Structure-activity relationships in nucleotide oligomerization domain 1 (Nod1) agonistic γ-glutamyldiaminopimelic acid derivatives

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pubs.acs.org/jmc

Structure-Activity Relationships in Nucleotide Oligomerization

Domain 1 (Nod1) Agonistic γ-Glutamyldiaminopimelic Acid

Derivatives

Geetanjali Agnihotri, Rehman Ukani, Subbalakshmi S. Malladi, Hemamali J. Warshakoon,

Rajalakshmi Balakrishna, Xinkun Wang, and Sunil A. David*

Department of Medicinal Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, United States

S Supporting Information

b

ABSTRACT: N-Acyl-γ-glutamyldiaminopimelic acid is a prototype ligand for

Nod1. We report a detailed SAR of C₁₂-γ-D-Glu-DAP. Analogues with glutaric

or γ-aminobutyric acid replacing the glutamic acid show greatly attenuated

Nod1-agonistic activity. Substitution of the meso-diaminopimelic (DAP) acid

component with monoaminopimelic acid, ^L- or D-lysine, or cadaverine also

results in reduced activity. The free amine on DAP is crucial. However, the N-

acyl group on the ^D-glutamyl residue can be substituted with N-alkyl groups

with full preservation of activity. The free carboxylates on the DAP and Glu

components can also be esteriﬁed, resulting in more lipophilic but active

analogues. Transcriptomal proﬁling showed a dominant up-regulation of IL-19,

IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing

activity of these compounds and also implicate cell signaling mediated by

TREM-1. These results may explain the hitherto unknown mechanism of

synergy between Nod1 and TLR agonists and are likely to be useful in designing

vaccine adjuvants.

’ INTRODUCTION

chemotype has been extensively studied since the early

1970s^14-19(preceding by several decades the discovery of

Nod2 as its primary receptor^9,20), structure-activity relation-

ships and signaling pathways involving Nod1-agonistic γ-gluta-

myl-meso-diaminopimelic acid peptides^21,22have remained

sparse. Investigators at the Fujisawa Pharmaceutical Company

reported in the early 1980s that ^D-lactoyl-L-alanyl-γ-D-glu-

tamyl-(^L)-meso-diaminopimelyl-(L)-glycine and heptanoyl-γ-D-

glutamyl-(^L)-meso-diaminopimelyl-D-alanine enhanced host de-

fense in murine models of fatal E. coli sepsis.^23-27Subsequent to

the discovery that the γ-glutamyl-meso-diaminopimelic acid (iE-

DAP) fragment is the crucial pharmacophore mediating recogni-

tion of peptidoglycan fragments by Nod1,^21,22synthetic, lipo-

philic, N-acylglutamyl derivatives have been shown to be potent

Nod1 agonists.^28,29N-Dodecanoyl-γ-D-glutamyldiaminopimelic

acid (C₁₂-iE-DAP) is now available commercially as a reference

Nod1 agonist. Also noteworthy is work by Boons and colleagues

who have synthesized and evaluated DAP-containing muramyl

peptides.^30,31

Cellular pattern recognition receptors (PRRs) are ubiquitous

innate immune sensors that recognize speciﬁc molecular patterns

present in molecules that are broadly shared by pathogens but are

structurally distinct from host molecules.^1-4PRRs include not

only membrane-bound Toll-like receptors (TLRs), members of

which are expressed on the cell surface and the endocytoplasmic

reticulum, but also cytosolic receptors encompassing the Nod

and RIG-I families of proteins.^4,5Nod1 and Nod2 are prototype

members of the nucleotide oligomerization domain (NOD) and

ligand-recognizing leucine-rich repeat (NLR)-containing pro-

teins that serve to signal the presence of intracytoplasmic

peptidoglycan fragments by sensing diaminopimelic acid pep-

tides and muramyl dipeptides, respectively.^6-9Our interest in

understanding the structural determinants of the biological

properties of ligands of PRRs stems from the potential value in

harnessing innate immune stimulatory properties in marshalling

and speciﬁcally directing subsequent adaptive immune responses

with desired immunophenotypes and proﬁles.^10,11We had

previously reported SAR studies on TLR2 and TLR7,^12,13and

while studies on several other TLR ligands continue, our atten-

tion is focused also on the intracytoplasmic NOD family of

receptors, especially Nod1. Whereas the muramyldipeptide

We were keen to explore the structural space around the iE-

DAP scaﬀold for the reason that Nod1 agonists are associated

Received: November 30, 2010

Published: February 07, 2011

r

2011 American Chemical Society

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Scheme 1^a

^aReagents: (i) ROH, HBTU, TEA, DMAP, DMF; (ii) (a) CF₃COOH, (b) FmocCl, Na₂CO₃, dioxane, H₂O; (iii) (a) (Boc)₂O, TEA,

H₂O, (b) R⁰OH, HBTU, DMAP, TEA, DMF; (iv) (a) HCl-dioxane, (b) (Boc)₂O (1 equiv), TEA, MeOH; (v) PS-carbodiimide, PS-

DMAP, CH₂Cl₂.

Scheme 2^a

^aReagents: (i) 30% piperidine, CH₂Cl₂; (ii) C₁₁H₂₃COCl, pyridine, DMAP; (iii) (a) H₂, Pd(OH)₂/C, MeOH, 60 psi, (b) CF₃COOH;

(iv) (a) CF₃COOH, (b) C₁₁H₂₃COCl, TEA, CH₂Cl₂; (v) H₂, Pd(OH)₂/C, AcOH, MeOH, 60 psi; (vi) CF₃COOH; (vii)

(a) C₁₁H₂₃COCl, pyridine, CH₂Cl₂, (b) H₂, Pd(OH)₂/C, MeOH, 60 psi; (viii) C₁₁H₂₃CHO (1 equiv), MP-CNBH₃, CH₂Cl₂, MeOH,

AcOH; (ix) RCHO (excess), MP-CNBH₃, CH₂Cl₂, MeOH, AcOH; (x) 1H-pyrazole-1-carboxamidine HCl, pyridine, microwave

3

irradiation, 60 °C; (xi) H₂, Pd(OH)₂/C, MeOH, 60 psi; (xii) N,N⁰-di-Boc-1H-pyrazole-1-carboxamidine HCl, pyridine, THF, 50 °C.

3

with the mobilization of adaptive immune responses with a

dominant Th2 bias, which is undesirable when a strong CD8þ

cytotoxic T lymphocytic response is required.³²We hypothe-

sized that covalent coupling to iE-DAP of the TLR7/8-agonistic

imidazoquinolines^12,33that we have found to be extraordinarily

Th1-polarizing^10,11could lead to vaccine adjuvants with balanced

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Scheme 3^a

^aReagents: (i) (a) 30% piperidine, CH₂Cl₂, (b) (Boc)₂O, TEA, MeOH; (ii) (a) chlorotris(triphenylphosphine)rhodium(I),

EtOH, H₂O, reﬂux, (b) C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF; (iii) (a) H₂, Pd(OH)₂/C, MeOH, 60 psi, (b) CF₃COOH;

(iv) (a) chlorotris(triphenylphosphine)rhodium(I), EtOH, H₂O, reﬂux, (b) C₁₁H₂₃NH₂, HBTU, TEA, DMAP, CH₂Cl₂;

(v) (a) chlorotris(triphenylphosphine)rhodium(I), EtOH, H₂O, reﬂux, (b) ROH, HBTU, DMAP, DMF; (vi) (a) 30% piperidine,

CH₂Cl₂, (b) C₁₁H₂₃COCl, pyridine, DMAP; (vii) (a) 30% piperidine, CH₂Cl₂, (b) 1H-pyrazole-1- carboxamidine HCl, pyridine,

3

microwave irradiation, 60 °C.

Th1/Th2 immunostimulatory phenotypes. In order to accom-

plish this goal, it was necessary to ﬁrst determine optimal

positions on the iE-DAP backbone that would permit the

generation of dual-active “hybrid” molecules, combining the

TLR7-agonistic imidazoquinoline and Nod1-active iE-DAP che-

motypes in a single covalently coupled construct. We report in

this paper an SAR study of Nod1-agonistic iE-DAP derivatives.

Secondary screens including transcriptomal proﬁling in ex vivo

models using whole human blood have revealed a possible basis

for the pronounced Th2 bias of this chemotype.

mono-N-Boc protection using stoichiometric equivalents of

Boc₂O. N-Fmoc-D-Glu-R-benzyl/allyl esters (4 or 5, re-

spectively) were coupled to the mono-N-Boc-diaminopimelic

acid diesters (9 or 10) using standard protocols to aﬀord the

orthogonally protected synthons (11-13, Scheme 1). N-Fmoc

(^D-Glu) deprotection followed by acylation with lauroyl chloride

and subsequent deprotection of the N-Boc and O-Bn groups

yielded C₁₂-iE-DAP (16, Scheme 2), the activity of which was

identical to that of the commercially available reference com-

pound with EC₅₀of ∼30 pM (Figure 1A).

The N⁰-acyl (DAP) compound (18) and the N,N⁰-diacyl (D-

Glu, and DAP, respectively) analogue (20), as well as N-alkyl

(22) and N,N-dialkyl analogues (26-28) were also synthesized

from synthon 11 (Scheme 2). Our initial attempts at converting

the N⁰amine to a guanidine group resulted in an undesired

cyclization-elimination reaction to form the aminoimidazolone

30; this was obviated by the use of N,N⁰-di-Boc-1H-pyrazole-1-

carboxamidine as the guanidination reagent, aﬀording the de-

sired product 32 (Scheme 2).

’ RESULTS AND DISCUSSION

The construction of orthogonally protected γ-D-glutamyldia-

minopimelic acid synthons (Scheme 1) allowed convenient

access to a number of analogues wherein the carboxyl and amino

substituents on both amino acids could be varied (Schemes 2-4).

For the sake of consistency, we designate the R-amino group on

the ^D-Glu as N, and the γ-amino group on the diaminopimelic

acid (DAP) residue as N⁰. We used the commercially available

diaminopimelic acid (6, mixture of four isomers), since deriva-

tives of all four isomers have been shown to be active.^28,29Esters

of DAP proved to be surprisingly diﬃcult to obtain by a number

of conventional methods, necessitating sequential N-Boc protec-

tion of the amines, esteriﬁcation (as benzyl or ethyl) of the

carboxylic acids, deprotection of the N-Boc groups, followed by

Next, analogues were synthesized from 12 with the acyl group

on the R-amine of ^D-Glu transposed to the R-carboxylic acid

group as the undecanoyl ester (35), the 1-aminoundecane-

derived amide (37), or the N-acyl long-chain ester 41

(Scheme 3). The N-guanidino ester analogue 43 and the R-^D-

Glu ethyl ester derivative of C₁₂-iE-DAP (45) were also obtained

from 12 (Scheme 3). The R-^D-Glu thioester DAP ethyl ester

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Scheme 4^a

^aReagents: (i) 30% piperidine, CH₂Cl₂;(ii) (a) (Boc)₂O, TEA, MeOH, (b) H₂,Pd(OH)₂/C, MeOH, 60 psi, (c) C₁₁H₂₃SH,HBTU,DMAP,TEA,

DMF; (iii) CF₃COOH; (iv) C₁₁H₂₃COCl, pyridine; (v) (a) H₂, Pd(OH)₂/C, MeOH, 60 psi, (b) CF₃COOH.

In order to examine the importance of the R-amino and

carboxyl groups of ^D-Glu, γ-aminobutyric acid (des-carboxyl, 53)

or glutaric acid (des-amino, 56) was coupled to the DAP

derivative 9 as depicted in Scheme 5. Similarly, suitably protected

monoaminopimelic acid 58, ^D- and L-lysine derivatives (62-65,

Scheme 6), and commercially available N-Boc-cadaverine and ^D,

L-R-amino-ε-caprolactam were used to examine the roles of the

amine and carboxyl groups of DAP (Scheme 7).

The N-acyl on the ^D-Glu residue appears to be optimal for

Nod1-agonistic activity, since the transposition of the acyl group

to the N⁰position on DAP (18) or diacylation at N and N⁰(20)

resulted in substantial decrease in activity (Table 1). The

substitution of the N-acyl (C₁₂) group with N-alkyl (22) led to

virtually identical activity (27 pM versus 23 pM, Table 1).

Compounds with C₈or C₁₆N,N-dialkyl groups (26, 28)

displayed attenuated potency, while the C₁₂N,N-dialkyl com-

pound 27 displayed a rather dramatic increase in potency, with

residual partial activity evident even at very high dilutions

(Table 1, Figure 1). Compound 27 was the most potent analogue

identiﬁed in this study. The primary amines on both the DAP and

D-Glu segments were found to be crucial; the guanidino analo-

gues 32 and 43 and the aminoimidazolone analogue 30 exhibited

diminished activity.

We next examined the importance of the free R-carboxyl

group on ^D-Glu. The potencies of the undecanoyl ester of iE-

DAP (35) and the ethyl ester of C₁₂-iE-DAP (45) were

comparable to that of the reference compound C₁₂-iE-DAP

(16). The undecanoyl ester of C₁₂-iE-DAP (41) and the

thioester analogue 48 also retained modest activity. The 1-ami-

noundecane-derived amide analogue (37), however, was sub-

stantially less active. Augmented activity in 35 and 45 appears to

be related to neither net charge nor bulk hydrophobicity, both of

which could be reasonably expected to modulate transmembrane

permeation and consequent presentation of the ligand to the

intracellular Nod1 receptor. The net charge at physiological pH

of 35 and 37 is zero, while that of 41 and 45 is -1. We surmised

that the higher potency of 45 relative to 41 could be due to the

more facile hydrolytic cleavage by intracellular esterases of the

shorter ethyl ester of 45, but our observation that the rate and

kinetics of NF-κB induction for both compounds were very

Figure 1. (A) NF-κB induction activity in stable transfectants of HEK-

293 cells expressing human Nod1. (B) Rank-order plot of potencies

(EC₅₀values) of iE-DAP analogues.

analogue (48) and DAP ethyl ester derivative of C₁₂-iE-DAP

(50) were synthesized from synthon 13 (Scheme 4).

similar (data not shown) is not in agreement with our conjecture,

and we do not yet understand the basis for the diﬀerence in

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Scheme 5^a

^aReagents: (i) Fmoc-GABA-OH, PS-carbodiimide, PS-DMAP, CH₂Cl₂; (ii) (a) 30% piperidine, CH₂Cl₂, (b) C₁₁H₂₃COCl, pyridine;

(iii) (a) H₂, Pd(OH)₂/C, MeOH, 60 psi, (b) CF₃COOH; (iv) glutaric anhydride, CH₂Cl₂; (v) C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF.

Scheme 6^a

^aReagents: (i) BnOH, p-TSA, reﬂux; (ii) (a) BnOH, HBTU, DMAP, DMF, (b) 30% piperidine, CH₂Cl₂; (iii) (a) PhCH₂OCOCl, Na₂CO₃,

dioxane, H₂O, (b) CF₃COOH; (iv) (a) (Boc)₂O, TEA, MeOH, (b) H₂, Pd(OH)₂/C, MeOH, 60 psi.

potencies of these analogues. Substitution of the N-acyl D-Glu

fragment with either N-acyl-γ-aminobutyric acid (53, des-R-

carboxyl analogue) or the undecanoyl ester of glutaric acid (56,

des-R-amino analogue) resulted only in considerable loss in

Nod1-agonistic activity. Taken together, the above SAR data

suggest that considerable “plasticity” exists in the recognition of

the ^D-Glu fragment. Indeed, a plot of the potencies of these

analogues displays an unexpectedly diﬀuse, near-uniform distri-

bution of EC₅₀values (Figure 1B), unlike the well-demarcated

and discrete SAR that we had previously observed for TLR2-¹³

and TLR7-active¹²compounds.

In contradistinction to the broad and diﬀuse SAR for ^D-Glu

fragment, the DAP portion of the molecule is far more stringent

in determining Nod1 agonism. Whereas esteriﬁcation of carboxyl

groups of DAP (50) is tolerated, substitution with monoamino-

pimelic acid (67, des-amino) or cadaverine (69, des-dicarboxyl)

resulted in abrogation of activity. The meso-diaminopimelic acid-

containing peptidoglycan is speciﬁc for Gram-negative bacteria,

and the corresponding homologue in Gram-positive organisms is

L-lysine coupled via its R-amine to γ-D-glutamyl residue (77), the

activity of which is ∼10-fold less than that of 16.^34,35The D-lysine

analogue 73 is bereft of any activity. The unnatural ε-amine-

coupled lysine analogues were also evaluated. The ^D-lysine

analogue 75 was found to be 4-fold more active than 79, its ^L-

lysine congener.

Primary assay readouts using cell-culture systems may not

always accurately reﬂect in vivo behavior owing to a variety of

reasons, including diﬀerential plasma protein binding (that we

ourselves have observed and characterized),³⁶and we therefore

wished to conﬁrm that 27 was indeed more potent than 16, the

reference Nod1 agonist, in ex vivo assays employing whole

human blood. Although Nod1 agonists have previously been

shown to enhance neutrophil recruitment in mice,³⁴a recent

report employing synthetic, nonacylated, γ-glutamyl-meso-dia-

minopimelic acid dipeptide indicated that Nod1-speciﬁc re-

sponses could not be elicited in isolated human neutrophils.³⁷

By use of an ex vivo system using fresh, whole human blood that

we have established for immunoproﬁling innate immune

stimuli,^10,13,38both p38 MAPK³⁹and CD11b⁴⁰were up-regu-

lated in a dose-dependent manner, with 27 being more potent

than 16 (Figure 2). Indeed, the nonacylated, γ-glutamyl-meso-

diaminopimelic acid dipeptide was inactive in our primary and

secondary screens (data not shown), emphasizing the require-

ment of an N-acyl functionality on glutamic acid.

Recent ﬁndings using murine models of immunization indi-

cate that Nod1 engagement drives B and T cell immunity with a

predominant Th2 polarization and additional Th17 priming.³²

However, mechanisms underlying the induction of strong Th2-

biased immune responses have remained obscure. Given the

signiﬁcant diﬀerences between species-dependent recognition of

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Scheme 7^a

^aReagents: (i) PS-carbodiimide, PS-DMAP, TEA, CH₂Cl₂; (ii) 30% piperidine, CH₂Cl₂; (iii) C₁₁H₂₃COCl, pyridine; (iv) H₂,

Pd(OH)₂/C, MeOH, 60 psi; (v) CF₃COOH.

innate immune ligands,¹¹we desired to evaluate whether surro-

gate markers of Th2-skewed responses could be observed in

human ex vivo model systems, in the hope that these studies may

also help uncover signaling events determining Th2 polarization.

Transcriptomal proﬁling experiments using human blood re-

vealed a very prominent up-regulation of class II helical cytokines

by 27, especially members of the IL-10 superfamily⁴¹including

IL-19, IL-20, IL-22, and IL-24 (Table 2); these cytokines,

especially IL-19,^42-45have been shown to drive Th2-polarized

responses. We have never before observed this particular con-

stellation of cytokines in our earlier¹⁰(and ongoing) immuno-

proﬁling studies, and we are excited in no small measure that

quantifying the induction of IL-19 and related cytokines may

have predictive value as biomarkers and surrogate signatures for

Th2 induction in immunoproﬁling vaccine adjuvants and may

perhaps supplant the necessity of long and laborious immuniza-

tion experiments that typically involve large numbers of animals.

Also observed, as could be expected in the context of Th17

induction,³²was the up-regulation of IL-17. Similar responses

were observed with 16 but of lower magnitude, consistent with

the lower potency of 16 (data not shown).

triggering receptor expressed on myeloid cells 1 (TREM-1,

Figure 3). TREM-1 is a recently discovered receptor expressed

on the cell surface of monocytes and neutrophils and plays an

important role in amplifying myeloid cell-activated inﬂammatory

responses.^46-48Our observation of the up-regulation of TREM-

1 signaling components may explain the hitherto unknown mecha-

nism of synergy between Nod1 and TLR agonists.^32,34,49,50

The overarching objective that led to our work on Nod1

ligands was not so much to synthesize higher potency com-

pounds but rather to understand SAR that would permit us to

covalently couple other innate immune ligands^12,13,33to the

iE-DAP scaﬀold in order to test the hypothesis that such

hybrids may imbue Th1-Th2 balanced, yet eﬀective vaccine

adjuvants. The studies reported here indicate that various

substitutions and modiﬁcations on the γ-^D-glutamyl moiety

are tolerated whereas the diaminopimelic acid fragment is far

more stringent. The R-carboxylic acid of ^D-Glu appears to be

optimal for coupling with other innate immune ligands, and

work in this area is in progress. Secondary and tertiary screens,

including transcriptomal proﬁling, have shed light on the

possible mechanisms underlying the strong Th2 bias of

Nod1 ligands and of synergy with other innate immune

stimuli.

Further analyses of gene transcripts induced by 27 yielded the

unexpected ﬁnding that exposure of human blood to 27 results in

the activation of pathways involving signaling mediated by

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Table 1. EC₅₀Values of hNod1 Agonistic Activities of Analogues

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Table 1. Continued

’ EXPERIMENTAL SECTION

Synthesis of Compound 2: (R)-1-Benzyl 5-tert-Butyl 2-(-

(tert-butoxycarbonyl)amino)pentanedioate. To a solution of

1 (1 g, 3.29 mmol) in anhydrous DMF were added HBTU (1.38 g, 3.62

mmol), triethylamine (0.5 mL, 3.62 mmol), benzyl alcohol (682 μL,

6.59 mmol), and a catalytic amount of DMAP. The reaction mixture was

stirred for 4 h, followed by evaporation of the solvent under reduced

pressure. The residue was then dissolved in ethyl acetate and washed

with water. The organic solvent was dried over anhydrous sodium

sulfate, filtered, evaporated under reduced pressure to obtain the crude

product which was purified using column chromatography (10%

EtOAc/hexanes) to obtain compound 2 (1.14 g, 88%). ¹H NMR (500

MHz, CDCl₃) δ 7.39-7.31 (m, 5H), 5.16 (dt, J = 23.2, 11.5 Hz, 3H),

4.35 (dd, J = 13.0, 8.2 Hz, 1H), 2.35-2.22 (m, 2H), 2.13 (td, J = 13.3, 7.0

Hz, 1H), 1.92 (tt, J = 14.7, 7.5 Hz, 1H), 1.43 (s, 18H). ¹³C NMR (126

MHz, CDCl₃) δ 172.09, 172.06, 155.39, 131.57, 118.82, 80.74, 79.93,

65.95, 53.11, 31.60, 28.30, 28.06, 27.89, 27.68. MS (ESI) calculated for

C₂₁H₃₁NO₆, m/z 393.22, found 416.22 (M þ Na)^þ.

Chemistry. All of the solvents and reagents used were obtained

commercially and used as such unless noted otherwise. Moisture- or

air-sensitive reactions were conducted under nitrogen atmosphere in

an oven-dried (120 °C) glass apparatus. The solvents were removed

under reduced pressure using standard rotary evaporators. Flash

column chromatography was carried out using RediSep Rf “Gold”

high performance silica columns on CombiFlash Rf (Teledyne-Isco,

Lincoln, NE) instrument unless otherwise mentioned, while thin-

layer chromatography was carried out on silica gel CCM precoated

aluminum sheets. Purity for all final compounds was confirmed to be

at least 97% by LC-MS using a Zorbax Eclipse Plus 4.6 mm ꢀ 150

mm, 5 μm analytical reverse phase C₁₈column with H₂O-isopro-

panol or H₂O-CH₃CN gradients (with 0.1% CF₃COOH in both

mobile phases) and an Agilent ESI-TOF mass spectrometer (mass

accuracy of 5 ppm) operating in the positive ion (or negative ion, as

appropriate) acquisition mode. Total ion current from 150 to 3500

Da was measured.

Synthesis of Compound 3: (R)-1-Allyl 5-tert-Butyl 2-((tert-

butoxycarbonyl)amino)pentanedioate. To a solution of 1 (1 g,

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drying under vacuum to obtain the trifluoroacetate salt. To the solution

of trifluoroacetate salt in dioxane, the aqueous solution of sodium

carbonate (810 mg, 7.64 mmol) was added at 10 °C. The reaction

mixture was stirred at room temperature for 10 min, followed by the

addition of FmocCl (722 mg, 2.79 mmol) solution in dioxane. The

reaction mixture was stirred at room temperature for 16 h, followed by

removal of the solvent under reduced pressure. The residue was

dissolved in ethyl acetate and water, and the solution was acidified using

10% HCl until the pH was ∼1. The aqueous layer was washed with ethyl

acetate. The ethyl acetate was dried over anhydrous sodium sulfate,

filtered, and evaporated under reduced pressure to obtain the crude

product which was purified using column chromatography (40%

1

EtOAc/hexanes) to obtain compound 4 (1.0 g, 91%). H NMR (500

MHz, CDCl₃) δ 7.73 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.33

(qd, J = 15.0, 7.2 Hz, 9H), 5.47 (d, J = 8.2 Hz, 1H), 5.19-5.06 (m, 2H),

4.53-4.30 (m, 3H), 4.16 (dd, J = 15.6, 8.8 Hz, 1H), 2.48-2.12 (m, 3H),

1.94 (dt, J = 14.5, 7.7 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 177.59,

171.91, 156.27, 143.99, 143.84, 141.50, 141.49, 135.19, 128.88, 128.81,

128.56, 127.95, 127.30, 125.29, 125.26, 120.21, 120.20, 67.73, 67.34,

53.42, 47.32, 29.91, 27.64. MS (ESI) calculated for C₂₇H₂₅NO₆, m/z

459.16, found 482.17 (M þ Na)^þ.

Synthesis of Compound 5: (R)-4-((((9H-Fluoren-9-yl)meth-

oxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic Acid.

The solution of compound 3 (1 g, 2.91 mmol) in trifluoroacetic acid

was stirred for 1 h, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt. To

the solution of trifluoroacetate salt in dioxane, the aqueous solution of

sodium carbonate (926 mg, 8.74 mmol) was added at 10 °C. The

reaction mixture was stirred at room temperature for 10 min, followed by

the addition of FmocCl (830 mg, 3.2 mmol) solution in dioxane. The

reaction mixture was stirred at room temperature for 16 h, followed by

removal of the solvent under reduced pressure. The residue was

dissolved in ethyl acetate and water, and the solution was acidified using

10% HCl until the pH was ∼1. The aqueous layer was washed with ethyl

acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate,

filtered, and evaporated under reduced pressure to obtain the crude

product which was purified using column chromatography (40%

1

EtOAc/hexanes) to obtain compound 5 (1.1 g, 92%). H NMR (400

MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.1 Hz, 2H), 7.40

(t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.90 (ddd, J = 22.5, 11.0, 5.7

Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.30 (dd, J = 27.7, 13.7 Hz, 2H), 4.65

(d, J = 5.5 Hz, 2H), 4.58-4.35 (m, 3H), 4.21 (t, J = 6.8 Hz, 1H), 2.58-

2.17 (m, 3H), 2.07-1.92 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ

172.70, 172.54, 155.72, 155.57, 135.49, 128.74, 128.57, 128.43, 80.07,

67.19, 67.14, 53.27, 32.24, 28.47, 28.45, 21.48, 21.19, 21.08. MS (ESI)

calculated for C₂₃H₂₃NO₆, m/z 409.15, found 432.14 (M þ Na)^þ.

Synthesis of Compound 7: Dibenzyl 2,6-Bis((tert-butoxy-

carbonyl)amino)heptanedioate. To a solution of compound 6 (1

g, 5.25 mmol) in water were added di-tert-butyl dicarbonate (4.58 g, 21.0

mmol) and triethylamine (2.92 mL, 21.0 mmol). The reaction mixture

was stirred for 6 h, followed by removal of the solvent under reduced

pressure to obtain the residue. To the solution of residue in anhydrous

DMF were added HBTU (4.38 g, 11.55 mmol), triethylamine (1.6 mL,

11.55 mmol), benzyl alcohol (1.63 mL, 15.75 mmol), and a catalytic

amount of DMAP. The reaction mixture was stirred for 8 h, followed by

evaporation of the solvent under reduced pressure. The residue was then

dissolved in ethyl acetate and washed with water. The organic solvent

was dried over anhydrous sodium sulfate, filtered, and evaporated under

reduced pressure to obtain the crude product which was purified using

column chromatography (8% EtOAc/hexanes) to obtain compound 7

Figure 2. Dose-responses of p38 MAP kinase (A) and CD11b

(B) induction in the granulocytic population in whole human blood

by 16 and 27 determined by ﬂow cytometry.

3.29 mmol) in anhydrous DMF were added HBTU (1.38 g, 3.62

mmol), triethylamine (0.5 mL, 3.62 mmol), allyl alcohol (450 μL,

6.59 mmol), and a catalytic amount of DMAP. The reaction mixture

was stirred for 4 h, followed by evaporation of the solvent under

reduced pressure. The residue was then dissolved in ethyl acetate

and washed with water. The organic solvent was dried over

anhydrous sodium sulfate, filtered, and evaporated under reduced

pressure to obtain the crude product which was purified using

column chromatography (10% EtOAc/hexanes) to obtain com-

pound 3 (1.0 g, 90%). ¹H NMR (500 MHz, CDCl₃) δ 5.91 (ddt, J =

16.3, 10.6, 5.8 Hz, 1H), 5.34 (dd, J = 17.2, 1.3 Hz, 1H), 5.26 (dd, J =

10.4, 0.7 Hz, 1H), 5.11 (d, J = 8.0 Hz, 1H), 4.64-4.63 (m, 2H), 4.33

(dd, J = 13.2, 8.3 Hz, 1H), 2.38-2.26 (m, 2H), 2.17-2.11 (m, 1H),

1.96-1.88 (m, 1H), 1.45 (s, 9H), 1.44 (s, 9 H). ¹³C NMR (126

MHz, CDCl₃) δ 172.05, 172.02, 155.35, 131.53, 118.78, 80.70,

79.89, 65.91, 53.07, 31.56, 28.26, 28.02, 27.85, 27.64. MS (ESI)

calculated for C₁₇H₂₉NO₆, m/z 343.20, found 366.19 (M þ Na)^þ.

Synthesis of Compound 4: (R)-4-((((9H-Fluoren-9-yl)me-

thoxy)carbonyl)amino)-5-(benzyloxy)-5-oxopentanoic Acid.

The solution of 2 (1 g, 2.54 mmol) in trifluoroacetic acid was stirred

for 1 h, followed by removal of the solvent by purging nitrogen and

1

(2.48 g, 83%). H NMR (500 MHz, CDCl₃) δ 7.44-7.29 (m, 10H),

5.13 (dt, J = 12.4, 8.3 Hz, 6H), 4.29 (s, 2H), 1.85-1.71 (m, 2H), 1.68 (s,

2H), 1.49-1.31 (m, 20H). ¹³C NMR (126 MHz, CDCl₃) δ 172.70,

172.54, 155.72, 155.57, 135.49, 128.74, 128.57, 128.43, 80.07, 67.19,

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Table 2. Interleukin Transcriptomal Responses in Human PBMCs Exposed to 20 μg/mL 27

probe set

fold change regulation gene symbol

gene title

220745_at

205207_at

210118_s_at

220322_at

216876_s_at

216244_at

212659_s_at

216243_s_at

207901_at

207906_at

212657_s_at

39402_at

30.39

29.54

14.34

11.10

10.56

8.27

8.19

7.54

6.13

5.99

4.94

4.45

4.14

4.00

3.87

3.62

3.57

3.41

3.17

3.15

2.29

2.05

2.03

4.05

3.69

2.75

2.06

2.02

up

IL19

interleukin 19

up

IL6

interleukin 6 (interferon, β2)

interleukin 1, R

up

IL1A

up

IL1F9

IL17A

IL1RN

IL12B

IL3

interleukin 1 family, member 9

interleukin 17A

up

interleukin 1 receptor antagonist

up

interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40)

up

interleukin 3 (colony-stimulating factor, multiple)

interleukin 1 receptor antagonist

interleukin 1, β

up

IL1RN

IL1B

up

205067_at

224071_at

221165_s_at

207433_at

211269_s_at

206341_at

206569_at

227997_at

222223_s_at

211506_s_at

224262_at

206295_at

1555431_a_at

220056_at

207008_at

220663_at

205227_at

up

IL1B

interleukin 1, β

up

IL20

interleukin 20

up

IL22

interleukin 22

up

IL10

interleukin 10

up

IL2RA

IL24

interleukin 2 receptor, R

interleukin 24

up

IL17RD

IL1F5

IL8

interleukin 17 receptor D

interleukin 1 family, member 5 (δ)

interleukin 8

up

IL1F10

IL18

interleukin 1 family, member 10 (θ)

interleukin 18 (interferon-γ-inducing factor)

interleukin 31 receptor A

interleukin 22 receptor, R1

interleukin 8 receptor, β

up

down

IL31RA

IL22RA1

IL8RB

IL1RAPL1 interleukin 1 receptor accessory protein-like 1

IL1RAP interleukin 1 receptor accessory protein

Figure 3. Pathway analysis of transcriptomal activation patterns in human PBMCs exposed to 20 μg/mL 16 and 27. The dashed line corresponds to the

threshold of -log (base 10) for P = 0.05 (after Benjamini-Hochberg multiple testing correction).

67.14, 53.27, 32.24, 28.47, 28.45, 21.48, 21.19, 21.08. MS (ESI)

Synthesis of Compound 8: Diethyl 2,6-Bis((tert-butoxy-

carbonyl)amino)heptanedioate. To a solution of compound

calculated for C₃₁H₄₂N₂O₈, m/z 570.29, found 593.27 (M þ Na)^þ.

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6 (1 g, 5.25 mmol) in water were added di-tert-butyl dicarbonate (4.58 g,

21.0 mmol) and triethylamine (2.92 mL, 21.0 mmol). The reaction

mixture was stirred for 6 h, followed by removal of the solvent under

reduced pressure to obtain the residue. To the solution of residue in

anhydrous DMF were added HBTU (4.38 g, 11.55 mmol), triethyla-

mine (1.6 mL, 11.55 mmol), ethyl alcohol (0.92 mL, 15.75 mmol), and a

catalytic amount of DMAP. The reaction mixture was stirred for 6 h,

followed by evaporation of the solvent under reduced pressure. The

residue was then dissolved in ethyl acetate and washed with water. The

organic solvent was dried over anhydrous sodium sulfate, filtered, and

evaporated under reduced pressure to obtain the crude product which

was purified using column chromatography (8% EtOAc/hexanes) to

obtain compound 8 (2.0 g, 85%). ¹H NMR (500 MHz, CDCl₃) δ 5.17-

4.99 (m, 2H), 4.30-4.08 (m, 6H), 1.84-1.74 (m, 2H), 1.72-1.55 (m,

4H), 1.48-1.31 (m, 18H), 1.25 (t, J = 7.1 Hz, 6H). ¹³C NMR (126

MHz, CDCl₃) δ 173.24, 173.06, 156.07, 155.92, 80.33, 68.61, 61.80,

53.58, 39.17, 32.74, 32.67, 30.80, 29.38, 28.81, 28.79, 28.71, 24.19, 23.45,

21.82, 21.47, 14.64, 14.52. MS (ESI) calculated for C₂₁H₃₈N₂O₈, m/z

446.26, found 469.26 (M þ Na)^þ.

(500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.3 Hz, 2H),

7.39-7.30 (m, 19H), 6.59-6.49 (m, 0.50H), 6.28 (s, 0.50H), 5.71 (dd,

J = 25.1, 7.7 Hz, 1H), 5.24-5.04 (m, 7H), 4.56 (d, J = 6.6 Hz, 1H), 4.50-

4.33 (m, 3H), 4.33-4.15 (m, 2H), 2.31-2.16 (m, 2H), 2.03-1.90 (m,

2H), 1.85-1.75 (m, 2H), 1.70-1.51 (m, 2H), 1.40-1.37 (m, 11H). ¹³

C

NMR (126 MHz, CDCl₃) δ 172.65, 172.57, 172.16, 172.09, 171.97,

171.82, 156.53, 155.78, 144.10, 143.85, 141.51, 141.48, 135.55, 135.36,

128.85, 128.83, 128.81, 128.73, 128.64, 128.60, 128.53, 128.45, 127.92,

127.30, 125.36, 120.18, 80.12, 67.59, 67.41, 67.33, 67.24, 53.63, 53.53,

53.33, 52.21, 47.37, 47.34, 32.32, 32.22, 31.73, 28.93, 28.51, 21.54, 21.43,

21.28, 21.13. MS (ESI) calculated for C₅₃H₅₇N₃O₁₁, m/z 911.40, found

934.39 (M þ Na)^þ.

Synthesis of Compound 12: (5R)-5-Allyl 10,14-Dibenzyl-

1-(9H-fluoren-9-yl)-18,18-dimethyl-3,8,16-trioxo-2,17-

dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate. To

a solution of 5 (500 mg, 1.22 mmol) in anhydrous dichloromethane

were added 9 (585 mg, 1.24 mmol), polystyrene bound carbodiimide

(1.09 g, 1.34 mmol), and a catalytic amount of polystyrene bound

DMAP. The reaction mixture was stirred at room temperature for 4 h,

followed by filtration to remove the solid resin. The filtrate was

evaporated under reduced pressure to obtain the residue which was

then purified using column chromatography (35% EtOAc/hexanes) to

obtain compound 12 (830 mg, 79%). ¹H NMR (400 MHz, CDCl₃) δ

7.79 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 6.6 Hz, 2H), 7.46-7.30 (m, 14H),

6.69-6.30 (m, 1H), 5.91 (dd, J = 19.3, 12.8 Hz, 1H), 5.72 (dd, J = 20.3,

7.6 Hz, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.27 (d, J = 10.4 Hz, 1H), 5.22-

5.04 (m, 5H), 4.70-4.56 (m, 3H), 4.52-4.36 (m, 3H), 4.35-4.19 (m,

2H), 2.38-2.20 (m, 3H), 1.98 (dd, J = 19.2, 8.3 Hz, 1H), 1.91-1.56 (m,

4H), 1.44 (s, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 172.66, 172.56,

172.20, 172.12, 171.99, 171.85, 156.56, 155.83, 144.09, 143.86, 141.51,

135.54, 135.45, 131.59, 128.83, 128.71, 128.64, 128.51, 127.94, 127.31,

125.35, 120.19, 119.40, 119.36, 119.32, 80.13, 67.43, 67.33, 67.23, 66.42,

53.61, 53.53, 53.33, 52.24, 47.37, 32.41, 32.34, 31.84, 31.75, 31.61, 29.00,

28.51, 21.46, 21.27, 21.14. MS (ESI) calculated for C₄₉H₅₅N₃O₁₁, m/z

861.38, found 884.33 (M þ Na)^þ.

Synthesis of Compound 9: Dibenzyl 2-Amino-6-((tert-

butoxycarbonyl)amino)heptanedioate. Compound 7 (2 g, 3.5

mmol) was dissolved in 10 mL of HCl-dioxane and stirred for 2 h. The

solvent was then removed under vacuum to obtain the hydrochloride

salt, which was then dissolved in methanol. Triethylamine was added to

the solution until pH ∼7 was obtained, followed by gradual addition of

di-tert-butyl dicarbonate (764 mg, 3.5 mmol). The reaction mixture was

stirred for 30 min, followed by evaporation of the solvent under reduced

pressure to obtain the crude product, which was then purified using

column chromatography (5% MeOH/CH₂Cl₂) to obtain compound 9

(640 mg, 39%). ¹H NMR (500 MHz, CDCl₃) δ 7.45-7.28 (m, 10H),

5.23-5.09 (m, 4H), 5.03 (t, J = 7.3 Hz, 1H), 4.41-4.23 (m, 1H), 3.41

(td, J = 8.1, 5.2 Hz, 1H), 1.85-1.50 (m, 6H), 1.49-1.34 (m, 11H). ¹³C

NMR (126 MHz, CDCl₃) δ 176.17, 176.14, 173.01, 155.83, 136.10,

135.84, 129.25, 129.09, 129.06, 128.88, 128.78, 80.36, 67.46, 67.16,

67.14, 54.70, 54.68, 53.80, 53.74, 34.73, 34.66, 32.77, 32.74, 28.77, 21.93.

MS (ESI) calculated for C₂₆H₃₄N₂O₆, m/z 470.24, found 471.24 (M þ

H)^þ.

Synthesis of Compound 13: (5R)-5-Benzyl 10,14-Diethyl-

1-(9H-fluoren-9-yl)-18,18-dimethyl-3,8,16-trioxo-2,17-di-

Synthesis of Compound 10: Diethyl 2-Amino-6-((tert-bu-

toxycarbonyl)amino)heptanedioate. Compound 8 (1 g, 2.24

mmol) was dissolved in 5 mL of HCl-dioxane and stirred for 2.5 h. The

solvent was then removed under vacuum to obtain the hydrochloride

salt, which was then dissolved in methanol. Triethylamine was added to

the solution until pH ∼7 was obtained, followed by gradual addition of

di-tert-butyl dicarbonate (490 mg, 2.24 mmol). The reaction mixture

was stirred for 30 min, followed by evaporation of the solvent under

reduced pressure to obtain the crude product, which was then purified

using column chromatography (5% MeOH/CH₂Cl₂) to obtain com-

pound 10 (271 mg, 35%). ¹H NMR (500 MHz, CDCl₃) δ 5.31 (dd, J =

31.4, 7.9 Hz, 1H), 4.28-4.16 (m, 5H), 3.96 (bs, 1H), 2.04-1.88 (m,

2H), 1.88-1.76 (m, 1H), 1.73-1.62 (m, 1H), 1.62-1.43 (m, 2H), 1.42

(s, 9H), 1.32-1.24 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 176.13,

176.08, 172.94, 155.59, 80.00, 61.50, 61.08, 54.40, 53.50, 53.44, 34.56,

34.53, 32.65, 32.62, 28.50, 21.73, 21.67, 14.42, 14.37. MS (ESI)

calculated for C₁₆H₃₀N₂O₆, m/z 346.21, found 347.21 (M þ H)^þ.

Synthesis of Compound 11: (5R)-Tribenzyl 1-(9H-Fluoren-

9-yl)-18,18-dimethyl-3,8,16-trioxo-2,17-dioxa-4,9,15-triaza-

nonadecane-5,10,14-tricarboxylate. To a solution of 4 (500 mg,

1.08 mmol) in anhydrous dichloromethane were added 9 (522 mg, 1.11

mmol), polystyrene bound carbodiimide (967 mg, 1.19 mmol), and a

catalytic amount of polystyrene bound DMAP. The reaction mixture

was stirred at room temperature for 4 h, followed by filtration to remove

the solid resin. The filtrate was evaporated under vacuum to obtain the

residue which was then purified using column chromatography (35%

oxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate. To

a

solution of 4 (195 mg, 0.425 mmol) in anhydrous dichloromethane

were added 10 (150 mg, 0.43 mmol), polystyrene bound carbodiimide

(367 mg, 0.467 mmol), and a catalytic amount of polystyrene bound

DMAP. The reaction mixture was stirred at room temperature for 4 h,

followed by filtration to remove the solid resin. The filtrate was

evaporated under reduced pressure to obtain the residue which was

then purified using column chromatography (30% EtOAc/hexanes) to

obtain compound 13 (232 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ

7.79-7.69 (m, 2H), 7.59 (dd, J = 21.4, 11.5 Hz, 2H), 7.55-7.26 (m,

9H), 6.55 (dd, J = 27.8, 7.3 Hz, 1H), 6.31 (dd, J = 18.6, 7.1 Hz, 1H), 5.75

(dd, J = 27.6, 7.7 Hz, 1H), 5.27-5.05 (m, 3H), 4.61-4.33 (m, 4H),

4.28-4.04 (m, 6H), 2.31-2.20 (m, 2H), 2.04-1.93 (m, 1H), 1.89-

1.52 (m, 5H), 1.46-1.36 (m, 9H), 1.34-1.21 (m, 7H). ¹³C NMR (126

MHz, CDCl₃) δ 156.50, 144.11, 143.88, 141.52, 135.38, 131.11, 129.02,

128.87, 128.76, 128.66, 128.59, 127.93, 127.31, 125.37, 120.19, 80.07,

68.37, 67.61, 67.29, 61.75, 61.57, 53.29, 52.13, 47.36, 38.92, 32.48, 32.37,

31.74, 30.56, 29.13, 28.53, 23.94, 23.20, 21.44, 14.37, 14.28, 11.12. MS

(ESI) calculated for C₄₃H₅₃N₃O₁₁, m/z 787.37, found 810.37 (M þ

Na)^þ.

Synthesis of Compound 14: Dibenzyl 2-((R)-4-Amino-

5-(benzyloxy)-5-oxopentanamido)-6-((tert-butoxycarbo-

nyl)amino)heptanedioate. Compound 11 (500 mg, 0.55 mmol)

was dissolved in 10 mL of dichloromethane, followed by the addition of

3 mL of piperidine. The reaction mixture was stirred for 15 min, followed

by removal of the solvent under reduced pressure. The crude was

purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain

1

EtOAc/hexanes) to obtain compound 11 (748 mg, 76%). H NMR

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compound 14 (347 mg, 92%). ¹H NMR (400 MHz, CDCl₃) δ 7.46-

7.31 (m, 15H), 6.57 (dt, J = 36.1, 8.8 Hz, 1H), 5.25-5.05 (m, 7H), 4.59

(d, J = 5.6 Hz, 1H), 4.28 (s, 1H), 3.52 (dd, J = 8.2, 5.1 Hz, 1H), 2.36 (qt,

J = 21.1, 6.8 Hz, 2H), 2.13 (dd, J = 12.5, 6.0 Hz, 1H), 1.93-1.50 (m, 7H),

1.49-1.32 (m, 11H). ¹³C NMR (101 MHz, CDCl₃) δ 176.03, 172.79,

172.67, 136.10, 135.85, 129.15, 129.12, 129.01, 128.94, 128.85, 128.76,

67.53, 67.32, 54.38, 54.01, 53.70, 52.40, 33.12, 33.00, 32.17, 30.48, 30.31,

28.82, 21.78, 21.59. MS (ESI) calculated for C₃₈H₄₇N₃O₉, m/z 689.33,

found 690.33 (M þ H)^þ.

7.68-7.52 (m, 2H), 7.46-7.27 (m, 19H), 6.27-6.06 (m, 1H), 5.93-

5.74 (m, 1H), 5.30-5.04 (m, 6H), 4.67-4.28 (m, 5H), 4.28-4.18 (m,

1H), 2.33-2.26 (m, 2H), 2.22-2.13 (m, 2H), 2.01 (dd, J = 15.1, 7.4 Hz,

1H), 1.92-1.79 (m, 2H), 1.79-1.66 (m, 3H), 1.64-1.56 (m, 2H),

1.48-1.17 (m, 19H), 0.90 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 172.48, 172.36, 172.06, 172.02, 156.61, 143.74, 141.50,

135.39, 128.84, 128.70, 128.47, 127.92, 127.30, 125.35, 120.17, 67.55,

67.41, 67.31, 53.83, 51.87, 51.64, 47.35, 36.69, 32.19, 32.11, 31.62, 29.83,

29.69, 29.54, 29.46, 25.81, 22.88, 21.67, 14.32. MS (ESI) calculated for

C₆₀H₇₁N₃O₁₀, m/z 993.51, found 1016.53 (M þ Na)^þ.

Synthesis of Compound 15: (15R)-Tribenzyl 2,2-Dimethyl-

4,12,17-trioxo-3-oxa-5,11,16-triazaoctacosane-6,10,15-tri-

carboxylate. To a solution of 14 (200 mg, 0.29 mmol) in pyridine

were added lauroyl chloride (82.6 μL, 0.34 mmol) and a catalytic

amount of DMAP. The reaction mixture was stirred for 6 h, followed

by evaporation of the solvent under reduced pressure to obtain the

residue, which was then dissolved in dichloromethane and washed with

water. The organic solvent was dried over anhydrous sodium sulfate,

filtered, and evaporated under reduced pressure to obtain the crude

product which was purified using column chromatography (50%

Synthesis of Compound 18: 2-((R)-4-Amino-4-carboxy-

butanamido)-6-dodecanamido)heptanedioic Acid. Com-

pound 17 (70 mg, 0.07 mmol) was dissolved in methanol, followed

by the addition of Pd(OH)₂/C. The solution was subjected to

catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed

by filtration through Celite bed. The solvent was removed under

reduced pressure to obtain the crude product which was purified

using column chromatography (50% MeOH/CH₂Cl₂) to obtain

1

compound 18 (32 mg, 92%). H NMR (500 MHz, MeOD) δ 4.34

1

EtOAc/hexanes) to obtain compound 15 (209 mg, 83%). H NMR

(s, 2H), 3.78-3.69 (m, 2H), 2.50 (t, J = 17.3 Hz, 2H), 2.23 (t, J = 7.4

Hz, 2H), 2.17-2.06 (m, 2H), 1.86-1.66 (m, 5H), 1.64-1.55 (m,

2H), 1.50-1.41 (d, J = 15.2 Hz, 2H), 1.39-1.19 (m, 14H), 0.88 (t, J =

6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.56, 53.81, 36.98,

33.24, 32.21, 30.92, 30.82, 30.64, 30.47, 27.13, 25.50, 23.90, 23.51,

14.59. MS (ESI) calculated for C₂₄H₄₃N₃O₈, m/z 501.31, found

502.32 (M þ H)^þ.

(500 MHz, CDCl₃) δ 7.42-7.27 (m, 15H), 6.98 (dd, J = 16.0, 7.8 Hz,

0.50H), 6.57-6.46 (m, 1.50H), 5.22-5.07 (m, 7H), 4.76-4.70 (m,

0.50H), 4.61-4.51 (m, 1.50H), 4.31-4.20 (m, 1H), 2.28-2.16 (m,

5H), 1.98-1.57 (m, 11H), 1.50-1.36 (m, 6H), 1.32-1.20 (m, 17H),

0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.68, 172.61,

172.46, 172.38, 172.33, 172.24, 172.20, 172.12, 172.04, 135.57, 135.45,

135.40, 128.85, 128.84, 128.82, 128.71, 128.65, 128.62, 128.58, 128.53,

128.48, 128.47, 80.10, 67.53, 67.23, 53.39, 52.29, 51.86, 36.83, 36.77,

32.53, 32.40, 32.12, 31.58, 29.84, 29.82, 29.70, 29.56, 29.51, 29.18, 28.54,

25.86, 25.79, 22.90, 14.34. MS (ESI) calculated for C₅₀H₆₉N₃O₁₀, m/z

871.50, found 894.50 (M þ Na)^þ.

Synthesis of Compound 19: Dibenzyl 2-Amino-6-((R)-

5-(benzyloxy)-4-dodecanamido-5-oxopentanamido)heptan-

edioate. Compound 15 (200 mg, 0.23 mmol) was dissolved in 10 mL

of trifluoroacetic acid and stirred for 30 min, followed by removal of the

solvent by purging nitrogen and drying under vacuum to obtain the

trifluoroacetate salt of compound 19 (203 mg, quantitative yield). MS

(ESI) calculated for C₄₅H₆₁N₃O₈, m/z 771.44, found 772.48 (M þ

H)^þ.

Synthesis of Compound 16: 2-Amino-6-((R)-4-carboxy-4-

dodecanamidobutanamido)heptanedioic Acid. Compound

15 (50 mg, 57.3 μmol) was dissolved in methanol, followed by the

addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through Celite bed. The solvent was removed under reduced pressure to

obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid.

The mixture was stirred for 30 min, followed by removal of the solvent

by purging nitrogen and drying under vacuum to obtain the trifluor-

oacetate salt of compound 16 (35 mg, quantitative yield). ¹H NMR (500

MHz, MeOD) δ 4.46-4.30 (m, 2H), 3.78-3.68 (m, 1H), 2.35 (t, J = 5.6

Hz, 2H), 2.27-2.10 (m, 3H), 1.88 (ddd, J = 24.9, 20.5, 16.8 Hz, 4H),

1.75-1.66 (m, 1H), 1.65-1.47 (m, 4H), 1.36-1.21 (m, 16H), 0.87 (t,

J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.64, 175.50, 175.02,

173.29, 55.13, 54.97, 53.42, 53.16, 37.09, 36.99, 33.25, 33.04, 32.52,

32.45, 32.38, 31.64, 31.53, 31.47, 30.92, 30.82, 30.65, 30.50, 28.82, 28.58,

28.30, 27.14, 27.11, 23.89, 23.06, 22.71, 22.63, 14.59. MS (ESI)

calculated for C₂₄H₄₃N₃O₈, m/z 501.31, found 502.33 (M þ H)^þ.

Synthesis of Compound 17: (5R)-Tribenzyl 1-(9H-Fluoren-

9-yl)-3,8,16-trioxo-2-oxa-4,9,15-triazaheptacosane-5,10,14-

tricarboxylate. Compound 11 (100 mg, 0.10 mmol) was dissolved in

5 mL of trifluoroacetic acid and stirred for 30 min, followed by removal

of the solvent by purging nitrogen and drying under vacuum to obtain

the trifluoroacetate salt. To the solution of trifluoroacetate salt in

anhydrous dichloromethane were added lauroyl chloride (39 μL, 0.16

mmol), triethylamine (30 μL, 0.22 mmol), and a catalytic amount of

DMAP. The reaction mixture was stirred for 2 h, followed by complete

removal of the solvent under reduced pressure. The residue was then

dissolved in dichloromethane and washed with water. The organic

solvent was dried over sodium sulfate, filtered, and evaporated under

reduced pressure to obtain the crude product which was purified using

column chromatography (40% EtOAc/hexanes) to obtain compound

17 (103 mg, 95%). ¹H NMR (400 MHz, CDCl₃) δ 7.83-7.70 (m, 2H),

Synthesis of Compound 20: 2-((R)-4-Carboxy-4-dodec-

anamidobutanamido)-6-dodecanamidoheptanedioic Acid.

To a solution of 19 (50 mg, 56.4 μmol) in pyridine were added lauroyl

chloride (16.1 μL, 67.7 μmol) and a catalytic amount of DMAP. The

reaction mixture was stirred for 6 h, followed by evaporation of the

solvent under reduced pressure. The residue was then dissolved in

dichloromethane and washed with water. The organic solvent was dried

over anhydrous sodium sulfate, filtered, and evaporated under reduced

pressure to obtain the crude product. The crude was purified using

column chromatography (35% EtOAc/hexanes) to obtain the pure

product, which was dissolved in methanol and subjected to catalytic

hydrogenolysis using Pd(OH)₂/C at 60 psi hydrogen pressure for 2 h,

followed by filtration through Celite bed. The solvent was removed

1

under reduced pressure to obtain compound 20 (34.3 mg, 89%). H

NMR (500 MHz, MeOD) δ 4.45-4.25 (m, 3H), 2.34 (t, J = 6.7 Hz,

2H), 2.28-2.07 (m, 5H), 1.98-1.79 (m, 3H), 1.70 (dd, J = 19.9, 14.8

Hz, 2H), 1.64-1.55 (m, 4H), 1.49-1.39 (m, 3H), 1.37-1.15 (m, 31H),

0.87 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, MeOD) δ 176.64, 175.98,

175.72, 175.21, 53.63, 36.99, 33.35, 33.25, 32.27, 30.92, 30.83, 30.65,

30.49, 28.88, 28.73, 27.10, 23.89, 23.54, 14.59. MS (ESI) calculated for

C₃₆H₆₅N₃O₉, m/z 683.47, found 682.49 (M - H)^-.

Synthesis of Compound 21: (15R)-Tribenzyl 2,2-Dimethyl-

4,12-dioxo-3-oxa-5,11,16-triazaoctacosane-6,10,15-tricar-

boxylate. To a solution of 14 (190 mg, 0.28 mmol) in anhydrous

dichloromethane (10 mL) and methanol (2 mL) were added dodecyl

aldehyde (51.5 mg, 0.28 mmol), 3-4 drops of acetic acid, and macro-

porous resin bound sodium cyanoborohydride (150 mg, 0.33 mmol).

The solution was stirred for 8 h, followed by filtration to remove the

resin. The solvent was removed under vacuum to obtain the residue,

which was purified using column chromatography (15% EtOAc/hexanes)

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to obtain compound 21 (124 mg, 51%). ¹H NMR (500 MHz, CDCl₃)

δ 7.43-7.27 (m, 15H), 5.25-5.09 (m, 6H), 4.50 (dd, J = 12.7, 7.7 Hz,

1H), 4.35-4.16 (m, 1H), 2.73-2.64 (m, 1H), 2.62-2.13 (m, 4H), 2.02

(t, J = 20.0 Hz, 1H), 1.91-1.72 (m, 3H), 1.71-1.50 (m, 4H), 1.41 (d, J =

12.6 Hz, 7H), 1.38-1.17 (m, 25H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 172.53, 172.11, 155.94, 155.65, 155.52, 135.36,

134.56, 130.91, 128.62, 128.43, 128.29, 79.94, 68.16, 67.97, 67.06, 60.40,

59.91, 53.22, 52.10, 47.94, 32.38, 32.20, 32.04, 31.95, 31.43, 30.99, 30.37,

29.64, 29.58, 29.36, 29.09, 28.92, 28.66, 28.34, 28.12, 27.43, 27.00, 23.76,

23.00, 22.72, 21.45, 21.24, 14.16. MS (ESI) calculated for C₅₀H₇₁N₃O₉,

m/z 857.52, found 858.53 (M þ H)^þ.

mmol) in anhydrous dichloromethane (10 mL) and methanol (5 mL)

were added hexadecanal (86.4 mg, 0.36 mmol), 3-4 drops of acetic acid,

and macroporous resin bound sodium cyanoborohydride (163.5 mg,

0.36 mmol). The solution was then stirred for 14 h, followed by filtration

to remove the resin. The solvent was removed under vacuum to obtain

the residue, which was purified using column chromatography (12%

1

EtOAc/hexanes) to obtain compound 25 (112 mg, 68%). H NMR

(500 MHz, CDCl₃) δ 7.45-7.28 (m, 15H), 5.31-4.96 (m, 7H), 4.48

(d, J = 5.2 Hz, 1H), 4.26-4.18 (m, 1H), 3.24-2.06 (m, 9H), 1.77-1.55

(m, 8H), 1.46-1.33 (m, 11H), 1.33-1.07 (m, 53H), 0.88 (t, J = 6.8 Hz,

6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.62, 172.24, 172.11, 171.63,

155.80, 155.69, 135.57, 131.10, 128.93, 128.83, 128.64, 128.57, 128.47,

128.41, 80.10, 68.36, 67.24, 67.18, 63.06, 62.51, 53.47, 53.37, 52.40,

52.14, 38.92, 32.82, 32.32, 32.14, 30.55, 29.92, 29.87, 29.86, 29.76, 29.74,

29.70, 29.58, 29.42, 29.36, 29.12, 28.51, 27.25, 25.07, 23.93, 23.19, 22.90,

21.78, 21.65, 14.34, 14.27. MS (ESI) calculated for C₇₀H₁₁₁N₃O₉, m/z

1137.83, found 1138.85 (M þ H)^þ.

Synthesis of Compound 22: 2-Amino-6-((R)-4-carboxy-

4-(dodecylamino)butanamido)heptanedioic Acid. Com-

pound 21 (50 mg, 58.3 μmol) was dissolved in methanol, followed

by the addition of Pd(OH)₂/C. The solution was subjected to

catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed

by filtration through Celite bed. The solvent was removed under

reduced pressure to obtain the residue, which was dissolved in 5 mL

of trifluoroacetic acid and stirred for 30 min, followed by removal of

the solvent by purging nitrogen and drying under vacuum to obtain

the trifluoroacetate salt of the compound 22 (35 mg, quantitative

yield in two steps). ¹H NMR (500 MHz, MeOD) δ 4.40 (d, J = 3.5 Hz,

1H), 3.88-3.56 (m, 3H), 3.21-3.07 (m, 1H), 3.06-2.94 (m, 2H),

2.53 (t, J = 6.7 Hz, 2H), 2.14 (d, J = 6.0 Hz, 2H), 1.91 (s, 3H), 1.79-

1.43 (m, 6H), 1.43-1.20 (m, 15H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR

(126 MHz, MeOD) δ 175.33, 62.44, 62.15, 54.58, 53.37, 46.97, 46.32,

45.72, 45.40, 44.90, 33.10, 32.83, 32.34, 31.97, 31.13, 30.78, 30.68,

30.52, 30.24, 27.61, 27.46, 26.59, 26.28, 23.75, 23.09, 22.55, 22.34,

14.44. MS (ESI) calculated for C₂₄H₄₅N₃O₇, m/z 487.33, found

488.34 (M þ H)^þ.

Synthesis of Compound 26: 2-Amino-6-((R)-4-carboxy-

4-(dioctylamino)butanamido)heptanedioic Acid. The crude

of compound 23 was dissolved in methanol, followed by the addition of

Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at

60 psi hydrogen pressure for 2 h, followed by filtration through Celite

bed. The solvent was removed under reduced pressure to obtain the

residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred

for 30 min, followed by removal of the solvent by purging nitrogen and

drying under vacuum to obtain the residue which was further purified

using column chromatography to obtain the trifluoroacetate salt of

1

compound 26 (60 mg, 64%). H NMR (500 MHz, 10% CDCl₃in

MeOD) δ 4.34 (dd, J = 9.2, 4.6 Hz, 1H), 3.91-3.79 (m, 2H), 3.21-3.00

(m, 4H), 2.51 (ddd, J = 29.4, 15.8, 6.3 Hz, 2H), 2.20-2.09 (m, 1H),

2.08-1.95 (m, 1H), 1.95-1.78 (m, 3H), 1.78-1.39 (m, 8H), 1.34-

1.16 (m, 19H), 0.82 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, 10%

CDCl₃in MeOD) δ 175.14, 174.73, 174.41, 171.93, 53.92, 53.35, 33.09,

32.93, 32.48, 31.97, 31.09, 30.27, 30.21, 30.19, 27.71, 25.66, 23.75, 23.42,

22.53, 14.55. MS (ESI) calculated for C₂₈H₅₃N₃O₇, m/z 543.39, found

544.41 (M þ H)^þ.

Synthesis of Compound 23: (15R)-Tribenzyl 2,2-Di-

methyl-16-octyl-4,12-dioxo-3-oxa-5,11,16-triazatetraco-

sane-6,10,15-tricarboxylate. To a solution of 14 (100 mg, 0.15

mmol) in anhydrous dichloromethane (10 mL) and methanol (5 mL)

were added octylaldehyde (56.5 μL, 0.36 mmol), 3-4 drops of acetic

acid, and macroporous resin bound sodium cyanoborohydride (163.5

mg, 0.36 mmol). The reaction mixture was then stirred for 10 h, followed

by filtration to remove the resin. The solvent was removed under

vacuum to obtain compound 23 as a crude. MS (ESI) calculated for

C₅₄H₇₉N₃O₉, m/z 913.58, found 914.60 (M þ H)^þ.

Synthesis of Compound 27: 2-Amino-6-((R)-4-carboxy-

4-(didodecylamino)butanamido)heptanedioic Acid. Com-

pound 24 (50 mg, 0.048 mmol) was dissolved in methanol, followed

by the addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through a Celite bed. The solvent was removed under reduced pressure

to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid

and stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

compound 27 (37 mg, quantitative yield). ¹H NMR (500 MHz, MeOD)

δ 4.43-4.37 (m, 1H), 3.87 (dd, J = 29.3, 7.5 Hz, 2H), 3.20 (dd, J = 19.5,

10.2 Hz, 2H), 3.15-3.05 (m, 2H), 2.73-2.46 (m, 3H), 2.27-1.99 (m,

3H), 1.98-1.83 (m, 3H), 1.72 (s, 5H), 1.63-1.43 (m, 3H), 1.31 (d, J =

38.3 Hz, 33H), 0.88 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, MeOD) δ

33.24, 30.92, 30.80, 30.69, 30.64, 30.34, 27.78, 23.90, 14.60. MS (ESI)

calculated for C₃₆H₆₉N₃O₇, m/z 655.51, found 656.51 (M þ H)^þ.

Synthesis of Compound 28: 2-Amino-6-((R)-4-carboxy-

4-(dihexadecylamino)butanamido)heptanedioic Acid.

Compound 25 (50 mg, 0.043 mmol) was dissolved in methanol,

followed by the addition of Pd(OH)₂/C. The solution was subjected

to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed

by filtration through a Celite bed. The solvent was removed under

reduced pressure to obtain the residue, which was dissolved in 5 mL of

trifluoroacetic acid and stirred for 30 min, followed by removal of the

solvent by purging nitrogen and drying under vacuum to obtain the

trifluoroacetate salt of the compound 28 (38 mg, quantitative yield). ¹H

NMR (500 MHz, 20% CDCl₃in MeOD) δ 4.48-4.20 (m, 2H), 3.86-

3.73(m, 3H),3.29-3.04 (m, 4H), 2.72-2.44 (m, 3H), 2.31-1.80 (m, 7H),

Synthesis of Compound 24: (15R)-Tribenzyl 16-Dodecyl-

2,2-dimethyl-4,12-dioxo-3-oxa-5,11,16-triazaoctacosane-

6,10,15-tricarboxylate. To the solution of 14 (100 mg, 0.15 mmol)

in anhydrous dichloromethane (10 mL) and methanol (5 mL) were

added dodecylaldehyde (66.8 mg, 0.36 mmol), 3-4 drops of acetic acid,

and macroporous resin bound sodium cyanoborohydride (163.5 mg,

0.36 mmol). The solution was then stirred for 14 h, followed by filtration

to remove the resin. The solvent was removed under vacuum to obtain

the residue, which was purified using column chromatography (15%

1

EtOAc/hexanes) to obtain compound 24 (105 mg, 71%). H NMR

(500 MHz, CDCl₃) δ 7.45-7.28 (m, 15H), 5.28 (dd, J = 12.0, 3.7 Hz,

1H), 5.23-5.01 (m, 6H), 4.44 (bs, 1H), 4.34-4.11 (m, 2H), 3.22-2.85

(m, 4H), 2.58-2.24 (m, 4H), 1.85-1.74 (m, 2H), 1.72-1.53 (m, 6H),

1.48-1.34 (m, 11H), 1.34-1.12 (m, 37H), 0.97-0.82 (m, 6H). ¹³C

NMR (126 MHz, CDCl₃) δ 172.69, 172.23, 171.94, 171.40, 162.45,

135.64, 135.61, 134.64, 131.10, 129.21, 129.16, 129.03, 129.01, 128.92,

128.89, 128.82, 128.78, 128.62, 128.45, 128.38, 128.35, 80.10, 68.36,

68.11, 67.20, 63.20, 62.48, 53.51, 53.40, 52.72, 52.55, 38.92, 32.71, 32.12,

30.55, 29.82, 29.69, 29.63, 29.61, 29.54, 29.22, 29.20, 29.13, 28.51, 27.11,

24.94, 23.93, 23.20, 22.90, 21.88, 21.71, 14.34. MS (ESI) calculated for

C₆₂H₉₅N₃O₉, m/z 1025.71, found 1026.73 (M þ H)^þ.

Synthesis of Compound 25: (15R)-Tribenzyl 16-Hexade-

cyl-2,2-dimethyl-4,12-dioxo-3-oxa-5,11,16-triazadotriacon-

tane-6,10,15-tricarboxylate. To a solution of 14 (100 mg, 0.15

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ARTICLE

1.80-1.48 (m, 8H), 1.46-1.12 (m, 46H), 0.89 (t, J = 6.9 Hz, 6H).

¹³C NMR (126 MHz, 20% CDCl₃in MeOD) δ 31.40, 29.14, 29.11,

29.09, 29.04, 28.97, 28.94, 28.88, 28.82, 28.53, 26.03, 22.10, 13.07. MS

(ESI) calculated for C₄₄H₈₅N₃O₇, m/z 767.64, found 768.65 (M þ

H)^þ.

Synthesis ofCompound30: (2R)-5-((4-(2-Amino-4-oxo-4,5-

dihydro-1H-imidazol-5-yl)-1-carboxybutyl)amino)-2-dode-

canamido-5-oxopentanoic Acid. To a solution of 19 (100 mg,

172.58, 172.32, 172.18, 171.85, 156.06, 155.83, 135.55, 135.53, 135.47,

132.32, 132.24, 131.68, 128.83, 128.81, 128.69, 128.64, 128.61, 128.51,

128.46, 119.23, 119.13, 80.34, 80.17, 80.08, 77.48, 77.23, 76.98, 67.37,

67.27, 67.22, 67.18, 66.26, 53.34, 53.16, 53.04, 52.25, 52.18, 32.47, 32.24,

31.85, 31.76, 31.64, 29.88, 29.26, 29.01, 28.86, 28.50. MS (ESI)

calculated for C₃₉H₅₃N₃O₁₁, m/z 739.37, found 762.37 (M þ Na)^þ.

Synthesis of Compound 34: (6R)-11,15-Dibenzyl 6-Unde-

cyl 2,2,19,19-tetramethyl-4,9,17-trioxo-3,18-dioxa-5,10,16-

triazaicosane-6,11,15-tricarboxylate. To a solution of com-

pound 33 (80 mg, 0.10 mmol) in ethanol/water (9:1) was added

Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was

refluxed for 3 h at 90 °C, followed by the removal of the solvent under

reduced pressure. The crude was purified using column chromatography

(5% MeOH/CH₂Cl₂) and dried under vacuum. To the solution of the

afforded product (52 mg, 0.07 mmol) in anhydrous DMF were added

1-undecanol (30 μL, 0.15 mmol), HBTU (33.7 mg, 0.09 mmol),

triethylamine (20 μL, 0.15 mmol), and a catalytic amount of DMAP.

The reaction mixture was stirred at room temperature for 14 h, followed

by removal of the solvent under reduced pressure. The residue was

dissolved in ethyl acetate and washed with water. The organic fraction

was dried over anhydrous sodium sulfate, filtered, concentrated, and

purified using column chromatography (35% EtOAc/hexanes) to obtain

compound 34 (44.4 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ 7.40-

7.30 (m, 10H), 5.27 (dd, J = 22.5, 8.4 Hz, 1H), 5.20-4.96 (m, 5H),

4.65-4.49 (m, 1H), 4.41-4.20 (m, 2H), 4.16-4.07 (m, 2H), 2.30 (t, J =

6.8 Hz, 2H), 2.24-2.13 (m, 1H), 1.93-1.72 (m, 3H), 1.67-1.59 (m,

3H), 1.43 (d, J = 2.7 Hz, 20H), 1.35-1.18 (m, 18H), 0.88 (t, J = 6.9 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.93, 170.59, 170.19, 154.13,

133.94, 127.20, 127.03, 126.85, 115.67, 78.69, 78.48, 65.63, 64.37, 51.77,

51.70, 51.47, 51.42, 50.64, 50.59, 30.87, 30.48, 30.17, 28.18, 28.07, 27.91,

27.82, 27.09, 26.90, 24.36, 21.27, 12.95. MS (ESI) calculated for

C₄₇H₇₁N₃O₁₁, m/z 853.51, found 876.47 (M þ Na)^þ.

0.13 mmol) in pyridine was added 1H-pyrazole-1-carboxamidine HCl

3

(55.2 mg, 0.37 mmol). The reaction mixture was heated in microwave at

65 °C for 1 h. The solvent was removed under vacuum, and the residue

was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The

solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen

pressure for 2 h, followed by filtration through Celite bed. The solvent

was removed under reduced pressure to obtain the crude product which

was purified using column chromatography (30% MeOH/CH₂Cl₂) to

1

obtain compound 30 (38 mg, 58%). H NMR (500 MHz, MeOD) δ

4.40-4.27 (m, 2H), 4.21-4.18 (m, 1H), 2.34 (t, J = 5.9 Hz, 2H), 2.24

(dd, J = 13.3, 6.9 Hz, 2H), 2.20-1.95 (m, 2H), 1.95-1.65 (m, 5H),

1.64-1.54 (m, 2H), 1.54-1.43 (m, 2H), 1.37-1.21 (m, 15H), 0.88 (t,

J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.67, 176.56, 176.20,

175.20, 61.16, 54.19, 54.01, 53.67, 37.11, 37.03, 33.35, 33.25, 32.54,

32.46, 31.92, 31.80, 30.92, 30.81, 30.65, 30.51, 28.87, 27.09, 23.90, 22.56,

22.33, 22.19, 14.60. MS (ESI) calculated for C₂₅H₄₃N₅O₇, m/z 525.32,

found 524.32 (M - H)^-.

Synthesis of Compound 32: 2-((R)-4-Carboxy-4-dodecana-

midobutanamido)-6-guanidinoheptanedioic Acid. To a so-

lution of 19 (50 mg, 0.06 mmol) in THF were added N,N⁰-di-Boc-1H-

pyrazole-1-carboxamidine HCl (57.4 mg, 0.19 mmol) and pyridine (1

3

mL). The reaction mixture was then stirred at 50 °C for 3 h. The solvent

was removed under reduced pressure, and the residue was dissolved in

methanol, followed by the addition of Pd(OH)₂/C. The solution was

subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 1 h,

followed by filtration through Celite bed. The solvent was removed

under reduced pressure to obtain the crude, which was purified using

column chromatography (8% MeOH/CH₂Cl₂). The product was

dissolved in 5 mL of trifluoroacetic acid, and the reaction mixture was

stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

compound 32 (30 mg, 63%). ¹H NMR (500 MHz, MeOD) δ 4.34 (s,

2H), 4.00 (s, 1H), 2.38-2.28 (m, 2H), 2.24 (dd, J = 14.4, 7.0 Hz, 2H),

2.18-2.08 (m, 1H), 2.07-1.80 (m, 4H), 1.79-1.65 (m, 2H), 1.61-

1.55 (m, 2H), 1.55-1.36 (m, 3H), 1.36-1.19 (m, 14H), 0.88 (t, J = 6.9

Hz, 3H). ¹³C NMR (126 MHz, 5% CDCl₃in MeOD) δ 176.46, 175.71,

175.05, 158.34, 57.03, 54.25, 37.11, 33.35, 33.20, 33.11, 32.83, 32.64,

30.88, 30.78, 30.62, 30.51, 29.28, 27.06, 23.86, 22.90, 22.58, 14.59. MS

(ESI) calculated for C₂₅H₄₅N₅O₈, m/z 543.33, found 544.35 (M

þ H)^þ.

Synthesis of Compound 35: 2-Amino-6-((R)-4-amino-5-

oxo-5-(undecyloxy)pentanamido)heptanedioic Acid. Com-

pound 34 (40 mg, 46.9 μmol) was dissolved in methanol, followed by

the addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through a Celite bed. The solvent was removed under reduced pressure

to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid

and stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

compound 35 (32.8 mg, quantitative yield). ¹H NMR (500 MHz,

CDCl₃) δ 4.40 (dd, J = 9.2, 4.8 Hz, 1H), 4.32-4.22 (m, 2H), 4.11

(dd, J = 11.8, 6.0 Hz, 1H), 3.82-3.74 (m, 1H), 2.53 (t, J = 6.9 Hz, 2H),

2.23 (dt, J = 13.3, 6.7 Hz, 1H), 2.18-2.09 (m, 1H), 1.90 (ddd, J = 21.0,

13.9, 5.3 Hz, 3H), 1.80-1.67 (m, 3H), 1.64-1.46 (m, 2H), 1.43-1.22

(m, 16H), 0.91 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

176.90, 172.93, 70.30, 57.13, 56.15, 56.04, 55.92, 55.86, 35.59, 34.66,

34.59, 33.84, 33.76, 33.26, 33.17, 32.99, 32.88, 32.08, 29.78, 29.71, 29.39,

26.26, 25.15, 24.95, 16.95. MS (ESI) calculated for C₂₃H₄₃N₃O₇, m/z

473.31, found 474.31 (M þ H)^þ.

Synthesis of Compound 33: (6R)-6-Allyl 11,15-Dibenzyl

2,2,19,19-tetramethyl-4,9,17-trioxo-3,18-dioxa-5,10,16-triazai-

cosane-6,11,15-tricarboxylate. Compound 12 (250 mg, 0.29

mmol) was dissolved in 10 mL of dichloromethane, followed by the

addition of 3 mL of piperidine. The reaction mixture was stirred for 15

min, followed by removal of the solvent under reduced pressure. The

residue was dissolved in dichloromethane, followed by the addition of di-

tert-butyl dicarbonate (95 mg, 0.43 mmol) and triethylamine (80 μL,

0.58 mmol). The reaction mixture was stirred at room temperature for 1

h, followed by removal of the solvent under reduced pressure. The crude

was purified using column chromatography (30% EtOAc/hexanes) to

obtain compound 33 (147 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ

7.41-7.29 (m, 10H), 5.97-5.82 (m, 1H), 5.30 (ddd, J = 37.2, 23.6, 5.8

Hz, 3H), 5.21-5.06 (m, 5H), 4.68-4.51 (m, 3H), 4.38-4.20 (m, 2H),

2.31 (dd, J = 14.5, 7.3 Hz, 2H), 2.25-2.13 (m, 1H), 1.99-1.54 (m, 6H),

1.43 (s, J = 2.9 Hz, 20H). ¹³C NMR (126 MHz, CDCl₃) δ 172.67,

Synthesis of Compound 36: Dibenzyl 2-((tert-Butoxycarbo-

nyl)amino)-6-((R)-4-((tert-butoxycarbonyl)amino)-5-oxo-

5-(undecylamino)pentanamido)heptanedioate. To a solu-

tion of 33 (240 mg, 0.32 mmol) in ethanol/water (9:1) was added

Wilkinson’s catalyst (30 mg, 0.03 mmol). The reaction mixture was

refluxed for 3 h at 90 °C, followed by removal of the solvent under

reduced pressure. The crude was purified using column chromatography

(5% MeOH/CH₂Cl₂). To the solution of afforded product (193 mg,

0.27 mmol) in anhydrous dichloromethane were added undecylamine

(118 μL, 0.55 mmol), HBTU (157 mg, 0.41 mmol), triethylamine (77

μL, 0.55 mmol), and a catalytic amount of DMAP. The reaction mixture

was stirred at room temperature for 14 h, followed by removal of the

solvent under reduced pressure. The residue was dissolved in ethyl

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ARTICLE

acetate and washed with water. The organic fraction was dried over

anhydrous sodium sulfate, filtered, concentrated, and purified using

column chromatography (25% EtOAc/hexanes) to obtain compound

a solution of 12 (100 mg, 0.11 mmol) in ethanol/water (9:1) was added

Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was

refluxed for 3 h at 90 °C, followed by removal of the solvent under

reduced pressure. The crude was purified using column chromatog-

raphy (5% MeOH/CH₂Cl₂) and dried under vacuum. To the

solution of the afforded product (75 mg, 91.3 μmol) in anhydrous

DMF were added ethyl alcohol (11 μL, 182 μmol), HBTU (41.6 mg,

109.5 μmol), and a catalytic amount of DMAP. The reaction mixture

was stirred at room temperature for 14 h, followed by removal of the

solvent under reduced pressure. The residue was dissolved in ethyl

acetate and washed with water. The organic fraction was dried over

anhydrous sodium sulfate, filtered, concentrated, and purified using

column chromatography (15% EtOAc/hexanes) to obtain com-

1

36 (188 mg, 81%). H NMR (500 MHz, CDCl₃) δ 7.44-7.27 (m,

10H), 6.65-6.49 (m, 1H), 5.63 (d, J = 8.1 Hz, 1H), 5.62-5.52 (m, 1H),

5.23-4.92 (m, 5H), 4.60-4.45 (m, 2H), 4.27 (d, J = 8.2 Hz, 1H), 4.09

(d, J = 6.7 Hz, 1H), 3.31-3.14 (m, 2H), 2.38-2.22 (m, 1H), 2.00-1.53

(m, 5H), 1.49-1.32 (m, 23H), 1.31-1.21 (m, 16H), 0.90-0.84 (m,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.07, 173.52, 172.99, 172.68,

171.76, 171.40, 157.18, 156.56, 135.51, 135.42, 128.88, 128.81, 128.74,

128.66, 128.59, 128.50, 128.38, 80.44, 80.17, 67.43, 67.28, 67.19, 53.47,

53.21, 52.65, 52.28, 39.89, 39.73, 32.98, 32.75, 32.47, 32.11, 31.96, 31.51,

30.67, 29.82, 29.76, 29.54, 28.53, 27.12, 22.89, 22.03, 21.87, 21.48, 21.35,

14.34. MS (ESI) calculated for C₄₇H₇₂N₄O₁₀, m/z 852.52, found 875.53

(M þ Na)^þ.

1

pound 39 (58.9 mg, 76%). H NMR (500 MHz, CDCl₃) δ 7.79-

7.69 (m, 2H), 7.57-7.52 (m, 2H), 7.57-7.26 (m, 14H), 6.61 (d, J =

6.9 Hz, 0.50H), 6.37 (t, J = 6.9 Hz, 0.50H), 5.65 (dd, J = 29.3, 7.3 Hz,

1H), 5.23-5.00 (m, 5H), 4.64-4.54 (m, 1H), 4.45-4.37 (m, 2H),

4.29-4.15 (m, 4H), 2.36-2.17 (m, 3H), 1.98-1.88 (m, 1H), 1.87-

1.74 (m, 2H), 1.73-1.62 (m, 2H), 1.48-1.33 (m, 10H), 1.33-1.23

(m, 5H). ¹³C NMR (126 MHz, CDCl₃) δ 172.67, 172.18, 171.99,

156.58, 155.85, 144.11, 143.88, 141.52, 135.55, 135.46, 131.10,

129.00, 128.83, 128.71, 128.64, 128.53, 127.95, 127.31, 125.36,

120.20, 80.12, 68.37, 67.43, 67.24, 61.98, 53.55, 53.35, 52.22,

47.39, 38.92, 32.45, 31.79, 30.56, 29.12, 28.82, 28.52, 23.94, 23.20,

21.47, 21.29, 21.14, 14.36, 14.29, 11.17. MS (ESI) calculated for

C₄₈H₅₅N₃O₁₁, m/z 849.38, found 872.37 (M þ Na)^þ.

Synthesis of Compound 37: 2-Amino-6-((R)-4-amino-5-

oxo-5-(undecylamino)pentanamido)heptanedioic Acid.

Compound 36 (40 mg, 46.9 μmol) was dissolved in methanol, followed

by the addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through a Celite bed. The solvent was removed under reduced pressure

to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid

and stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

compound 37 (32.8 mg, quantitative yield). ¹H NMR (500 MHz,

MeOD) δ 4.47-4.32 (m, 1H), 3.92-3.79 (m, 1H), 3.64-3.54 (m,

1H), 3.28-3.13 (m, 2H), 2.56-2.34 (m, 2H), 2.19-2.01 (m, 2H),

2.00-1.60 (m, 4H), 1.58-1.41 (m, 4H), 1.30 (d, J = 21.4 Hz, 16H),

0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 175.94, 175.78,

174.26, 174.03, 169.72, 55.49, 53.98, 53.77, 53.47, 40.75, 33.08, 32.29,

31.95, 31.82, 31.51, 31.31, 30.75, 30.46, 30.32, 28.74, 28.49, 28.04, 23.74,

22.62, 22.07, 14.44. MS (ESI) calculated for C₂₃H₄₄N₄O₆, m/z 472.33,

found 473.33 (M þ H)^þ.

Synthesis of Compound 40: (15R)-10,6-Dibenzyl 15-Un-

decyl-2,2-dimethyl-4,12,17-trioxo-3-oxa-5,11,16-triazaoc-

tacosane-6,10,15-tricarboxylate. Compound 38 (50 mg, 0.05

mmol) was dissolved in 10 mL of dichloromethane, followed by the

addition of 3 mL of piperidine. The reaction mixture was stirred for 15

min, followed by removal of the solvent under reduced pressure. The

residue (0.05 mmol) was dissolved in pyridine, followed by the addition

of lauroyl chloride (14.6 μL, 0.06 mmol) and a catalytic amount of

DMAP. The reaction mixture was stirred for 4 h, followed by evapora-

tion of the solvent under reduced pressure. The residue was then

dissolved in dichloromethane and washed with water. The organic

solvent was dried over anhydrous sodium sulfate, filtered, and evapo-

rated under reduced pressure to obtain the crude product which was

purified using column chromatography (18% EtOAc/hexanes) to obtain

compound 40 (36.4 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 7.74-

7.29 (m, 10H), 7.18-7.06 (m, 1H), 6.59-6.41 (m, 2H), 5.31-5.08 (m,

5H), 4.73-4.63 (m, 1H), 4.54 (ddd, J = 12.1, 10.0, 5.6 Hz, 2H), 4.30-

4.20 (m, 1H), 4.15-4.07 (m, 2H), 2.38-2.14 (m, 5H), 1.97-1.54 (m,

17H), 1.41 (d, J = 19.6 Hz, 7H), 1.34-1.19 (m, 26H), 0.98-0.81 (m,

6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.71, 172.50, 172.32, 172.18,

135.62, 128.86, 128.82, 128.65, 128.52, 128.46, 80.11, 67.31, 67.24,

66.15, 53.36, 52.27, 51.86, 36.87, 32.12, 29.82, 29.72, 29.56, 29.52, 29.44,

28.70, 28.55, 25.99, 25.90, 22.90, 14.34. MS (ESI) calculated for

C₅₄H₈₅N₃O₁₀, m/z 935.62, found 958.59 (M þ Na)^þ.

Synthesis of Compound 38: (5R)-10,14-Dibenzyl 5-Unde-

cyl 1-(9H-fluoren-9-yl)-18,18-dimethyl-3,8,16-trioxo-2,17-

dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate. To

a solution of 12 (100 mg, 0.11 mmol) in ethanol/water (9:1) was added

Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was

refluxed for 3 h at 90 °C, followed by removal of the solvent under

reduced pressure. The crude was purified using column chromatography

(5% MeOH/CH₂Cl₂) and dried under vacuum. To the solution of the

afforded product (70 mg, 85.2 μmol) in anhydrous DMF were added

1-undecanol (35 μL, 170 μmol), HBTU (38.8 mg, 102.2 μmol), and a

catalytic amount of DMAP. The reaction mixture was stirred at room

temperature for 14 h, followed by removal of the solvent under reduced

pressure. The crude was dissolved in ethyl acetate and washed with

water. The organic fraction was dried over anhydrous sodium sulfate,

filtered, concentrated, and purified using column chromatography (15%

EtOAc/hexanes) to obtain the compound 38 (63 mg, 75%). ¹H NMR

(500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 6.4 Hz, 2H),

7.50-7.26 (m, 14H), 6.64 (s, 0.50H), 6.37 (s, 0.50H), 5.67 (dd, J = 32.4,

7.9 Hz, 1H), 5.21-4.99 (m, 5H), 4.62-4.53 (m, 1H), 4.47-4.31 (m,

3H), 4.30-4.19 (m, 2H), 4.13 (td, J = 6.5, 2.6 Hz, 2H), 2.34-2.16 (m,

3H), 1.99-1.89 (m, 1H), 1.87-1.74 (m, 2H), 1.72-1.56 (m, 6H), 1.39

(d, J = 20.0 Hz, 10H), 1.33-1.20 (m, 15H), 0.90 (dt, J = 13.9, 7.3 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.77, 172.70, 172.64, 144.54,

144.29, 141.96, 135.98, 129.25, 129.14, 129.06, 128.95, 128.88, 128.36,

127.73, 125.79, 120.62, 80.57, 67.85, 67.75, 67.67, 66.58, 53.76, 52.66,

47.80, 32.91, 32.54, 32.19, 30.23, 30.13, 29.96, 29.85, 29.59, 29.13, 28.94,

26.41, 23.32, 21.58, 14.77. MS (ESI) calculated for C₅₇H₇₃N₃O₁₁, m/z

975.52, found 998.46 (M þ Na)^þ.

Synthesis of Compound 41: 2-Amino-6-((R)-4-dodecana-

mido-5-oxo-5-(undecyloxy)pentanamido)heptanedioic Acid.

Compound 40 (30 mg, 32.0 μmol) was dissolved in methanol, followed

by the addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through a Celite bed. The solvent was removed under reduced pressure

to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid

and stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

compound 41 (24.6 mg, quantitative yield). ¹H NMR (500 MHz,

CDCl₃) δ 4.51-4.26 (m, 2H), 4.21 (ddd, J = 19.6, 10.9, 5.9 Hz, 1H),

4.15-3.95 (m, 2H), 2.51-2.13 (m, 4H), 2.12-1.48 (m, 21H), 1.48-

1.38 (m, 2H), 1.37-1.05 (m, 21H), 0.96-0.80 (m, 6H). ¹³C NMR (126

MHz, CDCl₃) δ 168.30, 68.63, 50.65, 50.48, 50.31, 50.14, 49.97, 39.16,

Synthesis of Compound 39: (5R)-10,14-Dibenzyl 5-Eth-

yl-1-(9H-fluoren-9-yl)-18,18-dimethyl-3,8,16-trioxo-2,17-

dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate. To

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ARTICLE

32.38, 30.79, 30.15, 29.86, 29.83, 29.37, 26.21, 24.17, 23.44, 23.14, 14.53,

14.51, 11.41. MS (ESI) calculated for C₃₅H₆₅N₃O₈, m/z 655.48, found

654.51 (M - H^þ)^-.

1.30-1.07 (m, 18H), 0.81 (dt, J = 13.9, 4.4 Hz, 3H). ¹³C NMR (126 MHz,

MeOD) δ 173.46, 62.57, 53.58, 53.23, 37.02, 36.92, 33.23, 33.13, 32.83,

31.46, 30.91, 30.82, 30.63, 30.46, 30.44, 28.53, 28.23, 27.18, 27.11, 23.89,

14.65, 14.59. MS (ESI) calculated for C₂₆H₄₇N₃O₈, m/z 529.33, found

528.36 (M - H)^-.

Synthesis of Compound 46: Diethyl 2-((R)-4-Amino-

5-(benzyloxy)-5-oxopentanamido)-6-((tert-butoxycarbo-

nyl)amino)heptanedioate. Compound 13 (100 mg, 0.12 mmol)

was dissolved in 10 mL of dichloromethane, followed by the addition of

3 mL of piperidine. The reaction mixture was stirred for 15 min, followed

by removal of the solvent under reduced pressure to obtain the crude

product which was purified using column chromatography (5% MeOH/

CH₂Cl₂) to obtain compound 46 (66 mg, 92%). MS (ESI) calculated for

C₂₈H₄₃N₃O₉, m/z 565.30, found 588.30 (M þ Na)^þ.

Synthesis of Compound 43: 2-Amino-6-((R)-4-guanidino-

5-oxo-5-(undecyloxy)pentanamido)heptanedioic Acid. Com-

pound 38 (50 mg, 0.05 mmol) was dissolved in 10 mL of dichloromethane,

followed by the addition of 3 mL of piperidine. The reaction mixture was

stirred for 15 min, followed by removal of the solvent under reduced

pressure. The crude mixture was purified using column chromatography

(5% MeOH/CH₂Cl₂) to obtain the free primary amine derivative, which

was dissolved in pyridine, followed by the addition of 1H-pyrazole-1-

carboxamidine HCl (22.2 mg, 0.15 mmol). The sealed reaction mixture

3

was then heated in microwave at 65 °C for 1 h. The solvent was removed

under vacuum, and the residue was dissolved in methanol, followed by the

addition of Pd(OH)₂/C. The solution was subjected to catalytic hydro-

genolysis at 60 psi hydrogen pressure for 4 h, followed by filtration through

Celite bed. The solvent was removed under reduced pressure to obtain the

crude product which was purified using column chromatography (30%

MeOH/CH₂Cl₂) to obtain the free carboxylic acid derivative, which was

dissolved in 5 mL of trifluoroacetic acid and stirred for 30 min, followed by

removal of the solvent by purging nitrogen and drying under vacuum to

obtain the trifluoroacetate salt of compound 43 (20.3 mg, 47% in four

steps). ¹H NMR (500 MHz, MeOD) δ 4.43-4.08 (m, 5H), 3.58 (bs, 1H),

2.46-2.27 (m, 3H), 2.12-1.90 (m, 2H), 1.84 (bs, 3H), 1.66 (dd, J = 13.7,

6.9 Hz, 2H), 1.53-1.41 (m, 2H), 1.40-1.19 (m, 15H), 0.88 (t, J = 6.8 Hz,

3H). ¹³C NMR (126 MHz, MeOD) δ 67.16, 33.09, 30.76, 30.68, 30.49,

30.39, 29.66, 26.97, 23.75, 14.46. MS (ESI) calculated for C₂₄H₄₅N₅O₇, m/

z 515.33, found 516.31 (M þ H)^þ.

Synthesis of Compound 48: Diethyl 2-Amino-6-((R)-4-

amino-5-oxo-5-(undecylthio)pentanamido)heptanedioate.

To a solution of compound 46 (50 mg, 0.08 mmol) in dichloromethane

were added di-tert-butyl dicarbonate (28.9 mg, 0.13 mmol) and triethy-

lamine (24.6 μL, 0.17 mmol). The reaction mixture was stirred for 30

min, followed by complete removal of the solvent under reduced

pressure. The residue was dissolved in methanol, followed by the

addition of Pd(OH)₂/C. The solution was then subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through a Celite bed. The solvent was removed under reduced pressure

to obtain the residue, which was dissolved in anhydrous DMF, followed

by the addition of 1-undecanethiol (40 μL, 0.17 mmol), HBTU (40.2

mg, 0.10 mmol), triethylamine (24.7 μL, 0.17 mmol), and a catalytic

amount of DMAP. The reaction mixture was stirred at room tempera-

ture for 14 h, followed by removal of the solvent under reduced pressure.

The residue was dissolved in ethyl acetate and washed with water. The

organic fraction was dried over anhydrous sodium sulfate, filtered,

concentrated, and purified using column chromatography (30%

EtOAc/hexanes) to obtain compound 47 (41 mg, 63%). Compound

47 (40 mg, 0.05 mmol) was dissolved in 5 mL of trifluoroacetic acid and

stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

Synthesis of Compound 44: (15R)-10,6-Dibenzyl 15-Ethyl-

2,2-dimethyl-4,12,17-trioxo-3-oxa-5,11,16-triazaoctaco-

sane-6,10,15-tricarboxylate. Compound 39 (100 mg, 0.11

mmol) was dissolved in 10 mL of dichloromethane, followed by the

addition of 3 mL of piperidine. The reaction mixture was stirred for 15

min, followed by removal of the solvent under reduced pressure to

obtain the residue which was dissolved in pyridine, followed by the

addition of lauroyl chloride (41.9 μL, 0.18 mmol) and a catalytic amount

of DMAP. The reaction mixture was stirred for 4 h, followed by

evaporation of the solvent under reduced pressure. The residue was

then dissolved in dichloromethane and washed with water. The organic

solvent was dried over anhydrous sodium sulfate, filtered, and evapo-

rated under reduced pressure to obtain the crude product which was

purified using column chromatography (35% EtOAc/hexanes) to obtain

compound 44 (65.4 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ 7.73-

7.27 (m, 10H), 7.06 (dd, J = 17.7, 8.8 Hz, 1H), 6.65-6.53 (m, 1H),

6.47-6.36 (m, 1H), 5.20-5.05 (m, 4H), 4.53-4.50 (m, 3H), 4.33-

4.09 (m, 4H), 2.35-2.11 (m, 5H), 1.93-1.69 (m, 4H), 1.68-1.65 (m,

3H), 1.47-1.33 (m, 9H), 1.32-1.17 (m, 18H), 0.92-0.83 (m, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 172.42, 172.26, 135.58, 131.11, 129.02,

128.82, 128.65, 128.52, 128.47, 80.10, 68.37, 67.24, 61.95, 53.43, 52.32,

51.83, 38.93, 36.85, 32.12, 31.64, 30.56, 29.83, 29.71, 29.56, 29.51, 29.13,

28.55, 25.89, 23.94, 23.20, 22.91, 14.34, 14.28, 11.18. MS (ESI)

calculated for C₄₅H₆₇N₃O₁₀, m/z 809.48, found 832.51 (M þ Na)^þ.

Synthesis of Compound 45: 2-Amino-6-((R)-4-dodecana-

mido-5-ethoxy-5-oxopentanamido)heptanedioic Acid. Com-

pound 44 (30 mg, 37.0 μmol) was dissolved in methanol, followed by

the addition of Pd(OH)₂/C. The solution was subjected to catalytic

hydrogenolysis at 60 psi hydrogen pressure for 2 h, followed by filtration

through Celite bed. The solvent was removed under reduced pressure to

obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and

stirred for 30 min, followed by removal of the solvent by purging nitrogen

and drying under vacuum to obtain the trifluoroacetate salt of compound 45

(23.4 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.35-4.26

(m, 2H), 4.08(qd,J= 7.1, 3.1 Hz, 2H), 3.73 (d, J= 11.0 Hz, 1H), 2.32-2.24

(m, 2H), 2.20-2.01 (m, 3H), 1.92-1.74 (m, 4H), 1.74-1.31 (m, 6H),

1

compound 48 (41 mg, quantitative yield). H NMR (500 MHz, 20%

CDCl₃in MeOD) δ 4.32-4.26 (m, 1H), 4.26-4.13 (m, 3H), 4.09 (q,

J = 7.1 Hz, 2H), 3.91 (dt, J = 2.0, 6.5 Hz, 1H), 2.95 (t, J = 7.3 Hz, 2H),

2.50-2.35 (m, 2H), 2.28-1.69 (m, 6H), 1.69-1.59 (m, 1H), 1.57-

1.35 (m, 4H), 1.34-1.11 (m, 21H), 0.80 (t, J = 6.8 Hz, 3H). ¹³C NMR

(126 MHz, 20% CDCl₃in MeOD) δ 197.50, 174.15, 63.80, 62.71, 62.66,

59.98, 53.86, 53.83, 53.78, 53.74, 33.18, 31.98, 31.79, 31.65, 31.19, 31.16,

30.84, 30.71, 30.58, 30.30, 29.94, 28.52, 28.44, 23.85, 22.77, 22.63, 14.64,

14.59, 14.56. MS (ESI) calculated for C₂₇H₅₁N₃O₆S, m/z 545.35, found

568.36 (M þ Na)^þ.

Synthesis of Compound 50: (2R)-5-((6-Amino-1,7-dietho-

xy-1,7-dioxoheptan-2-yl)amino)-2-dodecanamido-5-ox-

opentanoic Acid. To a solution of 46 (36 mg, 0.06 mmol) in

pyridine were added lauroyl chloride (22.6 μL, 0.09 mmol) and a

catalytic amount of DMAP. The reaction mixture was stirred for 4 h,

followed by evaporation of the solvent under reduced pressure. The

residue was then dissolved in dichloromethane and washed with water.

The organic solvent was dried over anhydrous sodium sulfate, filtered,

and evaporated under reduced pressure to obtain the crude product

which was purified using column chromatography (18% EtOAc/

hexanes) to obtain compound 49 (39.0 mg, 82%). Compound 49 (30

mg, 0.04 mmol) was dissolved in methanol, followed by the addition of

Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at

60 psi hydrogen pressure for 2 h, followed by filtration through a Celite

bed. The solvent was removed under reduced pressure to obtain the

residue, which was then dissolved in 5 mL of trifluoroacetic acid and

stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt of

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compound 50 (26.4 mg, quantitative yield in two steps). ¹H NMR (500

MHz, MeOD) δ 4.45-4.37 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 4.16 (q,

J = 6.6 Hz, 2H), 4.05-3.97 (m, 1H), 2.34 (d, J = 2.6 Hz, 2H), 2.28-2.11

(m, 3H), 2.01-1.80 (m, 4H), 1.78-1.40 (m, 6H), 1.40-1.19 (m, 21H),

0.90 (dt, J = 13.6, 4.7 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.49,

175.06, 174.90, 173.29, 170.44, 63.70, 62.49, 53.88, 53.80, 53.33, 53.13,

52.83, 49.52, 49.35, 49.18, 49.01, 48.84, 48.67, 48.50, 36.96, 36.87, 33.10,

32.79, 32.09, 32.04, 31.95, 31.02, 30.89, 30.78, 30.68, 30.51, 30.38, 28.84,

28.47, 27.01, 26.97, 23.76, 22.65, 22.42, 22.32, 14.44. MS (ESI)

calculated for C₂₈H₅₁N₃O₈, m/z 557.37, found 558.40 (M þ H)^þ.

Synthesis of Compound 51: Dibenzyl 2-(4-((((9H-Fluoren-

9-yl)methoxy)carbonyl)amino)butanamido)-6-((tert-butox-

ycarbonyl)amino)heptanedioate. To a solution of 9 (221 mg,

0.47 mmol) in anhydrous dichloromethane were added 4-Fmoc-ami-

nobutyric acid (150 mg, 0.46 mmol), polystyrene bound carbodiimide

(382.11 mg, 0.47 mmol), and a catalytic amount of polystyrene bound

DMAP. The reaction mixture was stirred at room temperature for 8 h,

followed by filtration to remove the solid resin. The filtrate was

evaporated under vacuum to obtain the residue which was then purified

using column chromatography (50% EtOAc/hexanes) to obtain com-

pound 51 (230 mg, 63%). ¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J =

7.5 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.44-7.25 (m, 14H), 6.47 (d, J =

7.2 Hz, 1H), 5.21-5.00 (m, 6H), 4.61-4.50 (m, 1H), 4.46-4.33 (m,

2H), 4.31-4.15 (m, 2H), 3.29-3.11 (m, 2H), 2.26-2.12 (m, 2H), 1.76

(dd, J = 19.9, 6.1 Hz, 4H), 1.70-1.52 (m, 2H), 1.38 (d, J = 13.3 Hz,

11H). ¹³C NMR (126 MHz, CDCl₃) δ 172.70, 172.59, 172.45, 172.29,

157.10, 155.76, 144.15, 141.53, 135.54, 128.85, 128.82, 128.72, 128.64,

128.57, 128.45, 127.88, 127.25, 125.28, 120.16, 80.19, 67.42, 67.34,

67.24, 66.76, 53.34, 52.18, 47.49, 40.21, 33.33, 32.45, 32.30, 31.65, 28.53,

26.21, 21.59, 21.28. MS (ESI) calculated for C₄₅H₅₁N₃O₉, m/z 777.36,

found 800.33 (M þ Na)^þ.

vacuum to obtain the trifluoroacetate salt of compound 53 (38 mg,

quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.39 (dd, J = 9.3, 4.7

Hz, 1H), 3.80-3.73 (m, 1H), 3.20 (t, J = 7.0 Hz, 2H), 2.32-2.24 (m,

2H), 2.21-2.14 (m, 2H), 2.00-1.68 (m, 7H), 1.66-1.42 (m, 5H),

1.36-1.24 (m, 14H), 0.90 (dd, J = 13.1, 6.2 Hz, 3H). ¹³C NMR (126

MHz, MeOD) δ 176.66, 175.79, 175.46, 173.04, 54.83, 53.43, 39.81,

37.36, 34.18, 33.23, 32.45, 32.39, 31.61, 31.48, 30.90, 30.80, 30.63,

30.52, 27.24, 26.85, 23.89, 23.07, 22.75, 14.58. MS (ESI) calculated

for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.39 (M þ H)^þ.

Synthesis of Compound 54: 5-((1,7-Bis(benzyloxy)-6-(-

(tert-butoxycarbonyl)amino)-1,7-dioxoheptan-2-yl)amino)-

5-oxopentanoic Acid. To a solution of 9 (100 mg, 0.21 mmol) in

anhydrous dichloromethane was added glutaric anhydride (25.5 mg,

0.22 mmol). The reaction mixture was stirred at room temperature for 1

h, followed by removal of the solvent under reduced pressure. The

residue was then purified using column chromatography (5% MeOH/

CH₂Cl₂) to obtain compound 54 (118 mg, 95%). ¹H NMR (500 MHz,

CDCl₃) δ 7.73-7.29 (m, 10H), 6.87-6.34 (m, 1H), 5.22-5.08 (m,

4H), 4.60 (bs, 1H), 4.32-3.92 (m, 1H), 2.54-2.17 (m, 4H), 2.13-1.54

(m, 6H), 1.52-1.20 (m, 13H). ¹³C NMR (126 MHz, CDCl₃) δ 176.46,

172.80, 172.69, 172.41, 135.41, 131.11, 129.01, 128.86, 128.84, 128.74,

128.69, 128.54, 80.46, 68.37, 67.51, 67.43, 67.15, 53.35, 52.10, 51.82,

38.92, 35.67, 35.08, 33.61, 33.49, 32.99, 32.44, 31.80, 31.66, 31.00, 30.55,

29.91, 29.13, 28.53, 28.31, 23.93, 23.20, 21.55, 21.21, 20.61, 19.92. MS

(ESI) calculated for C₃₁H₄₀N₂O₉, m/z 584.27, found 607.26 (M þ

Na)^þ.

Synthesis of Compound 55: Dibenzyl 2-((tert-Butoxycarbo-

nyl)amino)-6-(5-oxo-5-(undecyloxy)pentanamido)heptan-

edioate. To a solution of 54 (90 mg, 0.15 mmol) in anhydrous DMF

were added 1-undecanol (63 μL, 0.30 mmol), HBTU (70 mg, 0.18

mmol), triethylamine (43 μL, 0.30 mmol), and a catalytic amount of

DMAP. The reaction mixture was stirred at room temperature for 12 h,

followed by removal of the solvent under reduced pressure. The residue

was dissolved in ethyl acetate and washed with water. The organic

fraction was dried over anhydrous sodium sulfate, filtered, concentrated,

and purified using column chromatography (10% EtOAc/hexanes) to

obtain compound 55 (86.4 mg, 78%). ¹H NMR (500 MHz, CDCl₃) δ

7.40-7.29 (m, 10H), 6.15 (d, J = 6.3 Hz, 1H), 5.20-5.02 (m, 5H),

4.62-4.54 (m, 1H), 4.32-4.19 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 2.40-

2.32 (m, 2H), 2.30-2.22 (m, 2H), 2.00-1.90 (m, 2H), 1.87-1.57 (m,

7H), 1.43 (s, 9H), 1.33-1.19 (m, 17H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 173.49, 173.48, 172.70, 172.57, 172.42,

172.27, 172.26, 155.85, 155.70, 135.55, 135.42, 128.86, 128.82, 128.73,

128.66, 128.55, 128.47, 80.20, 80.09, 67.43, 67.35, 67.25, 64.90, 53.31,

52.03, 51.99, 35.39, 33.47, 32.49, 32.26, 32.11, 31.96, 31.84, 29.80, 29.79,

29.73, 29.54, 29.47, 28.82, 28.52, 26.11, 22.89, 21.48, 21.20, 20.97, 14.33.

MS (ESI) calculated for C₄₂H₆₂N₂O₉, m/z 738.45, found 761.43

(M þ Na)^þ.

Synthesis of Compound 52: Dibenzyl 2-((tert-Butoxycarbo-

nyl)amino)-6-(4-dodecanamidobutanamido)heptanedio-

ate. Compound 51 (140 mg, 0.18 mmol) was dissolved in 10 mL of

dichloromethane, followed by the addition of 3 mL of piperidine. The

reaction mixture was stirred for 15 min, followed by removal of the

solvent under reduced pressure to obtain the residue which was

dissolved in pyridine, followed by the addition of lauroyl chloride

(64.0 μL, 0.27 mmol) and a catalytic amount of DMAP. The reaction

mixture was stirred for 4 h, followed by evaporation of the solvent under

reduced pressure. The residue was then dissolved in dichloromethane

and washed with water. The organic solvent was dried over anhydrous

sodium sulfate, filtered, and evaporated under reduced pressure to

obtain the crude product which was purified using column chromatog-

raphy (30% EtOAc/hexanes) to obtain compound 52 (100 mg, 75%).

¹H NMR (500 MHz, CDCl₃) δ 7.41-7.29 (m, 10H), 6.84-6.76 (m,

1H), 5.97-5.88 (m, 1H), 5.26-5.09 (m, 5H), 4.59-4.50 (m, 1H), 4.26

(d, J = 8.5 Hz, 1H), 3.40-3.30 (m, 1H), 3.25-3.17 (m, 1H), 2.29-2.22

(m, 2H), 2.17-2.14 (m, 2H), 1.87-1.76 (m, 4H), 1.66 (s, 4H), 1.63-

1.58 (m, 2H), 1.42 (d, J = 14.4 Hz, 9H), 1.33-1.20 (m, 16H), 0.88 (t, J =

7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.14, 173.13, 172.73,

172.62, 172.43, 172.27, 155.92, 155.80, 135.57, 128.84, 128.69, 128.65,

128.56, 128.46, 80.12, 67.27, 53.39, 52.34, 38.80, 37.10, 33.62, 32.30,

32.12, 31.57, 29.84, 29.82, 29.72, 29.57, 29.55, 28.55, 26.06, 25.93, 22.90,

21.66, 21.34, 14.34. MS (ESI) calculated for C₄₂H₆₃N₃O₈, m/z 737.46,

found 760.41 (M þ Na)^þ.

Synthesis of Compound 56: 2-Amino-6-(5-oxo-5-(undecy-

loxy)pentanamido)heptanedioic Acid. Compound 55 (35 mg,

0.04 mmol) was dissolved in methanol, followed by the addition of

Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis

at 60 psi hydrogen pressure for 2 h, followed by filtration through a

Celite bed. The solvent was removed under reduced pressure to

obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid

and stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate salt

of the compound 56 (26 mg, quantitative yield). ¹H NMR (500 MHz,

MeOD) δ 4.34 (dd, J = 8.7, 5.0 Hz, 1H), 4.02 (t, J = 6.7 Hz, 2H),

3.82-3.73 (m, 1H), 2.34 (t, J = 7.5 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H),

1.97-1.80 (m, 5H), 1.74-1.63 (m, 1H), 1.62-1.42 (m, 4H), 1.29-

1.25 (m, 16H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD)

δ 175.63, 175.60, 175.35, 175.32, 175.12, 172.76, 65.83, 54.68, 54.54,

53.40, 53.36, 35.86, 34.37, 33.19, 32.40, 32.32, 31.51, 31.39, 30.85,

Synthesis of Compound 53: 2-Amino-6-(4-dodecanami-

dobutanamido)heptanedioic Acid. Compound 52 (50 mg, 0.06

mmol) was dissolved in methanol, followed by the addition of Pd-

(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60

psi hydrogen pressure for 2 h, followed by filtration through a Celite bed.

The solvent was removed under reduced pressure to obtain the residue,

which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 min,

followed by removal of the solvent by purging nitrogen and drying under

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30.84, 30.78, 30.59, 30.51, 29.86, 27.16, 23.86, 22.99, 22.73, 22.27,

14.60. MS (ESI) calculated for C₂₃H₄₂N₂O₇, m/z 458.30, found

459.32 (M þ H)^þ.

(t, J = 7.5 Hz, 2H), 7.37-7.30 (m, 5H), 5.18 (q, J = 12.1 Hz, 2H), 4.46-

4.32 (m, 3H), 4.25-4.17 (m, 2H), 3.42 (dd, J = 5.5, 3.9 Hz, 1H), 3.18

(ddd, J = 20.5, 13.5, 7.0 Hz, 2H), 3.07 (s, 2H), 2.20-2.18 (m, 3H),

1.52-1.38 (m, 10H), 1.37-1.27 (m, 7H). ¹³C NMR (126 MHz, CDCl₃) δ

168.20, 141.65, 135.35, 131.45, 129.30, 129.14, 129.03, 128.87, 128.24,

127.59, 125.59, 120.48, 79.88, 68.70, 67.86, 50.32, 50.15, 49.98, 47.61, 39.76,

39.20, 30.83, 30.05, 29.41, 28.88, 24.34, 24.21, 23.47. MS (ESI) calculated

for C₃₇H₄₅N₃O₇, m/z 643.33, found 666.34 (M þ Na)^þ.

General Procedure for the Syntheses of Compounds

66-79. Synthesis of Compound 66: Dibenzyl 2-((R)-4-(((-

(9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzylo-

xy)-5-oxopentanamido)heptanedioate. To a solution of 4 (100

mg, 0.21 mmol) in dichloromethane were added 58 (78.8 mg, 0.22

mmol), polystyrene bound carbodiimide (170 mg, 0.22 mmol), triethy-

lamine (60 μL, 0.43 mmol), and a catalytic amount of polystyrene bound

DMAP. The reaction mixture was stirred at room temperature for 8 h,

followed by filtration to remove the solid resin. The filtrate was

evaporated under vacuum to obtain the residue which was then purified

using column chromatography (55% EtOAc/hexanes) to obtain the

compound 66 (154 mg, 89%). ¹H NMR (500 MHz, CDCl_3,) δ 7.74 (d,

J = 7.5 Hz, 2H), 7.57 (d, J = 7.0 Hz, 2H), 7.41-7.24 (m, 19H), 6.47 (d,

J = 7.8 Hz, 0.50H), 6.19 (d, J = 7.8 Hz, 0.50H), 5.67 (dd, J = 20.4, 8.0 Hz,

1H), 5.20-5.03 (m, 6H), 4.59 (ddd, J = 13.0, 7.5, 5.3 Hz, 1H), 4.49-

4.33 (m, 3H), 4.18 (t, J = 6.8 Hz, 1H), 2.27-2.19 (m, 5H), 2.02-1.77

(m, 3H), 1.63-1.61 (m, 1H), 1.38-1.16 (m, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 173.17, 171.06, 170.13, 170.04, 169.40, 153.91, 141.79,

141.42, 139.22, 133.04, 126.56, 126.55, 126.53, 126.51, 126.46, 126.31,

126.24, 126.21, 126.13, 126.09, 125.63, 125.00, 123.03, 117.90, 65.30,

65.07, 64.95, 64.08, 51.35, 50.01, 45.06, 31.74, 29.88, 26.66, 26.14, 22.55,

22.27. MS (ESI) calculated for C₄₈H₄₈N₂O₉, m/z 796.34, found 819.31

(M þ Na)^þ.

Synthesis of Compound 69: (R)-5-((5-Aminopentyl)am-

ino)-2-dodecanamido-5-oxopentanoic Acid. ¹H NMR (400

MHz, MeOD) δ 4.21 (dd, J = 8.4, 4.9 Hz, 1H), 3.30-3.18 (m, 2H),

2.92 (t, J = 6.7 Hz, 2H), 2.27-2.20 (m, 4H), 2.08 (td, J = 13.5, 6.5 Hz,

1H), 1.94 (td, J = 13.6, 7.3 Hz, 1H), 1.72-1.39 (m, 8H), 1.37-1.20 (m,

16H), 0.88 (dd, J = 12.0, 5.4 Hz, 3H). ¹³C NMR (101 MHz, 10% CDCl₃

in MeOD) δ 174.03, 53.83, 38.93, 37.98, 36.02, 32.27, 31.46, 29.16,

29.04, 28.87, 27.91, 26.29, 25.40, 22.64, 22.17, 13.26. MS (ESI)

calculated for C₂₂H₄₃N₃O₄, m/z 413.33, found 414.37 (M þ H)^þ.

Synthesis of Compound 70: (2R)-Benzyl 2-((((9H-Fluoren-

9-yl)methoxy)carbonyl)amino)-5-oxo-5-((2-oxoazepan-3-

yl)amino)pentanoate. ¹H NMR (500 MHz, CDCl₃, ethyl acetate)

δ 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.35 (qd, J = 14.7, 7.3

Hz, 9H), 6.98-6.90 (m, 1H), 6.06 (bs, 1H), 5.88-5.81 (m, 1H), 5.23-

5.13 (m, 2H), 4.54-4.48 (m, 1H), 4.46-4.33 (m, 3H), 4.21 (t, J = 7.1

Hz, 1H), 3.30-3.18 (m, 2H), 2.37-2.18 (m, 3H), 1.99-1.97 (m, 1H),

1.84-1.76 (dd, J = 14.6, 9.4 Hz, 1H), 1.62 (bs, 3H), 1.49-1.35 (m, 2H).

¹³C NMR (126 MHz, CDCl₃) δ 175.57, 172.14, 171.16, 156.34, 144.15,

143.99, 141.49, 135.45, 128.85, 128.71, 128.54, 127.90, 127.30, 125.43,

120.17, 67.51, 67.29, 53.98, 53.91, 52.46, 47.38, 42.38, 32.37, 32.32,

31.76, 31.70, 29.05, 28.10, 27.98, 27.93. MS (ESI) calculated for

C₃₃H₃₅N₃O₆, m/z 569.25, found 592.23 (M þ Na)^þ.

Synthesis of Compound 67: 2-((R)-4-Carboxy-4-dodeca-

namidobutanamido)heptanedioic Acid. Compound 66 (147

mg, 0.18 mmol) was dissolved in 10 mL of dichloromethane, followed by

the addition of 3 mL of piperidine. The reaction mixture was stirred for

15 min, followed by removal of the solvent under reduced pressure to

obtain the residue, which was dissolved in pyridine, followed by the

addition of lauroyl chloride (64.0 μL, 0.27 mmol) and a catalytic amount

of DMAP. The reaction mixture was stirred for 4 h, followed by

evaporation of the solvent under reduced pressure. The residue was

then dissolved in dichloromethane and washed with water. The organic

solvent was dried over anhydrous sodium sulfate, filtered, and evapo-

rated under reduced pressure to obtain the crude product which was

purified using column chromatography (25% EtOAc/hexanes) to obtain

the protected intermediate of compound 67 (100 mg, 73%). The

protected intermediate (50 mg, 0. 06 mmol) was then dissolved in

methanol, followed by the addition of Pd(OH)₂/C. The solution was

subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 h,

followed by filtration through a Celite bed. The solvent was removed

under reduced pressure to obtain compound 67 (29 mg, quantitative

yield). ¹H NMR (500 MHz, MeOD) δ 4.42-4.28 (m, 2H), 2.33 (t, J =

6.8 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.24-2.18 (m, 2H), 2.18-2.10 (m,

1H), 1.93 (dt, J = 13.8, 6.3 Hz, 1H), 1.85-1.82 (m, 1H), 1.72-1.54 (m,

5H), 1.41 (dd, J = 14.5, 7.4 Hz, 3H), 1.30-1.26 (m, 15H), 0.87 (t, J = 6.9

Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 177.59, 176.63, 176.63,

175.03, 54.04, 53.66, 37.03, 34.85, 33.36, 33.24, 32.50, 32.45, 30.91,

30.81, 30.64, 30.48, 28.97, 28.79, 27.07, 26.66, 25.78, 23.90, 14.60. MS

(ESI) calculated for C₂₄H₄₂N₂O₈, m/z 486.29, found 485.29

(M - H)^-.

Synthesis of Compound 71: (2R)-2-Dodecanamido-5-oxo-

5-((2-oxoazepan-3-yl)amino)pentanoic Acid. ¹H NMR (500

MHz, MeOD) δ 4.54 (dd, J = 11.4, 1.5 Hz, 1H), 4.41-4.31 (m, 1H), 3.30-

3.13 (m, 3H), 2.34 (tt, J = 6.4, 5.0 Hz, 2H), 2.27-2.11 (m, 3H), 2.02-1.72

(m, 5H), 1.65-1.49 (m, 3H), 1.43-1.22 (m, 16H), 0.88 (t, J = 7.0 Hz, 3H).

¹³C NMR (126 MHz, MeOD) δ 177.24, 176.35, 176.30, 174.12, 174.02,

53.35, 49.46, 49.29, 42.47, 36.94, 33.37, 33.27, 33.10, 32.21, 32.16, 30.78, 30.77,

30.67, 30.51, 30.50, 30.36, 29.94, 29.16, 28.87, 28.80, 26.92, 23.75, 14.45. MS

(ESI) calculated for C₂₃H₄₁N₃O₅, m/z 439.30, found 462.31 (M þ Na)^þ.

Synthesis of Compound 72: (R)-Benzyl 2-((R)-4-((((9H-

Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-

oxopentanamido)-6-((tert-butoxycarbonyl)amino)hexano-

ate. ¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d,

J = 7.2 Hz, 2H), 7.45-7.28 (m, 14H), 6.32 (d, J = 6.9 Hz, 1H), 5.71

(d, J = 8.0 Hz, 1H), 5.22-5.08 (m, 4H), 4.59 (t, J = 10.9 Hz, 2H), 4.40

(d, J = 6.9 Hz, 3H), 4.20 (t, J = 6.9 Hz, 1H), 3.02 (bs, 2H), 2.24 (s,

3H), 1.95 (dd, J = 15.4, 7.9 Hz, 1H), 1.83 (ddd, J = 15.7, 10.6, 5.3 Hz,

1H), 1.67 (d, J = 8.6 Hz, 1H), 1.41 (s, 11H), 1.36-1.23 (m, 2H). ¹³C

NMR (126 MHz, CDCl₃) δ 172.37, 172.01, 171.81, 156.51, 156.28,

144.10, 143.86, 141.53, 135.48, 135.36, 128.87, 128.84, 128.78,

128.73, 128.63, 128.56, 127.94, 127.31, 125.34, 120.20, 120.18,

79.31, 67.61, 67.36, 67.27, 53.63, 52.36, 47.37, 40.17, 32.26, 32.02,

29.71, 28.62, 22.50. MS (ESI) calculated for C₄₅H₅₁N₃O₉, m/z

777.36, found 800.35 (M þ Na)^þ.

Synthesis of Compound 73: (R)-6-Amino-2-((R)-4-car-

boxy-4-dodecanamidobutanamido)hexanoic Acid. ¹H

NMR (500 MHz, MeOD) δ 4.31 (dd, J = 9.1, 4.4 Hz, 1H), 4.26

(dd, J = 9.6, 4.3 Hz, 1H), 2.89-2.79 (m, 2H), 2.26 (t, J = 7.2 Hz,

2H), 2.21-2.08 (m, 3H), 1.88-1.75 (m, 2H), 1.68-1.48 (m, 5H),

1.47-1.08 (m, 18H), 0.80 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz,

MeOD) δ 176.55, 174.81, 53.42, 53.24, 40.56, 36.97, 33.10, 33.04,

32.34, 30.79, 30.77, 30.68, 30.52, 30.50, 30.38, 28.64, 27.95, 27.00,

23.78, 23.75, 14.45. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z

457.32, found 458.33 (M þ H)^þ.

In the case of N-Boc protected intermediates (compounds 68, 72, 76,

and 78), the compounds after hydrogenolysis were dissolved in triﬂuor-

oacetic acid and stirred for 30 min, followed by removal of the solvent by

purging nitrogen and drying under vacuum to obtain the triﬂuoroacetate

salts of compound 69, 73, 77, and 79.

Synthesis of Compound 68: (R)-Benzyl 2-((((9H-Fluoren-9-

yl)methoxy)carbonyl)amino)-5-((5-((tert-

butoxycarbonyl)amino)pentyl)amino)-5-oxopentanoate.

1

H NMR (500 MHz, CDCl₃) δ 7.77 (d, J = 7.5 Hz, 2H), 7.71 (dt, J = 7.3,

3.6 Hz, 1H), 7.61 (t, J = 6.8 Hz, 2H), 7.54 (dd, J = 5.7, 3.3 Hz, 1H), 7.40

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Synthesis of Compound 74: (R)-Benzyl 6-((R)-4-((((9H-

Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-

oxopentanamido)-2-(((benzyloxy)carbonyl)amino)hexa-

noate. ¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H),

7.61-7.52 (m, 2H), 7.38-7.25 (m, 19H), 5.80 (bs, 1H), 5.71 (d, J =

7.8 Hz, 1H), 5.45-5.41 (m, 1H), 5.19-5.09 (m, 4H), 5.06 (s, 2H),

4.44-4.30 (m, 4H), 4.16 (t, J = 6.8 Hz, 1H), 3.14 (dt, J = 13.8, 6.8 Hz,

2H), 2.17-2.11 (m, 3H), 1.93 (dd, J = 17.2, 11.2 Hz, 1H), 1.80 (dt,

J = 10.2, 7.3 Hz, 1H), 1.70-1.62 (m, 1H), 1.49-1.38 (m, 2H), 1.33-

1.24 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.41, 172.02,

156.59, 156.24, 144.10, 143.81, 141.53, 141.48, 136.40, 135.46,

135.37, 128.86, 128.84, 128.74, 128.72, 128.62, 128.53, 128.41,

128.30, 127.94, 127.29, 125.35, 125.31, 120.20, 67.58, 67.37, 67.28,

67.20, 53.88, 53.75, 47.34, 39.29, 32.59, 32.34, 29.00, 28.82, 22.53.

MS (ESI) calculated for C₄₈H₄₉N₃O₉, m/z 811.35, found 834.34 (M

þ Na)^þ.

31.12, 30.81, 27.32, 26.97, 26.73, 26.40, 20.95. MS (ESI) calculated for

C₄₂H₅₃N₃O₉, m/z 743.38, found 766.36 (M þ Na)^þ.

Synthesis of Compound 79: (S)-2-Amino-6-((R)-4-car-

boxy-4-dodecanamidobutanamido)hexanoic Acid. ¹H

NMR (500 MHz, MeOD) δ 4.35 (dd, J = 8.9, 4.9 Hz, 1H), 3.74

(t, J = 5.9 Hz, 1H), 3.24-3.12 (m, 2H), 2.30-2.13 (m, 5H), 1.95-

1.80 (m, 3H), 1.66-1.37 (m, 7H), 1.31-1.27 (m, 15H), 0.88 (t, J =

6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.28, 175.26,

174.74, 173.09, 54.56, 53.07, 39.70, 36.88, 33.15, 32.90, 31.17,

30.61, 30.59, 30.50, 30.35, 30.31, 30.23, 30.16, 29.73, 28.66, 26.78,

23.59, 23.11, 14.54. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z

457.32, found 458.35 (M þ H)^þ.

NF-KB Induction. The induction of NF-κB was quantified using

HEK-Blue cells as previously described by us.^10,51HEK293 cells

stably transfected with human Nod1 and alkaline phosphatase (sAP)

were obtained from InvivoGen (San Diego, CA) and were maintained

in HEK-Blue Selection medium containing zeocin and normocin.

Stable expression of secreted alkaline phosphatase (sAP) under

control of NF-κB/AP-1 promoters is inducible by Nod1 agonists,

and extracellular sAP in the supernatant is proportional to Nod1-

mediated NF-κB induction. HEK-Blue cells were incubated at a

density of ∼10⁵cells/mL in a volume of 80 μL/well in 384-well,

flat-bottomed, cell culture-treated microtiter plates until confluency

was achieved and in subsequently graded concentrations of com-

pounds. sAP was assayed spectrophotometrically using an alkaline

phosphatase-specific chromogen (present in the HEK-detection

medium as supplied by the vendor) at 620 nm.

Synthesis of Compound 75: (R)-2-Amino-6-((R)-4-carboxy-4-

dodecanamidobutanamido)hexanoic Acid. ¹H NMR (500

MHz, MeOD) δ 4.34 (dd, J = 9.1, 4.9 Hz, 1H), 3.80 (t, J = 6.1 Hz,

1H), 3.22 (dt, J = 13.3, 6.7 Hz, 1H), 3.13 (dt, J = 13.5, 6.7 Hz, 1H),

2.30-2.12 (m, 5H), 1.97-1.79 (m, 3H), 1.64-1.56 (m, 2H), 1.52 (dd,

J = 13.8, 6.7 Hz, 2H), 1.49-1.39 (m, 2H), 1.30-1.26 (d, J = 17.6 Hz,

16H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.53,

174.85, 54.57, 53.08, 39.85, 36.89, 33.27, 33.10, 31.40, 30.78, 30.68,

30.52, 30.50, 30.34, 30.00, 28.69, 26.96, 23.76, 23.40, 14.46. MS (ESI)

calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 456.33 (M - H)^-.

Synthesis of Compound 76: (S)-tert-Butyl 2-((R)-4-((((9H-

Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-oxo-

pentanamido)-6-(((benzyloxy)carbonyl)amino)hexanoate

Experiments Involving Human Blood. Human blood was

obtained from healthy adults by antecubital venipuncture in accordance

with University of Kansas Human Subjects Experimentation protocols

(Protocol No. HSCL 12397).

.

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.61-7.54

(m, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.35-7.25 (m, 12H), 6.30 (dd, J =

84.8, 7.6 Hz, 1H), 5.74 (dd, J = 17.5, 7.9 Hz, 1H), 5.14 (t, J = 12.2 Hz,

2H), 5.08-5.01 (m, 2H), 4.88 (bs, 1H), 4.42-4.30 (ddt, J = 31.5, 17.9,

7.6 Hz, 4H), 4.17 (t, J = 6.8 Hz, 1H), 3.14 (dd, J = 12.7, 6.4 Hz, 2H),

2.31-2.13 (m, 3H), 1.98 (dd, J = 14.5, 7.7 Hz, 1H), 1.76 (ddd, J = 15.5,

10.2, 5.3 Hz, 1H), 1.64-1.61 (m, 1H), 1.51-1.44 (m, 2H), 1.44-1.42

(m, 9H), 1.37-1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.11,

171.84, 171.77, 156.72, 156.51, 144.12, 143.85, 141.50, 136.79, 135.36,

128.85, 128.73, 128.70, 128.57, 128.28, 127.92, 127.30, 125.35, 120.19,

120.17, 82.33, 67.57, 67.31, 66.76, 53.67, 52.76, 47.34, 40.77, 32.41,

32.16, 29.58, 28.70, 28.19, 22.29. MS (ESI) calculated for C₄₅H₅₁N₃O₉,

m/z 777.36, found 800.36 (M þ Na)^þ.

Phosflow Flow Cytometric Assay for p38MAPK. Assays were

performed as described by us previously.^10,38,52Briefly, 1 mL aliquots of

fresh whole blood anticoagulated with heparin were incubated with 25 μLof

an equal volume of graded concentrations of compounds diluted in saline

for 15 min at 37 °C. Erythrocytes were lysed and leukocytes were fixed in

one step by mixing 200 μL of the samples in 4 mL of prewarmed whole

blood lyse/fix buffer (Becton-Dickinson Biosciences, San Jose, CA). After

the cells at 500 g were washed for 8 min in buffer, the cells were

permeabilized in ice-cold methanol for 30 min, washed twice in phos-

phate-buffered saline, and transferred to a Millipore MultiScreen BV 1.2 μm

filter plate and stained with either phycoerythrin (PE)-conjugated mouse

anti-p38MAPK (pT180/pY182, BD Biosciences) mAb or a matched PE-

labeled mouse IgG₁κ isotype control mAb for 60 min. The cells were

washed twice in the plate by aspiration according to protocols supplied by

the vendor. Cytometry was performed using a BD FACSArray instrument

in the single-color mode for PE acquisition on 20 000 gated events.

Postacquisition analyses were performed using FlowJo, version 7.0, software

(Treestar, Ashland, OR).

CD11b Flow Cytometric Assay. Assays were performed as des-

cribed by us previously.^10,38,52Briefly, 1 mL aliquots of fresh antic-

oagulated whole blood were incubated with 25 μL of graded dilutions of

the compounds for 1 h at 37 °C. Negative (saline) controls were

included in each experiment. Samples were placed on ice for 15 min

before an amount of 20 μL of anti-CD11b/Mac-1 antibody (Becton-

Dickinson) was added to each sample tube and allowed to incubate on

ice for 30 min. This 0 °C incubation step prevented internalization of

antibody and ensured staining of only extracellularly expressed CD11b.

Erythrocytes were lysed, and leukocytes were fixed in one step by mixing

200 μL of the samples in 4 mL of prewarmed whole blood lyse/fix buffer

(Becton-Dickinson Biosciences, San Jose, CA). After the cells were

washed twice at 200 g for 5 min in CBA buffer, the cells were transferred

to a 96-well plate. Flow cytometry was performed using a BD FACSAr-

ray instrument in the single-color mode for PE acquisition on 20 000

Synthesis of Compound 77: (S)-6-Amino-2-((R)-4-carboxy-4-

dodecanamidobutanamido)hexanoic Acid. ¹H NMR (500

MHz, MeOD) δ 4.35 (bs, 2H), 2.91 (t, J = 7.4 Hz, 2H), 2.32 (t, J =

6.7 Hz, 2H), 2.22 (t, J = 7.5 Hz, 2H), 2.17-2.08 (m, 1H), 1.96-1.87 (m,

2H), 1.77-1.55 (m, 5H), 1.48 (dd, J = 15.2, 7.6 Hz, 2H), 1.30-1.27 (m,

16H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.53,

175.19, 53.81, 40.68, 37.07, 33.41, 33.23, 32.47, 32.36, 30.92, 30.81,

30.65, 30.50, 29.17, 28.86, 28.12, 27.10, 23.95, 23.89, 14.60. MS (ESI)

calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.32 (M þ H)^þ.

Synthesis of Compound 78: (S)-tert-Butyl 6-((R)-4-((((9H-

Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-oxo-

pentanamido)-2-((tert-butoxycarbonyl)amino)hexanoate

.

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.61-7.55

(m, 2H), 7.41-7.25 (m, 9H), 5.89 (bs, 1H), 5.78 (d, J = 7.9 Hz, 1H),

5.16 (q, J = 12.2 Hz, 2H), 5.07 (d, J = 8.1 Hz, 1H), 4.37 (ddd, J = 28.8,

10.6, 7.1 Hz, 3H), 4.19 (t, J = 7.0 Hz, 1H), 3.19 (ddt, J = 25.6, 13.1, 6.5

Hz, 2H), 2.18 (t, J = 10.6 Hz, 2H), 2.01-1.92 (m, 1H), 1.78-1.62 (m,

3H), 1.58 (ddd, J = 11.4, 9.3, 5.7 Hz, 1H), 1.53-1.45 (m, 2H), 1.41 (d,

J = 8.0 Hz, 18H), 1.38-1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ

170.29, 170.24, 170.19, 154.78, 153.95, 142.30, 142.03, 139.72, 139.67,

133.58, 127.06, 126.95, 126.81, 126.13, 126.12, 125.48, 123.57, 123.54,

118.40, 118.38, 80.27, 78.07, 65.77, 65.53, 52.05, 52.00, 45.54, 37.78,

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ARTICLE

gated events. Postacquisition analyses were performed using FlowJo,

version 7.0, software.

helper T lymphocyte type 2; TLR, Toll-like receptor; TREM-1,

triggering receptor expressed on myeloid cells 1

Transcriptomal Profiling in Whole Human Blood. Assays

were performed as described by us previously.¹⁰Briefly, peripheral blood

mononuclear cells (PBMCs) isolated from fresh heparin-anticoagulated

human blood were stimulated with 20 μg/mL of the compounds for 2 h,

and total RNA was extracted from treated and negative control PBMC

samples with QIAamp RNA Blood Mini Kit (Qiagen). An amount of 4

μg of each of the RNA samples was used for transcriptomal profiling,

employing the human genome GeneChip U133 Plus 2.0 oligonucleotide

array (Affymetrix, Santa Clara, CA). Established standard protocols at

the KU Genomics Facility were performed on cRNA target preparation,

array hybridization, washing, staining, and image scanning. The micro-

array data were collected using the Affymetrix GeneChip Command

Console software (AGCC) and then subjected to quality assessment

before further analyses. QC criteria included low background, low noise,

detection of positive controls, and a 5⁰/3⁰ratio of <3.0. To facilitate

direct comparison of gene expression data between different samples,

the GeneChip data were first subjected to preprocessing, which included

scaling of data from all chips to a target intensity value of 500 (in

Affymetrix Expression Console software) and further normalization in

GeneSpring GX (Agilent Technologies, Santa Clara, CA). Prior to

identification of target genes, genes that were detected as nonexpressed

in all samples, i.e., those with absence (A) cells, were filtered out. Genes

whose expression was changed by our compounds by at least 2-fold

(compared to the negative control) were identified to be differentially

expressed. Pathway analyses of gene expression were performed using

IPA (Ingenuity Systems, Redwood, CA).

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’ ASSOCIATED CONTENT

Supporting Information. ¹H NMR, ¹³C NMR, and MS

S

b

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’ AUTHOR INFORMATION

Corresponding Author

*Phone: 785-864-1610. Fax: 785-864-1961. E-mail: sdavid@

ku.edu.

’ ACKNOWLEDGMENT

This work was supported by NIH/NIAID Contract HHS-

N272200900033C.

’ ABBREVIATIONS USED

AP-1, activator protein-1; CD, cluster of diﬀerentiation; DAP, dia-

minopimelic acid; DMAP, 4-dimethylaminopyridine; EC₅₀, half-

maximal eﬀective concentration; ESI-TOF, electrospray ionization

time of ﬂight; FmocCl, ﬂuorenylmethyloxycarbonyl chloride; Glu,

glutamic acid; HBTU, O-benzotriazole-N,N,N,N⁰-tetramethyluro-

nium hexaﬂuorophosphate; HEK, human embryonic kidney; iE-

DAP, γ-^D-glutamyldiaminopimelic acid; IL, interleukin; NF-κB, nu-

clear factor κB; Nod1, nucleotide oligomerization domain 1; Nod2,

nucleotide oligomerization domain 2; NLR, Nod-like receptor;

p38MAPK, p38 mitogen activated protein kinase; PBMCs, periph-

eral blood mononuclear cells; PE, phycoerythrin; PRRs, pattern

recognition receptors; RIG-I, retinoic acid inducible gene I; RNA,

ribonucleic acid; sAP, secreted alkaline phosphatase; SAR, struc-

ture-activity relationship; Th1, helper T lymphocyte type 1; Th2,

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Journal of Medicinal Chemistry

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dx.doi.org/10.1021/jm101535e |J. Med. Chem. 2011, 54, 1490–1510

Article Doi

Structure-activity relationships in nucleotide oligomerization domain 1 (Nod1) agonistic γ-glutamyldiaminopimelic acid derivatives

DOI: 10.1021/jm101535e

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Full text of DOI:10.1021/jm101535e

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