drug design and combinatorial chemistry) into a focused program that emphasizes the strengths of

each individual method. We have used this philosophy to direct our Src

SH2 program toward achieving novel proprietary Src SH2 inhibitors such

as AP22209, which exhibit promising antiresorptive activity both in an in

vivo animal model and in cell-based osteoclast assays. The use of structure-based, small-molecule libraries allowed us to rationally design compounds relative to predicted binding interactions, while taking advantage

of parallel synthesis to rapidly advance lead optimization. By adopting a

synthetic strategy that utilizes both solid and solution phase chemistries,

we were able to achieve the necessary chemical purity and diversity for

SAR interpretation at all stages of the drug discovery process. This

integrated drug design and combinatorial chemistry strategy is currently

being adapted to other drug discovery programs at ARIAD.


The authors thank all our colleagues at ARIAD Pharmaceuticals,

including Chi Vu, Virginia Jacobsen, Michael Yang, William Shakespeare, Regine Bohacek, Joseph Eyermann, Berkley Lynch, Shelia

Violette, and Manfred Weigele, and especially Mayumi Uesugi, Vaibhav

Varkhedkar, and Chad Haraldson, whose contributions were significant

to the success of this work. We also thank Chris Stearns for her help with

the figures, David Dalgarno for his editorial suggestions, and Jay

LaMarche for allowing us to buy all our expensive toys.


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Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.











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Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.


Three-Dimensional Structure of

the Inhibited Catalytic Domain

of Human Stromelysin-1 by

Heteronuclear NMR Spectroscopy

Paul R. Gooley

University of Melbourne, Parkville, Victoria, Australia



With the aid of isotopic enrichment it is now routine to determine the

structure of moderate to large proteins (20 to 40 kDa) by multidimensional

heteronuclear nuclear magnetic resonance (NMR) spectroscopy [1]. The

advantages these heteronuclear experiments offer are spectral simplification and a reduced dependence on narrow proton linewidths. By spreading

the 1H– 1H correlations of a 2-D NMR spectrum into a third and, perhaps,

a fourth dimension, according to the chemical shift of the attached 13C or


N nucleus, considerable spectral simplification is achieved. As the proton

of interest is now correlated with its bound 13C or 15N, the information

content for assignment is increased, and as the individual planes of the 3-D

or 4-D spectra contain relatively fewer overlapping peaks, problems with

assignment ambiguities are reduced. These experiments are more efficient

than their homonuclear counterparts because transfer of magnetization

relies on the large one-bond heteronuclear couplings (11 to 140 Hz). The

first stage in solving the structure of a protein requires the acquisition of a

large number of three-dimensional experiments for sequence-specific as-

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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