Structure-affinity relationships of glutamine mimics incorporated into phosphopeptides targeted to the SH2 domain of signal transducer and activator of transcription 3

6126 J. Med. Chem. 2009, 52, 6126–6141

DOI: 10.1021/jm901105k

Structure-Affinity Relationships of Glutamine Mimics Incorporated into Phosphopeptides

Targeted to the SH2 Domain of Signal Transducer and Activator of Transcription 3

Pijus K. Mandal,^†Zhiyong Ren,^‡Xiaomin Chen,^‡Chiyi Xiong,^§and John S. McMurray*^,†

^†Department of Experimental Therapeutics and ^‡Department of Biochemistry and Molecular Biology and ^§Department of Experimental

Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030

Received July 24, 2009

In cancer cells, signal transducer and activator of transcription 3 (Stat3) participates in aberrant growth,

survival, angiogenesis, and invasion signals and is a validated target for anticancer drug design. We are

targeting its SH2 domain to prevent docking to cytokine and growth factor receptors and subsequent

signaling. One of the important elements of the recognition sequence, pTyr-Xxx-Xxx-Gln, is glutamine.

We incorporated novel Gln mimics into a lead peptide, pCinn-Leu-Pro-Gln-NHBn, and found that a

linear, unconstrained side chain and carboxamide are necessary for high affinity, and the benzamide can

be eliminated. Replacement of Gln-NHBn with (R)-4-aminopentanamide or 2-aminoethylurea pro-

duced inhibitors with equal or greater potency than that of the lead, as judged by fluorescence

polarization (IC₅₀values were 110 and 130 nM, respectively). When Pro was replaced with cis-3,4-

methanoproline, the glutamine mimic, (4R,5S)-4-amino-5-benzyloxyhexanamide resulted in an IC₅₀of

69 nM, the highest affinity Stat3 inhibitor reported to date.

Introduction

hydrophilic nature of this amino acid is likely to impart

specificity for inhibitors of Stat3 since non-Stat SH2 domains

typically recognize hydrophobic residues at this position.^25-30

In a screen of putative receptor docking sites for Stat3, our

laboratory found that pTyr-Leu-Pro-Gln-Thr-Val-NH₂(1)

was a high-affinity ligand and it possessed glutamine at

pYþ3.⁷Extensive studies that probed the interactions be-

tween each amino acid and Stat3 were conducted.^7-11Mod-

ification of glutamine, for example, by side chain N-

methylation, conversion to carbamoyl threonine, replacement

with methionine sulfoxide, etc., provided information that

supported a model of phosphopeptide-Stat3 binding in

which the side chain fits tightly into a groove on the surface

of the protein at the junction of β-strand D and the STAT

protein-specific helix, RB⁰(Figure 1).³¹Direct hydrogen

bonds are hypothesized to occur between the NH₂group of

Gln and main chain oxygens of Glu638 and Pro639 as well as

between the CdO of Gln and the side chain NH₂of Gln644. A

water-mediated hydrogen bond may also form between the

CdO of Gln and the side chain carboxyl group of Glu638. In

addition, a hydrogen bond is predicted to exist between the

backbone NH of the residue C-terminal to Gln and the side

chain hydroxyl group of Tyr640 of Stat3.

Signal transducer and activator of transcription 3 (Stat3)

transmits signals from IL-6 family cytokines, epidermal

growth factor, plate-derived growth factor, leptin, and vas-

cular epithelial growth factor directly from their receptors on

the cell surface to the nucleus.^1-5On binding of these extra-

cellular signaling proteins, Stat3 is recruited to phosphotyr-

osine residues on their receptors to which it binds via its SH2

domain. Stat3 then becomes phosphorylated on Tyr705, a

process known as activation, either by the tyrosine kinase

activity of the receptor or that of associated Janus or Src-

family kinases. The phosphorylated protein dimerizes via

reciprocal interactions between the SH2 domains and

pTyr705 residues, and the activated complex is translocated

to the nucleus where it binds to promoters of genes involved in

cell survival, cell cycling, invasion and migration, and angio-

genesis. Constitutively activated Stat3 has been detected in

tumor samples from numerous cancers.⁶Inhibition of Stat3

activity by antisense oligonucleotides, decoy oligonucleotides,

and siRNA results in apoptosis and reduced cell growth

of tumor cells. Thus Stat3 is a target for antitumor drug

design.^2,4

We^7-11and others^12-21have been targeting the SH2 do-

main of Stat3 with phosphopeptides and related peptidomi-

metics to break up preformed dimers and to prevent initial

docking to receptors and the subsequent events of activation,

dimerization, nuclear transport, and expression of down-

stream genes. The recognition determinant for this target is

pTyr-Xxx-Xxx-Gln.^7,22-24Of particular importance is the

requirement for glutamine three residues C-terminal to the

phosphotyrosine, pYþ3. From a drug design perspective, the

Glutamine can be considered to be 4-carboxy-4-aminobu-

tyramide. In this paper, we incorporated a library of novel 4-

aminobutyramide (Aba)^aderivatives into a phosphopeptide

^aAbbreviations: Aba, 4-aminobutyramide; 4-Abu, 4-aminobutyric

acid; DIEA, diisopropylethylamine; DIPCDI, diisopropylcarbodiimide;

Fmoc, 9-fluorenylmethoxycarbonyl; FMPB, 4-(4-formyl-3-methyoxyphe-

noxy)butyryl; HOBt, 1-hydroxybenzotriazole; homoGlu, homoglutamic

acid; Met(O), methionine sulfoxide; Met(O2), methionine sulfone; mPro,

cis-3,4-methanoproline; pCinn, 4-phosphoryloxycinnamic acid; PyBOP,

1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate;

SAR, structure-activity relationship; TES, triethylsilane; TIS, triisopropyl-

silane.

*To whom correspondence should be addressed. E-mail: jmcmurra@

mdanderson.org. Telephone: 713-745-3763. Fax: 713-792-1204.

r

pubs.acs.org/jmc

Published on Web 09/03/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6127

Figure 1. Hydrogen-bonding interactions between Ac-pTyr-Leu-Pro-Gln-NHBn and Stat3 from the model described in ref 31. (A) Direct

hydrogen bonds between the side chain NH₂and the main chain carbonyl groups of Glu638 and Pro639, the side chain CdO of Gln and

Gln644, and the NH of benzylamide and Tyr640. (B) A water-mediated hydrogen bond between the side chain CdO of Gln and the carboxyl

group of Glu638. Peptide 2 is depicted in the green coloring scheme: carbon is in green, nitrogen is in blue, oxygen is in red, and hydrogen is in

white. Amino acids from Stat3 are depicted in the white coloring scheme with carbon being white and the other elements the same as in the

ligand.

Chart 1. Structures of the Starting Phosphopeptide Inhibitor of

Stat3, 1, and the Modified Lead, 2^a

Scheme 1. General Synthesis Scheme for Inhibitors Possessing

the New, Modified Glutamine Mimics

^aThe IC₅₀value of 1 was reported in ref 8 and that of 2 in ref 10.

to serve as Gln mimics to (1) further understand pepti-

de-protein interactions and (2) reduce the peptidic nature

of the inhibitor with the goal of increased stability to proteases

and possibly glutaminases. The lead inhibitor for these stu-

dies, pCinn-Leu-Pro-Gln-NHBn (2)^10,11(Chart 1), was cho-

sen for its ease of synthesis and for its high affinity. In 2 the

phosphotyrosine of peptide 1 was replaced with 4-phosphor-

yloxycinnamic acid (pCinn), and the C-terminal Thr-

Val-NH₂was substituted with a benzyl group. Conversion

of the pTyr to pCinn increased affinity,^10,11and the benzyl

group likely fits into a hydrophobic pocket on the surface

of Stat3.³¹Peptide 2 exhibited an IC₅₀of 138 nM in a

fluorescence polarization assay, as compared to 290 nM for

peptide 1.^10,11The studies described herein revealed that

replacement of the C-terminal Gln-NHBn unit of 2 with

simpler mimics, 4-aminopentanamide or 2-aminoethylurea,

led to inhibitors that were equipotent with the lead. However,

when proline was substituted with cis-3,4-methanoproline,⁸

replacement of the C-terminal CONHBn with the isosteric 1-

benzyloxyethyl group resulted in an IC₅₀value of 69 nM, the

highest affinity inhibitor of Stat3 reported to date.

Chemistry³²

Peptide Synthesis. The novel glutamine mimics were de-

rived from corresponding N^R-Fmoc glutamic acid analo-

gues, the syntheses of which are described below. Phos-

phopeptides were synthesized manually via attachment of

the side chain of the glutamic acid derivatives to Rink amide

resin so that upon cleavage from the support the final

products possessed glutamine mimics (Scheme 1). The Fmoc

protection scheme was used. Couplings were mediated with

either diisopropylcarbodiimide (DIPCDI)/1-hydroxybenzo-

triazole (HOBt) or 1H-benzotriazol-1-yloxytripyrrolidino-

phosphonium hexafluorophosphate (PyBOP)/HOBt/DIEA.

6128 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Mandal et al.

Table 1. Effect of the Length of the Side Chain of Glutamine Analogues

in the Peptide pCinn-Leu-Pro-NHCH(R)CO-NHBn

Scheme 3^a

peptide

R

IC₅₀(nM)

2

3

4

5

(CH₂)₂CONH₂

H

138 ( 8

4940 ( 1220

874 ( 189

1400 ( 189

^aReagents and conditions:(i) Fmoc-OSu, 10% Na₂CO₃, 1,4-dioxane.

CH₂CONH₂

(CH₂)₃CONH₂

Scheme 4^a

Scheme 2^a

^aReagents and conditions: (i) DMSO, oxalyl chloride, DIEA,

t

CH₂Cl₂, -78 °C; (ii) PPh₃CHCO₂Bu, CH₂Cl₂; (iii) TFA.

Fmoc removal was accomplished with three treatments of

20% piperidine in DMF for 6 min each. All sequences were

capped with 4-(di-tert-butylphosphoryl)cinnamic acid.¹¹

Peptides were cleaved from solid supports with TFA/triiso-

propylsilane/H₂O (TFA/TIS/H₂O) (95:2.5:2.5)³³and were

purified by reverse-phase HPLC.

For the synthesis of inhibitor 3 in which Gln-NHBn was

replaced with Gly-NHBn (Table 1), benzylamine was at-

tached to FMPB aldehyde resin through reductive amination

with NaBH₃CN and 1% AcOH/DMF.³⁴The resulting resin-

bound secondary amine was then acylated with Fmoc-Gly-

OH using PyBOP/HOBt/DIEA. The remaining amino acids

were coupled using DIPCDI/HOBt. Inhibitors 2, 4, and 5

(Table 1) were initiated by coupling Fmoc-Glu-NHBn (50),

Fmoc-Asp-NHBn (51), and Fmoc-homoGlu-NHBn (52,

homoGlu=homoglutamic acid), respectively, to Rink amide

resin via their side chains using PyBOP/HOBt/DIEA. Ami-

no acids 51 and 52 were synthesized as described for 50.⁸

Synthesis of Inhibitors Possessing Constrained Glutamine

Mimics. (E)-4-Amino-2-butenamide was used as a con-

strained glutamine mimic in inhibitor 9 (Table 3). The N-

Fmoc protected amino acid precursor, 56, was prepared as

depicted in Scheme 2. Commercially available Fmoc-ami-

noethanol (53) was oxidized to the aldehyde (54) by Swern

^aReagents and conditions: (i) (a) 2-mercaptopyridine, DCC, EtOAc;

(b) NaBH₄, THF-H₂O; (ii) DMSO, oxalyl chloride, DIEA, CH₂Cl₂,

-78 °C; (iii) PPh₃CHCO₂Bn, CH₂Cl₂; (iv) TES, Pd/C, MeOH.

t-

oxidation³⁵followed by Wittig coupling with PPh₃CHCO₂

Bu to give 55. Hydrolysis of the tert-butyl ester with TFA

gave 56, which was attached to Rink amide resin to start the

synthesis of 9.

treating the sodium salt of Fmoc-3-aminopropanesulfonic

acid (60) with SOCl₂/DMF (cat).³⁷Peptide 15 was initiated

by coupling of Fmoc-4-Abu-OH to hydroxylamine resin

using DIPCDI/HOBt, followed by assembly of the rest of

the amino acids. For inhibitor 16, FMPB aldehyde resin was

first treated with hydrazine to make the corresponding

hydrazone, which was then coupled with Fmoc-4-Abu-OH

followed by assembly of the remainder of the amino acids.

The peptide was cleaved from the resin with 6 N HCl/MeCN

and purified by HPLC.

Synthesis of Inhibitors Possessing 4-Alkyl Aba Analogues.

We demonstrated that during triethylsilane (TES)/Pd-C

mediated hydrogenation, the Fmoc group is stable, which

allowed selective deprotection of benzyl-based protecting

groups.³⁸This observation formed the basis of our strategy

for the synthesis of the 4-alkyl-substituted Aba analogues

used in inhibitors 17-24 (Table 5) and 42-49 (Table 7). A

modification of the procedure of Loukas et al.³⁹was em-

ployed for the synthesis of 4-alkyl-substituted Aba analo-

gues 64a-h and 68 (Scheme 4). The carboxyl groups of

Fmoc-4-aminotetrolic acid (58), used in the synthesis of

inhibitor 10 (Table 3), was prepared by protecting the amino

group of 4-aminotetrolic acid (57)³⁶with Fmoc-OSu/10%

Na₂CO₃/1,4-dioxane (Scheme 3). Throughout the synthesis

of inhibitors 9 and 10, Fmoc removal was accomplished with

5% DBU/DMF to avoid Michael addition with piperidine.

For inhibitors 12 and 13 (Table 3), commercially available

Fmoc-(2S,4S)-4-amino-1-Boc-pyrrolidine-2-carboxylic acid

and Fmoc-(2S,4R)-4-amino-1-Boc-pyrrolidine-2-carboxylic

acid, respectively, were coupled to Rink amide resin using

DIPCDI/HOBt.

Substitution of the Side Chain Carboxamide of 4-Amino-

butyramide (Aba). Inhibitors 8 (Table 3) and 14 (Table 4)

were synthesized by initial attachment of Fmoc-4-Abu-OH

(Abu = 4-aminobutyric acid) and Fmoc-3-aminopropane-

sulfonyl chloride (59), respectively, to Rink amide resin

followed by assembly of the remainder of the amino acids

using standard coupling methods. The latter was prepared by

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6129

Scheme 5^a

^aReagents and conditions: (i) (a) 2-mercaptopyridine, DCC, EtOAc; (b) NaBH₄, THF-H₂O; (ii) PPh₃/I₂/imidazole/CH₂Cl₂(91%); (iii) piperidine or

morpholine or N-methylpiperazine, THF; (iv) (a) TES/Pd-C, 2% CHCl₃/MeOH; (b) Fmoc-OSu, 10% Na₂CO₃, THF; (v) TFA.

Fmoc-D-amino acids, 61a-i, were converted to their corre-

sponding alcohols (62a-i) via formation of 2-thiopyridyl

esters followed by reduction with sodium borohydride. The

alcohols were oxidized to aldehydes 63a-i by Swern oxida-

tion,³⁵and these were elongated by Wittig coupling with

Ph₃PCHCO₂Bn to 64a-i. Concurrent reduction of the

double bond and hydrogenolysis of the benzyl group with

TES and Pd/C in MeOH gave Fmoc-protected glutamic acid

analogues (65a-i) ready for coupling to Rink amide resin. In

this scheme, the side chains of ^D-amino acids end up as

carboxyl group replacements in ^L-Gln analogues. This meth-

od was also used to prepare the constrained glutamic acid

analogue, Fmoc-3-(2-pyrrolidino)propionate (70), used in

the synthesis of 11 (Table 3).

Scheme 6^a

^aReagents and conditions: (i) (a) 2-mercaptopyridine, DCC, EtOAc;

(b) NaBH₄, THF-H₂O; (ii) PPh₃/I₂/imidazole/CH₂Cl₂(88%); (iii)

nBu₄NN₃/CH₂Cl₂(81%); (iv) (a) TES/Pd-C, 2% CHCl₃/MeOH; (b)

allyl chloroformate, DIEA, CH₂Cl₂(64%); (v) TFA.

Syntheses of inhibitors 26-28 (Table 5) were initiated by

coupling of 5-piperidino-4-(9-fluorenylmethoxycarbonyla-

mino)pentanoic acid and analogues (76a-c) to Rink resin.

To synthesize 76a-c (Scheme 5), the carboxyl group of Z-^L-

Glu(OtBu)-OH (71) was reduced to an alcohol (72) via

sodium borohydride mediated reduction of the intermediate

2-thiopyridine ester. Intermediate 72 was then transformed

to the corresponding iodide (73) by treatment with the PPh₃/

I₂/imidazole complex.⁴⁰Nucleophilic substitution with pi-

peridine, morpholine, and 4-methylpiperazine afforded tert-

butyl 5-piperidino-4-benzyloxyaminopentanoate 74a, tert-

butyl 5-morpholino-4-benzyloxyaminopentanoate 74b, and

tert-butyl 5-piperazino-4-benzyloxyaminopentanoate 74c,

respectively. TES/Pd-C mediated hydrogenation of 74a-c

in 2% CHCl₃/MeOH followed by Fmoc protection

yielded 75a-c. Treatment of 75a-c with neat TFA followed

by triturating with ether-hexane gave 76a-c as white

powders.

For the synthesis of substituted 4-aminomethyl Aba-con-

taining inhibitors (29-31, Table 5), tert-butyl 4-(9-fluorenyl-

oxycarbonylamino)-5-iodopentanoate (79) was synthesized

from Fmoc-Glu(OtBu)-OH using an analogous scheme as

for 73 (Scheme 6). Iodide 79 was treated with tetrabutylam-

monium azide, and the resulting azide, 80, was reduced with

TES/Pd-C to give the free amine, which was then protected

with the Alloc group to give 81. Removal of the side chain

tert-butyl group with neat TFA gave 82, which was then

attached to Rink amide resin. After addition of the pCinn,

Leu, and Pro units, the Alloc group was removed with

Pd(PPh₃)₄/CHCl₃/AcOH/NMM⁴¹, and the resin was split

into three portions. The first was not derivatized and led to

inhibitor 30. The second was acetylated with acetic anhy-

dride, leading to inhibitor 31. Treatment of the final portion

with a 3-fold excess of benzaldehyde in the presence of

NaCNBH₃/AcOH resulted in complete alkylation of the

amine, leading to inhibitor 29.

Syntheses of Carbamate-Containing Phosphopeptides.

Fmoc-amino alcohols 62a, 62g, and 62h (Scheme 4) and

Fmoc-aminoethanol (62j) were converted to the correspond-

ing 4-nitrophenyl carbonates (83a,g,h,j) by treatment with

nitrophenyl chloroformate¹⁰(Scheme 7). These were coupled

to Rink amide resin in the presence of DIEA to initiate the

synthesis of the inhibitors. After addition of the pCinn, Leu,

and Pro residues, cleavage from the resin yielded the carba-

mate-containing inhibitors 34-37 (Table 6).

Inhibitor 25 (Table 5), possessing a 4-amino-5-carbamoyl-

pentamide on the C-terminus, was synthesized by initial

attachment of 86 to Rink amide resin, followed by subse-

quent coupling of the remainder of the amino acids. Nitro-

phenyl carbonate 86 was obtained from reducing the

carboxyl group of Fmoc-Gln(Trt) (84) to the corresponding

alcohol 85, followed by treatment with 4-nitrophenyl chlor-

oformate (Scheme 8).

Synthesis of Urea-Containing Inhibitors 38 and 39. Synth-

eses of urea-containing inhibitors 38 and 39 (Table 6) started

with the coupling of Fmoc-aminoethylnitrophenylurethanes

90a,j to Rink amide resin to give resin-bound ureas. As

shown in Scheme 7, commercially available Boc-diami-

noethane (91) was treated with Fmoc-OSu followed by

HCl in EtOAc to prepare Fmoc-diaminoethane, 89j. Treat-

ment with nitrophenyl chloroformate provided the corre-

sponding nitrophenylurethane, 90j. The synthesis of 2-

methyl-2-aminonitrophenylurethane, 90a, was carried out

by a different route than that reported by Boeijen et al.⁴²

Fmoc-alaninol, 62a (Scheme 4), was converted to the iodide,

87a, as described for the 4-piperidinomethyl Aba analogues

6130 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Mandal et al.

Scheme 7^a

^aReagents and conditions: (i) nitrophenyl chloroformate, pyridine, CH₂Cl₂; (ii) PPh₃/I₂/imidazole/CH₂Cl₂(91%); (iii) Bu₄NN₃/CH₂Cl₂(84%);

(iv) TES, Pd/C, 2% CHCl₃/MeOH; (v) (a) Fmoc-OSu, NaHCO₃; (b) HCl/EtOAc.

Table 2. Probes of Potential Interactions of the Backbone Atoms at

Main Chain Interactions at Position pYþ3. To probe for

pYþ3 and Stat3

peptide

main chain interactions between the pYþ3 residue and Stat3,

sequence

IC₅₀(nM)

Gln was either removed (peptide 6) or replaced with Ala

(peptide 7). Peptide 7 displayed higher affinity than 6, which

is consistent with hydrogen bonding of the peptide bond C-

terminal to the pYþ3 residue and Tyr640 of Stat3 (Figure 1,

Table 2).³¹As in the case of 3, the absence of the Gln side

chain accounts for the reduced affinity of 7.

2

6

7

pCinn-Leu-Pro-Gln-NHBn

pCinn-Leu-Pro-NH₂

pCinn-Leu-Pro-Ala-NH₂

138 ( 8

11400 ( 1600

7800 ( 730

described above, which was then followed by azide substitu-

tion with tetrabutylammonium azide to give 88a. The use of

tetrabutylammonium azide resulted in a higher yield than

sodium azide³²for this step. Reduction of 88a with TES/

Pd-C³⁸gave the diamine, 89a, which was converted to the

nitrophenylurethane 90a with nitrophenyl chloroformate.

The urethane intermediates were added to Rink amide resin

to give solid-supported ureas. After adding the remainder of

the amino acids, cleavage from the resin with TFA gave urea-

containing inhibitors 38 and 39.

Synthesis of Inhibitor 47. Inhibitor 47 (Table 7) was

initiated by coupling of 4-[[(9H-fluoren-9-ylmethoxy)car-

bonyl]amino]-5-acetoxypentanoate 93 (Scheme 9) to Rink

resin. Hydroxypentanoate 78 (Scheme 6) was acetylated with

acetic anhydride and the tert-butyl ester deprotected to give

the starting amino acid.

Conformationally Constrained Aba Analogues. Peptides

with a properly constrained side chain will exhibit higher

affinity due to reduced entropy penalty on binding. The side

chain of glutamine has three rotatable C-C bonds. We

prepared a series of constrained Aba analogues as models

to probe the effect of constraining the Gln side chain

(Table 3). Note that Aba (8), exhibiting an IC₅₀value of

0.57 μM, results in a 3-fold loss of affinity as compared to

Gln-NHBn in peptide 2. Incorporation of double and triple

bonds in Aba (9 and 10, respectively) reduced affinity ca. 2-

fold compared to 8. Cyclizing C(4)-N(4) and C(2)-C(4)

caused 6-fold reductions compared to Aba (11-13). Taken

together with the loss in affinity observed when Gln-Thr-

NH₂was replaced with 3-acetamidopyrrolidine, nipecota-

mide, aminocyclohexane-3-carboxamide, and 4-acetamido-

piperazine reported previously,⁸we conclude that when

bound to Stat3, the alkyl chain of Gln adopts a conformation

not readily mimicked by chemical modification.

Results

Alteration of the Length of the Glutamine Side Chain. To

establish the importance of the distance of the side chain

carboxamide group from the main chain peptide, Gln was

replaced with glycine, asparagine, and homoglutamine

(Table 1). Substitution with glycine (3), without a side

chain, decreased affinity 50-fold, reiterating the impor-

tance of the alkylcarboxamide at pYþ3. Peptides contain-

ing asparagine (4), with a one carbon contraction, and

homoglutamine (5), with a one carbon extension with

respect to glutamine, showed reduced activity by 7- and

10-fold, respectively, indicating that the position of the

amide group is critical for attaining high affinity. The

reduced affinity of 4 is in keeping with a similar result in

which Asn substitution for Gln in 1 reduced the ability of

the peptide to inhibit DNA binding in an electrophoretic

mobility shift assay.⁷

Importance of the Side Chain Carboxamide Group. Pre-

viously,⁸we found that replacement of Gln by the isosteric

analogue, methionine sulfoxide, as well as the oxidized

version, methionine sulfone, resulted dramatic losses of

inhibition. Mono- and dimethylation of the side chain

nitrogen of glutamine also reduced affinity. Taken together,

these results suggested a role for the amide hydrogens in

hydrogen-bonding interactions with the protein. To deter-

mine if sulfonamide protons could interact with Stat3, the

side chain carboxamide of Aba was substituted with a

SO₂NH₂group (14). This modification resulted in very low

affinity (IC₅₀= 68 μM). In an attempt to mimic the water

molecule involved in the water-mediated hydrogen bond

observed in the computational model (Figure 1),³¹one of

the amide protons of Aba was substituted with either a

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6131

Table 3. Effect of Constrained Gaba Analogues on the Affinity of pCinn-Leu-Pro-Xxx

Scheme 8^a

Scheme 9^a

^aReagents and conditions: (i) Ac₂O, DIEA, DMAP (cat), CH₂Cl₂; (ii)

TFA.

Table 4. Importance of the Side Chain Carboxamide Group in pCinn-

Leu-Pro-Xxx

peptide

Xxx

IC₅₀(nM)

^aReagents and conditions: (i) (a) 2-mercaptopyridine, DCC, EtOAc;

(b) NaBH₄, THF-H₂O; (ii) 4-nitrophenyl chloroformate, pyridine,

CH₂Cl₂(62%).

8

NH(CH₂)₃CONH₂

NH(CH₂)₃SO₂NH₂

NH(CH₂)₃CONHOH

NH(CH₂)₃CONHNH₂

574 ( 130

68200 ( 23500

2640 ( 45

14

15

16

1830 ( 770

hydroxyl group (peptide 15) or an amino group (peptide 16).

In both cases, 3-5-fold reduced affinity was observed

(Table 4). The reduction of affinity may be due to steric

crowding in the glutamine binding pocket or loss of inter-

molecular H-bonding. Thus, a carboxamide group with both

amide protons intact is optimal for high affinity.

the unsubstituted methyl group. Piperidinomethyl Gaba

analogues were synthesized with the long-term goal of

solubility of prodrug versions of phosphopeptide inhibitors

of Stat3. Compounds 26-28, with IC₅₀values ranging from

1.2 to 1.5 μM, showed almost 10-fold reduced binding

affinity compared to the unsubstituted methyl group. The

acyclic tertiary amine containing inhibitor (29) also came out

with 7-fold decreased affinity. However, acetylation of the

amino group of 31 partially restored activity (compound 31).

It appears that a charged amine at this position may be

deleterious for activity. Addition of a carbamate at the C-

terminus, 25, gave an IC₅₀value of 612 nM, similar to the

acetamide 31.

Taken together, these results suggest that the binding

surface for the backbone CONH atoms of glutamine of 2 is

polar and that the alkyl groups do not make good contact.

This is in keeping with the proposed model in which the

phenolic hydroxyl group of Tyr640 is within hydrogen-

bonding distance of this group (Figure 1). However, in spite

of the polar surface, formal positive charge provided by

amines is not tolerated well.

4-Alkyl Aba Analogues. To further probe for interactions

between the main chain atoms at pYþ3 and Stat3, and to

search for new ones, a series of glutamine analogues in which

the backbone carboxyl group was substituted with alkyl

groups was used to replace Gln-NHBn in peptide 2

(Table 5). A methyl group produced the most potent inhi-

bitor, increasing affinity 5-fold (17) over the hydrogen of

Aba (8). Interestingly, compound 17 (IC₅₀= 110 nM) was

equipotent with 2. The configuration of C(4) is important as

the S enantiomer of 4-methyl Aba (inhibitor 18) resulted in

lower affinity than 17. The larger, simple alkyl substitutions

in 19-22 were not tolerated as well as the methyl group.

Benzyloxymethyl and benzyloxyethyl groups were appended

to C(4) of Aba, which resulted in IC₅₀values of 294 (23) and

272 nM (24), respectively. These groups are nearly isosteric

to the CONHBn group in compound 2, and 23 and 24 reduce

affinity by ca. 2-fold.

Substitution of Glutamine with Carbamate and Ureas.

Previously, we reported the replacement of the γ-methylene

group of glutamine with oxygen to give side chain carbamate

Attachment of an amino group to the γ-methyl carbon

(30) resulted in an IC₅₀of 1.2 μM, 10-fold lower affinity than

6132 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Table 5. Effect of Substitutions at C(4) of Aba in pCinn-Leu-Pro-Xxx

Mandal et al.

analogues.¹⁰O-Carbamoylserine (32) and O-carbamoyl-

threonine (33) resulted in 3- and 6-fold losses of affinity

compared to peptide 2. To further study this functional

group, a series of Aba analogues was converted to 2-ami-

noethylcarbamates (Table 6). In contrast to the loss in

binding of Aba vs Gln-NHBn (2 and 8) discussed above

(Table 5), the Aba analogue, 2-aminoethylcarbamate (34)

results in affinity equal to that of the carbamoylserine

benzylamide (32). Substitution of C(2) of the ethyl group

(C(4) of Aba) results in loss of activity, even those possessing

benzyloxymethyl and benzyloxyethyl groups, 36, and 37,

respectively. Replacement of Gln-NHBn with unsubstituted

2-aminoethylurea (38) results in equal affinity, 131 nM.

Addition of a methyl group (39) resulted in a 10-fold loss

in activity.

substitution of proline with cis-3,4-methanoproline (mPro)

increased affinity by a factor of 2 and that hydrocinnamoyl-

pTyr-Leu-mPro-Gln-NHBn exhibited an IC₅₀value of 125

nM in our FP assay.⁸mPro is a rather expensive starting

material so the studies discussed above were conducted on

peptides containing proline. Adding to the cost is the fact

commercially available Fmoc-cis-3,4-methanoPro is sold as

a racemic mixture of (2S,3R,4S) and (2R,3S,4R) enantio-

mers, and syntheses of peptides with this amino acid produce

two diastereomers. Only one-half of the material gives the

high-affinity interaction with Stat3. To test the effect of some

of the Gaba analogues discussed above, we substituted mPro

for Pro. Only the high-affinity diastereomers are reported

here.

Incorporation of cis-3,4-methanoPro in peptide2 resulted in

a 2-fold increase in affinity consistent with our previous report⁸

(40, Table 7). pCinn-Leu-cis-3,4-methanoPro-Gln-NHBn

Glutamine Analogues in Peptides Containing cis-3,4-

Methanoproline. In an earlier publication we reported that

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6133

Table 6. Inhibition of Stat3 by Carbamates and Ureas as Glutamine Mimics in pCinn-Leu-Pro-Xxx

^aFrom ref 10.

exhibited an IC₅₀value of 68 nM, the highest affinity Stat3

inhibitor reported to date. γ-Alkyl Gaba analogues were also

appended onto the C-terminus of pCinn-Leu-cis-3,4-metha-

noPro and the IC₅₀’s determined by FP. Substituting the

benzylamide moiety with hydrogen (compound 41) resulted

in a loss of affinity. In contrast to the proline analogues, γ-

methyl Gaba (42) was not equipotent to the Gln-NHBn

analogue, 40. Larger alkyl groups reduced affinity. The ben-

zyloxymethyl group (48) restored binding to 94 nM. However,

the isosteric benzyloxyethyl group (49) was equipotent with the

benzylamide, exhibiting an IC₅₀value of 69 nM. Thus com-

pound 49 is a very high affinity peptidomimetic inhibitor of

Stat3 in which pTyr is replaced by pCinn, proline is replaced

with 3,4-cis-methanoproline, and glutamine is replaced with

γ-(2-benzyloxyethyl) Aba. With only one natural amino acid

remaining, leucine, we have made great strides in the develop-

ment of a nonpeptide inhibitor of Stat3.

leads to a modified peptide with enhanced affinity; the IC₅₀

value of 49 was 69 nM. Conversion of these phosphopeptide

mimics into cell permeable inhibitors will be reported under

separate cover.

Experimental Section

Chemistry: General. N^R-Protected amino acids were pur-

chased from Advanced Chemtech, NovaBiochem, ChemImpex,

or AnaSpec. HOBt was from ChemImpex. Fmoc-cis-3,4-metha-

noPro was from EMD Biosciences (formerly NovaBiochem).

Rink amide resin was purchased from Advanced Chemtech,

loaded between 0.6 and 0.7 mmol/g. Anhydrous DMF for

amino acid solutions was from Baker. Other solvents were of

reagent grade and were used without further purification. Pep-

tides were purified by reverse-phase HPLC on a Rainin rabbit

HPLC or a Varian Dynamax HPLC using a Vydac 2.5 ꢀ 25 cm

peptide and protein C18 column or a 2.1 ꢀ 25 cm Phenomenex

Luna 10 μM C18(2). Gradients of MeCN in H₂O (both contain-

ing 0.1% TFA) or MeCN in 0.01 M NH₄OAc (pH 6.5) at 10 mL/

min were employed. Peptides were tested for purity by reverse-

phase HPLC on an Agilent 1090 HPLC or an Agilent 1100

HPLC using a Vydac 4.6 ꢀ 250 mm C18 peptide/protein column

or a 4.6 ꢀ 250 mm Phenomenex 5 μM C18(2) in two systems: (A)

10-50% MeCN in H₂O with 0.1% TFA in both solvents and

(B) 0-40% MeCN in 0.01 M NH₄OAc, pH 6.5. The flow rate

for both was 1.5 mL/min. Peptides were always >95% pure and

were often >98% pure as judged by analytical HPLC. Before

evaluation by fluorescence polarization, peptides were dried in

vacuo over P₂O₅at 37 °C for 24 h. NMR spectra were recorded

on Bruker 300 MHz DPX or 500 MHz DRX spectrometers.

Solid-Phase Peptide Synthesis: Manual Method. Manual so-

lid-phase synthesis was carried out on aliquots of 0.20 g of Rink

resin (0.6 mmol/g). Fmoc groups were removed with 2 ꢀ 5 mL of

20% piperidine for 3 and 7 min each. After removing the Fmoc

group from the resin, synthesis of inhibitors was initiated by

coupling of the 3-fold excesses of the new Fmoc-glutamic acid

Conclusions

We have probed the glutamine binding pocket of Stat3 with

a series of glutamine and Aba analogues. A nonconstrained,

linear side chain is necessary for high affinity, as is the

carboxamide group. These findings are consistent with the

model of Gln fitting into a tight pocket on the surface of the

Stat3 SH2 domain and the side chain amide participating in

hydrogen-bonding interactions.³¹Glutamine can be substi-

tuted with nonpeptide analogues. Replacement of the C-

terminal Gln-NHBn unit of pCinn-Leu-Pro-Gln-NHBn with

either (R)-4-aminopentamide or 2-aminoethylurea leads to

modified peptides with IC₅₀values of ca. 110 and 130 nM,

respectively, equipotent with the lead inhibitor. However,

replacement of proline with cis-3,4-methanoproline and

Gln-NHBn with (4R,5S)-4-amino-5-benzyloxyhexamide

6134 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Table 7. Effect of Substitution in pCinn-Leu-cis-3,4-methanoPro-Xxx

Mandal et al.

analogues, PyBop and HOBt, along with 6-fold excesses of

DIEA in 5 mL of (1:1 v/v) DMF/CH₂Cl₂. The nitrophenyl

carbonates (83a,h,g,j) and nitrophenyl carbamates (90a,j) were

added to the resin in 3-fold excess along with a 3-fold excess of

DIEA in 5 mL of (1:1 v/v) DMF/CH₂Cl₂as described.¹⁰

Reactions were monitored with ninhydrin. After coupling and

deprotection steps, resins were washed with 5 ꢀ 5 mL of DMF/

CH₂Cl₂. Cleavage was accomplished with three treatments of

the resins with 5 mL of TFA/TIS/H₂O (95:2.5:2.5)³³for 10 min

each. The volumes of the solvents were reduced, and the solu-

tions were dropped into ice-cold Et₂O. After 30 min the pre-

cipitates were collected by filtration and were washed 2ꢀ more

with Et₂O. Crude peptides were dried, and peptides were

purified by reverse-phase HPLC as described in the general

methods. All peptides were dried over P₂O₅in vacuo at 37 °C

for 24 h before testing. Peptide yields, HPLC retention

times, and mass spectra are tabulated in Supporting Informa-

tion Table S1.

Fmoc-homoGlu-NHBn (52). Starting with 0.5 g of Fmoc-

homoGlu(tBu)-OH the procedure described by Coleman

et al.⁸for Fmoc-Glu-NHBn was employed. Yield 0.49 g

(82%), white powder. ¹H NMR (DMSO-d₆, 500 MHz) δ

1.56-1.74 (m, 4H), 2.72 (t, J = 7.0 Hz, 2H), 4.1 (m, 1H),

4.26-4.37 (m, 5H), 7.25-7.38 (m, 7H), 7.47 (t, J = 7.5 Hz, 2H),

7.58 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 7.0 Hz, 2H), 7.94 (d, J =

7.5 Hz, 2H), 8.47 (t, J = 5.5 Hz, 1H). ¹³C NMR (DMSO-d₆, 125

MHz) δ 21.6, 31.9, 33.8, 42.1, 47.2, 54.9, 66.1, 120.6, 125.8,

127.2, 127.6, 128.1, 128.7, 139.8, 141.2, 144.3, 144.4, 156.5,

172.3, 174.7. Anal. (C₂₈H₂₈N₂O₅) C, H, N. Calcd: C, 71.17; H,

5.97; N, 5.93. Found: C, 70.41; H, 5.92; N, 5.87. HRMS (M þ H)

calcd, 473.2076; found, 473.2115. Note: % C is >0.4%.

Synthesis of 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-2-

butenoic Acid (56). Commercially available Fmoc-glycinol (53)

was oxidized to Fmoc-glycinal (54) via Swern oxidation.⁴³

A

solution of 54 (1.0 g, 3.55 mmol) and tert-butyl (triphenyl-

phosphoranylidene)acetate (1.3 g, 3.91 mmol) in dry CH₂Cl₂

(20 mL) was stirred for 2 h. The solvent was removed in vacuo,

and the residue was purified by silica gel column chromatogra-

phy (15% EtOAc-hexane, v/v) to get 55. Yield 85% (1.20 g). ¹H

NMR (CDCl₃, 500 MHz) δ 1.4 (s, 9H), 3.86 (m, 2H), 4.13 (t, J =

6.5 Hz, 1H), 4.35 (d, J = 6.5 Hz, 2H), 4.9 (m, 1H), 5.76 (d, J =

15.5 Hz, 1H), 6.71 (m, 1H), 7.22 (m, 2H), 7.31 (m, 2H), 7.5 (d,

J = 7.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃,

125 MHz) δ 28.1, 41.7, 47.2, 66.9, 80.7, 120.1, 123.5, 125.0,

127.1, 127.8, 141.4, 142.8, 143.8, 156.2, 165.3. HRMS (M þ H)

calcd, 380.1862; found, 380.1856.

Synthesis of Fmoc-Asp-NHBn (51). Starting with 0.5 g of

Fmoc-Asp(tBu)-OH the procedure described by Coleman et al.⁸

for Fmoc-Glu-NHBn was employed. Yield 0.48 g (89%), white

1

powder. H NMR (DMSO-d₆, 500 MHz) δ 2.56 (dd, J = 9.0,

16.5 Hz, 1H), 2.27 (dd, J = 5.5, 16.5 Hz, 1H), 4.22-4.33 (m,

5H), 4.42 (m, 1H), 7.2-7.35 (m, 7H), 7.43 (t, J = 7.0 Hz, 2H),

7.7 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.0 Hz, 2H), 7.9 (d, J = 8.0

Hz, 2H), 8.42 (t, J = 6.0 Hz, 1H). ¹³C NMR (DMSO-d₆, 125

MHz) δ 36.9, 42.6, 47.1, 51.9, 66.3, 120.6, 125.8, 127.1, 127.4,

127.6, 128.1, 128.6, 139.8, 141.2, 144.3, 156.3, 171.2, 172.3.

HRMS (M þ H) calcd, 445.1763; found, 445.1772. Anal.

(C₂₆H₂₄N₂O₅) C, H, N. Calcd: C, 70.26; H, 5.44; N, 6.30.

Found: C, 69.11; H, 5.35; N, 6.24. Note: % C is >0.4%.

Compound 55 (1.0 g) was treated with 5.0 mL of neat TFA for

1 h. The TFA was removed under vacuum, and residual acid was

removed by the addition and evaporation of toluene (3 ꢀ 5 mL).

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6135

Trituration with ether-hexane resulted in a white precipitate

which was collected by filtration and dried over P₂O₅yielding

0.81 g of 56 as a white powder, 95%. ¹H NMR (DMSO-d₆, 500

MHz) δ 3.8 (m, 2H), 4.25 (m, 1H), 4.33 (d, J = 6.5 Hz, 2H), 5.81

(d, J = 15.5 Hz, 1H), 6.76 (m, 1H), 7.34 (m, 2H), 7.42 (m, 2H),

7.66 (t, J = 5.5 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.9 (d, J = 7.5

Hz, 2H). ¹³C NMR (DMSO-d₆, 125.0 MHz) δ 41.4, 47.2, 66.0,

120.6, 122.0, 125.6, 127.5, 128.1, 141.2, 144.3, 145.5, 156.6,

167.4. Anal. (C₁₉H₁₇NO₄) C, H, N. Calcd: C, 70.58; H, 5.30;

N, 4.33. Found: C, 70.38; H, 5.56; N, 4.25. HRMS (M þ H)

calcd, 324.1236; found, 324.1164.

Synthesis of 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]tetro-

lic Acid (58). 4-Aminotetrolic acid (1.0 g, 7.37 mmol) (57), prepared

as described by Ahern et al.,³⁶was treated with 10 mL of 10%

Na₂CO₃and Fmoc-OSu (2.2 g, 6.6 mmol) in 20 mL of 1,4-dioxane

overnight. The solution was washed with 20 mL of EtOAc and the

aqueous layer then acidified with concentrated HCl. It was the

extracted with EtOAc (3 ꢀ 30 mL), and the combined organic

layers were washed with brine, dried (MgSO₄), and evaporated.

The residue was triturated with ether-hexane, and the precipitate

was collected by filtration and dried over P₂O₅. Yield 1.3 g (41%).

¹H NMR (DMSO-d₆, 500 MHz) δ4.04 (d, J= 5.0 Hz, 2H), 4.29 (t,

J = 6.5 Hz, 1H), 4.41 (d, J = 6.5 Hz, 2H), 7.39 (m, 2H), 7.47 (m,

2H), 7.75 (d, J = 7.5 Hz, 2H), 7.93-7.95 (m, 3H). ¹³C NMR

(DMSO-d₆, 125 MHz) δ 30.3, 47.1, 66.3, 75.4, 84.7, 120.6, 125.6,

127.6, 128.1, 141.2, 144.2, 154.4, 156.5. Anal. (C₁₉H₁₅NO₄) C, H,

N. Calcd: C, 71.02; H, 4.71; N, 4.36. Found: C, 70.37; H, 4.67; N,

4.37. HRMS (M þ H) calcd, 322.1079; found, 322.1083.

(m, 1H), 3.55 (m, 1H), 3.72 (m, 1H), 4.11 (m, 1H), 4.33 (m, 2H),

4.78 (br s, 1H), 7.21 (m, 2H), 7.3 (m, 2H), 7.48 (d, J = 7.5 Hz,

2H), 7.66 (d, J = 7.5 Hz, 2H). HRMS (M þ H) calcd, 298.1443;

found, 298.1356.

Fmoc-^D-valinol (62c). Yield 1.5 g (91%), white powder, mp

126 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.93 (m, 6H), 1.84 (m,

1H), 3.64 (m, 2H), 4.2 (t, J = 6.6 Hz, 1H), 4.43 (m, 2H), 4.9 (m,

1H), 7.28-7.42 (m, 4H), 7.58 (d, J = 7.2 Hz, 2H), 7.75 (d, J =

7.2 Hz, 2H). HRMS (M þ H) calcd, 326.1756; found, 326.1629.

Fmoc-^D-leucinol (62d). Yield 1.55 g (90%), white powder, mp

138 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.92 (d, J = 5.7 Hz, 6H),

1.32 (m, 2H), 1.63 (m, 1H), 3.46-3.76 (m, 3H), 4.21 (m, 1H),

4.44 (m, 2H), 4.73 (m, 1H), 7.28-7.42 (m, 4H), 7.58 (d, J = 7.5

Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ

22.1, 23.0, 24.8, 40.4, 47.4, 66.5, 120.0, 125.0, 127.0, 127.7, 141.4,

143.9. HRMS (M þ H) calcd, 340.1913; found, 340.1699.

Fmoc-^D-norleucinol (62e). Yield 1.6 g (92%), white powder,

mp 140 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.92 (m, 3H),

1.34-1.6 (m, 6H), 3.58-3.68 (m, 3H), 4.24 (m, 1H), 4.46 (m,

2H), 4.82 (m, 1H), 7.31-7.45 (m, 4H), 7.61 (d, J = 7.5 Hz, 2H),

7.78 (d, J = 7.5 Hz, 2H). HRMS (M þ H) calcd, 340.1913;

found, 340.1739.

Fmoc-^D-tyrosinol tert-Butyl Ether (62f). Yield 2.0 g (92%), mp

1

108 °C. H NMR (CDCl₃, 300 MHz) δ 1.34 (s, 9H), 2.82 (m,

2H), 3.67 (m, 2H), 3.91 (m, 1H), 4.21 (m, 1H), 4.42 (m, 2H), 4.94

(m, 1H), 6.92 (d, J = 8.4 Hz, 2H), 7.3-7.45 (m, 4H), 7.58 (d, J =

7.5 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H). HRMS (M þ H) calcd,

446.2331; found, 446.2057.

1

Synthesis of Sodium 3-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]propanesulfonate (60). To a stirred aqueous solution (30

mL) of 3-aminopropanesulfonic acid (1 g, 7.18 mmol) and NaOH

(0.3 g, 7.5 mmol) was added Fmoc-OSu (2.9 g, 8.6 mmol) in

portions over 10 min. Stirring was continued overnight. Excess

Fmoc-OSu was removed by washing with EtOAc (2 ꢀ 10 mL). The

aqueous layer was lyophilized, and the residual solid was triturated

with ether-hexane. The product was collected by filtration and

dried in vacuo over P₂O₅. Yield 1.9 g (72%). ¹H NMR (DMSO-d₆,

500 MHz) δ 1.77 (m, 2H), 2.5 (t, J = 7.5 Hz, 2H), 3.1 (m, 2H), 4.26

(m, 1H),4.3(d, J= 7.0 Hz, 2H), 7.37-7.41 (m, 3H), 7.46 (t, J=7.5

Fmoc-^D-serinol Benzyl Ether (62g). Yield 1.61 g (78%). H

NMR (CDCl₃, 300 MHz) δ 3.46-3.83 (m, 5H), 4.2 (t, J = 6.6

Hz, 1H), 4.4 (m, 2H), 4.5 (s, 2H), 5.43 (m, 1H), 7.23-7.41 (m,

9H), 7.58 (d, J = 7.2 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H). ¹³C

NMR (CDCl₃, 75 MHz) δ 47.3, 52.0, 66.8, 70.5, 73.6, 120.0,

125.1, 127.1, 127.7, 128.0, 128.6, 137.6, 141.4, 143.9. HRMS

(M þ H) calcd, 404.1862; found, 404.1855.

Fmoc-^D-threoninol Benzyl Ether (62h). Yield 1.74 g (81%). ¹H

NMR (CDCl₃, 300 MHz) δ 1.22 (d, J = 7.2 Hz, 3H), 2.65 (m,

1H), 3.7 (m, 2H), 3.83 (m, 1H), 4.2 (t, J = 6.6 Hz, 1H), 4.35-

4.45 (m, 3H), 4.61 (d, J = 11.4, 1H), 5.32 (m, 1H), 7.28-7.4 (m,

9H), 7.58 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H). ¹³C

NMR (CDCl₃, 75 MHz) δ 16.2, 47.3, 56.7, 63.9, 66.9, 70.9, 74.4,

120.0, 125.1, 127.1, 127.7, 127.9, 128.0, 128.6, 138.0, 141.4,

144.0. HRMS (M þ H) calcd, 418.2018; found, 418.1696.

Fmoc-^D-threoninol tert-Butyl Ether (62i). Yield 1.6 g (82%).

¹H NMR (CDCl₃, 500 MHz) δ 1.06 (d, J = 6.0 Hz, 3H), 1.11 (s,

9H), 3.52-3.57 (m, 3H), 3.85 (m, 1H), 4.12 (t, J = 7.0 Hz, 1H),

4.26-4.35 (m, 2H), 5.22 (br s, 1H), 7.21 (m, 2H), 7.29 (m, 2H),

7.5 (d, J = 7.5 Hz, 2H), 7.65 (d, J = 7.5 Hz, 2H). ¹³C NMR

(CDCl₃, 125 MHz) δ 20.1, 28.7, 47.3, 57.4, 66.9, 74.3, 120.0,

125.1, 127.1, 127.7, 141.4, 143.9, 157.1. HRMS (M þ H) calcd,

384.2175; found, 384.1983.

Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H), 7.93 (d, J = 7.5 Hz, 2H). ¹³

C

NMR (DMSO-d₆, 125 MHz) δ 26.3, 47.2, 49.6, 65.8, 120.6, 125.7,

127.6, 128.1, 141.2, 144.4, 156.6. HRMS (M þ H) calcd, 384.0882;

found, 384.0914.

Synthesis of 3-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]pro-

panesulfonyl Chloride (59).³⁷To a mixture of 60 (1.00 g, 2.6 mmol)

and thionyl chloride (10 mL) was added DMF (1 mL) dropwise.

The mixture was heated at reflux for 5 h. The volatiles were removed

by evaporation, followed by coevaporation with toluene 3 ꢀ 5 mL.

Compound 59 was coupled directly to Rink amide resin.

General Procedure for N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino Alcohols (62a-i and 67). A solution of Fmoc-amino acid

(5.0 mmol), DCC (6.0 mmol), and 2-mercaptopyridine (5.5 mmol)

in 100 mL of EtOAc was stirred for 3 h. The white precipitate was

filtered off, and the filtrate was concentrated under vacuum. It was

thendiluted with 50 mLof THF, and the solution was added slowly

to a suspension of NaBH₄(10.0 mmol) in 20 mL of THF and 10 mL

of water at 0 °C. After 30 min, the reaction was quenched by slow

addition of ice-cold 5% HCl(aq) (50 mL) and extracted with ether

(3 ꢀ 150 mL). The combined organic layers were washed with

aqueous 10% NaHCO₃(3 ꢀ 40 mL), water (2 ꢀ 50 mL), and brine

(1 ꢀ 40 mL). After drying (Na₂SO₄) and concentration under

vacuum the crude residue was purified either by recrystallization

from hexane-ether or by silica gel column chromatography.

Fmoc-^D-alaninol (62a). Yield 1.32 g (90%). ¹H NMR (CDCl₃,

500 MHz) δ 1.07 (m, 3H), 3.43 (m, 1H), 3.55 (m, 1H), 3.73 (m,

1H), 4.11 (m, 1H), 4.33 (m, 2H), 4.75 (br s, 1H), 7.22 (m, 2H), 7.3

(m, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H).

HRMS (M þ H) calcd, 298.1443; found, 298.1247.

Fmoc-^D-prolinol (67). Yield 1.45 g (89%). ¹H NMR (CDCl₃,

500 MHz) δ 1.6-1.9 (m, 4H), 3.3-3.4 (m, 2H), 3.6 (d, J = 4.5

Hz, 2H), 3.9 (m, 1H), 4.1 (m, 1H), 4.2 (t, J = 6.5 Hz, 1H), 4.3-

4.44 (m, 2H), 7.24-7.35 (m, 4H), 7.54 (d, J = 7.5 Hz, 2H), 7.7 (d,

J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 125 MHz) δ 24.0, 28.4, 47.2,

60.5, 63.5, 66.1, 67.5, 120.0, 125.1, 127.1, 127.8, 141.4, 144.0,

156.8. HRMS (M þ H) calcd, 324.1600; found, 324.1588.

General Procedure for the Synthesis of N-[(9H-Fluoren-9-

ylmethoxy)carbonyl]amino Aldehydes (63a-i and 68). Fmoc-

amino aldehydes were synthesized by Swern oxidation of corre-

sponding Fmoc-^D-amino alcohols.³⁵To a solution of oxalyl

chloride (8.0 mmol) in 30 mL of dry CH₂Cl₂, stirred at -78 °C

under argon, was added DMSO (16.0 mmol) via syringe drop-

wise with vigorous stirring. After 20 min, a solution of Fmoc-^D-

amino alcohol (5.0 mmol) in 20 mL of dry CH₂Cl₂was added

while maintaining the bath temperature at -78 °C. Stirring was

continued further for 30 min. Dry and distilled DIEA (30 mmol)

was then added using a syringe and the reaction mixture then

allowed to warm to room temperature without removing the

Fmoc-^L-alaninol (62b). Yield 1.4 g (92%), white powder, mp

100-102 °C. ¹H NMR (CDCl₃, 500 MHz) δ 1.07 (m, 3H), 3.43

6136 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Mandal et al.

bath. The reaction mixture then quenched with 20 mL of ice-

cold water and extracted with CH₂Cl₂(3 ꢀ 80 mL). The

combined organic layers were washed with 1 N HCl (3 ꢀ 30

mL) and brine (1 ꢀ 30 mL), dried (MgSO₄), and concentrated

under vacuum. The crude aldehydes were used immediately for

the next step without characterization.

General Procedure for Synthesis of N-[(9H-Fluoren-9-ylmeth-

oxy)carbonyl]-γ-amino-r,β-unsaturated Benzyl Esters (64a-i,

69). A mixture of Fmoc-amino aldehyde (4.0 mmol) and benzyl

(triphenylphosphoranylidene)acetate (4.4 mmol) in dry CH₂Cl₂

(20 mL) was stirred for 3 h. The progress of reaction was

monitored by thin-layer chromatography. After completion of

reaction, the solvent was removed in vacuo, and the residue was

purified by silica gel chromatography using EtOAc in hexane.

Benzyl (R)-4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-(E)-2-

pentenoate (64a). Prepared as in Mandal et al.³⁸

C, 77.09; H, 6.46; N, 2.54. HRMS (M þ H) calcd, 576.2750;

found, 576.2092.

Benzyl (S)-5-Benzyloxy-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]-

amino]-(E)-2-pentenoate (64g). Yield 1.53 g (72%). ¹H NMR

(CDCl₃, 500 MHz) δ 3.5 (m, 2H), 4.15 (t, J = 7.0 Hz, 1H),

4.4-4.6 (m, 2H), 4.5 2 (m, 1H), 5.15 (s, 2H), 5.34 (d, J = 8.0 Hz,

1H), 6.0 (d, J = 15.5 Hz, 1H), 6.93 (m, 1H), 7.24-7.34 (m, 14 H),

7.54 (d, J = 7.0 Hz, 2H), 7.7 (d, J = 7.0 Hz, 2H). ¹³C NMR

(CDCl₃, 125 MHz) δ 47.3, 52.00, 66.5, 70.9, 73.4, 120.1, 122.0,

122.1, 125.1, 127.2, 127.8, 128.1, 128.2, 128.4, 128.5, 128.6, 128.7,

136.0, 137.5, 141.4, 143.9, 144.0, 146.2, 146.3, 155.9, 165.9. Anal.

(C₃₄H₃₁NO₅) C, H, N. Calcd: C, 76.53; H, 5.86; N, 2.62. Found: C,

76.42; H, 5.96; N, 2.66. HRMS (M þ H) calcd, 534.2280; found,

534.2291.

Benzyl (4S,5S)-5-Benzyloxy-4-[[(9H-fluoren-9-ylmethoxy)car-

bonyl]amino]-(E)-2-hexenoate (64h). Yield 1.55 g (68%), white

1

Benzyl (S)-4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-(E)-

(87%), white powder,

powder. H NMR (CDCl₃, 300 MHz) δ 1.22 (d, J = 5.7 Hz,

2-pentenoate (64b). Yield 1.52

g

3H), 3.7 (m, 1H), 4.2 (t, J = 6.6 Hz, 1H), 4.3-4.45 (m, 4H), 4.58

(d, J = 11.7 Hz, 1H), 5.2 (s, 2H), 5.98 (d, J = 15.3 Hz, 1H), 6.98

(m, 1H), 7.28-7.38 (m, 14H), 7.58 (d, J = 7.5 Hz, 2H), 7.74 (d,

J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ 16.5, 47.3, 66.3,

66.9, 71.1, 75.1, 120.0, 121.7, 125.0, 127.0, 127.7, 127.8, 127.9,

128.3, 128.5, 128.6, 135.9, 137.7, 141.3, 143.8, 143.9, 147.2, 165.8.

Anal. (C₃₅H₃₃NO₅) C, H, N. Calcd: C, 76.76; H, 6.07; N, 2.56.

Found: C, 76.61; H, 6.06; N, 2.55. HRMS (M þ H) calcd,

548.2437; found, 548.2451.

1

mp 130-131 °C. H NMR (CDCl₃, 500 MHz) δ 1.18 (m, 3H),

4.1 (t, J = 7.0 Hz, 1H), 4.35 (m, 2H), 4.65 (br s, 1H), 5.1 (s,

2H), 5.84 (d, J = 16.0 Hz, 1H), 6.81 (d, J = 15.0 Hz, 1H), 7.2-

7.3 (m, 9H), 7.48 (d, J = 7.0 Hz, 2H), 7.65 (d, J = 7.0 Hz, 2H).

¹³C NMR (CDCl₃, 125 MHz) δ 20.2, 47.3, 66.4, 120.0, 120.2,

125.0, 127.1, 127.7, 128.3, 128.4, 128.6, 135.8, 141.4, 143.8, 143.9,

149.3, 155.4, 166.0. HRMS (M þ H) calcd, 428.1862; found,

428.1876.

Benzyl (R)-5-Methyl-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]-

amino]-(E)-2-hexenoate (64c). Yield 1.55 g (85%), white powder,

mp 141 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.94 (m, 6H), 1.89 (m,

1H), 4.23 (m, 2H), 4.48 (m, 2H), 4.75 (d, J = 9.0 Hz, 1H), 5.21 (s,

2H), 5.96 (d, J = 15.9 Hz, 1H), 6.93 (m, 1H), 7.31-7.42 (m, 9H),

7.60 (d, J = 7.2 Hz, 2H), 7.77 (d, J = 7.2 Hz, 2H). ¹³C NMR

(CDCl₃, 75 MHz) δ 18.0, 18.9, 32.2, 47.3, 66.4, 120.0, 121.5,

124.9, 127.1, 127.7, 128.3, 128.6, 135.8, 141.4, 143.8, 147.4, 155.8,

165.9. Anal. (C₂₉H₂₉NO₄) C, H, N. Calcd: C, 76.46; H, 6.42; N,

3.07. Found: C, 75.74; H, 6.45; N, 3.32. HRMS (M þ H) calcd,

456.2175; found, 456.1540.

Benzyl (R)-6-Methyl-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]-

amino]-(E)-2-heptenoate (64d). Yield 1.6 g (82%), white powder,

mp 118 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.94 (d, J = 6.3 Hz,

6H), 1.42 (m, 2H), 1.67 (m, 1H), 4.22 (t, J = 6.3 Hz, 1H),

4.41-4.5 (m, 3H), 4.65 (m, 1H), 5.2 (s, 2H), 5.96 (d, J = 15.3 Hz,

1H), 6.88 (m, 1H), 7.31-7.42 (m, 9H), 7.60 (d, J = 7.2 Hz, 2H),

7.77 (d, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ 22.7,

24.7, 43.7, 47.3, 50.4, 66.4, 120.0, 120.4, 124.9, 127.1, 127.7, 128.3,

128.4, 128.6, 135.9, 141.4, 143.8, 148.9, 155.6, 166.0. Anal.

(C₃₀H₃₁NO₄) C, H, N. Calcd: C, 76.73; H, 6.65; N, 2.98. Found:

C, 76.56; H, 6.71; N, 3.02. HRMS (M þ H) calcd, 470.2331;

found, 470.2342.

Benzyl (4S,5S)-5-tert-Butoxy-4-[[(9H-fluoren-9-ylmethoxy)-

carbonyl]amino]-(E)-2-hexenoate (64i). Yield 1.4 g (65%) as an

oil. ¹H NMR (CDCl₃, 500 MHz) δ 0.99-1.06 (m, 12H), 3.72 (m,

1H), 4.14 (m, 1H), 4.2 (m, 1H), 4.35-4.42 (m, 2H), 5.1 (s, 2H),

5.16 (d, J = 9.0 Hz, 1H), 5.87 (d, J = 15.5 Hz, 1H), 6.9 (m, 1H),

7.22-7.3 (m, 9H), 7.51 (d, J = 7.0 Hz, 2H), 7.66 (d, J = 7.0 Hz,

2H). ¹³C NMR (CDCl₃, 125 MHz) δ 20.4, 28.6, 47.3, 57.5, 66.4,

66.9, 68.2, 74.3, 120.0, 121.2, 125.1, 127.1, 127.8, 128.2, 128.3,

128.6, 136.0, 141.4, 143.8, 147.9, 156.4, 166.0. HRMS (M þ H)

calcd, 514.2593; found, 514.1597.

N-[[(9H-Fluoren-9-ylmethoxy)carbonyl]benzyl]-3-pyrrolidin-2-

yl-(E)-propenoate (69). Yield 1.2 g (62%) as an oil. H NMR

1

(CDCl₃, 500 MHz) δ 1.7-1.96 (m, 4H), 3.36 (m, 2H), 4.1

(m, 0.5H), 4.14-4.2 (m, 1H), 4.3 (m, 0.5H), 4.4 (m, 1.5H),

4.48 (m, 0.5H), 5.57 (d, J = 15.5 Hz, 0.5H), 5.8 (d, J = 15.5

Hz, 0.5H), 6.6 (m, 1H), 6.68 (m, 0.5H), 6.8 (m, 0.5H), 7.1-7.3

(m, 9H), 7.42 (m, 1H), 7.5 (m, 1H), 7.6-7.68 (m, 2H). ¹³C NMR

(CDCl₃, 125 MHz) δ 22.6, 23.5, 30.8, 31.7, 46.5, 46.8, 47.3,

57.9, 58.2, 66.5, 67.5, 120.0, 120.4, 120.7, 125.0, 127.1, 127.7,

128.5, 135.8, 141.3, 143.7, 143.9, 147.9, 148.1, 155.1, 155.4, 158.2,

158.6, 166.1, 166.3. HRMS (M þ H) calcd, 454.2018; found,

454.1068.

General Procedure for the Synthesis of 4-Alkyl-Substituted

4-(9-Fluorenylmethyloxycarbonylamino)butyric Acids. To a stir-

red suspension of Fmoc-protected γ-amino-R,β-unsaturated

benzyl esters (2.0 mmol) and 10% Pd-C (10% by wt) in 15

mL of methanol-THF (4:1 v/v) was added TES (20.0 mmol)

dropwise under argon atmosphere. The reaction started with

evolution of hydrogen gas. After completion of the reaction

(TLC), the mixture was filtered through Celite, and the solvent

was removed in vacuo. The crude product was purified either by

triturating with ether-hexane or by short silica gel column

chromatography, eluting with 2% hexane-EtOAc, followed

by hexane-EtOAc-MeOH (3:6:1 v/v/v).

(R)-4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-2-pentanoic

Acid (65a). Yield 0.6 g (88%), white powder, mp 140-141 °C.

Prepared as in Mandal et al.³⁸

(S)-4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-2-pentanoic

Acid (65b). Yield 0.59 g (87%), white powder, mp 128-130 °C.

¹H NMR (DMSO-d₆, 500 MHz) δ 0.94 (d, J = 6.5 Hz, 3H), 1.52

(m, 2H), 2.1 (m, 2H), 3.41 (m, 1H), 4.1 (m, 1H), 4.14-4.23 (m,

2H), 7.05 (d, J = 8.5 Hz, 1H), 7.22 (m, 2H), 7.31 (m, 2H), 7.59 (d,

J = 7.5 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H). ¹³C NMR (DMSO-d₆,

125.0 MHz) δ 21.3, 31.0, 31.7, 46.4, 47.3, 65.5, 120.6, 125.5, 127.5,

Benzyl (R)-4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-(E)-

2-octenoate (64e). Yield 1.52 g (78%), white powder, mp 116 °C.

¹H NMR (CDCl₃, 300 MHz) δ 0.93 (m, 3H), 1.33 (m, 4H), 1.53

(m, 2H), 4.23 (m, 1H), 4.35 (m, 1H), 4.48 (m, 2H), 4.7 (m, 1H), 5.21

(s, 2H), 5.96 (d, J = 15.6 Hz, 1H), 6.9 (m, 1H), 7.3-7.43 (m, 9H),

7.6 (d, J = 7.5 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H). ¹³C NMR

(CDCl₃, 75.0 MHz) δ 13.9, 22.3, 27.7, 34.2, 47.3, 52.0, 66.4, 120.0,

120.6, 124.9, 127.1, 127.7, 128.3, 128.4, 128.6, 135.9, 141.4, 143.8,

143.9, 148.7, 155.7, 166.0. Anal. (C₃₀H₃₁NO₄) C, H, N. Calcd: C,

76.73; H, 6.65; N, 2.98. Found: C, 76.61; H, 6.71; N, 2.99. HRMS

(M þ H) calcd, 470.2331; found, 470.2338.

Benzyl (R)-5-(4-tert-Butoxyphenyl)-4-[[(9H-fluoren-9-ylmeth-

oxy)carbonyl]amino]-(E)-2-pentenoate (64f). Yield 1.9 g (81%),

white powder, mp 82 °C. ¹H NMR (CDCl₃, 300 MHz) δ 1.33 (s,

9H), 2.87 (m, 2H), 4.2 (t, J = 6.6 Hz, 1H), 4.4 (m, 2H), 4.63-4.74

(m, 2H), 5.2 (s, 2H), 5.88 (d, J = 15.9 Hz, 1H), 6.91-6.94 (m,

3H), 7.02-7.1(m, 2H), 7.30-7.42 (m, 10H), 7.55 (m, 2H), 7.77(d,

J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ 28.8, 47.2, 66.4,

66.7, 78.4, 120.0, 121.1, 124.2, 124.9, 127.1, 127.7, 128.2, 128.3,

128.6, 129.8, 135.8, 141.3, 143.7, 147.7, 154.5, 155.5, 165.8. Anal.

(C₃₇H₃₇NO₅) C, H, N. Calcd: C, 77.19; H, 6.48; N, 2.43. Found:

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6137

(4S,5S)-5-tert-Butoxy-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]-

amino]-2-hexanoic Acid (65i). Yield 0.71 g (83%) as an oil. H

128.1, 141.2, 144.4, 156.1, 174.8. HRMS (M þ H) calcd,

340.1549; found, 340.1566.

1

(S)-5-Methyl-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

2-hexanoic Acid (65c). Yield 0.66 g (89%), white powder,

mp 120 °C. ¹H NMR (CDCl₃, 300 MHz) δ 0.9 (m, 6H),

1.5-1.78 (m, 3H), 2.36 (t, J = 7.5 Hz, 2H), 3.5 (m, 1H), 4.22

(m, 1H), 4.42 -4.6 (m, 3H), 7.3-7.43 (m, 4H), 7.60 (d, J = 7.5

Hz, 2H), 7.77 (d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz)

17.8, 19.0, 27.4, 31.1, 32.5, 47.5, 56.1, 66.4, 1120.0, 125.0,

127.0, 127.7, 141.4, 143.9, 156.6, 178.8. Anal. (C₂₂H₂₅NO₄) C,

H, N. Calcd: C, 71.91; H, 6.86; N, 3.81. Found: C, 71.60; H,

6.82; N, 3.80. HRMS (M þ H) calcd, 368.1862; found,

368.1276.

NMR (CDCl₃, 500 MHz) δ 1.03 (d, J = 6.0 Hz, 3H), 1.12 (s, 9H),

1.65-1.76 (m, 2H), 2.31 (t, J = 7.0 Hz, 2H), 3.48 (m, 1H), 3.6 (m,

1H), 4.14 (m, 1H), 4.34-4.41 (m, 2H), 4.5 (m, 1H), 5.0 (d, J = 9.5

Hz, 1H), 7.25 (m, 2H), 7.32 (m, 2H), 7.53 (d, J = 7.0Hz, 2H), 7.68

(d, J = 7.5 Hz, 2H), 10.1 (s, 1H). ¹³C NMR (CDCl₃, 125 MHz) δ

20.0, 27.7, 28.7, 31.0, 47.4, 56.0, 66.7, 68.4, 73.9, 120.0, 125.1,

127.1, 127.7, 141.4, 143.8, 157.2, 178.9. HRMS (M þ Na) calcd,

448.2100; found, 448.2134.

N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-3-pyrrolidin-2-ylpropio-

nic Acid (70). Yield 0.59 g (80%) as an oil. ¹H NMR (CDCl₃, 500

MHz) δ 1.53-2.07 (m, 6H), 2.36 (m, 1H), 3.3-3.45 (m, 2.5H),

3.95 (m, 0.5H), 4.0 (m, 1H), 4.35-4.46 (m, 1H), 4.58 (m,

1H), 7.28-7.38 (m, 4H), 7.77 (m, 2H), 7.74 (m, 2H), 9.4 (m,

2H). ¹³C NMR (CDCl₃, 125 MHz) δ 22.8, 23.6, 28.9, 29.4, 30.2,

31.2, 46.4, 47.3, 57.1, 66.8, 67.4, 119.95, 125.0, 127.1, 127.7, 141.4,

143.9, 155.8, 178.5. HRMS (M þ H) calcd, 366.1705; found,

366.1651.

Synthesis of tert-Butyl 4-(Benzyloxycarbonylamino)-5-hydro-

xypentanoate (72). To a stirred solution of Z-Glu(OtBu)-OH

(5.0 g, 14.8 mmol) and DCC (3.46 g, 16.8 mmol) in 150 mL of

EtOAc was added 2-mercaptopyridine (1.81 g, 16.33 mmol).

Stirring was continued for 4 h at which time the precipitate was

filtered off and the solvent was removed under vacuum. The

residue was dissolved in 100 mL of 1,4-dioxane, cooled to 0 °C

and treated with NaBH₄(2.0 g). When the reaction was finished,

as monitored by TLC, approximately after 30 min, it was then

quenched slowly by adding 2% KHSO₄(aq). The mixture was

extracted with Et₂O (3 ꢀ 150 mL). The combined organic layers

were washed with 5% NaHCO₃(2 ꢀ 50 mL) and brine, dried

(MgSO₄), and concentrated under vacuum. The crude product

was purified by silica gel column chromatography eluting with

35% EtOAc-hexane (v/v) to give 4.2 g of 72 as colorless oil.

Yield 88%. ¹H NMR (CDCl₃, 500 MHz) δ 1.45 (s, 9H), 1.77 (m,

1H), 1.87 (m, 1H), 2.34 (m, 2H), 2.76 (br s, 1H), 3.6 (m, 1H),

3.65-3.7 (m, 2H), 5.1 (s, 2H), 5.25 (d, J = 6.5 Hz, 1H), 7.36 (s,

5H). ¹³C NMR (CDCl₃, 125 MHz) δ 28.0, 28.1, 66.8, 80.9, 128.0,

128.2, 128.4, 128.6, 136.4, 156.6, 173.3. HRMS (M þ H) calcd,

324.1811; found, 324.0416.

Synthesis of tert-Butyl 4-(Benzyloxycarbonylamino)-5-iodo-

pentanoate (73). A solution of PPh₃(4.8 g, 18.6 mmol), I₂(2.4

g, 18.6 mmol), and imidazole (2.1 g, 31.0 mmol) in dry CH₂Cl₂

(30 mL) was stirred for 30 min under inert atmosphere at room

temperature. To this solution was added a solution of 62 (2.0 g,

6.2 mmol) in 15 mL of dry CH₂Cl₂, and stirring continued for 3

h. The solvent was removed, and the crude product was purified

by silica gel chromatography eluting 15% EtOAc-hexane (v/v).

A white solid was obtained (2.45 g, 91%). ¹H NMR (CDCl₃, 500

MHz) δ 1.45 (s, 9H), 1.85 (m, 2H), 2.3 (m, 2H), 3.33 (m 1H), 3.42

(m, 1H), 3.5 (m, 1H), 5.1 (br s, 3H), 7.37 (s, 5H). ¹³C NMR

(CDCl₃, 125 MHz) δ 28.1, 30.0, 31.8, 66.9, 80.8, 127.9, 128.1,

128.2, 128.3, 128.5, 128.7, 136.3, 155.7, 172.3. HRMS (M þ H)

calcd, 434.0828; found, 434.0541.

(S)-6-Methyl-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

2-heptanoic Acid (65d). Yield 0.68 g (88%), white powder, mp

1

150 °C. H NMR (CDCl₃, 300 MHz) δ 0.91 (d, J = 6.3 Hz,

6H), 1.24 (m, 2H), 1.53-1.62 (m, 2H), 1.85 (m, 1H), 2.35 (t,

J = 7.2 Hz, 2H), 3.73 (m, 1H), 4.2 (m, 1H), 4.44-4.5 (m, 3H),

7.31-7.42 (m, 4H), 7.60 (d, J = 7.2 Hz, 2H), 7.77 (d, J = 7.2

Hz, 2H). ¹³C NMR (CDCl₃, 75.0 MHz) δ 22.2, 23.0, 24.8, 30.8,

45.0, 47.4, 49.1, 66.3, 119.95, 125.0, 127.0, 127.7, 141.4, 143.9,

156.3, 178.2. Anal. (C₂₃H₂₇NO₄) C, H, N. Calcd: C, 72.42; H,

7.13; N, 3.67. Found: C, 72.14; H, 7.25; N, 3.80. HRMS (M þ

H) calcd, 382.2018; found, 382.1545.

(R) 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-2-octanoic

Acid (65e). Yield 0.67 g (87%), white powder. ¹H NMR

(DMSO-d₆, 500 MHz) δ 0.85 (s, 3H), 1.2-1.4 (m, 6H), 1.53

(m, 1H), 1.66 (m, 1H), 2.18 (m, 2H), 3.4 (m, 1H), 4.22 (m, 1H),

4.3 (m, 2H), 7.1 (d, J = 8.0 Hz, 1H), 7.3-7.43 (m, 4H), 7.7 (d,

J = 7.0 Hz, 2H), 7.9 (d, J = 7.0 Hz, 2H). ¹³C NMR (DMSO-d₆,

125 MHz) δ 14.0, 22.5, 27.9, 30.5, 30.8, 35.3, 47.4, 51.0, 66.4,

120.0, 125.0, 127.1, 127.7, 141.4, 143.9, 156.4, 178.2. HRMS

(M þ H) calcd, 382.2018; found, 382.1975. Anal. (C₂₃H₂₇NO₄)

C, H, N. Calcd: C, 72.42; H, 7.13; N, 3.67. Found: C, 72.65; H,

7.25; N, 4.19.

(R)-5-(4-tert-Butoxyphenyl)-4-[[(9H-fluoren-9-ylmethoxy)car-

bonyl]amino]-2-pentanoic Acid (65f). Yield 0.86 g (89%), white

powder, mp 127 °C. ¹H NMR (CDCl₃, 500 MHz) δ 1.22 (s, 9H),

1.53 (m, 1H), 1.76 (m, 1H), 2.3 (m, 2H), 2.6 (m, 2H), 3.8 (m, 1H),

4.3 (m, 2H), 4.6 (d, J = 9.0 Hz, 1H), 6.8 (d, J = 8.0 Hz, 2H), 6.9

(d, J = 8.0 Hz, 2H), 7.2-7.3 (m, 4H), 7.5 (d, J = 7.0 Hz, 2H), 7.7

(d, J = 7.0 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ 29.1, 30.8,

40.8, 47.3, 51.9, 66.5, 78.4, 120.0, 124.1, 125.0, 127.1, 127.7,

129.8, 132.1, 141.3, 143.8, 154.0, 156.1, 178.3. Anal.

(C₃₀H₃₃NO₅) C, H, N. Calcd: C, 73.90; H, 6.82; N, 2.87. Found:

C, 72.71; H, 6.69; N, 2.95. HRMS (M þ H) calcd, 488.2437;

found, 488.2423.

(S)-5-Benzyloxy-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

2-pentanoic Acid (65g). Yield 0.69 g (77%), white powder. H

1

NMR (DMSO-d₆, 500MHz) δ1.58(m1H), 1.80(m,1H), 2.24(m,

2H), 3.38 (m, 2H), 3.67 (m, 1H), 4.23 (m, 1H), 4.3-4.37 (m, 2H),

4.48 (s, 2H), 7.23-7.35 (m, 8H), 7.42 (m, 2H), 7.71 (d, J = 7.0 Hz,

2H), 7.89 (d, J = 7.5 Hz, 2H), 12.1 (s, 1H). ¹³C NMR (DMSO-d₆,

125 MHz) δ 26.9, 30.7, 47.2, 50.3, 65.7, 72.3, 72.4, 120.5, 120.6,

125.7, 127.4, 127.6, 127.8, 128.0, 128.2, 128.5, 128.8, 138.9, 141.2,

144.4, 156.4, 174.7. Anal. (C₂₇H₂₇NO₅) C, H, N. Calcd: C, 72.79;

H, 6.11; N, 3.14. Found: C, 72.76; H, 6.23; N, 3.09. HRMS (M þ

H) calcd, 446.1967; found, 446.1984.

(4S,5S)-5-Benzyloxy-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]-

amino]-2-hexanoic Acid (65h). Yield 0.75 g (82%), white pow-

der. ¹H NMR (DMSO-d₆, 500 MHz) δ 1.07 (d, J = 6.9 Hz, 3H),

1.63 (m, 1H), 1.8 (m, 1H), 2.2-2.3 (m, 2H), 3.53 (m, 1H), 3.62

(m, 1H), 4.22 (m, 1H), 4.26-4.31 (m, 3H), 4.5 (m, 2H), 7.2 (d,

J = 5.4 Hz, 1H), 7.27-7.43 (m, 9 H), 7.72 (d, J = 7.5 Hz, 2H),

7.9 (d, J = 7.5 Hz, 2H), 12.04 (s, 1H). ¹³C NMR (DMSO-d₆, 125

MHz) δ 15.6, 24.9, 31.0, 47.3, 53.9, 65.7, 70.4, 76.3, 120.6, 125.7,

127.5, 127.7, 127.8, 128.1, 128.6, 129.2, 139.4, 141.2, 144.4,

156.7, 174.8. Anal. (C₂₈H₂₉NO₅) C, H, N. Calcd: C, 73.18; H,

6.36; N, 3.05. Found: C, 72.52; H, 6.38; N, 3.01. HRMS (M þ

H) calcd, 460.2124; found, 460.2141.

Synthesis of tert-Butyl 4-(Benzyloxycarbonylamino)-5-(1-pi-

peridino)pentanoate (74a). A solution of 73 (1.0 g, 2.3 mmol) in

10 mL of dry THF was treated with piperidine (0.7 mL, 6.92

mmol) overnight under argon. The reaction mixture was diluted

with 100 mL of EtOAc and was washed with water and brine.

After drying (MgSO₄) and concentration under vacuum the

crude product was purified by silica gel chromatography eluting

with 50% EtOAc-hexane (v/v) to give the desired N-alkylated

product. Yield 0.74 g, 82%. ¹H NMR (CDCl₃, 500 MHz) δ 1.45

(s, 9H), 1.53-1.58 (m, 4H), 1.7 (m, 1H), 1.93 (m, 1H), 2.28-2.40

(m, 8H), 3.73 (m, 1H), 5.03 (br s, 1H), 5.12 (s, 2H), 7.37 (s, 5H).

¹³C NMR (CDCl₃, 125.0 MHz) δ 24.3, 26.0, 28.0, 28.1, 28.3,

32.0, 54.9, 66.5, 80.3, 127.8, 128.1, 128.4, 128.6, 129.1, 136.8,

156.4, 172.9. Anal. (C₂₂H₃₄N₂O₄) C, H, N. Calcd: C, 67.66; H,

8.78; N, 7.17. Found: C, 67.89; H, 8.91; N, 7.21. HRMS (M þ H)

calcd, 391.2597; found, 391.0992.

6138 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

tert-Butyl 4-(Benzyloxycarbonylamino)-5-(4-morpholii-

Mandal et al.

was treated with 95% TFA/CH₂Cl₂for 1 h, and the solvent

was removed under vacuum. Traces of TFA were removed by

addition and evaporation of toluene (2 ꢀ 10 mL). The product

then triturated with ether-hexane to give a white solid (0.56 g,

90%). ¹H NMR (DMSO-d₆, 500 MHz) δ 1.36 (m, 1H),

1.56-1.76 (m, 7H), 2.21 (m, 2H), 2.88 (m, 2H), 3.05 (m, 1H),

3.16 (m, 1H), 3.37 (m, 1H), 3.5 (m, 1H), 3.88 (m, 1H), 4.25 (m,

1H), 4.32 (m, 1H), 4.52 (m, 1H), 7.34 (m, 2H), 7.42 (m, 3H), 7.7

no)pentanoate (74b). The same procedure as for 74a was

used. Yield 0.76 g (84%). ¹H NMR (CDCl₃, 500 MHz) δ

1.45 (s, 9H), 1.67 (m, 1H), 1.94 (m, 1H), 2.3-2.48 (m, 8H),

3.65 (s, 4H), 3.77 (m, 1H), 4.9 (br s, 1H), 5.11 (s, 2H), 7.36 (s,

5H). ¹³C NMR (CDCl₃, 125 MHz) δ 28.0, 28.1, 32.0, 48.4,

53.9, 62.8, 66.9, 67.3, 80.4, 127.9, 128.1, 128.4, 128.6, 136.6,

156.4, 172.8. Anal. (C₂₁H₃₂N₂O₅) C, H, N. Calcd: C, 64.26;

H, 8.22; N, 7.14. Found: C, 63.85; H, 8.26; N, 7.07. HRMS

(M þ H) calcd, 393.2389; found, 393.1541.

(d, J = 7.5 Hz, 2H), 7.9 (d, J = 7.5 Hz, 2H), 9.24 (s, 1H). ¹³

C

NMR (DMSO-d₆, 125 MHz) δ 21.6, 22.5, 28.3, 30.2, 46.2, 47.3,

52.4, 53.6, 59.6, 65.8, 120.6, 125.5, 125.6, 127.5, 128.1, 141.3,

144.2, 144.3, 156.5, 158.5, 158.7, 174.3. HRMS (M þ H) calcd,

423.2284; found, 423.2716.

tert-Butyl 4-(Benzyloxycarbonylamino)-5-(4-methyl-1-pipera-

zino)pentanoate (74c). The same procedure as for 74a was used.

Yield 0.73 g (78%). ¹H NMR (CDCl₃, 500 MHz) δ 1.44 (s, 9H),

1.67 (m, 1H), 1.92 (m, 1H), 2.27 (s, 3H), 2.3-2.57 (m, 12H), 3.74

(m, 1H), 4.94 (m, 1H), 5.13 (s, 2H), 7.36 (s, 5H). ¹³C NMR

(CDCl₃, 125 MHz) δ 28.0, 28.1, 32.0, 45.4, 45.9, 46.4, 48.6, 53.2,

55.0, 62.1, 66.5, 80.4, 127.9, 128.1, 128.4, 128.5, 128.6, 136.7,

4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-5-(1-morpholi-

no)pentanoic Acid (76b). The same procedure as for 76a was

employed. Yield 0.56 g (90%). ¹H NMR (DMSO-d₆, 500 MHz)

δ 1.61 (m, 1H), 1.74 (m, 1H), 2.21 (m, 2H), 3.1-3.48 (m, 6H),

3.73 (m, 2H), 3.91 (m, 3H), 4.24-4.32 (m, 2H), 4.5 (m, 1H), 7.34

(m, 2H), 7.43 (m, 3H), 7.7 (d, J = 7.5 Hz, 2H), 7.9 (d, J = 7.5 Hz,

2H), 9.9 (br s, 1H). ¹³C NMR (DMSO-d₆, 125 MHz) δ 28.1,

30.2, 45.8, 47.2, 60.0, 63.5, 65.9, 120.6, 125.5, 125.6, 127.5, 128.1,

141.3, 144.2, 144.3, 156.5, 158.6, 158.8, 174.3. HRMS (M þ H)

calcd, 425.2076; found, 425.2117.

ꢀ

156.4, 172.8. Anal. (C₂₂H₃₅N₃O₄) C, H, N. Calcd: C, 65.16; H,

8.70; N, 10.36. Found: C, 64.50; H, 8.71; N, 10.05. HRMS (M þ

H) calcd, 406.2706; found, 406.1604.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-(1-piperidino)pentanoate (75a). To a suspension of 74a

(1.0 g, 2.56 mmol) and 10% Pd-C (0.2 g) in 2% CHCl₃-MeOH

(20 mL) was added TES (4.2 mL, 26.0 mmol) dropwise under argon

atmosphere. After completion (TLC monitoring), the catalyst was

filtered off through Celite and the filter cake washed with 10 mL of

methanol. The combined filtrates were concentrated under va-

cuum, and residual methanol was removed by the addition and

evaporation of toluene (2 ꢀ 10 mL). The residue was dissolved in 10

mL of 10% Na₂CO₃and 20 mL of 1,4-dioxane. Fmoc-OSu (1.3 g,

3.84 mmol) was added, and the mixture was stirred overnight. The

mixture was extracted with EtOAc (2 ꢀ 40 mL), and the combined

organic layers were washed with water followed by brine. The

organic part was dried (MgSO₄) and concentrated under vacuum.

The crude product was purified by silica gel column chromatogra-

phy eluting with 10% methanol-chloroform (v/v) to give desired

product. Yield 0.94 g, 76%. ¹HNMR(CDCl₃, 500 MHz) δ1.3-1.4

(m, 10H), 1.65 (m, 1H), 1.7-1.87 (m, 6H), 2.00 (m, 1H), 2.26 (m,

2H), 2.52 (m, 1H), 2.62 (m, 1H), 2.81 (m, 1H), 3.35 (m, 1H), 3.4 (m,

1H), 3.65 (m, 1H), 4.1-4.2 (m, 4H), 4.3 (m, 1H), 6.9 (d, J = 9.5 Hz,

1H), 7.23 (m, 2H), 7.3 (m, 2H), 7.54 (m, 2H), 7.66 (d, J = 7.5 Hz,

2H). ¹³C NMR (CDCl₃, 125 MHz) δ 22.0, 22.4, 28.0, 28.7, 31.1,

45.5, 47.0, 51.6, 55.6, 59.2, 67.4, 81.0, 119.8, 125.3, 125.5, 127.1,

127.6, 127.7, 141.3, 143.7, 144.1, 156.8, 172.1. HRMS (M þ H)

calcd, 479.2910; found, 479.1051.

tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-5-(1-

morpholino)pentanoate (75b). The same procedure as for 75a

was employed. Yield 0.91 g (74%). ¹H NMR (CDCl₃, 500 MHz)

δ 1.35 (s, 9H), 1.66 (m, 1H), 1.77 (m, 1H), 2.25 (m, 2H), 2.78-2.86

(m, 3H), 3.41 (m, 2H), 3.67 (m, 1H), 3.91 (m, 4H), 4.12 (m, 2H),

4.21-4.3 (m, 2H), 6.37 (d, J = 9.5 Hz, 1H), 7.24 (m, 2H), 7.31 (m,

2H), 7.52 (m, 2H), 7.68 (d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃,

125 MHz) δ 28.0, 28.1, 30.9, 45.2, 46.9, 51.1, 53.9, 60.3, 63.6, 67.5,

81.2, 114.8, 117.1, 119.9, 125.2, 125.3, 127.1, 127.7, 127.8, 141.2,

141.3, 143.5, 144.1, 156.7, 162.1, 162.3, 172.0. HRMS (M þ H)

calcd, 481.2702; found, 481.1066.

4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-5-(4-methy1-1-pi-

perazino)pentanoic Acid (76c). The same procedure as for 76a was

employed. Yield 0.45 g (72%). ¹H NMR (DMSO-d₆, 500 MHz) δ

1.55 (m, 1H), 1.78 (m, 1H), 2.22 (m, 2H), 2.66 (m, 2H), 2.8 (s, 3H),

2.9-3.5 (m, 8H), 3.71 (m, 1H), 4.24-4.3 (m, 2H), 4.41 (m, 1H),

7.25 (d, J = 8.0 Hz, 1H), 7.34 (m, 2H), 7.42 (m, 2H), 7.7 (m, 2H),

7.9 (d, J = 7.5 Hz, 2H). ¹³C NMR (DMSO-d₆, 125 MHz) δ 28.1,

30.5, 42.5, 47.3, 47.4, 49.6, 65.7, 115.6, 117.9, 120.6, 125.6, 127.5,

128.1, 141.3, 144.3, 144.4, 156.5, 158.6, 158.9, 159.2, 159.4, 174.6.

HRMS (M þ H) calcd, 438.2393; found, 438.2433.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-hydroxypentanoate (78). Fmoc-Glu(OtBu)-OH (77) (4.0

g, 9.4 mmol) was treated as described for the preparation of 72 to

1

give 78. Yield 3.2 g (83%). NMR same as in ref 44. H NMR

(CDCl₃, 500 MHz) δ 1.37 (s, 9H), 1.71 (m, 1H), 1.8 (m, 1H), 2.25

(m, 2H), 3.5 (m, 1H), 3.6 (m, 2H), 4.13 (m, 1H), 4.33 (m, 2H), 5.12

(br s, 1H), 7.24 (m, 2H), 7.33 (m, 2H), 7.52), 7.52 (d, J = 7.5 Hz,

2H), 7.68(d, J= 7.5 Hz, 2H). ¹³CNMR (CDCl₃, 125 MHz) δ25.7,

28.1, 31.9, 47.3, 53.0, 64.7, 66.7, 81.0, 120.0, 125.1, 127.1, 127.7,

141.3, 143.9, 155.9, 173.3. HRMS (M þ H) calcd, 412.2124; found,

412.2132.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-iodopentanoate (79). The same procedure was followed

as described for 73 starting with 2.0 g (4.86 mmol) of 78. Yield 2.3 g

(88%). ¹H NMR (500 MHz, CDCl₃) δ 1.38 (s, 9H), 1.78 (m, 2H),

2.2-2.3 (m, 2H), 3.24 (m, 1H), 3.35 (m, 1H), 3.4 (m, 1H), 4.15 (m,

1H), 4.3 (m, 1H), 4.4 (m, 1H), 4.95 (d, J = 7.5 Hz, 1H), 7.25 (m,

2H), 7.33 (m, 2H), 7.53 (d, J= 7.0 Hz, 2H), 7.7 (d, J =7.5Hz, 2H).

¹³C NMR (CDCl₃, 125 MHz) δ 28.1, 29.9, 31.8, 47.3, 50.5, 66.8,

80.9, 120.0, 125.0, 125.1, 127.1, 127.7, 141.3, 143.8, 155.7, 172.4.

HRMS (M þ H) calcd, 522.1141; found, 522.1157.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-azidopentanoate (80).⁴⁰To a solution of 79 (2.0 g, 3.84

mmol) in 30 mL of dry CH₂Cl₂was added nBu₄NN₃(3.3 g, 11.5

mmol) in portions. After completion of the reaction (ca. 8 h,

monitored by TLC), the solvent was removed in vacuo, and the

product was purified by silica gel chromatography (10%

tert-Butyl [[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-5-(4-

methy1-1-piperazino)pentanoate (75c). The same procedure as

for 75a was employed. Yield 0.78 g (64%). ¹H NMR (500 MHz,

CDCl₃) δ 1.35 (s, 9H), 1.6 (m, 1H), 1.75 (m, 1H), 2.22 (m, 2H),

2.74 (s, 3H), 2.88 (m, 1H), 3.13 (m, 1H), 3.33-3.5 (m, 8H), 3.97

(m, 1H), 4.11 (t, J = 6.5 Hz, 1H), 4.28 (d, J = 7.0 Hz, 2H), 5.7 (d,

J = 9.0 Hz, 1H), 7.23 (m, 2H), 7.32 (m, 2H), 7.5 (m, 2H), 7.68

(m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 27.8, 28.0, 28.3, 31.2,

43.3, 46.5, 46.9, 50.6, 60.6, 67.3, 81.2, 112.7, 115.0, 117.3, 119.9,

125.11, 127.2, 127.7,127.9, 141.3, 143.4, 144.1, 156.8, 162.1,

162.4, 162.7, 171.9. HRMS (M þ H) calcd, 494.3019; found,

494.2492.

1

EtOAc-hexane v/v). Yield 1.45 g (86%). H NMR (500 MHz,

CDCl₃) δ1.37 (s, 9H), 1.73 (m, 2H), 2.2-2.3 (m, 2H), 3.35 (m, 2H),

3.71 (m, 1H), 4.13 (m, 1H), 4.3-4.4 (m, 2H), 4.93 (d, J = 8.0 Hz,

1H), 7.24 (m, 2H), 7.33 (m, 2H), 7.51 (d, J = 7.5 Hz, 2H), 7.68 (d, J

= 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 125.0 MHz) δ 28.1, 32.0, 46.8,

51.2, 54.8, 66.8, 80.9, 120.1, 125.1, 127.1, 127.7, 127.8, 141.3, 143.8,

155.9, 172.5. HRMS (M þ H) calcd, 437.2189; found, 437.2208.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-(allyloxycarbonyl)aminopentanoate (81). To a stirred sus-

pension of 80 (1.0 g, 2.3 mmol) and 10% Pd-C (0.1 g) in 15 mL of

Synthesis of 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-

5-(1-piperidino)pentanoic Acid (76a). Compound 75a (0.7 g)

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6139

1% CHCl₃in MeOH was added TES (4.0 mL) dropwise under

argon atmosphere. After completion of ∼20 min, the mixture was

filtered through Celite and the filtrate then concentrated under

vacuum. The residue was dissolved in 15 mL of CH₂Cl₂and was

treated with allyl chloroformate (0.5 mL, 4.6 mmol) and DIEA (1.2

mL, 6.9 mmol) for 3 h at 0 °C. The solution was transferred to a

separatory funnel with an additional 50 mL of CH₂Cl₂and was

washed with 5% HCl (2 ꢀ 15 mL), 5% NaHCO₃(20 mL), and

brine and dried (MgSO₄). The solvent was removed, and the crude

product was purified by silica gel chromatography (25%

EtOAc-hexane, v/v) yielding 81 as a white powder. Yield 0.73 g

General Procedure for 4-Nitrophenyl Fmoc-aminocarbonates:

Synthesis of 4-Nitrophenyl 2-[(9H-Fluoren-9-ylmethoxy)car-

bonyl]aminoethylcarbonate (83j). 4-Nitrophenyl chloroformate

(1.6 g, 7.8 mmol) in 10 mL of dry CH₂Cl₂was added dropwise to

a stirred solution of Fmoc-aminoethanol (2.0 g, 7.06 mmol) and

pyridine (0.9 mL, 10.6 mmol) in 20 mL of dry CH₂Cl₂at 0 °C

under an atmosphere of argon. Upon completion (TLC), the

solution was transferred to a separatory funnel with an addi-

tional 20 mL of CH₂Cl₂. The organic solution was washed with

10% HCl (3 ꢀ 30 mL), 10% Na₂CO₃(3 ꢀ 30 mL), and brine (50

mL) and was dried (MgSO₄). The crude mixture was purified by

silica gel chromatography eluting with 40% EtOAc-hexane to

give 2.6 g (82%) of desired product as a white solid. ¹H NMR

(300 MHz, CDCl₃) δ 3.6 (m, 2H), 4.25-4.48 (m, 5H), 5.2 (m,

1H), 7.28-7.45 (m, 6H), 7.6 (d, J = 7.2 Hz, 2H), 7.8 (d, J = 7.5

Hz, 2H), 8.28 (d, J = 8.4 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ

39.9, 47.2, 66.9, 68.2, 120.0, 121.6, 121.7, 124.9, 125.3, 125.5,

127.0, 127.7, 141.4, 143.8, 145.5, 152.4, 155.4. Anal.

(C₂₄H₂₀N₂O₇) C, H, N, O. Calcd: C, 64.28; H, 4.50; N, 6.25;

O, 24.98. Found: C, 63.91; H, 4.42; N, 6.36. HRMS (M þ H)

calcd, 454.2230; found, 454.2240.

1

(64%). H NMR (CDCl₃, 500 MHz) δ 1.46 (s, 9H), 1.75 (m,

1H), 1.84 (m, 1H), 2.34 (m, 2H), 3.28-3.36 (m, 2H), 3.73 (m, 1H),

4.22 (m, 1H), 4.35-4.45 (m, 2H), 4.55 (m, 2H), 5.17-5.3 (m,

4H), 5.90 (m, 1H), 7.33 (m, 2H), 7.42 (m, 2H), 7.61 (d, J = 7.0 Hz,

2H), 7.78(d, J =7.0 Hz, 2H). ¹³CNMR(CDCl₃, 125MHz) δ27.3,

28.1, 32.0, 45.1, 47.3, 52.0, 65.7, 66.7, 80.8, 117.7, 120.0,

125.1, 127.1, 127.7, 132.8, 141.3, 143.9, 156.6, 156.9, 172.7. HRMS

(M þ H) calcd, 495.2495; found, 495.2478. Anal. (C₂₈H₃₅N₂O₆) C,

H, N. Calcd: C, 68.00; H, 6.93; N, 5.66. Found: C, 67.93; H, 6.88;

N, 5.62.

Synthesis of 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-

5-(allyloxycarbonyl)aminopentanoic Acid (82). Compound 81

(0.6 g, 1.2 mmol) was treated with neat TFA for 1 h. The TFA

was removed under vacuum, and the residue was triturated with

ether-hexane to form a white solid. The product was collected

by filtration and dried in vacuo over P₂O₅. Yield 0.44 g (82%).

¹H NMR (DMSO-d₆, 500 MHz) δ 1.5 (m, 1H), 1.7 (m, 1H), 2.2

(m, 2H), 3.03 (m, 2H), 3.5 (m, 1H), 4.19-4.3 (m, 3H), 4.45 (m,

2H), 5.14 (dd, J = 2.0, 17.0 Hz, 1H), 5.26 (dd, J = 2.0, 28.5 Hz,

1H), 5.87 (m, 1H), 7.15 (d, J = 14.5, 1H), 7.23 (t, J = 20.0 Hz,

1H), 7.32 (m, 2H), 7.42 (m, 2H), 7.68 (d, J = 12.0 Hz, 2H), 7.88

(d, J = 12.0 Hz, 2H), 12.04 (br s, 1H). ¹³C NMR (DMSO-d₆, 125

MHz) δ 27.3, 30.7, 44.6, 47.3, 50.8, 64.7, 65.7, 117.3, 120.6,

125.7, 127.5, 128.1, 134.2, 141.2, 144.3, 144.4, 156.4, 156.6,

174.6. HRMS (M þ H) calcd, 439.1869; found, 439.1836. Anal.

(C₂₄H₂₇N₂O₆) C, H, N. Calcd: C, 65.74; H, 5.98; N, 6.39.

Found: C, 65.56; H, 5.93; N, 6.28.

4-Nitrophenyl 2-[(9H-Fluoren-9-ylmethoxy)carbonyl]amino-

3-benzyloxybutylcarbonate (83g). The procedure for 83j was

used. Compound 62g (1.0 g, 2.4 mmol) yielded 1.1 g of 83g

(77%). ¹H NMR (CDCl₃, 500 MHz) δ 1.16 (d, J = 6.0 Hz, 3H),

3.68 (m, 1H), 3.97 (m, 1H), 4.11 (m, 1H), 4.20 (d, J = 6.5 Hz,

2H), 4.27-4.33 (m, 3H), 4.55 (m, 1H), 5.16 (d, J = 9.5 Hz, 1H),

7.17-7.27 (m, 11H), 7.47 (d, J = 7.0 Hz, 2H), 7.64 (d, J = 7.0

Hz, 2H), 8.08 (d, J = 8.5 Hz, 2 Hz). ¹³C NMR (CDCl₃, 125

MHz) δ 16.0, 47.3, 54.1, 67.1, 68.6, 70.8, 72.0, 120.1, 121.8,

125.1, 125.3, 127.2, 127.8, 128.0, 128.1, 128.6, 137.8, 141.4,

143.8, 145.4, 152.4, 155.5, 156.8. HRMS (M þ H) calcd,

449.1349; found, 449.1357.

4-Nitrophenyl 2-[(9H-Fluoren-9-ylmethoxy)carbonyl]amino-

3-benzyloxypropylcarbonate (83f). The procedure for 83j was

used. Compound 62f (1.0 g, 2.47 mmol) yielded 1.2 g of 83g

1

(81%). H NMR (CDCl₃, 300 MHz) δ 3.64 (m, 2H), 4.25 (m,

2H), 4.46 (m, 4H), 4.58 (s, 2H), 5.27 (d, J = 8.4 Hz, 1H),

7.31-7.45 (m, 13H), 7.61 (d, J = 7.5 Hz, 2H), 7.79 (d, J = 7.5

Hz, 2H), 8.24 (m, 2H). ¹³C NMR (CDCl₃, 75 MHz) δ 47.2, 49.5,

67.0, 68.1, 68.5, 73.5, 120.1, 121.7, 125.0, 125.3, 127.1, 127.8,

128.1, 128.6, 137.5, 141.4, 143.8, 145.4, 152.3, 155.4, 156.2.

Anal. (C₃₂H₂₈N₂O₈) C, H, N. Calcd: C, 67.60; H, 4.96; N,

4.93. Found: C, 67.62; H, 4.86; N, 4.96. HRMS (M þ H) calcd,

569.1924; found, 569.1937.

Synthesis of tert-Butyl 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]-

amino]-5-acetoxypentanoate (92). Acetic anhydride (1.4 mL, 14.6

mmol) was added to a solution of 78 (1.0 g, 2.43 mmol), DIEA (0.85

mL, 4.86 mmol), and DMAP (0.03 g, 0.24 mmol) in dry CH₂Cl₂(15

mL). After completion of the reaction (TLC), 10 mL of water was

added and the mixture extracted with CH₂Cl₂(3 ꢀ 30 mL). The

combined organic layers were washed with 5% HCl (2 ꢀ 20 mL)

and saturated NaHCO₃(2 ꢀ 20 mL). After drying (MgSO₄) and

concentration under vacuum, the crude product was purified by

silica gel column chromatography eluting with 25% EtOAc-

4-Nitrophenyl 2-[(9H-Fluoren-9-ylmethoxy)carbonyl]aminopro-

pylcarbonate (83a). The procedure for 83j was used. Compound

62a (1.0 g, 3.36 mmol) yielded 1.3 g of 83a (84%). H NMR

1

hexane (v/v) to give 1.0 g (93%) of 92. H NMR (CDCl₃, 500

(CDCl₃, 500 MHz) δ 1.20 (s, 3H), 4.09-4.22 (m 4H), 4.36 (m, 2H),

4.78 (br s, 1H), 7.22-7.34 (m, 6H), 7.51 (d, J= 7.5 Hz, 2H), 7.69 (d,

J= 7.5 Hz, 2H), 8.17 (d, J=9.0Hz, 2H). ¹³CNMR(CDCl₃, 125.0

MHz) δ 17.3, 45.8, 47.2, 66.8, 71.6, 120.1, 121.7, 124.9, 125.3, 127.1,

127.8, 141.4, 143.8, 145.5, 152.5, 155.4, 155.7. Anal. (C₂₅H₂₂N₂O₇)

C, H, N. Calcd: C, 64.93; H, 4.80; N, 6.06. Found: C, 64.78; H, 4.69;

N, 6.04. HRMS (M þ H) calcd, 463.1505; found, 463.1507.

Synthesis of N-Triphenylmethyl 4-[[(9H-Fluoren-9-ylmethoxy)-

carbonyl]amino]-5-hydroxypentanamide (85). Starting with 2.0 g

(3.27 mmol) of Fmoc-Gln(Trt)-OH (84), the same procedure as

described above for the preparation of amino alcohols 62a-i was

MHz) δ 1.37 (s, 9H), 1.64-1.77 (m, 2H), 1.98 (s, 3H), 2.23 (m, 2H),

3.84 (m, 1H), 4.00 (m, 2H), 4.13 (m, 1H), 4.34 (m, 2H), 4.88 (d, J =

9.0 Hz, 1H), 7.24 (m, 2H), 7.32 (m, 2H), 7.5 (d, J=7.5Hz,2H),7.68

(d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 125.0 MHz) δ 28.1, 31.9,

46.8, 47.2, 50.4, 66.0, 66.6, 80.8, 119.9, 120.1, 125.0, 127.0, 127.2,

127.6, 127.8, 141.3, 143.9, 143.8, 156.0, 170.9, 172.5. HRMS (M þ

H) calcd, 454.2230; found, 454.2240.

Synthesis of 4-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-5-

acetoxypentanoic Acid (93). Compound 92 (0.5 g) was treated

with 2 mL of neat TFA for 1 h. The TFA was removed in vacuo,

and residual acid was removed by addition and evaporation of

toluene (2 ꢀ 5 mL). Trituration with ether-hexane resulted in a

white solid which was collected by filtration and dried in vacuo

1

followed to give 85. Yield 1.2 g (63%). H NMR (CDCl₃, 500

MHz) δ 1.71 (m, 1H), 1.83 (m, 1H), 2.2-2.34 (m, 2H), 3.36-3.44

(m, 2H), 3.58 (m, 1H), 4.22 (t, J = 6.5 Hz, 1H), 4.43 (d, J = 6.5 Hz,

2H), 5.46 (d, J = 8.5 Hz, 1H), 7.1 (s, 1H), 7.24-7.32 (m, 17H),

7.4-7.42 (m, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.77 (d, J = 7.5 Hz,

2H). HRMS (M þ H) calcd, 597.2753; found, 597.2763.

1

over P₂O_5.Yield 0.4 g (90%). H NMR (CDCl₃, 500 MHz) δ

1.77 (m, 1H), 1.89 (m, 1H), 2.1 (s, 3H), 2.42 (m, 2H), 3.96 (m,

1H), 4.06 (m, 1H), 4.14 (m, 1H), 4.22 (m, 1H), 4.46 (m, 2H), 4.95

(d, J = 8.5 Hz, 1H), 7.33 (m, 2H), 7.42 (m, 2H), 7.6 (m, 2H), 7.78

(d, J = 7.5 Hz, 2H). ¹³C NMR (CDCl₃, 125 MHz) δ 26.6, 30.4,

31.6, 47.2, 50.1, 66.0, 66.7, 119.8, 120.0, 120.1, 125.0, 127.0,

127.2, 127.6, 127.9, 141.3, 143.8, 156.2, 171.0, 177.9. HRMS

(M þ Na) calcd, 420.1423; found, 420.1442.

Synthesis of 4-Nitrophenyl [N-Triphenylmethyl-4-[[(9H-fluoren-

9-ylmethoxy)carbonyl]amino]pentanamide-5-yl]carbonate (86). To

a stirred solution of 85 (1.0 g, 1.67 mmol) and pyridine (0.27 mL,

3.34 mmol) in 20 mL of dry CH₂Cl₂at 0 °C was added a solution of

nitrophenyl chloroformate (0.37 g, 1.84 mmol) in 10 mL of dry

6140 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

Mandal et al.

CH₂Cl₂dropwise. The reaction was slowly warmed to room

temperature and stirred for 4 h. It was then diluted with an

additional 100 mL of CH₂Cl₂and transferred to a separatory

funnel. The organic layer was washed with water (20 mL) and brine

(20 mL) and dried (MgSO₄). The solvent was removed, and the

concentrated crude product was purified by silica gel chromatog-

raphy (20% EtOAc-hexane, v/v). Yield 0.79 g (62%). ¹H NMR

(CDCl₃, 500 MHz) δ 1.74 (m, 1H), 1.8 (m, 1H), 2.26 (m, 2H), 3.88

(m, 1H), 4.1-4.15 (m, 3H), 4.33 (m, 1H), 4.40 (m, 1H), 5.08 (d, J=

8.5 Hz, 1H), 6.7 (s, 1H), 7.1-7.22 (m, 19H), 7.25-7.31 (m, 2H),

7.47 (d, J = 7.5 Hz, 2H), 7.64 (d, J = 7.5 Hz, 2H), 8.11 (d, J = 9.0

Hz, 2H). ¹³C NMR (CDCl₃, 125 MHz) δ 26.6, 33.4, 47.4, 49.7,

66.6, 70.7, 96.2, 120.0, 120.1, 121.6, 121.8, 125.0, 125.3, 127.0,

127.2, 127.7, 127.9, 128.1, 128.6, 128.8, 141.4, 143.7, 143.8, 144.6,

145.4, 152.4, 155.4, 156.4, 171.1. HRMS (M þ H) calcd, 762.2815;

found, 762.3604.

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Acknowledgment. We are grateful to the National Cancer

Institute (CA096652) and the M. D. Anderson Cancer Center

University Cancer Fund for support of this work. Partial

support by a grant from the Center for Targeted Therapy of

The University of Texas M. D. Anderson Cancer Center and

the Texas Institute of Drug and Diagnostic Development at

The University of Texas at Austin is acknowledged. We also

acknowledge NCI Cancer Center Support Grant CA016672

for the support of our NMR facilities and the M. D. Anderson

Cancer Center Chemistry Core Facility for some of the mass

spectrometry. Funding as an Odyssey Fellow (Z.R.) was

supported by the Odyssey Program and the Cockrell Founda-

tion Award for Scientific Achievement at UTMDACC.

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small-molecule inhibitor of STAT3 via “click chemistry”. Bioorg.

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Supporting Information Available: Mass spectral and HPLC

characterization of peptides 3-49. This material is available free

of charge via the Internet at http://pubs.acs.org.

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Article Doi

Structure-affinity relationships of glutamine mimics incorporated into phosphopeptides targeted to the SH2 domain of signal transducer and activator of transcription 3

DOI: 10.1021/jm901105k

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