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Figure 13 Series of de novo designed nonpeptides containing a benzamide

template (exemplified by compound 12, AP21733) designed to interact favorably

with Src SH2 and specifically to displace structural waters found in complexed

Src SH2 structures [14,27]. The Src SH2 binding IC50 is shown for each

compound, as well as a comparative IC50 for Ac-pTyr-Glu-Glu-Ile-NH2

(compound 9).

contacts with Lys182 (206 in Src) and Ile193 (217 in Src). The phenyl ring

of the benzamide template also forms favorable stacking interactions with

Tyr181 (205 in Src). Although the cyclohexylmethyl group interacts with

the pY+3 pocket, the contacts are primarily surface type and do not

extend as deeply into the pocket as the Ile of pTyr-Glu-Glu-Ile. Consequently, SAR exploration of the pY+3 pocket, which had not been

rigorously studied with nonpeptide (peptidomimetic) small molecules

[13,14], became the first objective to be investigated.

Parallel synthesis provides the means of rapidly preparing discrete

analogues for both lead generation and lead optimization strategies,

which makes it an attractive option for developing compound databases

for therapeutic targets. Furthermore, the incorporation of structure-based

methods into the design and evaluation of parallel synthetic libraries has

proven to be a successful strategy for integrating the two drug discovery

technologies [29]. For the synthesis of the benzamide-containing compounds, we devised a hitherto unreported solid phase, parallel synthetic

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

route focusing on pY+3 derivatives based on compound 13 and AP21733

[30]. Our synthetic philosophy adopts an integrated solid and solution

phase strategy that differs from the traditional unidirectional approach by

recognizing the strengths and limitations of each synthetic method and

then devising a route accordingly (Fig. 14) [31]. In addition, this strategy

provides chemical flexibility to incorporate, within the compound’s

molecular design, the necessary functional group complexity dictated by

our structure-based methods. The importance of the carboxamide group

guided our decision to exploit this functionality both as a solid support

attachment site and as a conserved binding element. A Rink amide linkage

was chosen to provide facile coupling of the template, via its benzoic acid,

and eventual generation of the critical benzamide binding moiety upon

cleavage from the solid support. The protected salicylic acid template 14

(synthesized using a modification of the solution phase literature procedure) [27] was coupled to Rink amide AM resin by means of standard

protocols (EDC/HOBt) to provide the benzamide-linked resin 15

Figure 14 Parallel synthetic approaches demonstrating a traditional (unidirectional) strategy and a multifaceted, integrated strategy; the latter utilizes both

solid and solution phase reactions.

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

(Scheme 3) [30]. The pY+3 diversity alcohols (R1)-OH (Fig. 15) were

attached to the template through a Mitsunobu coupling to provide ether

derivatives of 16. Palladium-mediated Alloc deprotection followed by

amide formation using the phosphate-ester-containing diversity acids

(R2)-CO2H provided the fully coupled resin-bound products of 17.

Cleavage from the resin with 95% TFA/H2O, which also afforded benzyl

phosphate deprotection, followed by reversed-phase (RP) semipreparative

Scheme 3 Abbreviations: EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide)hydrochloride; HOBt, 1-hydroxybenzotriazole; DEAD, diethyl


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

Figure 15 The diversity alcohols (R1)-OH and carboxylic acids (R2)-CO2H used

to synthesize compounds represented by 18 and 19. (From Ref. 30.)

Table 5 Src SH2 Binding (FP) for Analogs of Compound 18

Source: Ref. 30.

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

HPLC purification generated the final compounds represented by 18

(mixture of diastereomers) and 19.

The selection of the pY+3 diversity R1 groups was guided by a FLO

docking program [32], utilizing 800 commercially available alcohols

(prefiltered by MW, H-bond donors, and reactive groups outside the

OH). The R1 groups were computationally incorporated [33] into the

benzamide template, docked into our Src SH2 binding site model [34], and

then rank-ordered according to favorable fit. The final list of alcohols was

Figure 16 The predicted binding mode of compound 23 in the pY+3 pocket of

the Src SH2 model. The branch point in the pY+3 bisallyl group allows favorable

binding interactions to occur. (From Ref. 30.)

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

selected according to predicted binding as well as the ability of the R1

group to impart beneficial properties to the molecule as related to low

molecular weight, increase solubility, and other factors.

Table 5 contains the Src SH2 binding results for a selected set of

pY+3-modified nonpeptide analog. Relative to compound 20, which was

synthesized by means of our solid phase method to act as an internal

standard, increases in binding affinity appeared to track the degree of

hydrophobicity at the R1 group as demonstrated by compounds 21

(methyl) and 22 (isopropyl). From a drug design perspective, the result

of 22 is significant because a four-carbon reduction took place, relative to

the cyclohexylmethyl group (MW decrease by 54), without greatly compromising the binding affinity (four-fold).

An extension of the a-branch point of the isopropyl group to a

bisallyl resulted in the highest affinity analog, compound 23. Inspection of

the docked structure of 23 in our Src SH2 model reveals how the branch

point allows one allyl side chain to hug the surface of the protein, while the

other is able to extend deeply into the pY+3 pocket (Fig. 16). A significant

decrease in binding affinity occurs with the incorporation of a morpholine

group, as exemplified by compound 24. Presumably, this result reflects an

incompatibility of the positively charged morpholine group (at pH 7.2 of

the binding assay) in the hydrophobic pY+3 binding pocket of the Src

SH2 domain; structurally, the pY+3 pocket according to our Src SH2

model accommodates this compound.




The next logical step in the progression to a cellularly active Src SH2

inhibitor was to incorporate a high affinity, biologically stable pTyr

mimic into the benzamide template. Drug design efforts at ARIAD led to

a novel Src SH2 inhibitors containing 4-diphosphonomethylphenylalanine (Dmp), namely, compound 25 (AP21773; Fig. 17) [16]. The design

concept for the Dmp group evolved from a 1.5 A˚ x-ray structure of Src

SH2, crystallized from citrate buffer, that fortuitously contained a citrate

molecule bound in the pTyr pocket. The x-ray structure reveals a number

of additional hydrogen bonds that citrate makes compared with a pTyr

group; this inspired the design of the Dmp moiety as a novel mimic of the

citrate interactions. Armed with these designed hydrogen bond contact

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

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.

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