Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their in Vivo Adjuvant Activity

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Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists

Deﬁnes Their In Vivo Adjuvant Activity

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Samo Guzelj, Sanja Nabergoj, Martina Gobec, Stane Pajk, Veronika Klancic, Bram Slutter, Ruza Frkanec,

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Adela Stimac, Primoz Sket, Janez Plavec, Irena Mlinaric-Rascan, and Ziga Jakopin *

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ABSTRACT: We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-

domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We

identiﬁed two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows

superior adjuvant activity in vivo. Both compounds had immunostimulatory eﬀects on peripheral blood mononuclear cells at the

protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the

cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C₁₈lipophilic tail of 75 is identiﬁed as a pivotal

structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-

encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced

Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.

(ii) a centrally located nucleotide-binding domain that is

required for oligomerization; and (iii) a C-terminal leucine-

rich repeat domain that is implicated in ligand recognition.⁸

NOD2 is primarily expressed in leukocytes and intestinal

epithelial cells (especially Paneth cells), where it is required to

sense bacterial cell wall peptidoglycan fragments that enter the

cytosol.^8,9The minimal essential peptidoglycan substructure

that can still activate NOD2 is muramyl dipeptide (MDP), a

glycopeptide in the cell wall of both Gram-positive and Gram-

negative bacteria.¹⁰⁻¹³MDP comprises N-acetylmuramic acid

(MurNAc) and an ^L-alanine-D-isoglutamine dipeptide, which is

attached to the MurNAc via a lactic acid spacer. Recognition of

MDP is followed by self-oligomerization, through which

1. INTRODUCTION

Defense against invading pathogens in vertebrates is

accomplished through coordinated responses of the non-

speciﬁc innate and the antigen-speciﬁc adaptive immune

systems. The former orchestrates the ﬁrst line of defense

through the action of a superfamily of pattern recognition

receptors (PRRs). PRRs are responsible for recognition of

“nonself” features, which are conserved microbial components

that are also known as pathogen-associated molecular

patterns.^1,2As well as promotion of the immediate innate

immune response, PRRs are involved in shaping of the

gradually forming adaptive immune responses through engage-

ment of antigen-presenting cells.³Thus, both naturally and

synthetically derived modulators of PRRs have been of

considerable interest for medicinal chemists for development

as vaccine adjuvants.⁴⁻⁷

Received: April 8, 2021

Published: May 27, 2021

Nucleotide-binding oligomerization-domain-containing pro-

tein 2 (NOD2) belongs to the intracellular NOD-like receptor

family of PRRs, and it is composed of three motifs: (i) two

eﬀector N-terminal caspase recruitment domains (CARDs);

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Figure 1. MDP and its representative derivatives.

NOD2 recruits the adaptor protein receptor-interacting serine/

threonine kinase 2, RIP2, via CARD−CARD interactions. This

triggers the downstream signaling cascades that include the

mitogen-activated protein kinase and nuclear factor κB (NF-

κB) pathways, which results in a wide array of immune

responses.¹⁴These are characterized by release of pro-

inﬂammatory cytokines, chemokines, and antimicrobial factors

(including defensins), generation of reactive nitrogen species,

and recruitment and priming of neutrophils, inﬂammatory

monocytes, and dendritic cells (DCs).¹⁵⁻¹⁷Of note, NOD2

activation also facilitates autophagy, an essential process for

eﬃcient antigen processing and activation of the adaptive

immune system.^18,19

Due to their broad immunomodulatory eﬀects, NOD2

agonists are of signiﬁcant clinical relevance. They have been

highlighted for providing nonspeciﬁc protective eﬀects against

bacterial and viral infections.²⁰Furthermore, NOD2 is an

enticing target for expansion of the currently limited selection

of vaccine adjuvants.²¹As well as generating robust and

sustainable systemic immune responses, recent reports have

additionally extended the potential of NOD2-activating

adjuvants to mucosal vaccines.^22,23These represent an

attractive alternative to conventional vaccines, as they generate

mucosal immune responses, which are essential for protection

against pathogens transmitted through mucosal surfaces.

Finally, the developing ﬁeld of cancer immunotherapy has

highlighted NOD2 agonists as potential immunotherapeutics,

either as adjuvants in cancer vaccines, or by directly enhancing

immune cell antitumor activity.⁷

structure−activity relationships.²⁹⁻³¹Notably, two lipophilic

derivatives of MDP, known as romurtide³²and mifamurtide,³³

are currently in use for the treatment of leukopenia and

osteosarcoma, respectively, while a hydrophilic MDP deriva-

tive, murabutide, has been investigated in several clinical trials

as a vaccine adjuvant (Figure 1).^34,35

The discovery that the full glycopeptide scaﬀold is not

mandatory for NOD2 agonism led to the concept of

desmuramylpeptides, a class of compounds that lack the

MurNAc moiety. Replacement of this carbohydrate fragment

with suitable surrogates can simplify their synthesis and allow

for easier manipulation of their lipophilicity. Most desmur-

amylpeptides comprise the preserved or slightly varied MDP

dipeptide motif plus various lipophilic groups, which have

included carbocycles,³⁶adamantane,³⁷⁻³⁹and diverse aromatic

moieties.⁴⁰⁻⁴⁶Here, we describe the design, synthesis, and

biological evaluation of a series of novel acylated desmuramyl-

tripeptides that are based on the structure of 1, which is a

potent NOD2 agonist that was previously reported by our

group and contains a trans-feruloyl-glycine MurNAc mim-

etic.⁴⁶Our current study identiﬁed the structural requirements

for in vitro and in vivo immunostimulatory activity of 1, which

led to the surprising observation that NOD2 activation in vitro

does not necessarily coincide with its adjuvant activity in vivo.

We identiﬁed 68 as a potent in vitro NOD2 agonist with more

than 2-fold improved potency over 1 and 75 with superior

adjuvant activity in vivo. Compounds 68 and 75 induced

proinﬂammatory transcriptional changes and cytokine produc-

tion in peripheral blood mononuclear cells (PBMCs), both

alone and in combination with lipopolysaccharide (LPS), and

enhanced antigen presentation of DCs. Furthermore, 75

stimulated the cytotoxic activity of PBMCs against malignant

cells and, importantly, had promising in vivo adjuvant activity

with a balanced Th1/Th2 immune response in a mouse model

of adjuvanticity.

MDP was ﬁrst recognized as the minimal eﬀective

component of Freund’s complete adjuvant.²⁴While MDP

promotes both innate and adaptive immune responses, its use

in the clinic is hindered by its strong pyrogenicity,^25,26rapid

elimination,²⁷and metabolic instability.²⁸To circumvent these

issues and to improve its clinical utility, structural modiﬁca-

tions of MDP have been studied extensively, with several

reviews available that have comprehensively described their

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Figure 2. Design of novel desmuramylpeptides based on 1.

adjuvanticity.⁵³Likewise, chain elongation with L-lysine at the

C-terminus, which mimics the structure of peptidoglycan from

Gram-positive bacteria, achieved a similar immunostimulatory

eﬀect compared to that of MDP.⁵⁴⁻⁵⁶By applying both

principles to 1 (see Figure 2), derivatives were designed where

L-valine was replaced by L-serine and by its more lipophilic

congeners O-benzyl-^L-serine and L-threonine (compounds 30−

32; Table 1), as well as by compounds where the peptide was

elongated at the ω-carboxyl group of ^D-glutamic acid with a

methyl ester of either ^L-lysine or N⁶-stearoyl-L-lysine; this latter

closely resembles the structure of romurtide (compounds 41

and 82; Table 1).

While 1 had potent activity in in vitro assays, it induced less

pronounced increases in mouse serum titers of antigen-speciﬁc

IgG upon ovalbumin immunization. We hypothesized that the

disparity between the in vitro and in vivo data was a

consequence of poor pharmacokinetic properties, including

the metabolic instability of the ester groups. Compound 1 was

suggested to be a prodrug by in vitro experiments, which was

supported by in silico experiments, with the need for hydrolysis

of the ethyl esters for its activation. However, if this occurs

prior to 1 reaching its eﬀector cells, this will be detrimental to

its eﬀects. We also demonstrated that the hydrolyzed free acid

was less able to cross the cell membrane compared to its parent

diester compound.⁴⁶Additionally, while MDP enters cells

through the SLC15 peptide transporters and endocytosis,⁵⁷⁻⁶⁰

desmuramylpeptides cross the cell membrane by passive

absorption to reach their target receptor NOD2 in the

cytoplasm.⁶¹

2. RESULTS AND DISCUSSION

2.1. Design. Compound 1 comprises a trans-feruloyl-

glycine MurNAc mimetic attached to the ^L-valine-D-glutamate

dipeptide, and it showed good NOD2 agonistic activity with

twice the potency of MDP in a HEK293 cell assay with

overexpressed NOD2. It also enhanced the LPS-induced

release of proinﬂammatory cytokines and was devoid of

pyrogenicity, although it showed weak adjuvant activity in a

mouse model of adjuvanticity.

Recent biophysical data have suggested that MDP interacts

with NOD2 through the leucine-rich repeat domain.⁴⁷

Maekawa et al. (2016) solved the crystal structure of NOD2

apoprotein in its ADP-bound inactive form.⁴⁸To date,

however, no crystal structure of NOD2 with a bound ligand

has been reported, and thus the exact binding mechanisms of

MDP and its related compounds (including 1) remain to be

deﬁned. Previous studies of 1 have suggested that the aromatic

ring of the trans-feruloyl moiety contributes to NOD2 binding

with π−π stacking and cation−π interactions, while both the 4-

hydroxy and 3-methoxy groups form H-bonds with residues in

the putative binding site.⁴⁶To determine whether these

interactions can be optimized, we designed several derivatives

with modiﬁed substitutions of the aromatic ring (Figure 2).

Furthermore, we evaluated both increased ﬂexibility of (i.e.,

reduction of the feruloyl alkene bond) and conformational

restrictions to (i.e., introduction of the cyclopropyl ring) the

trans geometry. Notably, cyclopropyl fragments have an

established track record in drug design, in part due to their

“locking” of E/Z isomerizable alkene bonds in favorable

conformations.⁴⁹Exploration of the chemical space around the

MurNAc surrogate moiety thus yielded compounds 18−21

and 24−28 (Figure 2, Table 1).

Taking these aspects into account, we set out to modify the

pharmacokinetic properties of the parent molecule, according

to two diﬀerent approaches. First, the introduction of lipophilic

acyl groups to the carbohydrate⁶²and D-isoglutamine⁶³

moieties of MDP was previously shown not only to improve

the adjuvant and immunoprotective properties but also to

decrease the pyrogenicity of these derivatives. Analogous

transformations were applied to 1 by acylation of the phenolic

hydroxyl group on the trans-feruloyl moiety, as well as by

replacing the ethyl esters with bulkier groups, to yield a library

Recognition of MDP by NOD2 is highly stereospeciﬁc.

While deviations from the ^L−D conformation of the L-alanine-

D-isoglutamine pharmacophore can result in reduced or lack of

activity, slight variations of the amino acids are permissible. For

example, replacement of ^L-alanine with L-valine or L-serine

showed comparable activity.⁵⁰⁻⁵²Additionally, the L-threonine

derivative decoupled the pyrogenic activity of MDP from its

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a

Scheme 1. Synthesis of Compounds 1−32

a

Reagents and conditions: (i) Boc-L-Val or Boc-O-benzyl-L-Ser or Boc-L-Thr, DCC, HOBt, Et₃N, DMAP, EtOAc, rt; (ii) TFA/DCM (1:5), rt; (iii)

Boc-Gly, DCC, HOBt, Et₃N, DMAP, EtOAc, rt; (iv) RCOOH, EDC, HOBt, DIPEA, DMAP, DMF, rt; (v) H₂, Pd/C, CH₃COOH, rt.

a

Scheme 2. Synthesis of Cyclopropane Carrying trans-Ferulic Acid Derivatives

a

Reagents and conditions: (i) SOCl₂, EtOH, reﬂux; (ii) CH₃COCl, Et₃N, THF, rt; (iii) NaH, trimethylsulfoxonium iodide, DMSO, 50 °C; (iv) 1

M NaOH, EtOH, rt; (v) BnCl, K₂CO₃, DMF, 80 °C; (vi) H₂, Pd/C, rt; (vii) Pd(OAc)₂, Et₃SiH, Et₃N, DCM, rt.

of prodrug derivatives (56, 68, 72−81; Table 3). Among these,

the cyclopentyl derivatives might serve a dual role. In addition

to increasing the lipophilicity, chemical motifs that incorporate

cyclopentyl esters have been reported to be selectively cleaved

by human carboxylesterase-1, an enzyme that is restricted in

expression to hepatocytes and cells of monocyte−macrophage

lineage.⁶⁴As these latter cells express high levels of NOD2,

implementation of these esterase-sensitive chemical motifs

might lead to beneﬁcial buildup of the hydrolyzed active

compound only in these cells, given that charged acids would

have little possibility to leave the cells.

Second, we explored the chemical space of the ^D-glutamic

acid moiety with various mimetics of the carboxylic acid

functionality. Namely, we introduced bioisosteric replacements

of ester moieties with amides and esters of hydroxamic acid

(compounds 57−64; Table 2). Hydroxamates have previously

been used as successful bioisosteric replacements of carboxylic

acid groups.⁶⁵Amides, on the other hand, are well established

for their potential as prodrugs of carboxylic acids.⁶⁶

2.2. Chemistry. To prepare compounds with modiﬁcations

of the trans-ferulic acid moiety, we designed a scalable

divergent synthetic route that comprised the sequential

deprotection and amide bond formation steps shown in

Scheme 1. ^D-Glutamic acid was ﬁrst esteriﬁed with thionyl

chloride in ethanol, to produce the diester 2. Coupling of 2 to

commercially available Boc-protected ^L-valine with dicyclohex-

ylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) pro-

duced the dipeptide 3. Boc deprotection of 3 with triﬂuoro-

acetic acid (TFA) in dichloromethane (DCM) produced the

deprotected TFA salt, which was immediately coupled to Boc-

glycine using DCC/HOBt. Treatment of the resulting 6 with

TFA produced the deprotected salt, which allowed diversiﬁ-

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Scheme 3. Synthesis of Compound 41

a

Reagents and conditions: (i) EtOH, DCC, DMAP, DCM, rt; (ii) TFA/DCM (1:5), rt; (iii) Boc-L-Val, DCC, HOBt, Et₃N, DMAP, EtOAc, rt; (iv)

Boc-Gly, DCC, HOBt, Et₃N, DMAP, EtOAc, rt; (v) H₂, Pd/C, EtOH, rt; (vi) 37, COMU, DIPEA, DMF, rt; (vii) 39, NaHCO₃, THF/H₂O, rt;

(viii) DBU, 1-octanethiol, THF, rt.

cation via coupling to various carboxylic acids using the 1-

ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC)/

HOBt coupling strategy, to produce acyl tripeptides of 1,

19−23, and 25−29. Compound 1 was additionally reduced by

catalytic hydrogenation to produce the more ﬂexible congener

18, while 23 was deprotected with TFA to produce the free

amine 24.

Analogously, desmuramylpeptides with a modiﬁed central

amino acid were synthesized by coupling 2 to commercially

available Boc-protected ^L-serine (O-benzyl protected) and L-

threonine, to produce dipeptides 4 and 5. Following

deprotection with TFA, DCC/HOBt coupling to Boc-glycine

produced the tripeptides 7 and 9. Catalytic hydrogenation of 7

produced the deprotected 8, after which compounds 7, 8, and

9 were deprotected with TFA and coupled with trans-ferulic

acid using the EDC/HOBt coupling strategy, to produce, in

turn, the desmuramylpeptides 30, 31, and 32.

Johnson−Corey−Chaykovsky reaction resulted in cleavage of

the acetyl group, along with subsequent methylation of the in

situ liberated 4-phenol group, to produce 12, which then

underwent alkaline hydrolysis, to produce the 3,4-dimethoxy

derivative 13. To avoid deprotection of the hydroxyl group, the

less labile double benzyl protection was used, with trans-ferulic

acid reacted with benzyl chloride, to produce 14. Cyclo-

propanation of 14 produced 15. Removal of the benzyl groups

from 15 by catalytic hydrogenation over palladium/carbon

unexpectedly resulted in the opening of the cyclopropyl ring

(16), while a milder debenzylation method using palladium

acetate, triethylsilane, and triethylamine was used to produce

the desired compound, 17.⁶⁷

The synthesis of compounds with an additional L-lysine

residue incorporated is shown in Scheme 3. First, the 5-benzyl

ester of Boc-^D-glutamic acid was subjected to DCC/HOBt-

mediated coupling with ethanol, to produce compound 33.

TFA-mediated cleavage of the Boc protecting group and

subsequent DCC/HOBt coupling was performed twice, ﬁrst

with Boc-^L-valine, and then with Boc-glycine, to produce the

tripeptide 35. Following cleavage of the benzyl ester of 35 with

catalytic hydrogenation, the free acid 36 was coupled to Fmoc-

protected ^L-lysine (37; Supporting Information, Scheme S1)

Cyclopropane mimetics of trans-ferulic acid were synthe-

sized as shown in Scheme 2. trans-Ferulic acid was protected in

sequential steps, ﬁrst with thionyl chloride in ethanol to

produce the ethyl ester 10, which was reacted with acetyl

chloride to produce the doubly protected compound 11.

Cyclopropanation of the double bond of 11 using the

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Scheme 4. Synthesis of Compounds 56−64

a

Reagents and conditions: (i) trans-ferulic acid, EDC, HOBt, DIPEA, DMAP, DMF, rt; (ii) 1 M NaOH, EtOH, rt; (iii) 44, EDC, HOBt, DIPEA,

DMAP, DMF, rt; (v) 47−55, EDC, HOBt, DIPEA, DMAP, DMF, rt.

using (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-

dimethylamino-morpholino-carbenium hexaﬂuorophosphate

(COMU) as the coupling reagent, to produce 38. Then 38

was converted to 40 via a two-step sequence that involved

cleavage of the Boc protecting group with TFA, and

subsequent coupling to the N-hydroxysuccinimide-activated

trans-ferulic acid 39 (Supporting Information, Scheme S2).

Finally, Fmoc deprotection of the tetrapeptide 40 under

alkaline conditions generated by 1,8-diazabicyclo[5.4.0]undec-

7-ene (DBU) in tetrahydrofuran (THF), with 1-octanethiol

acting as the dibenzofulvene scavenger,⁶⁸produced the desired

desmuramylpeptide 41.

Assembly of the desmuramylpeptides with modiﬁcations of

the ^D-glutamic acid moiety was carried out as shown in Scheme

4. Glycine was ﬁrst esteriﬁed with thionyl chloride in ethanol

to produce the ethyl ester 42, which was then coupled to trans-

ferulic acid using the EDC/HOBt coupling strategy. Next, the

N-feruloyl-glycine 43 produced was deprotected with alkaline

hydrolysis, and subsequently coupled with the ethyl ester of ^L-

valine (44) using EDC/HOBt, to produce the acyl dipeptide

45. Alkaline hydrolysis of 45 produced the free acid 46.

Diversiﬁcation by EDC/HOBt-mediated coupling to various ^D-

glutamic acid derivatives (47−55; Supporting Information,

Scheme S3) produced the desmuramylpeptides 56−64, with

incorporated carboxylic acid bioisosteres.

A synthetic procedure similar to that described in Scheme 1

was used for the synthesis of lipophilic esters 68 and 72

(shown in Scheme 5). Boc-^D-glutamic acid was ﬁrst esteriﬁed

with cyclopentanol using EDC to produce the corresponding

diester 65. Following Boc deprotection with TFA, the ensuing

coupling to Boc-protected ^L-valine produced the dipeptide 66.

Similarly, acid-catalyzed esteriﬁcation of ^D-glutamic acid with

1-octadecanol produced the diester 69, which was coupled to

Boc-^L-valine to produce the dipeptide 70. Compounds 66 and

70 were then subjected to two iterative cycles of TFA-

mediated Boc deprotection with consecutive coupling, ﬁrst to

Boc-glycine, to produce 67 and 71, and ultimately to trans-

ferulic acid, to produce the ester congeners of 1, 68 and 72.

Compounds 1, 30, 31, 32, 41, and 68 were further acylated

with acyl chlorides of varying chain lengths in the presence of

triethylamine to produce the lipophilic ester derivatives 73−82

(Scheme 6).

2.3. Biological Studies. 2.3.1. NOD2 Agonistic Activity of

Synthesized Desmuramylpeptides. To determine the NOD2

agonistic potential of the synthesized desmuramylpeptides at

the cellular level, we used the validated and commercially

available HEK-Blue NOD2 cell line reporter assay. HEK-Blue

NOD2 cells were ﬁrst treated for 18 h with MDP, 1, or the

novel desmuramylpeptides at 2 μM. The NF-κB transcriptional

activity measured was normalized to that of the vehicle treated

(0.1% DMSO) control HEK-Blue NOD2 cells. The com-

pounds that showed signiﬁcant activity at 2 μM were further

assayed for their dose-dependent activities, for determination

of their EC₅₀values. None of the tested compounds were

cytotoxic toward the HEK-Blue NOD2 cells at the highest

tested concentration (20 μM), as determined by the (3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-

phenyl)-2H-tetrazolium) (i.e., MTS) cell viability assay

(Supporting Information, Figure S1).

Exploration around the chemical space of the cinnamoyl

moiety of the parent compound 1 (EC₅₀= 89 nM) produced

compounds 18−21 and 24−28 (Table 1). Increased ﬂexibility

through the reduction of the double bond (18; EC₅₀= 6.16

μM) resulted in decreased NOD2 agonistic activity by a factor

of 70. Similarly, the NOD2 agonistic activity was decreased by

a factor of 156 with the spacer prolonged to a propylene group

(19; EC₅₀= 13.9 μM), which indicated that 1 provides the

optimal positioning of the aromatic ring. The 4-hydroxy-3-

methoxy substitution pattern of the aromatic ring in 1 was also

seen to be advantageous; namely, the 3,4-dimethoxy (20; EC₅₀

= 763 nM), 3,4-dihydroxy (21; EC₅₀= 1.73 μM), and 4-amino

(24; EC₅₀= 542 nM) derivatives all showed lower activities.

Interestingly, the 4-isopropyl (25; EC₅₀= 71 nM) and the 4-

nitro (26; EC₅₀= 49 nM) derivatives showed similar NOD2

agonistic activity to 1, despite their contrasting electronic

properties, which indicated that the interactions with the

protein of the group in the 4-position are primarily of a

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a

cyclopropyl ring), and the relative levels of those two species in

the mixture could not be determined. Considering that it is

likely that only one of them binds ideally to NOD2, changes in

their proportions would inﬂuence the EC₅₀determined, which

provides a possible explanation of the disparate data obtained

after cyclopropanation of 1 and 20. Our previous study also

identiﬁed 29, a cyclopropyl derivative with a 3,4-diﬂuoro

substituted aromatic ring.⁴⁶With an EC₅₀of 49 nM, 29 was

the most potent derivative in the cyclopropyl series. With the

aim to ascertain the potencies of individual diastereomers, we

subjected 29 to chiral HPLC resolution (Supporting

Information, Figures S4, S5, and S6), which yielded very low

quantities of its pure diastereomers 29a (EC₅₀= 66 nM) and

29b (EC₅₀= 39 nM), with a relatively small diﬀerence between

Scheme 5. Synthesis of Compounds 68 and 72

1

their NOD2 activities. H−¹H NOESY analysis revealed that

both 29a and 29b contained a trans-conﬁgured cyclopropyl

ring (Supporting Information, Figure S9); however, as for 27

and 28, it was not possible to determine their absolute

stereochemical conﬁgurations here. Nonetheless, as this

approach provided only marginal improvements in the

NOD2 activity, we posited that their chiral resolution would

serve no direct purpose here and that the diastereomeric

mixtures of cyclopropane featuring derivatives do not need to

be separated.

Next, we examined the eﬀects of modiﬁcations to the amino-

acid structure (30−32, 41, 82; Table 1). The MDP analogs

with ^L-threonine and L-serine were previously shown to be

suitable substitutes for the ^L-alanine, as demonstrated by their

retained in vivo adjuvant activities;^50,51however, these

substitutions have yet to be evaluated in the context of

desmuramylpeptides. Interestingly, desmuramylpeptides with

both ^L-threonine (32; EC₅₀= 12.5 μM) and L-serine (31; EC₅₀

> 20 μM) showed relatively poor activities. O-Benzylation of L-

serine (30; EC₅₀= 5.56 μM) slightly improved the activity,

perhaps indicating the importance of the bulky nature of this

amino-acid side chain. Lower activity was also seen when the

peptide chain was extended by ^L-lysine (41; EC₅₀= 10.2 μM).

This might be attributable to decreased membrane perme-

ability at physiological pH, due to the presence of an ionized

amine group; however, amidation of this group with a

lipophilic stearoyl chain (82; EC₅₀= 11.5 μM) did little to

improve the activity. Similar data were obtained in a study by

Eﬀenberg et al. (2017), where norAbuMDP-Lys-L18 (a

derivative of MDP with stearoyl-^L-lysine) showed reduced

potency compared to MDP, despite the resemblance of both

compounds to romurtide.⁷⁰

a

Reagents and conditions: (i) cyclopentanol, EDC, DMAP, DCM, rt;

(ii) 1-octadecanol, pTsOH, toluene, reﬂux; (iii) TFA/DCM (1:5), rt;

(iv) Boc-^L-Val, DCC, HOBt, Et₃N, DMAP, EtOAc, rt; (v) Boc-L-Val,

EDC, HOBt, DIPEA, DMAP, DCM, rt; (vi) Boc-Gly, DCC, HOBt,

Et₃N, DMAP, EtOAc, rt, or Boc-Gly, EDC, HOBt, DIPEA, DMAP,

DCM, rt; (vii) trans-ferulic acid, EDC, HOBt, DIPEA, DMAP, DMF

or DCM, rt.

Exploration of the chemical space around the D-glutamic

acid moiety produced desmuramylpeptides 57−64 (Table 2).

Both functionalized carboxamates and amides were evaluated

as potential bioisosteric replacements of the carboxylic acid

functionality. When compared to the 2.83-fold and 3.06-fold

NOD2 activations of MDP and 1 in a single point assay, with

respect to untreated control cells, these compounds showed

considerably diminished NOD2 agonistic activity at 2 μM

(1.30-fold to 1.45-fold NOD2 activation). These data are in

agreement with the previously reported impaired activities of

MDP derivatives that have either a diamidated ^D-isoglutamine

moiety⁵²or a lipophilic amide attached to the α position of D-

isoglutamine.⁷¹Slightly better activities were expected for the

derivatives with hydroxamate, given that hydroxamic acids are

readily hydrolyzed to their corresponding carboxylic acids.⁷²

However, it is worth noting that HEK293 cells have low

hydrolytic activity due to low expression of carboxylesterases,

hydrophobic nature and are related to the size of the

substituent. A nitro-substituted cinnamic acid was previously

used in the design of MDP-C, which is a potent in vivo NOD2

agonist; however, in contrast to 26, the nitro group was

attached to the ^D-glutamic acid moiety.⁶⁹Cyclopropanation of

the alkene moiety in the structure of the 3,4-dimethoxy

derivative 20 produced 27 (EC₅₀= 322 nM) with a 2.4-fold

improved potency over 20, which suggests that the cyclo-

propane ring assists in directing the aromatic ring to a more

favorable position. Surprisingly, by applying the same concept

to 1, which thus produced 28 (EC₅₀= 369 nM), the potency

1

was reduced by a factor of 4. The H−¹H NOESY spectra

revealed that both 27 and 28 retained the trans orientation of

the substituents on the cyclopropane ring (Supporting

Information, Figures S7 and S8). This analysis, however, did

not diﬀerentiate between the two trans-conﬁgured diaster-

eomers (i.e., carrying the R,R and S,S conﬁgurations on the

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a

Scheme 6. Synthesis of Compounds 73−82

a

Reagents and conditions: (i) RCOCl, Et₃N, THF, rt; (ii) C₁₇H₃₅COCl, Et₃N, DMF, rt.

the enzymes involved in the hydrolysis of both hydroxamic

acids and amides.^73,74Higher susceptibility of ester groups to

both enzymatic and spontaneous hydrolytic processes might

explain the superior eﬀects of 1 and other ester derivatives in

the HEK-Blue NOD2 cell assays. Nonetheless, given that

direct bioisosteric replacement of both ethyl ester groups with

their ethyl amide counterparts resulted in substantially

diminished activity of 57, these data provide conﬁrmation of

our previous in vitro and docking studies that suggested that

the 1 ester moieties predominantly assist in compound

internalization and do not contribute to the NOD2 binding.⁴⁶

These data encouraged us to introduce the last group of

modiﬁcations, in which we retained the original pharmaco-

phore of 1 and increase its lipophilicity by introducing

cleavable ester groups of varying sizes to the cinnamoyl and

D-glutamic acid moieties. As the cellular assays are deﬁned by

both the crossing of the membrane by the compounds and

their activation of NOD2, the lipophilicity of these compounds

will have a major role in their cellular NOD2 activities. The

phenolic hydroxy group served as a useful attachment point for

the introduction of acetyl (C₂), lauroyl (C₁₂), and stearoyl

(C₁₈) groups through esteriﬁcation. Likewise, the two ethyl

ester functional groups were readily replaced with bulkier

cyclopentyl, tert-butyl, and stearyl groups. The NOD2

agonistic activities of the resulting prodrug derivatives are

summarized in Table 3.

Of note, there was an inverted U-shape correlation between

the compound NOD2 activities and their lipophilicities (as

calculated logP [ClogP] values; Figure 3). Namely, the

desmuramylpeptides with ClogP in the 1.7 to 3.8 range

showed increasing NOD2 activation, where compound 68 had

an EC₅₀of 40 nM, an over 2-fold improvement over 1 (EC₅₀

=

89 nM). Compound 74, a derivative with a lauroyl tail on the

aromatic ring, was the most active NOD2 agonist of this series

(EC₅₀= 30 nM), despite a signiﬁcantly increased ClogP of

7.08. Interestingly, lauric acid (C₁₂) was previously shown to

activate NOD2 and induce IL-8 secretion from HCT116 colon

epithelial cells.⁷⁵To determine whether the increased activity

of 74 here is a result of the release of lauric acid after

hydrolysis, the HEK-Blue NOD2 cells were also treated with

lauric acid, both alone and in combination with 1. In contrast

to previous indications, lauric acid did not show any NOD2

activation alone, and it did not enhance the NOD2 agonistic

activity of 1 (data not shown). Further increases in the

lipophilicity resulted in a sharp drop in the in vitro NOD2

agonistic activity. Extending the C₁₂chain of 74 to a longer C₁₈

chain, which produced 75 (EC₅₀= 2.83 μM), resulted in

markedly diminished activity, by a factor of 90, although the

stearoyl group is a structural motif that has been used

repeatedly in the preparation of potent MDP derivatives.

We postulated that the diﬀerences in NOD2 activation here

can be attributed to more facile cleavage of the shorter acetyl

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Table 1. NOD2 Agonistic Activities of Compounds with Structural Modiﬁcations of the MurNAc Mimetic, the Central Amino

Acid, and the Eﬀect of Chain Elongation by ^L-Lysine

a

SEAP activities were measured in NOD2-speciﬁc HEK-Blue cell supernatants after incubation for 18 h with MDP (2 μM) or the compounds of

interest (2 μM). The data are shown as fold increases of NF-κB transcriptional activity relative to the negative control (0.1% DMSO) and are

b

expressed as mean SEM of at least two independent experiments. EC₅₀values are expressed as mean SEM of at least two independent

c

experiments with 7 or 8 concentrations used (from 1 nM to 20 μM). Mixture of two diastereomers (R,R and S,S conﬁgurations of substituents on

d

the cyclopropyl ring). It was not possible to conclusively determine the absolute stereochemical conﬁgurations of 29a and 29b; however, both

contained a trans-conﬁgured cyclopropyl ring (R,R and S,S conﬁgurations).

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Table 2. NOD2 Agonistic Activities of the Desmuramylpeptides with a Modiﬁed D-Glutamic Acid Moiety

a

SEAP activities were measured in NOD2-speciﬁc HEK-Blue cell supernatants after incubation for 18 h with MDP (2 μM) or the compounds of

interest (2 μM). The data are shown as fold increases of NF-κB transcriptional activity relative to the negative control (0.1% DMSO) and are

b

expressed as mean SEM of at least two independent experiments. Mixture of two diastereomers with L and D conﬁgurations of valine.

and lauroyl esters, compared to the longer stearoyl esters, by

the intracellular enzymes in the HEK293 cell line used. We

thus expected that the introduction of the acetyl, lauroyl, and

stearoyl groups to the structure of the dicyclopentyl congener

68 would follow the same activity trend, with the lauroyl

derivative additionally improving the activity of 68. Un-

expectedly, the resulting C₁₂counterpart 77 (EC₅₀= 243 nM)

showed a sixth of the activity of 68. As exempliﬁed by 78

(EC₅₀= 6.16 μM), extending the lipophilic tail to C₁₈further

abrogated the NOD2 activation. These data demonstrate that

NOD2 activation by these compounds is most likely linked

directly to their lipophilicity. This is further supported by the

NOD2 agonistic activities of the two acetylated derivatives, the

diethyl ester 73 (EC₅₀= 62 nM), and its dicyclopentyl

surrogate 76 (EC₅₀= 63 nM). These two maintained similar

ClogP values and consequently retained the EC₅₀values of

their congeners with a free phenolic hydroxy group. Finally,

replacing both of the ethyl groups of 1 with stearyl esters (i.e.,

72) almost completely abrogated the NOD2 agonistic activity.

Similar structure−activity relationships were identiﬁed pre-

viously and attributed to the diﬀerent interactions of the

lipophilic MDP analogs with the biomembranes. While higher

lipophilicity facilitated membrane permeability, it also

increased the association of the compounds with the

membrane.⁷⁶The retention of the lipophilic derivatives in

the membrane might explain their reduced eﬀects in the cell

assays here, as they will then not be available to bind to NOD2,

which is located in the cytoplasm. Conversely, these same

eﬀects might be beneﬁcial and lead to enhanced activities of

such derivatives in in vivo assays, especially when liposomes are

used as the lipophilic delivery system.

As expected, similarly reduced NOD2 activation was shown

when a stearoyl chain was introduced into the structure of 30

(2.04-fold activation at 2 μM) to produce 79 (1.68-fold), a

derivative with O-benzyl-^L-serine replacement of the L-valine.

However, when a stearoyl chain was introduced into 31 (1.21-

fold) to produce 80 (the ^L-serine based analog; 1.21-fold)

there was almost no change to the NOD2 activation, while

when a stearoyl chain was introduced into 32 (the L-threonine

based analog; EC₅₀= 12.5 μM) to produce 81 (EC₅₀= 1.45

μM), this showed a 10-fold improvement in potency.

Compound 56 provided a notable deviation from the

ClogP−EC₅₀relationship, where there were two tert-butyl ester

groups on the ^D-glutamic acid. tert-Butyl esters are

considerably less hydrolyzable by intracellular esterases, and

especially by human carboxylesterase-1. On the other hand,

cyclopentyl esters were previously shown to be excellent

substrates for hydrolysis by human carboxylesterase-1.⁶⁴Given

the low carboxylesterase activity of HEK293 cells, it is no

surprise that 56 resulted in markedly reduced NOD2

activation, despite its favorable ClogP (3.30).

As NOD2 and NOD1 agonists share certain structural

characteristics, we wanted to determine whether these

compounds selectively target NOD2. Selectivity against

NOD1 of MDP and all of these synthesized desmuramylpep-

tides was analyzed in an analogous assay with the HEK-Blue

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Table 3. NOD2 Agonistic Activities of the Lipophilic Desmuramylpeptides

a

SEAP activities were measured in NOD2-speciﬁc HEK-Blue cell supernatants after incubation for 18 h with MDP (2 μM) or the compounds of

interest (2 μM). The data are shown as fold increases of NF-κB transcriptional activity relative to the negative control (0.1% DMSO) and are

b

expressed as mean SEM of at least two independent experiments. EC₅₀values are expressed as mean SEM of at least two independent

c

experiments with 7 or 8 concentrations used (from 1 nM to 20 μM). ND, not determined. ClogP values as calculated by the ChemDraw software.

without the NOD2 antagonist pretreatment. The comparative

reduction in NF-κB transcriptional activities indicated that the

eﬀects of these desmuramylpeptides are due to their NOD2

activation (Supporting Information, Figure S3).

We note here that the readouts from the HEK-Blue cell

assay system might not accurately represent the behaviors of

these synthesized desmuramylpeptides under in vivo con-

ditions. Thus, further biological evaluations were carried out

for the most potent in vitro NOD2 agonists, 68, 73, and 74,

also with the inclusion of two derivatives with a stearoyl group,

75 and 81. The C₁₈lipophilic tail was shown to be beneﬁcial

for development of MDP derivatives with activities in vivo, in

part due to its anchoring in the membrane of the liposomes⁷⁸

used as the delivery system of choice for the in vivo

applications of NOD2 agonists. As the experimental design

included the investigation of in vivo adjuvant activities of

NOD2 agonists encapsulated in a liposomal formulation,

inclusion of 75 and 81 in further testing was thus warranted,

despite their lower NOD2 activation in the HEK-Blue NOD2

cell assays.

Figure 3. ClogP−EC₅₀relationship of the compound 1 prodrug

derivatives.

NOD1 cell line. None of the compounds tested showed any

signiﬁcant activity at 2 μM, which thus conﬁrmed their

selectivity for NOD2 (Supporting Information, Figure S2).

Furthermore, the speciﬁcities were determined by pretreat-

ing HEK-Blue NOD2 cells with a previously reported NOD2

antagonist⁷⁷prior to stimulation with MDP (2 μM) or the

desmuramylpeptides (2 μM). The resulting NF-κB-induced

SEAP activities were compared to those of the controls

2.3.2. Immunostimulatory Eﬀect of Desmuramylpeptides

on PBMCs. The immunostimulatory eﬀects of the selected

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Figure 4. Eﬀects of the MDP and desmuramylpeptide treatments on the release of cytokines from human PBMCs. Cytokine concentrations were

measured after 18 h stimulation with MDP (2 μM) or the desmuramylpeptides (2 μM) in the absence or presence of LPS (10 ng/mL). Data are

expressed as mean SEM of 4 independent experiments. ^#p < 0.05, ^###p < 0.001 versus untreated controls; *p < 0.05, **p < 0.01, ***p < 0.001

versus LPS-treated PBMCs.

desmuramylpeptides were evaluated using human primary

peripheral blood mononuclear cells (PBMCs). This heteroge-

neous mixture of immune cells allowed the study of the eﬀects

of these NOD2 agonists in a physiologically relevant system,

where other NOD2-interacting and downstream signaling

proteins were present. The eﬀects of desmuramylpeptides were

ﬁrst determined for the secreted cytokine proﬁle using a

cytometric bead array cytokine kit. Moreover, considering that

activation of NOD2 is also an important ampliﬁcation signal

for Toll-like receptor (TLR)-induced inﬂammatory re-

sponses,⁷⁹⁻⁸¹we also examined the eﬀects on cytokine

secretion of the combination of desmuramylpeptides with

LPS, a well-known TLR4 agonist.

Figure 4 shows the eﬀects of the desmuramylpeptides (2

μM) on induction of the cytokines IL-1β, IL-6, IL-8, and TNF-

α, both alone and in combination with LPS (10 ng/mL).

These data show similar structure−activity relationships to the

data obtained in the SEAP reporter gene assays. Compounds

68, 73, and 74 by themselves induced small increases in the

levels of all four of these cytokines, with similar eﬀects to

MDP, while 75 and 81 induced lower responses. In line with

previous studies, the eﬀects of MDP and the desmuramylpep-

tides were most pronounced for the induction of the

chemokine IL-8.⁸²

described synergistic signal ampliﬁcation between NOD2 and

TLR4.^83,84On the other hand, the IL-8 produced appeared to

plateau following the combined stimulation with LPS and the

NOD2 agonists, which thus indicated the saturation of IL-8

production in these PBMCs.

Among the diverse PBMC subpopulations, natural killer

(NK) cells express high levels of functional NOD2.⁸⁵As innate

immune cells, NK cells have a central role in immunosurveil-

lance, by their detection and destruction of virus-infected and

cancer cells, through both direct cytolytic activity and release

of cytokines, which can further facilitate the recruitment and

activation of other innate and adaptive immune cell types.

Stimulation of NK cells with MDP activates the NF-κB

signaling pathway, which results in expression and release of

TNF-α and interferon (IFN)-γ, as the key regulators of the

Th1 cellular immune response.⁸⁵This is further enhanced

synergistically by IL-12 and IFN-α, which suggests a role for

accessory-cell-derived cytokines in the formation of an optimal

NK cell response.⁸⁶MDP-stimulated NK cells also show

enhanced cytotoxicity toward the Tu167 squamous cell

carcinoma of the head and neck cell line.⁸⁵Furthermore,

monocytes represent ∼10% of PBMCs, and they have a similar

nonspeciﬁc cytolytic activity against cancer cells, which is

potentiated after MDP stimulation, both alone and in

combination with IFN-γ.^87,88

In the present study, we examined these desmuramylpep-

tides in terms of potentiation of the cytotoxic activity of the

PBMCs against cancer cells. To this end, we used a previously

described ﬂow-cytometry-based cytotoxicity assay where

PBMCs are co-incubated with ﬂuorescently labeled cancer

cells.⁸⁹As indicated above, the NOD2-mediated NK cell

activity originates from both the direct eﬀects of NOD2

agonists on NK cells and the indirect activation through

Stimulation with LPS resulted in large increases in cytokine

production in general, which were further enhanced by MDP

and the desmuramylpeptides. When compared to stimulation

by LPS alone, all of the tested compounds signiﬁcantly

increased the levels of IL-1β, IL-6, and TNF-α. These eﬀects

were synergistic; i.e., the IL-1β, IL-6, and TNF-α produced

upon combined desmuramylpeptide and LPS stimulation were

higher than the sum of the eﬀects from these individual

immunostimulants. This is in agreement with the previously

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cytokine release by other NOD2-responsive cells, including

monocytes. Thus, we used the whole PBMC population as the

eﬀector cells, rather than isolated NK cells, to provide

conditions that would be closer to the in vivo environment,

wherein the immune responses can be reinforced by

interactions between the distinct immune cell subpopulations.

Two malignant cell lines were used as the target cells in a 40:1

eﬀector to target cell ratio: MEC1 B-chronic lymphocytic

leukemia cells, and K562 chronic myelogenous leukemia cells.

The K562 cells are considered a “classic” NK cell target, as

they lack the expression of major histocompatibility complex

(MHC) class I that is required for NK cell inhibition.⁹⁰

Interestingly, apart from LPS as the positive control,⁹¹only

75 showed any signiﬁcant boost to the cytotoxicity of the

PBMCs against both of these cancer cell lines at 10 μM

(Figure 5A). At 1 μM, this augmented cytotoxicity was still

acetic, lauric, stearic acids), these data indicated that the long

lipophilic tail might be a contributing factor for PBMC

cytotoxicity. Conversely, 81 also has a stearoyl tail but was

devoid of activity; however, the generally weaker NOD2

agonism of ^L-threonine based desmuramylpeptides compared

to ^L-valine derivatives has to be borne in mind. In parallel, the

PBMCs and cancer cells were also treated with the

desmuramylpeptides alone, to deﬁne any direct cytotoxicity

they might have toward the PBMCs or cancer cells. None of

these resulted in increased proportions of dead cells, thereby

conﬁrming that the enhanced activity of 75 can be attributed

to the stimulation of PBMC cytotoxic activity (data not

shown).

Similar lipophilicity-dependent eﬀects on immune cell

stimulation were described by Kalyuzhin et al. (1996).⁷⁶An

MDP derivative with a C₇lipophilic tail was shown to be a

potent stimulator of T cells, macrophages, and NK cells, with

the NK cell activity seen as increased cytotoxicity against YAC-

1 lymphoma cells. On the other hand, a C₁₆MDP derivative

showed suppressive eﬀects on the function of lymphocytes,

except for the release of IL-1 and TNF-α, which was

comparable after stimulation for both of these MDP

derivatives. Based on these data, where the tested desmur-

amylpeptides induced similar levels of cytokines in PBMCs, the

large diﬀerences in their cytotoxicity activation suggest that

there is no linear dependence between lipophilicity and

immunostimulatory eﬀects in the various PBMC cell

subpopulations. As for the HEK-Blue NOD2 cell assay, these

eﬀects might be ascribed to the hydrolipophilic balance and its

eﬀects on the interactions of these compounds with

biomembranes.⁷⁶Further studies are required to determine

the physicochemical properties that are optimal for the

induction of NK cell activity. Nonetheless, given the previously

reported link between NOD2-dependent NK cell activation

and in vivo antitumor activity,⁹²the data from the present

study demonstrate the potential of these desmuramylpeptides

in NK cell-dependent cancer immunotherapeutic approaches.

Furthermore, we carried out an analogous cytotoxicity assay

on macrophages that were produced by diﬀerentiation of

THP-1 cells using phorbol 12-myristate 13-acetate, for 3

days.⁹³Interestingly, the desmuramylpeptide treatments did

not enhance the tumoricidal activities of the resulting M0

macrophages against the MEC1 cancer cell line (data not

shown). THP-1-derived macrophages have been reported to

express functional NOD2; however, in line with our ﬁndings,

MDP treatment of these cells did not result in their activation,

as shown by the unchanged levels of their secreted cytokines.⁸⁵

Based on the potent in vitro NOD2 activation by 68 and the

PBMC cytotoxicity activation by 75, we explored the eﬀects of

68 and 75 in terms of induction at the transcriptional level.

Next-generation sequencing of RNA isolated from PBMCs was

carried out following their stimulation for 18 h with 68 or 75

(2 μM). After applying a low expression ﬁlter, the remaining

19046 genes from the desmuramylpeptide-treated samples

were compared to the vehicle-treated controls (0.1% DMSO).

As shown in Figure 6, 68 signiﬁcantly modulated the

expression of 445 genes (230 up-regulated, 215 down-

regulated, compared to the control), while 75 modulated the

expression of 270 genes (127 up-regulated, 143 down-

regulated, compared to the control). Diﬀerential expression

analysis between the 68- and 75-treated samples, however, did

not show any signiﬁcant diﬀerentially expressed genes (data

not shown). This thus indicates that 68 and 75 induced similar

Figure 5. Eﬀects of MDP and desmuramylpeptides on the cytotoxic

activities of PBMCs against K562 and MEC1 cells. (A) PBMCs were

treated for 18 h with MDP (10 μM), desmuramylpeptides (10 μM),

or LPS (1 μg/mL) before the addition of the K562 or MEC1 cells.

Cytotoxicity was determined after 4 h co-incubation. (B) Concen-

tration-dependent eﬀect of 75 on the induction of PBMC cytotoxicity.

Data are shown as activities relative to the negative control (NT, 0.1%

DMSO) and are expressed as mean SEM of three (MEC1) or four

(K562) independent experiments. *p < 0.05, **p < 0.01, ***p <

0.001 versus relevant negative controls.

signiﬁcant against the K562 cells, but not against the MEC1

cells, while at 100 nM, no eﬀects were seen against either cell

line (Figure 5B). These data are markedly in contrast with the

data obtained in the HEK-Blue NOD2 cell line, where 75 had

one-hundredth the NOD2 agonistic activity compared to

MDP, 1, 68, 73, and 74. As the desmuramylpeptides 73, 74,

and 75 belong to a series of 1 prodrug derivatives that contain

increasing lengths of lipophilic acids on the aromatic ring (i.e.,

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pathways involved in immune responses (Figure 7). The

pathway that was most signiﬁcantly overexpressed by both

compounds was the KEGG pathway “Cytokine-cytokine

receptor interaction” (hsa04060), within which there was up-

regulation of IL1B, IL6, and CXCL8 (IL-8), which conﬁrms

the observations made at the protein level (see Figure 4).

Compounds 68 and 75 also induced the transcription of

proinﬂammatory IL-1 cytokines and their antagonists (IL1A,

IL36B, IL1RN, IL36RN), IL-17 cytokines (IL17A, IL17F),

oncostatin M (member of the IL-6 cytokine family), and

IL12B (p40 subunit of IL-12) (Figure 8A). Furthermore, there

was marked up-regulation of several CC (CCL1, 3, 7, 20, 22,

3L3, 4L2) and CXC (CXCL1, 2, 3, 5, 6, 7, 8) chemokines.

Indeed, a study on the transcriptional signatures of 23 diﬀerent

TLR/NOD-like receptor agonists (including murabutide as a

representative NOD2 agonist) by Salyer and David (2018)

identiﬁed strong up-regulation of CC and CXC chemokines as

a shared characteristic of the majority of the innate immune

stimulants they tested.⁹⁵CXCL1, 2, 3, 5, 6, 7, and 8 are

primarily responsible for neutrophil traﬃcking, as these bind to

the cognate receptor CXCR2, and thus contribute to

neutrophil-mediated inﬂammation.⁹⁶Interestingly, while the

up-regulation of IL12B, IL17A, and IL17F indicated a Th1/

Th17 type response, 68 and 75 both induced the down-

regulation of Th1-associated chemokines CXCL9, CXCL10,

and CXCL11. These three chemokines share the same CXCR3

receptor that is mainly expressed on Th1 and NK cells, and

they are predominantly involved in IFN-γ-driven Th1 immune

responses. Conversely, the up-regulated CCL1 and CCL22 are

generally considered to be indicative of Th2 responses.⁹⁶The

shift toward Th2-type responses was further substantiated by

up-regulation of the co-stimulatory molecule TNFSF4 (also

known as OX40 ligand [OX40L]). Notably, the activation of

the OX40L−OX40 axis has previously been linked to NOD2

ligand-driven Th2 polarization.⁹⁷

Figure 6. Signiﬁcantly up-regulated and down-regulated genes in

PBMCs from three independent donors after 18 h of treatment with

68 and 75 (2 μM) treatment. A false discovery rate cutoﬀ of <0.05

and a gene expression fold-change >1.5 or <0.667 compared to the

untreated control (0.1% DMSO) was applied.

transcriptional changes, where those of 75 were of lower

magnitude, which is consistent with the weaker in vitro potency

of 75 compared to 68.

To explore the functional implications of the observed

transcriptional changes, gene enrichment analysis was

performed for the diﬀerentially expressed genes after the 68

and 75 treatments. This was based on the Gene Ontology

(GO) biological processes database and the Kyoto Encyclo-

pedia of Genes and Genomes (KEGG) database. The analysis

was performed using Metascape, which is a web-based

annotation tool that is distinguished from other similar tools

by hierarchical clustering of the overlapping enrichment

terms.⁹⁴Each cluster is then represented only by its most

signiﬁcant term, thus removing other redundant terms within

each speciﬁc cluster. The analysis of the 68 and 75 induced

transcriptomes revealed signiﬁcant enrichment of several

Figure 7. Top 20 most-enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. The

diﬀerentially expressed genes after 68 and 75 treatments (2 μM) were used as input in pathway enrichment analysis using Metascape.⁹⁴

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factors STAT2, IRF7, and IRF9, we did not detect the

transcription of type I IFN mRNA. However, it is known that

the expression of IFNs is both induced and shut oﬀ rapidly, to

ensure swift immunoprotective eﬀects prior to the onset of

detrimental eﬀects to the host.¹⁰²

2.3.3. In Vitro Adjuvant Properties of Desmuramylpep-

tides 68 and 75. Dendritic cells have an instrumental role in

bridging the innate and adaptive arms of immunity by

processing and presenting antigens to naive T cells, to thus

generate antigen-speciﬁc T-cell and B-cell immune re-

sponses.¹⁰³As DCs express a wide range of PRRs, including

NOD2, they serve as critical target cells for adjuvant functions.

NOD2 activation in DCs was previously shown to increase

expression of co-stimulatory molecules (e.g., CD80, CD86,

CD40) and production of inﬂammatory cytokines (e.g., TNF-

α, IL-6, IL-8, IL-12), indicators of DC maturation and

activation.^104,105NOD2 engagement by MDP in DCs also

induces autophagy, a vital process in the delivery of cytosolic

proteins for MHC class II antigen presentation, which

ultimately leads to induction of CD4⁺T helper cell

responses.^18,19Depending on the nature of the stimuli involved

in DC activation, these cells respond with the production of

diﬀerent cytokines, which leads to the polarization of T helper

cells toward distinct eﬀector functions.¹⁰³Among these, the

Th2 and Th1 subtypes are known to promote antigen-speciﬁc

humoral and cellular immunity, respectively. Cellular immune

responses are further characterized by induction of cytotoxic

CD8⁺T cells, which are instrumental in protective immunity

against intracellular pathogens and tumors. Notably, NOD2

activation functions as an eﬀective signal for DC-mediated

cross-priming of cytotoxic CD8⁺T cells, through up-regulation

of MHC class I-dependent antigen cross-presentation path-

ways.^106,107

To evaluate the adjuvant potential of the two most-

promising desmuramylpeptides, 68 and 75, in terms of their

eﬀects on DC-mediated activation of CD4⁺and CD8⁺T cells,

we used an in vitro antigen-presentation assay with C57BL/6

mouse bone-marrow-derived DCs (BMDCs). Following

stimulation with 68 and 75 (1 or 10 μM) in the presence of

ovalbumin soluble protein (50 μg/mL), BMDCs were

cocultured with carboxyﬂuorescein succinimidyl ester

(CFSE)-labeled naive ovalbumin-speciﬁc CD4⁺or CD8⁺T

cells, which were isolated from splenocytes of OT II and OT I

transgenic mice, respectively. After 3 days of coculture, CD25

(α subunit of IL-2 receptor)¹⁰⁸expression was determined,

along with the CFSE dilution, as markers of T-cell activation

and proliferation, respectively. Furthermore, we also examined

how this desmuramylpeptide-induced priming of T cells

aﬀected the secreted cytokine proﬁles in BMDC/T-cell

cocultures.

Figure 8. Heat maps of the (A) log₂(fold change) of the diﬀerentially

expressed genes in the “Cytokine-cytokine receptor interaction”

KEGG pathway and (B) log₂(fold change) of signiﬁcantly modulated

interferon-stimulated genes (ISGs) by 68 and 75.

While 68 and 75 induced similar transcriptional changes in

regards to cytokines and chemokines, there were pronounced

diﬀerences in the induction of genes related to the defense

response against viral pathogens. Speciﬁcally, there was a 68-

mediated up-regulation of several interferon-stimulated genes

(ISGs), which, conversely, were modulated by 75 to a lesser

degree (Figure 8B). Their transcription is triggered through

the JAK-STAT pathway following recognition of IFNs by their

cognate receptors. Several of the upregulated ISGs, such as

EIF2AK2 (protein kinase R), IFITM1, ISG20, MX1, OAS1,

and RSAD2 (viperin) were previously featured for their

antiviral eﬀector functions.⁹⁸Importantly, the activity of

endogenously induced type I IFNs and their respective ISGs

was also found to be a key step in the development of adaptive

immune responses induced by a wide range of currently used

vaccine adjuvants. Their eﬀects are especially essential in the

formation of Th1-type responses by promoting the diﬀer-

entiation of Th1 cells and the induction of cytotoxic T

lymphocytes, thus generating antiviral and antitumor protec-

tive immunity.^99,100A meta-analysis of the transcriptional

proﬁles induced by several diﬀerent vaccines linked LY6E,

MX1, OAS3, IFI44L, IFI6, and IFITM3 to the early phases

after vaccination.¹⁰¹With the exception of IFITM3, all of these

genes were signiﬁcantly up-regulated by 68. It is worth noting

that while we did observe the up-regulation of IFN

downstream signaling proteins, such as the transcription

As illustrated in Figure 9A,B, with 68 and 75 at 10 μM, both

signiﬁcantly enhanced BMDC-mediated CD4⁺T-cell activa-

tion and proliferation. In line with its superior in vitro NOD2

activation, 68 also showed this eﬀect at 1 μM. Enhanced T-cell

activation by 68, and to a lesser degree by 75, was additionally

characterized by elevated levels of IL-2, IL-6, IFN-γ, TNF-α,

and IL-17A (Figure 9C). The eﬀects of 68 were most

pronounced for the production of IL-2, a pleiotropic cytokine

that in addition to driving CD4⁺T-cell growth also augments

the activity of CD8⁺and NK cells. Activated CD4⁺T cells

produce large amounts of IL-2, although Th1 cells are

generally considered to be the major source of IL-2.¹⁰⁹IFN-

γ is characterized by its inductive eﬀects on CD8⁺T cells,

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Figure 9. CD4⁺and CD8⁺T cell activation, proliferation, and cytokine secretion in response to ovalbumin presentation by bone-marrow-derived

dendritic cells (BMDCs) pretreated with desmuramylpeptides 68 and 75. BMDCs from C57BL/6 mice were treated with 68 and 75 (1 and 10

μM) and ovalbumin (50 μg/mL). After 18 h, they were washed and cocultured for 72 h with carboxyﬂuorescein succinimidyl ester (CFSE)-labeled

ovalbumin-speciﬁc CD4⁺or CD8⁺T cells, isolated from OT II or OT I mouse splenocytes, respectively. (A) Representative dot plots of live

Thy1.2⁺/CD4⁺or Thy1.2⁺/CD8⁺T cells, showing CD25 expression and CFSE dilution. (B) Quantiﬁcation of the proportions of CD25⁺, CFSE^low

T cells. The analysis regions are shown in panel A. (C) Cytokine concentrations in coculture supernatants following co-incubation with BMDCs as

described in panel A. Data are expressed as mean SEM of duplicates of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

macrophages, and NK cells, and its up-regulation of MHC I

and MHC II antigen presentation pathways in DCs, and it is

similarly associated with a switch toward a Th1-like immune

response. Taken together with the unaﬀected levels of Th2-

associated IL-4 and IL-10, these data indicate that 68 induced

a primarily Th1 polarized response. Interestingly, NOD2

activation by MDP was previously identiﬁed as a driving force

toward Th2 polarization, with reduced levels of IFN-γ and

increased production of IL-4.¹¹⁰This previous study, however,

used human-monocyte-derived DCs, while the present study

used BMDCs from C57BL/6 mice, a strain that is generally

considered as Th1 dominant.¹¹¹Further studies on other mice

strains and human DCs would thus be required to accurately

determine the Th1/Th2 polarization promoted by the

desmuramylpeptides.

Compound 68 also signiﬁcantly increased the BMDC

processing and cross-presentation of ovalbumin to CD8⁺T

cells, which resulted in their enhanced activation, proliferation,

and cytokine secretion. It has been shown that activation of

NOD1 and NOD2 in a similar in vitro cross-priming assay

described by Asano et al. (2010) translated into enhanced

proliferation of IFN-γ-secreting CD8⁺T cells in vivo, which

resulted in increased antigen-speciﬁc antitumor and anti-

bacterial cytotoxic activities.¹⁰⁶Complementary data in

intranasally immunized mice were also reported for adaman-

tylamide dipeptide, another representative of the desmur-

amylpeptide class of adjuvants.¹¹²As most of the currently

licensed adjuvants almost exclusively induce antibody

responses, there is a pressing need for adjuvants that can

induce cellular immunity, especially in the cancer immuno-

therapy ﬁeld. Based on the data described above, 68 holds

great potential in this respect.

2.3.4. In Vivo Adjuvant Properties of Desmuramylpep-

tides 68 and 75 in Ovalbumin-Induced Antibody Responses.

There is clear evidence that NOD2 activation translates into

adjuvant activities in vivo.^31,52,113Due to the unfavorable

pharmacokinetic and toxicologic properties of MDP, signiﬁcant

eﬀort has been devoted to the development of more tolerable

MDP derivatives. During this research, in vivo experimental

data have revealed that both lipophilicity of the derivative and

context of application inﬂuence the intensity and type of the

provoked immune response. For example, MDP applied in

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Figure 10. Ovalbumin-speciﬁc IgG (left), IgG1 (middle), and IgG2a (right) responses in NIH/OlaHsd mice after immunization with ovalbumin-

loaded neutral liposomes (10 μg of ovalbumin per dose), with adjuvants MDP or the desmuramylpeptides (0.30 μmol of adjuvant per dose). The

concentrations were measured 1 week after the booster dose. Data are expressed as mean SEM of 5 mice per group. *p < 0.05; ***p < 0.001.

saline induces a predominantly Th2-biased humoral immune

response.¹⁵On the other hand, lipophilic MDP derivatives

promote a Th1-biased cellular immune response, especially

when used in conjunction with lipophilic carrier systems such

as liposomes.^70,114Notably, liposomes have repeatedly been

featured in the development of vaccines, due to their

versatility, biocompatibility, and enhancing eﬀects on the

generation of immune responses.¹¹⁵

To determine whether enhanced antigen presentation in

vitro is correlated to in vivo adjuvant activities, an

immunization study was performed in a murine model of

adjuvanticity. Speciﬁcally, the selected desmuramylpeptides

were investigated for induction of systemic immune responses

against the model antigen ovalbumin. Seven groups of NIH/

OlaHsd mice were immunized with ovalbumin-containing

neutral liposomes, either alone or with additional adjuvants of

MDP or the desmuramylpeptides 1, 68, 74, 75, or 81. After

immunization and one booster dose, the mice sera were

collected and assayed for ovalbumin-speciﬁc IgG antibodies

(Figure 10).

As expected, due to the low immunogenicity of ovalbumin,

the ovalbumin-loaded liposomes without added adjuvants

induced weak systemic responses and thus served as the

negative controls. In agreement with our previous experiments,

MDP moderately enhanced the production of anti-ovalbumin

IgG antibodies, while 1 showed only marginal adjuvant

activity.⁴⁶On the other hand, there was a signiﬁcant 5-fold

boost in the elicited IgG responses in the group immunized

with the addition of 75, followed by a 3-fold boost by 81,

where both of these derivatives had a C₁₈lipophilic tail on the

aromatic ring. The C₁₂-carrying derivative 74 showed similar

activity to 1, while 68, although it was the most potent in vitro

NOD2 agonist, was devoid of adjuvant activity under these

conditions.

To understand the nature of these induced immune

responses in terms of Th1/Th2 polarization, the levels of the

Th1-associated IgG1 and Th2-associated IgG2a antibody

isotypes were also measured (Figure 10).¹¹⁶In all of the

experimental groups, the levels of induced anti-ovalbumin

IgG1 closely resembled the total IgG levels; however, there

were notable diﬀerences in the desmuramylpeptide enhance-

ment of production of IgG2a. Consistent with previous reports,

MDP induced a predominantly Th2-biased response,^15,117with

signiﬁcant increases in IgG1 generation and only marginal

increases in IgG2a, compared to the control without adjuvant.

Likewise, a largely IgG1-based response was elicited by 1 and

81, while in the groups immunized by 68 and 74, there was

slight suppression of the IgG2a response. Finally, immuniza-

tion with the liposomes containing the 75 adjuvant

signiﬁcantly enhanced ovalbumin-speciﬁc IgG1 responses

and, importantly, also induced the highest levels of IgG2a

antibodies, which indicated a shift toward a more balanced

Th1/Th2 response.

It is clear that the in vivo activities of the desmuramylpep-

tides do not follow the same structure−activity relationship

rules as the in vitro activities. Given that 1, 68, 74, and 75 act

as prodrugs of the same active compound, their diﬀerential

eﬀects on the induction of humoral immune responses appear

to originate from their distinctive physicochemical properties.

Indeed, an evident increase in the adjuvant activities correlated

with the addition of a lipophilic C₁₈tail on the aromatic ring, as

for 75 and 81.

Entrapment and subsequent retention of both antigens and

adjuvants are important considerations in the design of

liposomal vaccines. The entrapment of soluble MDP in

liposomes was previously shown to be problematic, due to

the diﬀusive escape with a short half-life of retention of 5 h.¹¹⁸

Many lipophilic MDP derivatives have been designed to allow

their loading into liposomes to be increased, to provide

increased adjuvant activity and reduced side eﬀects. For

example, addition of lipophilic adamantane groups to

peptidoglycan fragments was shown to assist in the anchoring

of these derivatives into the liposomal lipid bilayer, using NMR

spectroscopy.¹¹⁹Likewise, straight-chain lipophilic anchors can

be used for the same purpose, due to the extensive network of

van der Waals interactions that they can form in the lipophilic

bilayer of liposomes. Surprisingly, a reduction in chain length

from C₁₈to C₁₆was shown to signiﬁcantly increase the

propensity for the undesired escape of lipids from liposomal

membranes.⁷⁸Analogously, the enhanced in vivo activities of

75, and to a lesser extent of 81, compared to their C₁₂

congener 74 and the compounds that lacked a lipophilic

anchor appeared to be due to their more eﬃcient liposome

encapsulation and subsequent retention, thus facilitating their

uptake by antigen-presenting cells. Stable incorporation into

liposomes additionally protects adjuvants from the actions of

hydrolytic enzymes. Given that desmuramylpeptides rely on

passive absorption to cross the cell membrane,⁶¹extracellular

hydrolysis would severely hamper their activation of the

cytosolic NOD2.

One of the key advantages of liposomes is their versatility.

The chemical properties of the lipid components and the

preparation procedures can be chosen to modulate charge, size,

size distribution, entrapment, and location of antigens and

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adjuvants, all of which can potentially inﬂuence the intensity

and form of the immune response against the antigen of

interest.¹¹⁵The net surface charge of liposomes, in particular,

was shown to signiﬁcantly inﬂuence the entrapment eﬃciency

of the antigen¹²⁰and the interactions of the liposomes with

antigen-presenting cells and other endogenous tissue compo-

nents.¹²¹Moreover, surface modiﬁcations of liposomes can be

exploited for targeted delivery of antigens and adjuvants to

speciﬁc immune-cell populations, to thus promote the desired

immune responses while avoiding adverse eﬀects due to

targeting of irrelevant cell types. Among the potential targets,

mannose receptors are highly expressed on the surface of DCs

and macrophages. Liposomes decorated with mannose

receptor ligands (i.e., mannosylated liposomes) enhance the

uptake and activation of DCs, to result in ampliﬁed immune

responses against the encapsulated antigen.¹²²Additionally,

MDP-containing mannosylated liposomes have been shown to

be eﬀective for inhibition of liver metastasis through their

targeted delivery to tumoricidal macrophages. This thus

expands the potential of these formulations to cancer

immunotherapies.¹²³

To determine whether the adjuvanticity of 75 can be

modulated through changes to the liposomal formulation, a

second in vivo experiment was designed in which the adjuvant

activities of three diﬀerent liposomal compositions were

compared. Neutral liposomes were prepared from egg

phosphatidylcholine and cholesterol. Addition of the anionic

dicetylphosphate resulted in liposomes with a negative net

surface charge, while monomannosyl−poly(ethylene glycol)−

palmitic acid derivative (Man-PEG-Pam) was added to

assemble mannosylated liposomes.

The measured ovalbumin-speciﬁc IgG, IgG1, and IgG2a

antibody levels after immunization with MDP or 75 adjuvants

in neutral, negatively charged, and mannosylated liposomes

(Figure 11) led to several observations: (i) Contrary to

previous reports,¹²⁴negatively charged liposomes without

adjuvant elicited a substantially weaker ovalbumin-speciﬁc

humoral response compared to both neutral and mannosylated

liposomes, both of which exhibited similar adjuvant activities.

It is worth noting that the entrapment eﬃciency of ovalbumin

was not evaluated in our study and might contribute to the

reduced adjuvanticity of negatively charged liposomes. (ii) The

composition of liposomes with MDP-adjuvant signiﬁcantly

inﬂuenced the intensity of the immune response. Neutral

liposomes had the weakest adjuvant activities, and interest-

ingly, this was even weaker than the neutral liposomes without

adjuvant. A stronger IgG response was observed with

negatively charged liposomes, while mannosylated liposomes

showed the strongest immunogenicity. The levels of Th2-

associated IgG1 antibodies followed the same trend. The Th1-

associated IgG2a response, however, remained unchanged,

compared to liposomes without adjuvant, which is consistent

with the predominantly Th2-biased adjuvant activity of

MDP.¹⁵(iii) The liposomal composition had little inﬂuence

on the intensity of the 75-elicited IgG response, although it did

alter the Th1/Th2 bias of the provoked immune response.

Namely, the negatively charged liposomes showed diminished

IgG1 activity. Mannosylated liposomes, on the other hand,

enhanced the production of IgG2a antibodies, which is in

agreement with previous reports of Th1-biased immune

responses upon immunization with mannosylated liposomes

with NOD2 agonist adjuvant.¹²⁵(iv) Finally, regardless of the

liposomal composition, 75 increased the levels of IgG

Figure 11. Ovalbumin-speciﬁc IgG (top), IgG1 (middle), and IgG2a

(bottom) responses in NIH/OlaHsd mice after immunization with

neutral (phosphatidylcholine−cholesterol, 7:5), anionic (phosphati-

dylcholine−cholesterol−dicetylphosphate, 7:5:1), and mannosylated

(phosphatidylcholine−cholesterol−mannose-PEG-PA, 7:5:0.5) oval-

bumin-loaded liposomes (10 μg of ovalbumin per dose), with

adjuvants MDP or 75 (0.15 μmol of adjuvant per dose). The

concentrations were measured 1 week after the second booster dose.

Data are expressed as mean SEM of 5 mice per group. *p < 0.05,

**p < 0.01.

antibodies compared to the controls without adjuvant, with

comparable or stronger adjuvant activities than MDP.

Additionally, while MDP elicited a predominantly Th2-biased

IgG1 response, 75 also augmented the production of

ovalbumin-speciﬁc IgG2a antibodies, resulting in a mixed

Th1/Th2 response.

The combined data from both of the in vivo experiments

showed that introduction of a C₁₈lipophilic anchor into the

structure of 1, to produce 75, substantially improved the 1 in

vivo adjuvant activity, which appears to be due to the stronger

incorporation and retention of 75 in the liposomal lipid bilayer.

Similar transformations were applied previously to MDP and

its derivatives, which resulted in higher immunoadjuvant

activities and, importantly, reduced pyrogenicity, one of the

main factors that has hindered the use of MDP in the clinic.⁷⁰

In line with previous reports, the increased lipophilicity

additionally augmented the production of IgG2a antibodies,

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binary pump system, a photodiode array detector, a thermostatted

column compartment, and the Chromeleon Chromatography data

system. The columns used were Waters Acquity UPLC BEH C18 (1.7

μm, 2.1 mm × 50 mm) or Waters Acquity UPLC CSH C18 (1.7 μm,

2.1 mm × 50 mm) with a ﬂow rate of 0.3 mL/min. The eluent was a

mixture of 0.1% TFA in water (A) and acetonitrile (B) with a gradient

(%B) as follows: 0−10 min, 5−95%; 10−12 min, 95%; 12−12.5 min,

95−5%. The columns were thermostatted at 40 °C. All of the

compounds tested were established to be ≥95% pure.

Compounds 1, 2, 3, 6, 29, 42, 43, 44, 45, and 46 were prepared as

previously described by our group.⁴⁶The NOD2 antagonist was

synthesized as described.⁷⁷The analytical data here were identical to

those reported previously. The assembly of the ﬁnal compounds was

as described below, while the preparation of other precursors is given

in the Supporting Information.

4.2. General Synthetic Procedures. 4.2.1. General Procedure

A: TFA-Mediated Acidolysis. The Boc-protected compound was

added to an ice-chilled stirred mixture of TFA and DCM (1:5), and

the mixture was allowed to warm to room temperature. After 3 h, the

solvent was evaporated in vacuo. The residue was washed three times

with diethyl ether.

4.2.2. General Procedure B: EDC-Mediated Coupling. To an ice-

chilled stirred solution of the requisite amine (1 equiv) and carboxylic

acid (1.0−1.2 equiv) in dry dimethylformamide (DMF), N,N-

diisopropylethylamine (DIPEA; 3 equiv) was added. After stirring

for 15 min, HOBt (1.0−1.2 equiv), EDC (1.0−1.2 equiv), and a

catalytic amount of 4-dimethylaminopyridine (DMAP) were added,

and the mixture was allowed to warm to room temperature. The

stirring was continued overnight, after which the mixture was diluted

with ethyl acetate (EtOAc; 40 mL) and washed with 1 M HCl (2 ×

20 mL), saturated NaHCO₃solution (2 × 20 mL), and brine (20

mL). The organic layer was dried over anhydrous Na₂SO₄and

concentrated in vacuo. If necessary, the residues were further puriﬁed

using column chromatography to produce suﬃciently pure com-

pounds.

4.2.3. General Procedure C: HCl-Mediated Acidolysis. To an ice-

chilled stirring solution of the Boc-protected compound in EtOAc (3

mL), 1 M HCl solution in acetic acid (2 mL) was added dropwise.

The mixture was allowed to warm to room temperature. The stirring

was continued for 2 h, after which the mixture was diluted with

EtOAc (30 mL) and washed with a 1 M NaOH solution (3 × 10 mL).

The organic layer was dried over anhydrous Na₂SO₄and concentrated

in vacuo to produce the deprotected amine.

4.2.4. General Procedure D: Acylation with Acyl Chlorides. To an

ice-chilled stirring solution of the alcohol or amine (1 equiv) in THF,

Et₃N (1.2 equiv) and the requisite acyl chloride (1.2 equiv) were

added dropwise, and the resulting mixture was allowed to warm to

room temperature. The stirring was continued for 1 h, after which the

mixture was diluted with EtOAc (25 mL) and washed with 1 M HCl

(2 × 10 mL), saturated NaHCO₃solution (2 × 10 mL), and brine

(10 mL). The organic layer was dried over anhydrous Na₂SO₄and

concentrated in vacuo. If necessary, the residues were further puriﬁed

using column chromatography, to produce suﬃciently pure

compounds.

which indicated a shift toward a balanced Th1/Th2 immune

response, especially when 75 was administered in the form of

mannosylated liposomes. Given that the majority of the

currently used adjuvants induce predominantly Th2-biased

responses, the eliciting of a Th1-biased or balanced response

remains a highly sought after trait for adjuvants; for example,

induction of Th1 responses is highly desirable for vaccines that

target intracellular pathogens or cancers. Furthermore, when

another representative MDP derivative was used in an

intranasal mucosal vaccine (i.e., murabutide), this induced

robust systemic and mucosal immunity that was characterized

by IgG and IgA levels higher than those after parenteral

vaccination with alum-adjuvant.²²We thus postulate that 75

shows similar potential, and propose 75-adjuvanted mucosal

vaccination as the next potential direction for further research.

3. CONCLUSIONS

In the present study, we performed a focused structure−

activity relationship optimization of 1, which resulted in a

library of novel desmuramylpeptide NOD2 agonists. In

particular, we have identiﬁed two promising compounds: 68,

as a potent in vitro NOD2 agonist with a more than 2-fold

improved potency over 1, and 75, which shows superior

adjuvant activity in vivo. Both 68 and 75 induced cytokine

production in PBMCs, both alone and in combination with

LPS, and enhanced antigen presentation of DCs. Furthermore,

75 stimulated the cytotoxic activity of PBMCs against

malignant cells. Interestingly, the in vitro NOD2 activation

and in vivo adjuvant activities do not necessarily correlate in a

linear fashion and are highly dependent on the lipophilicity of

the compounds. Speciﬁcally, we identiﬁed a C₁₈lipophilic

chain as a pivotal moiety, which conferred in vivo adjuvant

activity if administered with a liposomal delivery system. As a

C₁₈-lipidated derivative of 1, 75 showed promising adjuvant

activity in vivo in a mouse model of adjuvanticity, surpassing

that of MDP and achieving a more balanced Th1/Th2 immune

response. The 75 bona fide induction of an immune response

to a model antigen in mice thus highlights its potential as a

vaccine adjuvant. Taken together, these data provide deeper

understanding of the desmuramylpeptide structural features

required to achieve in vitro and in vivo immunostimulatory

activities.

4. EXPERIMENTAL SECTION

4.1. Materials. Chemicals were obtained from Sigma-Aldrich (St.

Louis, MO, U.S.A.), Tokyo Chemical Industry (Tokyo, Japan), Acros

Organics (Geel, Belgium), Enamine (Monmouth Junction, NJ,

U.S.A.), and Apollo (Stockport, U.K.) and were used without further

puriﬁcation. MDP, C12-iE-DAP, and LPS (from E. coli O55:B5) were

obtained from InvivoGen, Inc. (San Diego, CA, U.S.A.). Analytical

TLC was performed on Merck 60 F254 silica gel plates (0.25 mm),

with visualization using ultraviolet light, ninhydrin, and potassium

permanganate. Column chromatography was carried out on silica gel

4.3. Synthesis and Characterization of Compounds.

4.3.1. Diethyl (3-(4-Hydroxy-3-methoxyphenyl)propanoyl)glycyl-^L-

valyl-^D-glutamate (18). A solution of compound 1 (64 mg, 0.120

mmol) in acetic acid (10 mL) was hydrogenated over 10% palladium-

on-carbon for 2 h at room temperature and under atmospheric

pressure. The catalyst was removed by ﬁltration, and the ﬁltrate was

concentrated in vacuo to produce the title compound 18 as a white

1

60 (particle size 240−400 mesh). H and ¹³C NMR spectra were

recorded at 400 and 100 MHz, respectively, on an Avance III

spectrometer (Bruker Corporation, Billerica, MA, U.S.A.) in CDCl₃,

DMSO-d_6,or deuterated methanol (MeOD) with tetramethylsilane as

the internal standard. NOESY spectra were recorded at 800 MHz on

an Agilent Technologies NMR spectrometer (Santa Clara, CA,

U.S.A.). Mass spectra were obtained using an Exactive Plus orbitrap

mass spectrometer (Thermo Fisher Scientiﬁc, Waltham, MA, U.S.A.)

or on Expression CMS mass spectrometer (Advion Inc., Ithaca, NY,

U.S.A.). Analytical UHPLC analyses were performed on a Dionex

UltiMate 3000 Rapid Separation Binary System (Thermo Fisher

Scientiﬁc, Waltham, MA, U.S.A.) equipped with an autosampler, a

1

solid (65 mg, 100%). H NMR (400 MHz, DMSO-d₆) δ 8.71 (br,

1H), 8.38 (d, J = 7.6 Hz, 1H), 8.19−8.10 (m, 1H), 7.84 (d, J = 8.9

Hz, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.65 (d, J = 7.9 Hz, 1H), 6.57 (dd,

J = 8.1, 2.0 Hz, 1H), 4.30−4.19 (m, 2H), 4.12−3.99 (m, 4H), 3.90 (d,

J = 4.8 Hz, 1H), 3.78−3.70 (m, 4H), 2.74−2.64 (m, 2H), 2.43−2.31

(m, 4H), 2.06−1.92 (m, 2H), 1.90−1.76 (m, 1H), 1.22−1.12 (m,

6H), 0.90−0.76 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.57,

172.04, 171.54, 171.42, 169.50, 147.82, 145.03, 132.54, 120.63,

115.70, 112.76, 61.07, 60.42, 57.78, 55.90, 51.52, 42.49, 37.70, 31.23,

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31.14, 30.17, 26.35, 19.56, 18.17, 14.52, 14.45. HRMS calcd for

C₂₆H₄₀N₃O₉m/z: 538.2759 (M + H)⁺, found 538.2756.

42.63, 30.43, 29.86, 25.87, 18.34, 16.65, 13.06. HRMS calcd for

C₂₅H₃₇N₄O₇m/z: 505.2657 (M + H)⁺, found 505.2641.

4.3.6. Diethyl ((E)-3-(4-Isopropylphenyl)acryloyl)glycyl-^L-valyl-D-

glutamate (25). Compound 6 (92 mg, 0.200 mmol) was deprotected

using general procedure A and coupled to trans-4-isopropylcinnamic

acid (42 mg, 0.220 mmol) using general procedure B. The residue was

washed twice with diethyl ether to produce the title compound 25 as

a white solid (44 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.42−

8.31 (m, 2H), 7.94 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.40

(d, J = 15.7 Hz, 1H), 7.29 (d, J = 7.9 Hz, 2H), 6.69 (d, J = 15.8 Hz,

1H), 4.31−4.21 (m, 2H), 4.14−3.97 (m, 4H), 3.90 (d, J = 5.8 Hz,

2H), 2.96−2.84 (m, 1H), 2.35 (t, J = 7.5 Hz, 2H), 2.05−1.93 (m,

2H), 1.89−1.76 (m, 1H), 1.25−1.10 (m, 12H), 0.85 (t, J = 6.5 Hz,

6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.95, 171.44, 170.92,

168.82, 165.34, 149.99, 138.84, 132.40, 127.54, 126.79, 120.75, 60.48,

59.81, 57.27, 50.97, 42.10, 33.20, 30.66, 29.60, 25.81, 23.57, 19.02,

17.64, 13.95, 13.90. HRMS calcd for C₂₈H₄₂N₃O₇m/z: 532.3017 (M

+ H)⁺, found 532.3012.

4.3.7. Diethyl ((E)-3-(4-Nitrophenyl)acryloyl)glycyl-^L-valyl-D-glu-

tamate (26). Compound 6 (92 mg, 0.200 mmol) was deprotected

using general procedure A and coupled to trans-4-nitrocinnamic acid

(42 mg, 0.220 mmol) using general procedure B. The residue was

washed twice with diethyl ether to produce the title compound 26 as

a yellow solid (69 mg, 65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.51

(t, J = 5.8 Hz, 1H), 8.40 (d, J = 7.5 Hz, 1H), 8.27 (d, J = 8.7 Hz, 2H),

8.00 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 15.9

Hz, 1H), 6.95 (d, J = 15.9 Hz, 1H), 4.31−4.21 (m, 2H), 4.15−3.97

(m, 4H), 3.94 (d, J = 5.7 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.07−1.93

(m, 2H), 1.91−1.76 (m, 1H), 1.22−1.11 (m, 6H), 0.85 (t, J = 6.3 Hz,

6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.53, 172.01, 171.47,

169.12, 165.04, 148.00, 141.94, 137.14, 129.09, 126.59, 124.59, 61.05,

60.39, 57.87, 51.53, 42.69, 31.26, 30.18, 26.40, 19.58, 18.25, 14.52,

14.47. HRMS calcd for C₂₅H₃₅N₄O₉m/z: 535.2399 (M + H)⁺, found

535.2393.

4.3.2. Diethyl (4-(4-Hydroxy-3-methoxyphenyl)butanoyl)glycyl-^L-

valyl-^D-glutamate (19). Compound 6 (83 mg, 0.179 mmol) was

deprotected using general procedure A and coupled to 16 (42 mg,

0.200 mmol) using general procedure B. The residue was washed

twice with diethyl ether to produce the title compound 19 as an oﬀ-

white solid (46 mg, 46%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (s,

1H), 8.35 (d, J = 7.6 Hz, 1H), 8.07 (t, J = 5.9 Hz, 1H), 7.73 (d, J = 9.0

Hz, 1H), 6.73 (s, 1H), 6.65 (d, J = 7.9 Hz, 1H), 6.55 (d, J = 7.8 Hz,

1H), 4.27−4.21 (m, 2H), 4.11−4.01 (m, 4H), 3.77−3.69 (m, 5H),

2.45 (t, J = 7.8 Hz, 2H), 2.34 (t, J = 7.6 Hz, 2H), 2.12 (t, J = 7.5 Hz,

2H), 2.02−1.93 (m, 2H), 1.86−1.79 (m, 1H), 1.75 (p, J = 7.5 Hz,

2H), 1.19−1.14 (m, 6H), 0.85−0.80 (m, 6H). ¹³C NMR (100 MHz,

CDCl₃) δ 173.92, 172.86, 171.68, 171.35, 169.60, 146.54, 143.80,

133.38, 120.96, 114.30, 111.15, 61.64, 60.80, 58.44, 55.86, 51.86,

43.32, 35.33, 34.88, 31.01, 30.37, 27.31, 26.82, 19.26, 17.83, 14.14,

14.08. HRMS calcd for C₂₇H₄₂N₃O₉m/z: 552.2916 (M + H)⁺, found

552.2907.

4.3.3. Diethyl ((E)-3-(3,4-Dimethoxyphenyl)acryloyl)glycyl-^L-

valyl-^D-glutamate (20). Compound 6 (138 mg, 0.300 mmol) was

deprotected using general procedure A and coupled to trans-3,4-

dimethoxycinnamic acid (68 mg, 0.330 mmol) using general

procedure B. The residue was washed twice with diethyl ether to

produce the title compound 20 as an oﬀ-white solid (102 mg, 64%).

¹H NMR (400 MHz, MeOD) δ 7.51 (d, J = 15.6 Hz, 1H), 7.22−7.13

(m, 2H), 6.99 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 15.7 Hz, 1H), 4.49−

4.41 (m, 1H), 4.34−4.28 (m, 1H), 4.26−4.04 (m, 6H), 3.89 (s, 3H),

3.88 (s, 3H), 2.40 (t, J = 7.1 Hz, 2H), 2.27−2.13 (m, 2H), 2.09−1.97

(m, 1H), 1.32−1.18 (m, 6H), 1.00 (t, J = 6.9 Hz, 6H). ¹³C NMR

(100 MHz, MeOD) δ 172.94, 172.35, 171.55, 170.61, 168.07, 150.97,

149.31, 141.05, 127.82, 121.99, 117.56, 111.25, 110.02, 61.06, 60.28,

58.62, 55.02, 51.79, 42.58, 30.43, 29.85, 25.86, 18.35, 16.65, 13.07.

HRMS calcd for C₂₇H₄₀N₃O₉m/z: 550.2759 (M + H)⁺, found

550.2756.

4.3.4. Diethyl ((E)-3-(3,4-Dihydroxyphenyl)acryloyl)glycyl-^L-valyl-

D-glutamate (21). Compound 6 (78 mg, 0.170 mmol) was

deprotected using general procedure A and coupled to trans-caﬀeic

acid (34 mg, 0.187 mmol) using general procedure B. The residue was

washed twice with diethyl ether to produce the title compound 21 as

a cream-colored solid (43 mg, 49%). ¹H NMR (400 MHz, MeOD) δ

7.44 (d, J = 15.7 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.94 (dd, J = 8.2,

2.1 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 15.7 Hz, 1H),

4.49−4.41 (m, 1H), 4.31 (d, J = 6.1 Hz, 1H), 4.24−4.15 (m, 2H),

4.15−3.93 (m, 4H), 2.43 (t, J = 7.2 Hz, 2H), 2.30−2.11 (m, 2H),

2.08−1.93 (m, 1H), 1.32−1.15 (m, 6H), 1.00 (t, J = 7.0 Hz, 6H). ¹³C

NMR (100 MHz, MeOD) δ 172.95, 172.36, 171.54, 170.69, 168.37,

147.57, 145.36, 141.60, 126.75, 120.93, 116.29, 115.04, 113.69, 61.07,

60.30, 58.63, 51.78, 42.58, 30.43, 29.85, 25.85, 18.34, 16.65, 13.06.

HRMS calcd for C₂₅H₃₆N₃O₉m/z: 522.2446 (M + H)⁺, found

522.2445.

4.3.8. Diethyl (2-(3,4-Dimethoxyphenyl)cyclopropane-1-

carbonyl)glycyl-^L-valyl-D-glutamate (27). Compound 6 (92 mg,

0.200 mmol) was deprotected using general procedure A and coupled

to 13 (49 mg, 0.220 mmol) using general procedure B. Puriﬁcation by

ﬂash chromatography (5% MeOH in DCM) produced the title

1

compound 27 as a yellow solid (36 mg, 31%). H NMR (400 MHz,

DMSO-d₆) δ 8.43−8.36 (m, 2H), 7.85−7.77 (m, 1H), 6.84 (d, J = 8.3

Hz, 1H), 6.75−6.69 (m, 1H), 6.62 (dd, J = 8.2, 2.1 Hz, 1H), 4.29−

4.20 (m, 2H), 4.14−3.98 (m, 4H), 3.84−3.76 (m, 2H), 3.73 (s, 3H),

3.70 (s, 3H), 2.35 (t, J = 7.6 Hz, 2H), 2.22−2.14 (m, 1H), 2.06−1.88

(m, 3H), 1.88−1.77 (m, 1H), 1.32−1.24 (m, 1H), 1.21−1.11 (m,

7H), 0.89−0.79 (m, 6H). ¹³C NMR (100 MHz, MeOD) δ 174.23,

172.92, 172.27, 171.56, 170.55, 149.07, 147.75, 133.45, 117.96,

111.70, 110.29, 61.06, 60.31, 58.40, 55.13, 55.05, 51.78, 42.65, 30.57,

29.84, 25.88, 24.96, 24.58, 18.35, 16.64, 14.62, 13.10, 13.06. HRMS

calcd for C₂₈H₄₀N₃O₉m/z: 562.2770 (M − H)⁻, found 562.2769.

4.3.9. Diethyl (2-(4-Hydroxy-3-methoxyphenyl)cyclopropane-1-

carbonyl)glycyl-^L-valyl-D-glutamate (28). Compound 6 (111 mg,

0.242 mmol) was deprotected using general procedure A and coupled

to 17 (42 mg, 0.202 mmol) using general procedure B. White solid

(98 mg, 88%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (s, 1H), 8.41−

8.35 (m, 2H), 7.84−7.76 (m, 1H), 6.70−6.67 (m, 1H), 6.66 (d, J =

8.1 Hz, 1H), 6.49 (dd, J = 8.1, 2.0 Hz, 1H), 4.31−4.20 (m, 2H),

4.14−3.99 (m, 4H), 3.83−3.77 (m, 2H), 3.74 (s, 3H), 2.35 (t, J = 7.5

Hz, 2H), 2.19−2.09 (m, 1H), 2.03−1.93 (m, 2H), 1.92−1.86 (m,

1H), 1.85−1.77 (m, 1H), 1.31−1.22 (m, 1H), 1.22−1.12 (m, 7H),

0.83 (t, J = 7.5 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.52,

172.17, 172.14, 172.00, 171.48, 169.41, 147.94, 145.27, 132.10,

118.35, 115.81, 110.99, 61.05, 60.41, 57.65, 57.62, 56.03, 51.52, 42.68,

42.63, 31.34, 31.31, 30.17, 26.41, 25.50, 24.45, 19.56, 18.14, 15.38,

14.53, 14.46. HRMS calcd for C₂₇H₄₀N₃O₉m/z: 550.2759 (M + H)⁺,

found 550.2748.

4.3.5. Diethyl ((E)-3-(4-Aminophenyl)acryloyl)glycyl-^L-valyl-D-

glutamate (24). Compound 6 (276 mg, 0.600 mmol) was

deprotected using general procedure A and coupled to 22 (174 mg,

0.660 mmol) using general procedure B. The residue was washed

twice with diethyl ether to produce compound 23 as a yellow solid

(179 mg, 49%). This compound was deprotected using general

procedure A. The residue was dissolved in EtOAc (50 mL) and

washed with a saturated NaHCO₃solution (3 × 30 mL) and brine

(30 mL). The organic layer was dried over anhydrous Na₂SO₄and

concentrated in vacuo to produce the title compound 24 as an orange

1

solid (135 mg, 91%). H NMR (400 MHz, MeOD) δ 7.46 (d, J =

15.7 Hz, 1H), 7.34 (d, J = 8.6 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H), 6.42

(d, J = 15.7 Hz, 1H), 4.48−4.42 (m, 1H), 4.31 (d, J = 6.0 Hz, 1H),

4.23−4.14 (m, 2H), 4.14−3.92 (m, 4H), 2.43 (t, J = 7.3 Hz, 2H),

2.27−2.13 (m, 2H), 2.08−1.95 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H),

1.22 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.2 Hz, 6H). ¹³C NMR (100

MHz, MeOD) δ 172.95, 172.34, 171.54, 170.77, 168.77, 150.25,

141.92, 129.24, 123.70, 114.39, 114.16, 61.06, 60.29, 58.62, 51.80,

4.3.10. Diethyl ((E)-3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-

glycyl-^L-threonyl-D-glutamate (32). Compound 9 (388 mg, 0.841

mmol) was deprotected using general procedure A and coupled to

trans-ferulic acid (180 mg, 0.925 mmol) using general procedure B.

7828

https://doi.org/10.1021/acs.jmedchem.1c00644

J. Med. Chem. 2021, 64, 7809−7838

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Puriﬁcation by ﬂash chromatography (6% MeOH in DCM) produced

the title compound 32 as a yellow solid (263 mg, 58%). H NMR

= 8.4 Hz, J = 2.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.46 (d, J = 16.0

Hz, 1H), 4.29−4.25 (m, 2H), 4.02−3.91 (m, 2H), 3.85 (s, 3H), 2.26

(t, J = 7.6 Hz, 2H), 2.15−2.00 (m, 2H), 1.94−1.81 (m, 1H), 1.42 (s,

9H), 1.38 (s, 9H), 0.93 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz,

CDCl₃) δ 173.12, 172.28, 171.23, 170.71, 166.75, 165.00, 147.17,

146.93, 144.83, 144.20, 138.75, 127.17, 122.49, 117.24, 114.70,

110.20, 80.92, 58.47, 52.43, 38.61, 28.10, 28.01, 27.96, 27.32. HRMS

calcd for C₃₀H₄₄N₃O₉m/z: 590.3078 (M − H)⁻, found 590.3062.

4.3.15. (R)-N¹,N⁵-Diethyl-2-((S)-2-(2-((E)-3-(4-hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-

pentanediamide (57). Compound 48 (108 mg, 0.326 mmol) was

deprotected using general procedure C and coupled to 46 (139 mg,

0.391 mmol) using general procedure B to produce the title

compound 57 as an orange solid (99 mg, 57%). ¹H NMR (400

MHz, DMSO-d₆) δ 9.45 (s, 1H), 8.40−8.13 (m, 2H), 8.03−8.00 (m,

1H), 7.90−7.75 (m, 2H), 7.33 (d, J = 16.0, 1H), 7.14 (d, J = 1.6 Hz,

1H), 7.00 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H),

6.57 (d, J = 15.6 Hz, 1H), 4.28−4.05 (m, 2H), 3.92−3.82 (m, 2H),

3.80 (s, 3H), 3.07−3.02 (m, 4H), 2.18−1.86 (m, 3H), 1.83−1.60 (m,

2H), 1.19−1.00 (m, 6H), 0.98−0.96 (m, 6H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.05, 169.51, 169.48, 169.41, 166.32, 160.85, 160.10,

159.44, 148.30, 147.77, 126.67, 125.45, 118.57, 115.61, 110.81,

107.63, 107.07, 84.11, 55.47, 46.64, 33.28, 31.40, 29.96, 27.69, 27.60,

26.69, 24.43, 21.90, 14.69. HRMS calcd for C₂₆H₄₀N₅O₇m/z:

534.2990 (M + H)⁺, found 534.2986.

1

(400 MHz, CDCl₃) δ 7.74 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.1 Hz,

1H), 7.51 (d, J = 15.5 Hz, 1H), 7.14 (t, J = 5.3 Hz, 1H), 7.04−6.95

(m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.43−6.31 (m, 2H), 4.58−4.47 (m,

2H), 4.45−4.35 (m, 1H), 4.23−4.01 (m, 6H), 3.86 (s, 3H), 3.79 (d, J

= 6.4 Hz, 1H), 2.43 (t, J = 7.4 Hz, 2H), 2.29−2.14 (m, 1H), 2.13−

1.97 (m, 1H), 1.28−1.14 (m, 9H). ¹³C NMR (100 MHz, CDCl₃) δ

173.08, 172.23, 171.25, 170.31, 167.36, 147.69, 146.86, 142.03,

127.09, 122.31, 117.15, 114.84, 110.06, 67.30, 61.91, 60.84, 58.35,

55.92, 52.35, 43.70, 30.54, 26.26, 18.82, 14.12, 14.07. HRMS calcd for

C₂₅H₃₆N₃O₁₀m/z: 538.2395 (M + H)⁺, found 538.2396.

4.3.11. Diethyl O-Benzyl-N-(((E)-3-(4-hydroxy-3-

methoxyphenyl)acryloyl)glycyl)-^L-seryl-D-glutamate (30). Com-

pound 7 (227 mg, 0.420 mmol) was deprotected using general

procedure A and coupled to trans-ferulic acid (82 mg, 0.420 mmol)

1

using general procedure B. Yellow solid (177 mg, 69%). H NMR

(400 MHz, CDCl₃) δ 7.71 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 7.9 Hz,

1H), 7.52 (d, J = 15.6 Hz, 1H), 7.33−7.17 (m, 6H), 7.02−6.94 (m,

2H), 6.84 (d, J = 8.0 Hz, 1H), 6.36 (d, J = 15.5 Hz, 1H), 4.85−4.71

(m, 1H), 4.62−4.43 (m, 3H), 4.21−3.97 (m, 6H), 3.91−3.83 (m,

1H), 3.81 (s, 3H), 3.69−3.57 (m, 1H), 2.35 (t, J = 7.7 Hz, 2H),

2.26−2.11 (m, 1H), 2.07−1.94 (m, 1H), 1.26−1.11 (m, 6H). ¹³C

NMR (100 MHz, CDCl₃) δ 172.85, 171.70, 170.05, 169.86, 167.25,

147.78, 147.04, 141.84, 137.40, 128.43, 127.81, 127.73, 127.11,

122.20, 117.30, 115.01, 110.29, 73.40, 73.30, 69.54, 61.59, 60.64,

55.88, 53.03, 51.99, 43.60, 30.22, 26.93, 26.88. HRMS calcd for

C₃₁H₄₀N₃O₁₀m/z: 614.2708 (M + H)⁺, found 614.2695.

4.3.12. Diethyl ((E)-3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-

glycyl-^L-seryl-D-glutamate (31). Compound 8 (277 mg, 0.620

mmol) was deprotected using general procedure A and coupled to

trans-ferulic acid (120 mg, 0.620 mmol) using general procedure B.

Orange solid (150 mg, 46%). ¹H NMR (400 MHz, MeOD) δ 7.52 (d,

J = 15.7 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H),

6.83 (d, J = 8.2 Hz, 1H), 6.54 (d, J = 15.7 Hz, 1H), 4.54−4.45 (m,

2H), 4.25−4.01 (m, 6H), 3.92 (s, 3H), 3.89−3.79 (m, 2H), 2.49−

2.40 (m, 2H), 2.29−2.17 (m, 1H), 2.10−2.03 (m, 1H), 1.35−1.18

(m, 6H). ¹³C NMR (101 MHz, MeOD) δ 173.06, 171.69, 171.02,

170.72, 168.48, 148.68, 147.91, 141.59, 126.73, 122.00, 116.56,

115.08, 110.33, 61.46, 61.15, 60.26, 55.44, 55.00, 51.93, 42.91, 29.82,

29.37, 26.01, 13.04. HRMS calcd for C₂₄H₃₄N₃O₁₀m/z: 524.2239 (M

+ H)⁺, found 524.2231.

4.3.16. (R)-N¹,N⁵-Dibutyl-2-((S)-2-(2-((E)-3-(4-hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-

pentanediamide (58). Compound 49 (120 mg, 0.336 mmol) was

deprotected using general procedure A and coupled to 46 (130 mg,

0.370 mmol) using general procedure B to produce the title

1

compound 58 as an orange solid (115 mg, 58%). H NMR (400

MHz, DMSO-d₆) δ 9.45 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.16 (t, J =

5.6 Hz, 1H), 8.05−7.94 (m, 1H), 7.85−7.71 (m, 2H), 7.33 (d, J =

15.6, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 8.0 Hz, J = 1.2 Hz,

1H), 6.79 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 16.0 Hz, 1H), 4.18−4.11

(m, 2H), 3.92−3.86 (m, 2H), 3.80 (s, 3H), 3.06−3.00 (m, 4H),

2.10−1.99 (m, 2H), 1.98−1.81 (m, 2H), 1.78−1.63 (m, 1H), 1.36−

1.22 (m, 8H), 0.87−0.80 (m, 12H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 171.18, 171.17, 171.13, 169.45, 147.76, 139.38, 138.21, 121.54,

115.60, 110.81, 55.46, 52.37, 38.09, 31.87, 31.21, 31.13, 19.53, 19.45,

19.08, 18.26, 13.65. HRMS calcd for C₃₀H₄₈N₅O₇m/z: 590.3554 (M

+ H)⁺, found 590.3542.

4.3.17. tert-Butyl N⁵-Butyl-N²-((E)-3-(4-hydroxy-3-

methoxyphenyl)acryloyl)glycyl-^L-valyl-D-glutaminate (59). Com-

pound 50 (45 mg, 0.126 mmol) was deprotected using general

procedure A and coupled to 46 (53 mg, 0.151 mmol) using general

procedure B to produce the title compound 59 as a yellow solid (35

mg, 47%). ¹H NMR (400 MHz, MeOD) δ 7.42 (d, J = 15.6 Hz, 1H),

7.10 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.46

(d, J = 15.6 Hz, 1H), 4.24−4.14 (m, 2H), 4.09−3.89 (m, 2H), 3.85

(s, 3H), 3.15−3.00 (m, 2H), 2.22−2.03 (m, 4H), 1.94−1.79 (m, 2H),

1.41 (s, 9H), 1.33−1.20 (m, 3H), 0.97−0.82 (m, 9H). ¹³C NMR (100

MHz, CDCl₃) δ 172.46, 172.41, 171.41, 171.03, 169.98, 167.32,

167.11, 147.79, 146.92, 142.02, 141.72, 127.02, 122.29, 117.25,

114.91, 110.02, 82.22, 58.95, 55.93, 52.65, 43.81, 39.49, 32.63, 31.58,

31.52, 30.43, 27.96, 19.43, 17.64, 13.77. HRMS calcd for C₃₀H₄₇N₄O₈

m/z: 591.3394 (M + H)⁺, found 591.3403.

4.3.13. Methyl ((R)-5-Ethoxy-4-((S)-2-(2-((E)-3-(4-hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-5-

oxopentanoyl)-^L-lysinate (41). To a stirring solution of compound

40 (104 mg, 0.119 mmol) in THF (15 mL), 1-octanethiol (210 μL,

1.19 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 9 μL,

0.060 mmol) were added. The resulting mixture was stirred overnight

at room temperature. The solvent was evaporated in vacuo, and the

residue was washed twice with diethyl ether then recrystallized from

ethanol/diethyl ether to produce the title compound 41 as a yellow

solid (40 mg, 52%). ¹H NMR (400 MHz, MeOD) δ 7.47 (d, J = 15.7

Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.2, 2.0 Hz, 1H), 6.77

(d, J = 8.2 Hz, 1H), 6.48 (d, J = 15.6 Hz, 1H), 4.43−4.35 (m, 1H),

4.33−4.07 (m, 4H), 3.90 (s, 3H), 3.71 (s, 3H), 3.66−3.59 (m, 1H),

3.61−3.51 (m, 1H), 2.62 (t, J = 7.1 Hz, 2H), 2.40−2.18 (m, 4H),

2.11−2.01 (m, 2H), 1.84−1.70 (m, 2H), 1.67−1.50 (m, 1H), 1.49−

1.38 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H), 1.02 (t, J = 6.6 Hz, 6H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 173.24, 172.22, 171.97, 171.59,

169.51, 166.35, 165.79, 148.35, 140.00, 126.67, 122.12, 118.80,

116.16, 111.37, 60.93, 55.96, 53.76, 52.43, 52.28, 52.19, 48.32, 42.76,

38.08, 32.00, 28.74, 26.45, 23.88, 19.65, 19.41, 18.26, 14.50. HRMS

calcd for C₃₁H₄₈N₅O₁₀m/z: 650.3396 (M + H)⁺, found 650.3387.

4.3.14. Di-tert-butyl ((E)-3-(4-Hydroxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (56). Compound 47 (55 mg,

0.153 mmol) was deprotected using general procedure C and coupled

to 46 (64 mg, 0.184 mmol) using general procedure B. Puriﬁcation by

ﬂash chromatography (5% MeOH in DCM) produced the title

compound 56 as an orange solid (27 mg, 30%). ¹H NMR (400 MHz,

MeOD) δ 7.44 (d, J = 15.6, 1H), 7.11 (d, J = 1.6 Hz, 1H), 7.01 (dd, J

4.3.18. tert-Butyl N²-((E)-3-(4-Hydroxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-N⁵-methoxy-D-glutaminate (60). Compound

51 (62 mg, 0.187 mmol) was deprotected using general procedure C

and coupled to 46 (79 mg, 0.224 mmol) using general procedure B.

Puriﬁcation by ﬂash chromatography (3% MeOH in DCM) produced

1

the title compound 60 as an orange waxy solid (12 mg, 11%). H

NMR (400 MHz, MeOD) δ 7.44 (d, J = 15.6 Hz, 1H), 7.11 (d, J =

2.0 Hz, 1H), 7.01 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.76 (d, J = 8.0 Hz,

1H), 6.47 (d, J = 15.6 Hz, 1H), 4.34−4.33 (m, 1H), 4.13−4.09 (m,

1H), 4.05−3.93 (m, 2H), 3.85 (s, 3H), 3.68 (s, 3H), 2.45−2.20 (m,

2H), 2.15−2.02 (m, 2H), 1.88−1.76 (m, 1H), 1.44 (s, 9H), 0.92−

0.90 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.20, 169.08,

167.42, 166.65, 161.17, 146.74, 141.98, 127.12, 124.23, 122.54,

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117.21, 114.74, 114.18, 109.51, 82.51, 57.32, 55.96, 52.30, 43.49,

31.19, 29.32, 28.29, 27.98, 24.88, 19.00, 17.75. HRMS calcd for

C₂₇H₃₉N₄O₉m/z: 563.2717 (M − H)⁻, found 563.2722.

1.79 (m, 3H), 1.41−1.38 (m, 9H), 0.94−0.89 (m, 6H). ¹³C NMR

(100 MHz, CDCl₃) δ 172.20, 172.09, 171.01, 169.94, 169.57, 169.42,

166.85, 147.49, 146.81, 141.58, 141.40, 127.35, 122.16, 117.80,

114.79, 109.91, 80.79, 58.13, 55.92, 50.02, 46.60, 46.21, 43.41, 31.35,

30.97, 30.92, 30.88, 28.06, 27.24, 26.01, 24.12, 19.30, 17.99, 17.88.

*Some signals in the ¹³C spectrum are doubled due to the presence of

both ^L- and D-conﬁgured valine. HRMS calcd for C₃₀H₄₃N₄O₈m/z:

587.3081 (M − H)⁻, found 587.3078.

4.3.19. tert-Butyl N²-((E)-3-(4-Hydroxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-N⁵-methoxy-N⁵-methyl-D-glutaminate (61).

Compound 52 (78 mg, 0.225 mmol) was deprotected using general

procedure C and coupled to 46 (95 mg, 0.270 mmol) using general

procedure B to produce the title compound 61 as an orange solid (57

mg, 44%). ¹H NMR (400 MHz, MeOD) δ 7.44 (d, J = 15.6 Hz, 1H),

7.10 (d, J = 1.6 Hz, 1H), 7.01 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.76

(d, J = 8.0 Hz, 1H), 6.46 (d, J = 15.6 Hz, 1H), 4.34−4.20 (m, 2H),

4.05−3.90 (m, 2H), 3.85 (s, 3H), 3.70−3.57 (m, 3H), 3.13−3.07 (m,

3H), 2.58−2.42 (m, 2H), 2.18−2.01 (m, 2H), 1.98−1.84 (m, 1H),

1.42−1.41 (m, 9H), 0.97−0.90 (m, 6H). ¹³C NMR (100 MHz,

CDCl₃) δ 173.63, 173.44, 171.20, 170.65, 169.65, 169.40, 166.94,

147.62, 146.88, 141.82, 141.63, 127.22, 122.25, 122.22, 117.55,

114.86, 110.01, 82.03, 65.88, 61.22, 58.43, 58.37, 55.91, 53.06, 52.99,

43.57, 43.37, 32.19, 31.26, 31.14, 28.32, 26.16, 19.35, 19.16, 18.07,

15.28. *Some signals in the ¹³C spectrum are doubled due to the

presence of both ^L- and D-conﬁgured valine. HRMS calcd for

C₂₈H₄₁N₄O₉m/z: 577.2874 (M − H)⁻, found 577.2858.

4.3.23. Dicyclopentyl ((E)-3-(4-Hydroxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (68). Compound 67 (108 mg,

0.200 mmol) was deprotected using general procedure A and coupled

to trans-ferulic acid (43 mg, 0.220 mmol) using general procedure B.

The residue was washed twice with diethyl ether to produce the title

1

compound 68 as an oﬀ-white solid (93 mg, 76%). H NMR (400

MHz, CDCl₃) δ 7.56 (d, J = 15.6 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H),

7.09−6.97 (m, 3H), 6.92−6.81 (m, 2H), 6.37 (d, J = 15.6 Hz, 1H),

6.00 (s, 1H), 5.21−5.07 (m, 2H), 4.55−4.47 (m, 1H), 4.46−4.40 (m,

1H), 4.23−4.06 (m, 2H), 3.91 (s, 3H), 2.41−2.29 (m, 2H), 2.26−

2.08 (m, 2H), 2.06−1.93 (m, 1H), 1.87−1.76 (m, 4H), 1.74−1.51

(m, 12H), 1.00−0.88 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ

172.72, 171.46, 171.10, 169.47, 166.86, 147.57, 146.75, 141.86,

127.23, 122.31, 117.40, 114.73, 109.87, 78.65, 77.57, 58.50, 55.96,

52.03, 43.69, 32.70, 32.65, 32.60, 32.51, 30.80, 30.69, 26.93, 23.70,

23.64, 19.34, 17.74. HRMS calcd for C₃₂H₄₆N₃O₉m/z: 616.3229 (M

+ H)⁺, found 616.3221.

4.3.24. Dioctadecyl ((E)-3-(4-Hydroxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (72). Compound 71 (489 mg,

0.538 mmol) was deprotected using general procedure A and coupled

to trans-ferulic acid (125 mg, 0.646 mmol) using general procedure B,

using DCM instead of DMF as the reaction solvent. Puriﬁcation by

ﬂash chromatography (5% MeOH in DCM) produced the title

compound 72 as a yellow solid (376 mg, 71%). ¹H NMR (400 MHz,

CDCl₃) δ 7.56 (d, J = 15.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.06

(dd, J = 8.1, 1.9 Hz, 1H), 7.03−6.94 (m, 2H), 6.89 (d, J = 8.2 Hz,

1H), 6.77 (t, J = 5.2 Hz, 1H), 6.35 (d, J = 15.6 Hz, 1H), 5.95 (d, J =

7.8 Hz, 1H), 4.60−4.52 (m, 1H), 4.47−4.38 (m, 1H), 4.17−4.06 (m,

4H), 4.02 (t, J = 6.8 Hz, 2H), 3.91 (s, 3H), 2.48−2.32 (m, 2H),

2.30−2.13 (m, 2H), 2.10−1.97 (m, 1H), 1.64−1.52 (m, 4H), 1.25 (s,

60H), 1.00−0.91 (m, 6H), 0.88 (t, J = 6.8 Hz, 6H). ¹³C NMR (100

MHz, CDCl₃) δ 173.08, 171.78, 171.11, 169.50, 166.89, 147.59,

146.72, 141.97, 127.19, 122.36, 117.29, 114.73, 109.77, 65.88, 65.09,

58.48, 55.93, 51.99, 43.74, 31.94, 30.70, 30.40, 29.73, 29.68, 29.64,

29.57, 29.54, 29.38, 29.31, 29.26, 29.21, 28.56, 28.49, 26.84, 25.90,

25.81, 22.71, 19.37, 17.65, 14.14. HRMS calcd for C₅₈H₁₀₂N₃O₉m/z:

984.7611 (M + H)⁺, found 984.7609.

4.3.20. tert-Butyl (R)-2-((S)-2-(2-((E)-3-(4-Hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-5-

oxo-5-(pyrrolidin-1-yl)pentanoate (62). Compound 53 (70 mg,

0.196 mmol) was deprotected using general procedure C and coupled

to 46 (82 mg, 0.235 mmol) using general procedure B to produce the

1

title compound 62 as a yellow solid (65 mg, 57%). H NMR (400

MHz, MeOD) δ 7.44 (d, J = 16.0 Hz, 1H), 7.10 (s, 1H), 7.00 (d, J =

8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.46 (d, J = 15.6 Hz, 1H),

4.42−4.16 (m, 2H), 4.09−3.90 (m, 2H), 3.84 (s, 3H), 3.83−3.65 (m,

2H), 3.43−3.31 (m, 2H), 2.43−2.25 (m, 2H), 2.21−1.68 (m, 7H),

1.44−1.37 (m, 9H), 0.97−0.89 (m, 6H). ¹³C NMR (100 MHz,

CDCl₃) δ 172.27, 171.17, 171.08, 169.80, 167.08, 167.03, 147.81,

147.07, 146.98, 141.70, 127.28, 127.19, 122.29, 122.22, 117.66,

117.60, 115.10, 115.04, 114.94, 110.21, 110.12, 82.00, 80.92, 58.47,

58.44, 56.02, 56.00, 53.52, 53.39, 46.84, 46.77, 46.74, 46.12, 46.01,

43.49, 31.22, 28.08, 26.09, 24.37, 19.37, 19.33, 18.02. *Some signals

in the ¹³C spectrum are doubled due to the presence of both L- and D-

conﬁgured valine. HRMS calcd for C₃₀H₄₃N₄O₈m/z: 587.3081 (M −

H)⁻, found 587.3088.

4.3.21. tert-Butyl (R)-4-((S)-2-(2-((E)-3-(4-Hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-5-

(methoxy(methyl)amino)-5-oxopentanoate (63). Compound 54

(70 mg, 0.201 mmol) was deprotected using general procedure C

and coupled to 46 (84 mg, 0.241 mmol) using general procedure B to

1

produce the title compound 63 as an orange solid (40 mg, 34%). H

4.3.25. Diethyl ((E)-3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-

glycyl-^L-valyl-D-glutamate (73). Synthesized from 1 (64 mg, 0.120

mmol) and acetyl chloride (10 μL, 0.144 mmol) using general

procedure D. Puriﬁcation by ﬂash chromatography (EtOAc)

NMR (400 MHz, MeOD) δ 7.44 (d, J = 16.0 Hz, 1H), 7.10 (d, J =

2.0 Hz, 1H), 7.01 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.76 (d, J = 8.0 Hz,

1H), 6.47 (d, J = 15.6 Hz, 1H), 4.34−4.22 (m, 2H), 4.06−3.91 (m,

2H), 3.85 (s, 3H), 3.77 (s, 3H), 3.15 (s, 3H), 2.36−2.22 (m, 2H),

2.16−1.92 (m, 2H), 1.89−1.78 (m, 1H), 1.41−1.38 (m, 9H), 0.95−

0.90 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.29, 172.08,

171.22, 171.12, 169.46, 169.38, 166.85, 147.54, 146.82, 141.61,

127.29, 122.25, 122.19, 117.62, 114.81, 109.98, 109.91, 80.77, 65.88,

61.64, 58.39, 58.28, 55.92, 48.82, 48.73, 43.46, 43.41, 32.14, 31.23,

28.05, 26.95, 19.35, 19.20, 18.13, 17.87, 15.28. *Some signals in the

¹³C spectrum are doubled due to the presence of both L- and D-

1

produced the title compound 73 as a white solid (27 mg, 39%). H

NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J = 7.6 Hz, 1H), 8.32 (t, J =

5.8 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.43 (d, J = 15.8 Hz, 1H), 7.34

(d, J = 1.9 Hz, 1H), 7.17 (dd, J = 8.3, 1.8 Hz, 1H), 7.12 (d, J = 8.1 Hz,

1H), 6.76 (d, J = 15.8 Hz, 1H), 4.31−4.21 (m, 2H), 4.14−3.98 (m,

4H), 3.91 (d, J = 5.7 Hz, 2H), 3.82 (s, 3H), 2.35 (t, J = 7.5 Hz, 2H),

2.26 (s, 3H), 2.05−1.91 (m, 2H), 1.91−1.77 (m, 1H), 1.21−1.11 (m,

6H), 0.85 (t, J = 6.5 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ

172.55, 172.05, 171.50, 169.31, 168.94, 165.70, 151.53, 140.65,

138.89, 134.34, 123.75, 122.60, 120.58, 112.06, 61.06, 60.40, 57.82,

56.23, 51.52, 42.68, 31.26, 30.17, 26.37, 20.88, 19.58, 18.23, 14.52,

14.47. HRMS calcd for C₂₈H₄₀N₃O₁₀m/z: 578.2708 (M + H)⁺, found

578.2704.

conﬁgured valine. HRMS calcd for C₂₈H₄₃N₄O₉m/z: 579.3030 (M +

H)⁺, found 579.3036.

4.3.22. tert-Butyl (R)-4-((S)-2-(2-((E)-3-(4-Hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-5-

oxo-5-(pyrrolidin-1-yl)pentanoate (64). Compound 55 (65 mg,

0.182 mmol) was deprotected using general procedure C and coupled

to 46 (76 mg, 0.218 mmol) using general procedure B to produce the

4.3.26. Diethyl ((E)-3-(4-(Dodecanoyloxy)-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (74). Synthesized from 1 (64

mg, 0.120 mmol) and lauroyl chloride (34 μL, 0.144 mmol) using

general procedure D. Puriﬁcation by ﬂash chromatography (20%

hexanes in EtOAc) produced the title compound 74 as a white solid

1

title compound 64 as a yellow solid (30 mg, 28%). H NMR (400

MHz, MeOD) δ 7.44 (d, J = 15.6 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H),

7.01 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.47

(d, J = 15.6 Hz, 1H), 4.65−4.60 (m, 1H), 4.34−4.19 (m, 1H), 4.06−

3.91 (m, 2H), 3.84 (s, 3H), 3.70−3.59 (m, 2H), 3.44−3.31 (m, 2H),

2.34−2.22 (m, 2H), 2.14−2.04 (m, 1H), 2.02−1.91 (m, 3H), 1.86−

1

(45 mg, 52%). H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 15.6 Hz,

1H), 7.29 (d, J = 7.8 Hz, 1H), 7.13−7.04 (m, 3H), 7.01 (d, J = 7.9

Hz, 1H), 6.97−6.90 (m, 1H), 6.48 (d, J = 15.6 Hz, 1H), 4.60−4.50

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(m, 1H), 4.49−4.41 (m, 1H), 4.21−4.05 (m, 6H), 3.84 (s, 3H), 2.57

(t, J = 7.5 Hz, 2H), 2.47−2.32 (m, 2H), 2.26−2.15 (m, 2H), 2.10−

1.96 (m, 1H), 1.75 (p, J = 7.5 Hz, 2H), 1.47−1.18 (m, 22H), 1.01−

0.91 (m, 6H), 0.89 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

δ 172.97, 171.74, 171.66, 171.14, 169.35, 166.41, 151.38, 141.22,

141.19, 133.54, 123.21, 120.65, 120.03, 111.60, 61.67, 60.82, 58.52,

55.89, 51.97, 43.69, 34.04, 31.92, 30.83, 30.41, 29.63, 29.52, 29.36,

29.30, 29.06, 26.77, 25.01, 22.70, 19.35, 17.72, 14.14, 14.11. HRMS

calcd for C₃₈H₆₀N₃O₁₀m/z: 718.4273 (M + H)⁺, found 718.4260.

4.3.27. Diethyl ((E)-3-(3-Methoxy-4-(stearoyloxy)phenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (75). Synthesized from 1 (64

mg, 0.120 mmol) and stearoyl chloride (49 μL, 0.144 mmol) using

general procedure D. The residue was washed twice with diethyl ether

(m, 2H), 4.19−4.13 (m, 2H), 3.84 (s, 3H), 2.57 (t, J = 7.5 Hz, 2H),

2.48−2.27 (m, 2H), 2.25−2.09 (m, 2H), 2.06−1.94 (m, 1H), 1.88−

1.51 (m, 18H), 1.47−1.37 (m, 2H), 1.37−1.18 (m, 26H), 1.00−0.92

(m, 6H), 0.88 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

172.71, 171.70, 171.44, 171.07, 169.30, 166.36, 151.39, 141.20,

141.16, 133.58, 123.19, 120.64, 120.10, 111.66, 78.66, 77.57, 58.51,

55.90, 52.03, 43.64, 35.31, 34.05, 32.69, 32.65, 32.60, 32.52, 31.93,

30.91, 30.68, 29.70, 29.67, 29.64, 29.53, 29.37, 29.30, 29.20, 29.08,

28.88, 26.95, 25.01, 24.24, 23.69, 23.64, 22.70, 19.32, 17.79, 14.13.

HRMS calcd for C₅₀H₈₀N₃O₁₀m/z: 882.5838 (M + H)⁺, found

882.5810.

4.3.31. Diethyl O-Benzyl-N-(((E)-3-(3-methoxy-4-(stearoyloxy)-

phenyl)acryloyl)glycyl)-^L-seryl-D-glutamate (79). Synthesized from

30 (68 mg, 0.110 mmol) and stearoyl chloride (41 μL, 0.120 mmol)

1

to produce the title compound 75 (38 mg, 40%). H NMR (400

1

MHz, CDCl₃) δ 7.60 (d, J = 15.6 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H),

7.14−7.05 (m, 3H), 7.01 (d, J = 7.9 Hz, 1H), 6.95 (t, J = 5.2 Hz, 1H),

6.48 (d, J = 15.6 Hz, 1H), 4.60−4.50 (m, 1H), 4.49−4.40 (m, 1H),

4.22−4.05 (m, 6H), 3.84 (s, 3H), 2.57 (t, J = 7.5 Hz, 2H), 2.45−2.35

(m, 2H), 2.29−2.13 (m, 2H), 2.10−1.98 (m, 1H), 1.79−1.69 (m,

2H), 1.47−1.17 (m, 34H), 1.00−0.92 (m, 6H), 0.88 (t, J = 6.6 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃) δ 172.98, 171.74, 171.69, 171.16,

169.37, 166.43, 151.38, 141.22, 141.19, 133.54, 123.21, 120.65,

120.03, 111.61, 61.68, 60.83, 58.52, 55.89, 51.97, 43.70, 34.04, 31.94,

30.81, 30.41, 29.71, 29.68, 29.65, 29.54, 29.38, 29.31, 29.08, 26.77,

25.01, 22.71, 19.35, 17.71, 14.14, 14.11. HRMS calcd for

C₄₄H₇₂N₃O₁₀m/z: 802.5212 (M + H)⁺, found 802.5204.

4.3.28. Dicyclopentyl ((E)-3-(4-Acetoxy-3-methoxyphenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (76). Synthesized from 68 (69

mg, 0.112 mmol) and acetyl chloride (10 μL, 0.134 mmol) using

general procedure D. Puriﬁcation by ﬂash chromatography (3%

MeOH in DCM) produced the title compound 76 as an oﬀ-white

solid (59 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 15.6

Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.15−

7.06 (m, 3H), 7.01 (d, J = 8.7 Hz, 1H), 6.51 (d, J = 15.6 Hz, 1H),

5.20−5.08 (m, 2H), 4.56−4.42 (m, 2H), 4.20−4.10 (m, 2H), 3.85 (s,

3H), 2.41−2.29 (m, 5H), 2.26−2.08 (m, 2H), 2.03−1.92 (m, 1H),

1.85−1.77 (m, 4H), 1.74−1.60 (m, 8H), 1.59−1.51 (m, 4H), 1.01−

0.88 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.66, 171.54,

171.21, 169.40, 168.86, 166.40, 151.32, 140.99, 133.77, 123.15,

120.58, 120.32, 111.73, 78.66, 77.55, 58.54, 55.93, 52.01, 43.61, 32.69,

32.64, 32.60, 32.50, 30.93, 30.69, 26.94, 23.69, 23.63, 20.66, 19.33,

17.84. HRMS calcd for C₃₄H₄₈N₃O₁₀m/z: 658.3334 (M + H)⁺, found

658.3311.

using general procedure D as a white solid (93 mg, 96%). H NMR

(400 MHz, CDCl₃) δ 7.59 (d, J = 15.5 Hz, 1H), 7.39−7.22 (m, 6H),

7.13−6.98 (m, 4H), 6.62 (t, J = 5.2 Hz, 1H), 6.41 (d, J = 15.6 Hz,

1H), 4.67 (td, J = 6.4, 3.8 Hz, 1H), 4.62−4.47 (m, 3H), 4.21−3.98

(m, 6H), 3.97−3.89 (m, 1H), 3.84 (s, 3H), 3.65−3.58 (m, 1H), 2.58

(t, J = 7.5 Hz, 2H), 2.43−2.26 (m, 2H), 2.26−2.13 (m, 1H), 2.07−

1.94 (m, 1H), 1.76 (p, J = 7.5 Hz, 2H), 1.46−1.16 (m, 34H), 0.88 (t,

J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.97, 171.70,

171.46, 169.59, 169.07, 166.33, 151.39, 141.44, 141.26, 137.28,

133.47, 128.51, 127.97, 127.84, 123.21, 120.77, 119.80, 111.52, 73.51,

69.18, 61.63, 60.74, 55.90, 52.89, 52.08, 43.60, 34.05, 31.93, 30.24,

29.71, 29.67, 29.64, 29.53, 29.37, 29.30, 29.08, 26.87, 25.02, 22.70,

14.13. HRMS calcd for C₄₉H₇₄N₃O₁₁m/z: 880.5318 (M + H)⁺, found

880.5302.

4.3.32. Diethyl ((E)-3-(3-Methoxy-4-(stearoyloxy)phenyl)-

acryloyl)glycyl-^L-seryl-D-glutamate (80). Synthesized from 31 (75

mg, 0.140 mmol) and stearoyl chloride (53 μL, 0.160 mmol) using

general procedure D. Puriﬁcation by ﬂash chromatography (6%

MeOH in DCM) produced the title compound 80 as a white solid

1

(23 mg, 21%). H NMR (400 MHz, MeOD) δ 7.57 (d, J = 15.6 Hz,

1H), 7.30 (d, J = 1.8 Hz, 1H), 7.21 (dd, J = 8.2, 1.9 Hz, 1H), 7.06 (d,

J = 8.2 Hz, 1H), 6.69 (d, J = 16.0 Hz, 1H), 4.55−4.44 (m, 2H), 4.23−

4.03 (m, 6H), 3.88 (s, 3H), 3.87−3.84 (m, 2H), 2.60 (t, J = 7.3 Hz,

2H), 2.51−2.40 (m, 2H), 2.33−2.16 (m, 1H), 2.13−1.98 (m, 1H),

1.81−1.69 (m, 2H), 1.51−1.18 (m, 34H), 0.92 (t, J = 7.1 Hz, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 173.27, 172.12, 171.72, 170.89,

169.57, 166.69, 151.40, 141.53, 141.29, 133.41, 123.22, 120.75,

119.75, 111.61, 61.95, 60.91, 55.90, 54.87, 52.50, 43.75, 34.05, 31.93,

30.53, 29.71, 29.67, 29.53, 29.37, 29.30, 29.08, 25.01, 22.70, 14.13,

14.10. HRMS calcd for C₄₂H₆₈N₃O₁₁m/z: 790.4848 (M + H)⁺, found

790.4835.

4.3.29. Dicyclopentyl ((E)-3-(4-(Dodecanoyloxy)-3-

methoxyphenyl)acryloyl)glycyl-^L-valyl-D-glutamate (77). Synthe-

sized from 68 (60 mg, 0.097 mmol) and lauroyl chloride (28 μL,

0.117 mmol) using general procedure D. The residue was washed

twice with hexane to produce the title compound 77 as a white solid

4.3.33. Diethyl ((E)-3-(3-Methoxy-4-(stearoyloxy)phenyl)-

acryloyl)glycyl-^L-threonyl-D-glutamate (81). Synthesized from 32

(65 mg, 0.120 mmol) and stearoyl chloride (49 μL, 0.144 mmol)

using general procedure D. Puriﬁcation by ﬂash chromatography (5%

MeOH in DCM) produced the title compound 81 as an oﬀ-white

1

(77 mg, 97%). H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 15.6 Hz,

1H), 7.17−7.08 (m, 3H), 7.01 (d, J = 7.9 Hz, 1H), 6.90 (d, J = 8.7

Hz, 1H), 6.78 (t, J = 5.1 Hz, 1H), 6.46 (d, J = 15.6 Hz, 1H), 5.21−

5.09 (m, 2H), 4.56−4.46 (m, 1H), 4.46−4.38 (m, 1H), 4.23−4.06

(m, 2H), 3.85 (s, 3H), 2.57 (t, J = 7.5 Hz, 2H), 2.44−2.29 (m, 2H),

2.27−2.08 (m, 2H), 2.05−1.92 (m, 1H), 1.88−1.51 (m, 18H), 1.48−

1.38 (m, 2H), 1.36−1.25 (m, 14H), 1.00−0.91 (m, 6H), 0.88 (t, J =

6.7 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 172.79, 171.70, 171.41,

170.96, 169.22, 166.32, 151.40, 141.29, 141.24, 133.52, 123.21,

120.71, 119.97, 111.57, 78.69, 77.61, 58.48, 55.90, 52.06, 43.67, 34.04,

32.70, 32.65, 32.61, 32.52, 31.91, 30.78, 30.68, 29.62, 29.51, 29.34,

29.28, 29.06, 26.91, 25.01, 23.69, 23.64, 22.69, 19.33, 17.69, 14.12.

HRMS calcd for C₄₄H₆₈N₃O₁₀m/z: 798.4899 (M + H)⁺, found

798.4876.

1

solid (35 mg, 36%). H NMR (400 MHz, CDCl₃) δ 7.69 (d, J = 7.5

Hz, 1H), 7.57 (d, J = 15.5 Hz, 1H), 7.53−7.47 (m, 1H), 7.15−7.06

(m, 3H), 7.00 (d, J = 8.5 Hz, 1H), 6.47 (d, J = 15.5 Hz, 1H), 4.58−

4.44 (m, 2H), 4.41 (d, J = 6.5 Hz, 1H), 4.21−4.02 (m, 6H), 3.84 (s,

3H), 2.57 (t, J = 7.5 Hz, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.29−2.16 (m,

1H), 2.13−1.99 (m, 1H), 1.75 (p, J = 7.5 Hz, 2H), 1.49−1.06 (m,

37H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

173.16, 172.22, 171.79, 171.29, 170.13, 166.79, 151.38, 141.41,

141.23, 133.46, 123.20, 120.73, 119.83, 111.62, 67.28, 61.97, 60.89,

58.29, 55.88, 52.41, 34.04, 31.94, 30.55, 29.71, 29.68, 29.65, 29.54,

29.38, 29.31, 29.08, 26.16, 25.01, 24.95, 22.71, 18.78, 14.14, 14.08.

HRMS calcd for C₄₃H₇₀N₃O₁₁m/z: 804.5005 (M + H)⁺, found

804.4999.

4.3.30. Dicyclopentyl ((E)-3-(3-Methoxy-4-(stearoyloxy)phenyl)-

acryloyl)glycyl-^L-valyl-D-glutamate (78). Synthesized from 68 (62

mg, 0.101 mmol) and stearoyl chloride (41 μL, 0.121 mmol) using

general procedure D. The residue was washed twice with hexane to

4.3.34. Methyl N²-((R)-5-Ethoxy-4-((S)-2-(2-((E)-3-(4-hydroxy-3-

methoxyphenyl)acrylamido)acetamido)-3-methylbutanamido)-5-

oxopentanoyl)-N⁶-stearoyl-L-lysinate (82). Synthesized from 41 (30

mg, 0.0462 mmol) and stearoyl chloride (14 μL, 0.0462 mmol) using

general procedure D. Puriﬁcation by ﬂash chromatography (7%

MeOH in DCM) produced the title compound 82 as an oﬀ-white

1

produce the title compound 78 as a white solid (83 mg, 93%). H

NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 15.6 Hz, 1H), 7.25 (d, J = 9.5

Hz, 1H), 7.14−7.04 (m, 3H), 7.00 (d, J = 8.2 Hz, 1H), 6.94 (t, J = 5.2

Hz, 1H), 6.48 (d, J = 15.7 Hz, 1H), 5.22−5.09 (m, 2H), 4.56−4.40

1

solid (11 mg, 26%). H NMR (400 MHz, CDCl₃) δ 7.65−7.54 (m,

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2H), 7.37 (d, J = 7.3 Hz, 1H), 7.25−7.21 (m, 1H), 7.12−7.06 (m,

1H), 7.04 (d, J = 1.9 Hz, 1H), 6.94−6.84 (m, 2H), 6.41 (d, J = 15.6

Hz, 1H), 5.84 (s, 1H), 5.75 (d, J = 5.4 Hz, 1H), 4.41−4.34 (m, 2H),

4.29−4.12 (m, 3H), 4.03−3.93 (m, 2H), 3.93 (s, 3H), 3.72 (s, 3H),

3.34−3.18 (m, 2H), 2.34 (t, J = 6.5 Hz, 2H), 2.22−2.13 (m, 4H),

1.78 (s, 2H), 1.69−1.36 (m, 5H), 1.34−1.16 (m, 33H), 1.01 (d, J =

6.8 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.92−0.84 (m, 3H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 173.19, 172.41, 172.20, 171.92, 171.50,

169.42, 166.27, 148.83, 148.28, 139.95, 126.79, 122.05, 118.95,

116.10, 111.36, 60.94, 57.77, 55.98, 52.38, 52.25, 52.17, 38.49, 35.89,

31.75, 31.56, 31.29, 30.98, 29.49, 29.47, 29.40, 29.23, 29.16, 29.12,

27.15, 25.78, 23.23, 22.56, 19.64, 18.23, 14.50, 14.42. HRMS calcd for

C₄₉H₈₂N₅O₁₁m/z: 916.6005 (M + H)⁺, found 916.6002.

4.4. Mice. 4.4.1. Experiments with Bone-Marrow-Derived

Dendritic Cells and T Cells. C57BL/6, OT I (C57BL/6-Tg-

(TcraTcrb)1100Mjb/J), and OT II (C57BL/6-Tg(TcraTcrb)-

425Cbn/Crl) mice were purchased from Jackson Laboratory (Bar

Harbor, ME, U.S.A.) and bred at the University of Leiden (The

Netherlands). The mice were kept under standard laboratory

conditions, with food and water provided ad libitum. The mice

were euthanized while sedated by cervical dislocation. All animal work

was performed according to the guidelines of the European

Parliament Directive 2010/63EU, and the experimental work was

approved by the Animal Ethics Committee of Leiden University. For

culture conditions of BMDCs, see below.

4.4.2. In Vivo Experiments. NIH/OlaHsd inbred mice were raised

at the Institute of Immunology, Croatia. All mice used were females

from 2.0 to 2.5 months old. During the experimental period, the mice

were housed in the Animal Facility of the Institute of Immunology,

with food and water provided ad libitum. All animal work was

performed according to the Croatian Law on Animal Welfare (2017),

which complies strictly with the EC Directive (2010/63/EU).

4.5. Cell Cultures. 4.5.1. HEK-Blue NOD1 and NOD2 Cells. HEK-

Blue NOD1 and NOD2 cells (InvivoGen, San Diego, CA, U.S.A.)

were cultured according to the manufacturer instructions in

Dulbecco’s modiﬁed Eagle’s medium (Sigma-Aldrich, St. Louis,

MO, U.S.A.) supplemented with 10% heat-inactivated fetal bovine

serum (Gibco), 2 mM ^L-glutamine (Sigma-Aldrich), 100 U/mL

penicillin (Sigma-Aldrich), 100 μg/mL streptomycin (Sigma-Aldrich),

and 100 μg/mL normocin (InvivoGen) for two passages. All

subsequent passages were cultured in medium additionally

supplemented with 30 μg/mL blasticidin (InvivoGen) and 100 μg/

mL Zeocin (InvivoGen). The cells were incubated in a humidiﬁed

atmosphere at 37 °C and 5% CO₂.

4.5.2. Peripheral Blood Mononuclear Cells. Human PBMCs from

healthy and consenting donors were isolated from heparinized blood

by density gradient centrifugation with Ficoll-Paque (Pharmacia,

Sweden). The isolated cells were resuspended in RPMI 1640 medium

(Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% heat-

inactivated fetal bovine serum (Gibco), 2 mM ^L-glutamine (Sigma-

Aldrich), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL

streptomycin (Sigma-Aldrich) and used in the assays.

colony-stimulating factor (ImmunoTools, Friesoythe, Germany) for 7

days at 37 °C and 5% CO₂. The purity of the BMDCs was evaluated

with PE-labeled anti-mouse CD11c (Biolegend, San Diego, CA,

U.S.A.) by ﬂow cytometry with >90% shown to be CD11c positive.

4.6. Cytotoxicity. The tested compounds were dissolved in

DMSO and further diluted in culture medium to the desired ﬁnal

concentrations, such that the ﬁnal DMSO concentration did not

exceed 0.1%. HEK-Blue NOD2 cells were seeded (40 000 cells/well)

in 96-well plates in 100 μL of culture medium and treated with 20 μM

of each compound or with the corresponding vehicle (0.1% DMSO;

control cells). After 18 h of incubation (37 °C, 5% CO₂), the

metabolic activity was assessed using the CellTiter 96 Aqueous One

Solution cell proliferation assay (Promega, Madison, WI, U.S.A.),

according to the manufacturer instructions. The experiments were run

in duplicate and repeated as two independent biological replicates.

4.7. Measurement of NF-kB Transcriptional Activity (HEK-

Blue Detection). HEK-Blue NOD2 and NOD1 cell line reporter

assays are derived from HEK293 cells by cotransfection of the

hNOD2 or hNOD1 genes, respectively, and an NF-κB-inducible

secreted embryonic alkaline phosphatase (SEAP) reporter gene.

Following activation of NOD2 or NOD1, the resulting NF-κB induces

production of SEAP, the levels of which can be quantiﬁed

colorimetrically. HEK-Blue NOD2 or NOD1 cells were seeded (2.5

× 10⁵cells/mL) in 96-well plates in 200 μL of HEK-Blue detection

medium (InvivoGen, San Diego, CA, U.S.A.) and treated with the

compounds (2 μM for ﬁxed concentration assay; 7−8 diﬀerent

concentrations from 1 nM to 20 μM for EC₅₀determination) or with

the corresponding vehicle (0.1% DMSO). After 18 h of incubation

(37 °C, 5% CO₂), secreted embryonic alkaline phosphatase (SEAP)

activity was determined spectrophotometrically as absorbance at 630

nm (BioTek Synergy microplate reader; Winooski, VT, U.S.A.). EC₅₀

values were calculated using Prism software (version 9; GraphPad

Software, CA, U.S.A.). For determination of speciﬁcity, HEK-Blue

NOD2 cells (2.5 × 10⁵cells/mL) were ﬁrst pretreated for 1 h with a

10 μM NOD2 antagonist, before the addition of the compounds (2

μM), with incubation for 18 h. SEAP activity in the supernatants was

determined as above. The experiments were run in duplicate and

repeated as at least two independent biological replicates.

4.8. Cytokine Release from Peripheral Blood Mononuclear

Cells. Peripheral blood mononuclear cells were seeded (1 × 10⁶cells/

mL) in 48-well plates in 500 μL of growth medium and treated with

the compounds (2 μM) or with the corresponding vehicle (0.1%

DMSO) in the absence and presence of LPS (10 ng/mL). Cell-free

supernatants were collected after 18 h of incubation (37 °C, 5% CO₂)

and stored at −80 °C until tested. Cytokine production was

determined with the BD Cytometric Bead Array Human Inﬂamma-

tory Cytokines Kit (contents: IL-8, IL-1β, IL-6, IL-10, TNF, IL-

12p70; BD Bioscience) on an Attune NxT ﬂow cytometer (Thermo

Fisher Scientiﬁc, Waltham, MA, U.S.A.). Standard curves were

generated using recombinant cytokines contained in the kit. The

data were analyzed using the FlowJo (Tree Star, Inc., Ashland, OR,

U.S.A.) and Prism (GraphPad, San Diego, CA, U.S.A.) software. The

experiments were repeated as four independent biological replicates.

Statistical signiﬁcance was determined by repeated measures one-way

ANOVA followed with Dunnett’s multiple comparisons test.

4.5.3. Cancer Cell Lines. K562 cells are a chronic myelogenous

leukemia cell line (ATCC, Manassas, VA, U.S.A.),¹²⁶and MEC1 cells

are a B-chronic lymphocytic leukemia cell line (DSMZ GmbH,

Braunschweig, Germany).¹²⁷The K562 cells were cultured in RPMI

1640 medium (Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented

with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM ^L-

glutamine (Sigma-Aldrich), 100 U/mL penicillin (Sigma-Aldrich),

and 100 μg/mL streptomycin (Sigma-Aldrich). The MEC1 cells were

cultured in Iscove’s modiﬁed Dulbecco’s medium supplemented with

10% heat-inactivated fetal bovine serum (Gibco), 2 mM ^L-glutamine

(Sigma-Aldrich), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/

mL streptomycin (Sigma-Aldrich).

4.5.4. Bone-Marrow-Derived Dendritic Cells. Bone marrow cells

were isolated from the tibia of C57BL/6 mice and cultured in

Dulbecco’s modiﬁed Eagle’s medium (Lonza, Basel, Switzerland)

supplemented with 10% heat-inactivated fetal bovine serum (Lonza),

2 mM ^L-glutamine (Lonza), 100 U/mL penicillin (Lonza), 100 μg/

mL streptomycin (Lonza), and 20 ng/mL granulocyte-macrophage

4.9. Peripheral Blood Mononuclear Cell Cytotoxicity. The

PBMC cytotoxicity assays using K562 and MEC1 cells were

performed as described previously, with some modiﬁcations.⁸⁹

PBMCs were seeded (4 × 10⁵cells/well) in 96-well plates and

treated with compounds (0.1−10 μM) or vehicle (0.1% DMSO) for

18 h. The K562 or MEC1 cells were stained with CFSE (Invitrogen,

Carlsbad, CA, U.S.A.), washed twice with complete medium, and

added (1 × 10⁴cells/well) to pretreated PBMCs for a ﬁnal eﬀector

cell to target tumor cell ratio of 40:1. After a 4 h co-incubation (37

°C, 5% CO₂), cells were stained with Sytox blue dead cell stain

(Invitrogen) and analyzed using an Attune NxT ﬂow cytometer

(Thermo Fisher Scientiﬁc, Waltham, MA, U.S.A.) and the FlowJo

software (Tree Star, Inc., Ashland, OR, U.S.A.). Cells that were

positive for both CFSE and Sytox blue were deﬁned as dead K562 and

MEC1 cells. PBMCs alone and the CFSE-labeled cancer cells alone

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were also treated with the compounds at the same concentrations and

stained with Sytox blue to determine any direct cytotoxicity of the

compounds toward the PBMCs and cancer cells. The experiments

were run in duplicate and repeated as three independent biological

replicates. Statistical signiﬁcance was determined by one-way ANOVA

followed with Dunnett’s multiple comparisons test.

4.10. RNA Sequencing. Peripheral blood mononuclear cells from

three independent donors were seeded (2 × 10⁶cells/mL) in 24-well

plates in 1 mL of growth medium and treated with the compounds (2

μM) or vehicle (0.1% DMSO) for 18 h at 37 °C in 5% CO₂. The cells

were washed with phosphate-buﬀered saline, resuspended in

RNAlater RNA stabilization solution (Sigma-Aldrich, St. Louis,

MO, U.S.A.), and stored at −80 °C. RNA extraction, library

construction, and sequencing were conducted by Genewiz (Leipzig,

Germany).

expression. The experiments were run in duplicate and repeated as

two independent biological replicates. Statistical signiﬁcance was

determined by one-way ANOVA followed by Dunnett’s multiple

comparisons test.

4.12. T Cell Cytokine Release. Supernatants from CD4⁺and

CD8⁺T cells after 72 h of co-incubation with BMDCs (pretreated

with desmuramylpeptides [10 μM] and washed) were collected as

described above. The cytokine concentrations were determined with

the Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit

(contents: IL-2, IL-4, IL-6, IFN-γ, TNF, IL-17A, IL-10; BD

Bioscience) on an Attune NxT ﬂow cytometer (Thermo Fisher

Scientiﬁc, Waltham, MA, U.S.A.). Standard curves were generated

using recombinant cytokines contained in the kit. The data were

analyzed using the FlowJo (Tree Star, Inc., Ashland, OR, U.S.A.) and

Prism (GraphPad, San Diego, CA, U.S.A.) software. The experiments

were run in duplicate and repeated as two independent biological

replicates.

Brieﬂy, total RNA was extracted using the RNeasy mini kit

(Qiagen, Hilden, Germany) according to the manufacturer protocol.

RNA samples were quantiﬁed using Qubit 4.0 Fluorometer (Life

Technologies, Carlsbad, CA, U.S.A.), and RNA integrity was checked

with RNA Kit on a 5600 Fragment Analyzer (Agilent Technologies,

Palo Alto, CA, U.S.A.). All RNA samples were of high quality with an

RNA quality number of ≥9.4. RNA sequencing libraries were

prepared using NEBNext Ultra II RNA library prep kit for Illumina

according to the manufacturer instructions (New England Biolabs,

Ipswich, MA, U.S.A.). Libraries were loaded on the Illumina NovaSeq

6000 instrument, and clustering was performed directly on the

NovaSeq before sequencing according to the manufacturer

instructions. The samples were sequenced using a 2 × 150 paired

end conﬁguration. Image analysis and base calling were conducted by

the NovaSeq Control Software. Raw sequence data (.bcl ﬁles)

generated from the Illumina NovaSeq were converted into fastq ﬁles

and demultiplexed using Illumina’s bcl2fastq 2.19 software. One

mismatch was allowed for index sequence identiﬁcation. After

investigating the quality of the raw data, sequence reads were

trimmed to remove possible adapter sequences and nucleotides with

poor quality using Trimmomatic v.0.36. The trimmed reads were

mapped to the Homo sapiens reference genome as available on

ENSEMBL, using STAR aligner v.2.5.2b, thus generating BAM ﬁles.

Unique gene hit counts were calculated using feature counts from the

Subread package v.1.5.2. Only unique reads that fell within exon

regions were counted. After extraction of gene hit counts, the gene hit

counts table was used for downstream diﬀerential expression analysis.

Diﬀerential expression analysis was performed with iDEP.91.¹²⁸

First, a low expression ﬁlter was applied (0.5 counts per million in at

least 1 library). The remaining gene counts were normalized by

counts per million in the EdgeR package, with a pseudocount of 4.

Diﬀerential gene expression analysis was performed with the DESeq2

method, using a false discovery rate <0.05 and a gene expression fold

change >1.5 or <0.667 as the cutoﬀ values. The list of diﬀerentially

expressed genes was then used as input for gene annotation and

pathway enrichment analysis with Metascape.⁹⁴

4.11. Bone-Marrow-Derived Dendritic Cell Antigen Pre-

sentation. CD4⁺and CD8⁺T cells were puriﬁed from splenocytes of

OT II and OT I transgenic mice using CD4⁺and CD8⁺T cell

isolation kits (Miltenyi Biotec, Germany), according to manufacturer

instructions. Puriﬁed T cells were stained with CFSE (Invitrogen,

Carlsbad, CA, U.S.A.) and washed. Then, 5 × 10⁴T cells were mixed

with 1 × 10⁴BMDCs per well (pretreated with desmuramylpeptides

[1 or 10 μM] and 50 μg/mL ovalbumin [InvivoGen, San Diego, CA,

U.S.A.] for 18 h, and then washed). After 72 h of co-incubation (37

°C, 5% CO₂), the supernatants were collected and stored at −80 °C

for subsequent cytokine measurements. The cells were stained with

Fixable viability dye eFluor 780 (eBioscience, Thermo Fisher

Scientiﬁc, MA, U.S.A.), anti-Thy1.2-PE-Cy7 antibodies (Biolegend,

San Diego, CA, U.S.A.), anti-CD8-eFluor450 antibodies (eBio-

science), anti-CD4-eFluor450 antibodies (eBioscience), and anti-

CD25-APC antibodies (Biolegend) and analyzed using a Beckman

Coulter Cytoﬂex S ﬂow cytometer (CA, U.S.A.) and FlowJo software

(Tree Star, Inc., Ashland, OR, U.S.A.). Live Thy1.2⁺/CD4⁺and

Thy1.2⁺/CD8⁺were evaluated for CFSE dilution and CD25

4.13. In Vivo Induction of Ovalbumin-Speciﬁc Immune

Response. 4.13.1. Materials, Antigens, and Antibodies. Bovine

serum albumin, Tween 20, monoclonal anti-chicken egg albumin

(clone OVA-14 mouse IgG1 isotype), o-phenylenediaminedihydro-

chloride, and MDP were from Sigma (U.S.A.). Horseradish-

peroxidase-conjugated goat anti-mouse IgG (HRP-anti-mouse IgG)

was from Bio-Rad Laboratories (U.S.A.). Biotin-conjugated rat anti-

mouse IgG1 and anti-mouse IgG2a monoclonal antibodies and

streptavidin−peroxidase were from PharMingen, Becton Dickinson

(U.S.A.). Chemicals for buﬀers and solutions were from Kemika

(Croatia). Ovalbumin was from Serva (Germany). ^L-α-Phosphati-

dylcholine, type XI-E, from fresh egg yolk (egg-phosphatidylcholine)

was from Avanti Polar Lipids. Cholesterol from porcine liver and

dicetylphosphate were from Sigma (U.S.A.). Monomannosyl-PEG-

palmitic acid derivative (Man-PEG-Pam) was synthesized as

previously described.¹²⁹

4.13.2. Preparation of Liposomes. Multilamellar liposomes were

prepared by the modiﬁed thin lipid ﬁlm method as described

previously.^130,131For the preparation of neutral liposomes, a 7:5

molar ratio of egg-phosphatidylcholine and cholesterol was used. For

the preparation of negatively charged liposomes, a 7:5:1 molar ratio of

egg-phosphatidylcholine, cholesterol, and dicetylphosphate was used,

while a 7:5:0.5 molar ratio of egg-phosphatidylcholine, cholesterol,

and Man-PEG-Pam was used in the preparation of mannosylated

liposomes. Lipids (total lipid concentration, 4 mg/mL) were dissolved

in chloroform/methanol (2:1). A methanol or chloroform/methanol

solution of MDP or the desmuramylpeptides was added to the lipid

solution for a ﬁnal concentration of 2 mM and 1.5 mM for the ﬁrst

(Figure 10) and second (Figure 11) experiments, respectively. After

rotary evaporation of the solvent, the remaining lipid ﬁlm was dried in

vacuum for 1 h and then dispersed by gentle handshaking in an

ovalbumin solution in saline (0.0667 and 0.1 mg/mL for the ﬁrst and

second experiments, respectively). This was then left overnight at 4

°C to swell and stabilize. Liposome size was reduced by soniﬁcation.

Nonentrapped material was not separated from liposomes, and the

complete liposome suspension was used for immunizations.

4.13.3. Immunizations. For the ﬁrst experiment (Figure 10), sex-

matched NIH/OlaHsd mice (ﬁve per group) were immunized

subcutaneously in the tail base and boosted one time after 21 days.

The injection volume in all experimental groups was 0.15 mL per

mouse, which corresponds to 10 μg of OVA, 400 μg of lipids, and

0.30 μmol of MDP and desmuramylpeptides. For the second

experiment (Figure 11), sex-matched NIH/OlaHsd mice (ﬁve per

group) were immunized and boosted two times subcutaneously into

the tail base at 21-day intervals. The injection volume in all

experimental groups was 0.1 mL per mouse, which corresponds to

10 μg of OVA, 400 μg of lipids, and 0.15 μmol of MDP and 75. The

mice were anesthetized with ip application of ketamine/xylazine (25

mg/kg each) prior to blood collection from the axillary plexus on the

seventh day after the last booster dose. Individual serum from each

animal was decomplemented at 56 °C for 30 min and then stored at

−20 °C until tested.

7833

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J. Med. Chem. 2021, 64, 7809−7838

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4.13.4. Ovalbumin-Speciﬁc Serum Antibody Concentration

Determination by ELISA. ELISA assays were performed as detailed

previously.⁴⁶Brieﬂy, high-binding ELISA plates (Costar, U.S.A.) were

coated with a 15 μg/mL solution of ovalbumin in carbonate buﬀer,

pH 9.6, and incubated overnight at room temperature. Nonspeciﬁc

antibody binding was blocked with 0.5% w/v bovine serum albumin

in PBS-T (0.05% (v/v) Tween 20 in phosphate-buﬀered saline) for 2

h at 37 °C. After washing, ﬁve serial dilutions of mice sera and

standard preparations were added in duplicate. Plates were incubated

overnight at room temperature, washed, and analyzed for ovalbumin-

speciﬁc IgG levels by incubation with HRP-conjugated goat anti-

mouse IgG (2 h at 37 °C) and then, after washing, with 0.6 mg/mL o-

phenylenediaminedihydrochloride solution in citrate-phosphate buf-

fer, pH 5.0, with 0.5 μL of 30% H₂O₂per milliliter for 30 min at room

temperature in the dark. The enzymatic reaction was stopped with

12.5% H₂SO₄, and absorbance at 492 nm was measured using a

microplate reader (Thermo Fisher Scientiﬁc, Waltham, MA, U.S.A.).

For the determination of ovalbumin-speciﬁc IgG1 and IgG2a, the

plates were incubated with biotin-conjugated rat anti-mouse IgG1 or

IgG2a (2 h at 37 °C) and subsequently with streptavidin−peroxidase

for another 2 h at 37 °C. After washing, the substrate solution was

added and incubated for 30 min at room temperature in the dark, as

described above. The enzymatic reaction was stopped with 12.5%

H₂SO₄, and absorbance at 492 nm was measured using a microplate

reader. The relative quantities of antibodies were determined by

parallel line assays using appropriate standard preparations of anti-

ovalbumin IgG, anti-ovalbumin IgG1, and anti-ovalbumin IgG2a.

Statistical signiﬁcance was determined by one-way ANOVA followed

by Dunnett’s multiple comparisons test.

Authors

Samo Guzelj − Faculty of Pharmacy, University of Ljubljana,

SI-1000 Ljubljana, Slovenia

Sanja Nabergoj − Faculty of Pharmacy, University of

Ljubljana, SI-1000 Ljubljana, Slovenia

Martina Gobec − Faculty of Pharmacy, University of

Ljubljana, SI-1000 Ljubljana, Slovenia

Stane Pajk − Faculty of Pharmacy, University of Ljubljana, SI-

1000 Ljubljana, Slovenia

̌

Veronika Klancic − Faculty of Pharmacy, University of

Ljubljana, SI-1000 Ljubljana, Slovenia

Bram Slutter − Div. BioTherapeutics, Leiden Academic Centre

̈

for Drug Research, Leiden University, 2333 CC Leiden, The

Netherlands

̌

Ruza Frkanec − Centre for Research and Knowledge Transfer

in Biotechnology, University of Zagreb, 10000 Zagreb,

Croatia

̌

Adela Stimac − Centre for Research and Knowledge Transfer

in Biotechnology, University of Zagreb, 10000 Zagreb,

Croatia

Primoz Sket − Slovenian NMR Centre, National Institute of

Chemistry, SI-1000 Ljubljana, Slovenia

Janez Plavec − Slovenian NMR Centre, National Institute of

Chemistry, SI-1000 Ljubljana, Slovenia

Irena Mlinaric-Rascan − Faculty of Pharmacy, University of

Ljubljana, SI-1000 Ljubljana, Slovenia

̌

4.14. Screening against PAINS. All tested compounds were

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00644

screened against the PAINS ﬁlter¹³²as implemented in CANVAS

̈

(Schrodinger software, Release 2020-2, New York, U.S.A.).

Compound 21 was identiﬁed as an interfering compound due to

the presence of a catechol structure. Given that 21 was tested in two

independent analogous assays with HEK-Blue NOD1 and NOD2

cells and it showed no activity in the NOD1 assay, the activity

measured in the NOD2 assay did not arise from nonspeciﬁc

activation. Furthermore, the reduced NOD2 activation of 21 after

NOD2 antagonist pretreatment in the speciﬁcity assay provided an

additional argument for the absence of interference of the catechol

structure in 21.

4.15. Statistics. Data analysis was performed using Prism software

(version 9; GraphPad Software, CA, U.S.A.). Statistical diﬀerences

were determined as speciﬁed under individual experimental

procedures. A p value < 0.05 was considered statistically signiﬁcant.

Author Contributions

The study was designed by Z.J. and S.G. The synthetic work

and characterization of compounds was conducted by Z.J.,

S.G., and V.K. Chiral HPLC resolution was performed by S.P.

̌

NOESY experiments were conducted by S.G., P.S., and J.P. In

vitro assays of cytotoxicity, NOD1 and NOD2 stimulation,

PBMC isolation, and stimulation and determination of

cytotoxicity versus cancer cells were conducted by S.G., M.

G., and S.N. In vitro assays with BMDCs and T cells were

conducted by S.G. and B.S. Preparation of experimental

liposomal formulations and in vivo experiments (immunization

̌

experiments, analysis of sera) were conducted by A.S. and R.F.

̌

Z.J. and S.G. wrote the manuscript. Z.J., S.G., S.N., M.G., S.P.,

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00644.

■

̌

V.K., B.S., A.S., R.F., and I.M.R. analyzed the data, read the

sı

manuscript, and gave approval to the ﬁnal version.

Notes

The authors declare no competing ﬁnancial interest.

(Molecular formula strings CSV)

Synthesis and analytical data of intermediate compounds

4, 5, 7−17, 22, 33−40, 47−55, 65−67, and 69−71,

supporting schemes and ﬁgures, experimental procedure

ACKNOWLEDGMENTS

■

This research was funded by the Slovenian Research Agency

(Grants P1-0208 and J3-9256), the Croatian Science

Foundation (HrZZ) (Project No. IP-2018-01-6910), their

mutual bilateral contract (BI-HR/18-19-001), and the COST

action CA16231 “European Network of Vaccine Adjuvants

(ENOVA)” (Grant ID STSM-CA16231- 130120-114265).

The authors thank Dr. Chris Berrie for proofreading the

manuscript.

1

for chiral HPLC resolution of compound 29, H−¹H

NOESY determination of cyclopropane stereoconﬁgura-

1

tion in compounds 27, 28, 29a, and 29b, and H and

¹³C NMR spectra of representative tested compounds

(PDF)

AUTHOR INFORMATION

Corresponding Author

■

ABBREVIATIONS USED

■

̌

Ziga Jakopin − Faculty of Pharmacy, University of Ljubljana,

BMDC, bone-marrow-derived dendritic cell; CARD, caspase

recruitment domain; CFSE, carboxyﬂuorescein succinimidyl

ester; COMU, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-

dimethylamino-morpholino-carbenium hexaﬂuorophosphate;

SI-1000 Ljubljana, Slovenia; orcid.org/0000-0001-9384-

0858; Phone: +386 1 4769 646; Email: ziga.jakopin@

ﬀ a.uni-lj.si; Fax: + 386 1 4258 031

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J. Med. Chem. 2021, 64, 7809−7838

Journal of Medicinal Chemistry

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Article

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DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DC, dendritic cell;

DCC, dicyclohexylcarbodiimide; DIPEA, N,N-diisopropyle-

thylamine; DMAP, 4-dimethylaminopyridine; DMF, dimethyl-

formamide; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)-

carbodiimide; EtOAc, ethyl acetate; GO, Gene Ontology;

HOBt, 1-hydroxybenzotriazole; ISG, interferon-stimulated

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LPS, lipopolysaccharide; Man-PEG-Pam, monomannosyl−

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Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their in Vivo Adjuvant Activity

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