3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of m1 aminopeptidases with immune-regulating properties

Article

pubs.acs.org/jmc

3,4-Diaminobenzoic Acid Derivatives as Inhibitors of the Oxytocinase

Subfamily of M1 Aminopeptidases with Immune-Regulating

Properties

Athanasios Papakyriakou,^†,‡Efthalia Zervoudi,^†Soﬁa Tsoukalidou,^†Francois-Xavier Mauvais,^§

Georgia Sfyroera,^†Dimitrios C. Mastellos,^†Peter van Endert,^§Emmanuel A. Theodorakis,^‡

Dionisios Vourloumis,*^,†and Efstratios Stratikos*^,†

^†National Center for Scientiﬁc Research “Demokritos”, Aghia Paraskevi, 15310 Athens, Greece

^‡Department of Chemistry and Biochemistry, University of California−San Diego, 9500 Gilman Drive, San Diego, California

92093-0358, United States

^§Institut National de la Sante

National de la Recherche Scientiﬁque, Unite

́

et de la Recherche Med

́

icale, Unite

́ ́ ́

1151; Universite Paris Descartes, Sorbonne Paris Cite; Centre

́

8253, 75015 Paris, France

S

* Supporting Information

ABSTRACT: Members of the oxytocinase subfamily of M1

aminopeptidases (ERAP1, ERAP2, and IRAP) play important

roles in both the adaptive and innate human immune

responses. Their enzymatic activity can contribute to the

pathogenesis of several major human diseases ranging from

viral and parasitic infections to autoimmunity and cancer. We

have previously demonstrated that diaminobenzoic acid

derivatives show promise as selective inhibitors for this

group of aminopeptidases. In this study, we have thoroughly

explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors

for ERAP2 (IC₅₀= 237 nM) and IRAP (IC₅₀= 105 nM). Cell-based analysis indicated that the lead compounds can be eﬀective

in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone

marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of

immune responses.

and parasitic infections to cancer and inﬂammatory diseases

with autoimmune etiology (reviewed in refs 8 and 9). Recently,

a secreted form of ERAP1 has also been implicated in innate

immunity responses: macrophages activated by interferon-

gamma and liposaccharides secrete ERAP1 in a TLR-dependent

pathway,^10,11resulting in the enhancement of their phagocytic

activity and inﬂammatory potential. Similarly, ERAP1 knockout

mice show increased activation of NK and NKT cells,¹²

consistent with a role of ERAP1 in regulating both adaptive and

innate immune responses. ERAP1 polymorphic variation was

recently shown to quantitatively aﬀect innate immune

responses.¹³Since natural polymorphic variation of ERAP1

and ERAP2 can generate a range of enzymatic activities in the

population, it has been hypothesized that the activity of these

enzymes is an important regulator of immune responses in

humans.¹⁴

INTRODUCTION

■

The three members of the oxytocinase subfamily of M1

aminopeptidases,¹namely, endoplasmic reticulum amino-

peptidases 1 and 2 (ERAP1 and ERAP2) and insulin regulated

aminopeptidase (IRAP), henceforth referred to as antigen

processing aminopeptidases (APAs), have been, during recent

years, shown to have important biological functions, primarily

in the regulation of human adaptive and innate immune

responses. ERAP1 and ERAP2 act in intracellular antigen

processing and are responsible not only for the correct

generation of many antigenic epitopes but also for the

destruction of others.²⁻⁴By this function, these enzymes can

regulate cellular immune responses to infected or diseased cells

and contribute to immune evasion by pathogens and cancer

cells as well as to autoimmunity.⁵IRAP combines the speciﬁcity

of ERAP1 and ERAP2 and functions in a specialized pathway of

antigen processing and presentation called cross-presentation,

present in dendritic cells (DCs), possibly regulating early stages

of inﬂammatory immune responses.^6,7ERAP1 and ERAP2 are

polymorphic, and several coding single nucleotide poly-

morphisms in these genes have been associated with

predisposition to major human diseases, ranging from viral

Inhibition of APAs is a promising approach for selectively

regulating immune responses. ERAP1 knockout mice present a

distinct repertoire of antigenic peptides on their cell sur-

Received: December 3, 2014

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Figure 1. (a) Crystallographic structure of ERAP1 in the closed conformation (PDB ID: 2YD0), illustrating the domain organization and the active

site of the enzyme. The catalytic zinc is shown as a gray sphere, and protein residues are shown as sticks with carbon in cyan, nitrogen in blue, oxygen

in red, and sulfur in yellow. (b) Schematic representation of the 1,4-disubstituted DABA inhibitors, where R₁and R₂are L-α-amino acid side chains

targeting the S1 and Sn′ subsites of the aminopeptidases, respectively, and X = H, Me, or Bn. (c) Molecular model of the 1,4-disubstituted DABA

scaﬀold (carbon atoms shown in orange) docked at the active site of ERAP1. The α-amino terminal docking residues are E183 and E320; the zinc-

binding residues are H353, H357, and E376, whereas the catalytic E354 is expected to interact with the free aniline of the inhibitors. Catalytically

important Y438 and conserved F433 that forms the bottom of S1 are also shown.

face.¹⁵⁻¹⁷Genetic downregulation of ERAP1 can lead to novel

cellular immune responses, including nonclassical cellular

responses, through the activation of cytotoxic-T lymphocytes

and natural killer cells.¹⁸⁻²⁰A pseudophosphinic peptide APA

inhibitor has been demonstrated recently to enhance antigen

presentation and elicit potent anti-cancer CTL responses

toward a cryptic antigenic epitope that is normally destroyed

by ERAP1.²¹On the other hand, downregulation of ERAP1 has

been recently shown to reduce CTL responses against a viral

epitope associated with the pathogenesis of autoimmunity.²²As

a result, the pharmacological regulation of APA activity has

attracted signiﬁcant scientiﬁc interest over the past few years as

a promising area with therapeutic potential. Unfortunately,

most characterized APA inhibitors suﬀer either from low

potency or low selectivity, necessitating the exploration of other

approaches. Selectively targeting only one of the members of

this family of aminopeptidases may be highly desirable in order

to ﬁne-tune immune responses while minimizing potential side

eﬀects.

We recently demonstrated that using 3,4-diaminobenzoic

acid (DABA) as a scaﬀold is a valid approach for generating

inhibitors for this class of aminopeptidases with promising

selectivity proﬁles.²³In this study, we explored the DABA

derivatives more extensively, with the aim of improving their

potency and selectivity toward ERAP1, ERAP2, and IRAP. By

employing a rational, structure-based optimization procedure,

we identiﬁed potent inhibitors for ERAP2 (237 nM) and IRAP

(105 nM) as well as a highly selective ERAP2 inhibitor (755

nM) in comparison to ERAP1 (>100 μM).

results suggest that these compounds may hold promise upon

further development as targeted modulators of inﬂammatory

immune responses.

RESULTS AND DISCUSSION

■

Inhibitor Design. Selection of the amino acid side chains

used for inhibitor design was based on a previous study on the

selectivity proﬁle of the S1 subsite of ERAP1, ERAP2, and

IRAP using a library of 82 ﬂuorogenic substrates.²⁴Speciﬁcally,

it was suggested that ERAP1 displays a general preference for

substrates comprising long, aromatic or hydrophobic, R₁side

chains (Figure 1). In contrast, the S1 subsite of ERAP2 exhibits

higher selectivity for positively charged groups, especially Arg

and homo-Arg, whereas IRAP combines the speciﬁcity of

ERAP1 and ERAP2. All ^D-amino acid-based substrates were

poorly processed by the three enzymes, suggesting that the ^L

conﬁguration is a prerequisite for binding. In our initial

eﬀorts,²³we utilized homo-Phe (hPhe), which is predicted to

be ideally accommodated within the S1 speciﬁcity pocket of

ERAP1 due to π-stacking interactions with a conserved

aromatic residue (F433/450/544 for ERAP1/ERAP2/IRAP,

respectively). In combination with the positively charged side

chain of Lys for R₂, 1,3-substituted derivative 4a displayed low-

micromolar aﬃnity for ERAP1 (IC₅₀= 2.0 0.6 μM) and >10-

fold selectivity for ERAP2. In this study, we explored the

contribution of hydrophobic (Leu and nor-Leu), aromatic

(Phe, Tyr, homo-Tyr(OBn), Ser-OBn), and positively charged

Arg side chains at position R₁, while keeping lysine in the R₂

position (Figure 1). Subsequently, a series of 1,3-substituted

DABA derivatives was synthesized by combining the above-

mentioned R₁amino acids with hydrophobic (Val), polar

(Thr), aromatic (Tyr and Trp), and positively charged Arg side

chains at position R₂. Our collection of 1,3-disubstituted DABA

analogues was enriched with a selection of 1,4-disubstituted

DABA to evaluate the diﬀerent binding orientations of the

inhibitors. A subset of monosubstituted DABA derivatives was

also evaluated for direct comparison.

To evaluate the biological potential of the discovered

inhibitors, we utilized two specialized cellular assays relevant

to innate and adaptive inﬂammatory immune responses. The

low-micromolar ERAP1 inhibitor, 4a (2 μM), was able to

inhibit macrophage activation by inﬂammatory mediators with

submicromolar potency. The most potent IRAP inhibitor, 4u,

was able to block the IRAP-dependent cross-presentation

pathway in dendritic cells with low-nanomolar potency. These

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a

Scheme 1. Synthesis of the 1,3- and 1,4-Substituted 3,4-Diaminobenzoic Acid Derivatives

a

Reagents and conditions: (a) protected amino acid (1.1−1.5 equiv), HBTU (2.0−3.0 equiv), DIEA (3.0−4.0 equiv), DMF, 4−12 h, rt, 65−92%;

(b) LiOH 1 M (20 equiv), dioxane/H₂O (1:1), 3−6 h, rt, 87−95%. (c) LiOH 1 M (20 equiv), dioxane/H₂O (1:1), 3−6 h, rt, or cat. Pd/C (10 wt

%), H₂, MeOH, 1−2 h, rt, 90−95%. (d) TFA/CH₂Cl₂(1:2), 20−40 min, rt, 95−98%. (e) (Boc)₂O (1.0 equiv), Gu·HCl (15 mol %), EtOH, 40 min,

45 °C, 78%. (f) Protected amino acid (2.0 equiv), HATU (3.0 equiv), DBU (4.0 equiv), DMF, 24−36 h, rt, 45−72%. R₁: side chain of L-α-amino

acid selected from Leu, nor-Leu, Ser(OBn), Phe, homo-Phe, Tyr(OBn), homo-Tyr(OBn), or Arg(Z)₂. R₂: side chain of L-α-amino acid selected from

Val, Thr, Tyr(O-tBu), Trp, Lys(Boc), ornithine(Boc), or Arg(Z)₂. R₁′, R₂′: the corresponding deprotected L-amino acid side chains. X: H, OMe, or

OBn.

Chemistry. General synthetic routes for all new analogues

are presented in Scheme 1. As described previously,²³1,3-

disubstituted DABA derivatives 4 were obtained through the

coupling of N-Boc-protected amino acids, furnishing inter-

mediates 2, followed by saponiﬁcation of the corresponding

methyl ester, formation of the second amide bond using O-

methyl or O-benzyl protected amino acids, and global removal

of the protecting groups. Following an analogous route, our

approach was extended to furnish 1,4-disubstituted DABA

derivatives 10 starting from 3-N-Boc protected DABA

intermediate 7. In this case, the second amide bond formation

at the less reactive 4-NH₂was achieved in higher yields by

utilizing a combination of HATU as the coupling reagent and

DBU as the base. As mentioned before, monosubstituted

DABA derivatives 3, 6, and 9 were also prepared, as presented

in Scheme 1, for direct comparison.

In Vitro Evaluation. The inhibitory potency of the

synthesized compounds for the three enzymes was determined

using an established ﬂuorogenic assay.²³Regarding monosub-

stituted DABA derivatives 3a−3g′ (Table 1), 6a−6f, and 9a−

9d (Table 2), the in vitro evaluation did not reveal any low-

micromolar (<10 μM) ERAP1 inhibition. In contrast, 4-amino

substituted DABA derivatives 6e (R₁Nle) and 6f (R₁Arg)

displayed IC₅₀values of 1.1 and 2.5 μM for ERAP2,

respectively, whereas 6b (R₁Tyr(OBn)) exhibits IC₅₀= 1.7

μM for IRAP.

In the eﬀort to optimize our previous hit 4a, we initially

substituted R₂Lys for the shorter side chain of L-ornithine

(Orn). Surprisingly, both the methyl ester and the carboxylic

acid of 1,3-hPhe-DABA-Orn derivatives (4d and 4d′,

respectively) were found to be inactive (IC₅₀> 100 μM) for

all three aminopeptidases (Table 1), indicating a very important

interaction associated with the longer Lys side chain.

Substitution of hPhe by the shorter Phe side chain yielded

compound 4e, which is inactive for ERAP1, although it is more

potent than 4a for ERAP2 and IRAP, demonstrating the

importance of the longer hPhe side chain, as proposed

previously.²³From the remaining 1,3-disubstituted DABA-Lys

derivatives, 4e−4l, the in vitro evaluation revealed several

moderate inhibitors (IC₅₀= 2−8 μM) for ERAP2 and IRAP

only, with most compounds being inactive toward ERAP1

(Table 1).

The next generation of 1,3-disubstituted DABA derivatives,

4m−4y, was designed by combining Tyr, hTyr, Nle, and Arg

for R₁with Val, Tyr, and Trp for R₂(Table 1). Although Tyr in

the R₁position yielded low-aﬃnity inhibitors (4m−4p′) for all

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Table 1. In Vitro Evaluation of the Designed 1,3-Substituted

DABA Derivatives for ERAP1, ERAP2, and IRAP*

Table 2. In Vitro Evaluation of the Designed 1,4-Substituted

DABA Derivatives for ERAP1, ERAP2, and IRAP*

*

NI, no inhibition observed up to 100 μM; >100 indicates that a

limited inhibition is observed in the 50−100 μM range.

contrast, the inhibitory eﬀect of low-micromolar IRAP

inhibitors 4b (IC₅₀= 1.3 μM) and 4c (IC₅₀= 2.8 μM) is

marginally aﬀected at their corresponding para-isomers 10d

(IC₅₀= 2.4 μM) and 10c (IC₅₀= 1.3 μM), respectively.

By employing Tyr in the R₁position, we obtained two more

potent inhibitors, 10h, with IC₅₀= 1.1 μM for ERAP1, and

10h′, with IC₅₀= 1.3 μM for IRAP. In particular, 10h displayed

a 2-fold increase in selectivity for ERAP1 compared to ERAP2,

with respect to that of the initial hit, 4a. Interestingly,

compound 10j′ (R₁hTyr and R₂Trp), the topoisomer of

ERAP1-inactive 4r, exhibits an IC₅₀= 0.8 μM for ERAP1 with a

∼12-fold selectivity with respect to ERAP2. Similar to the 1,3-

analogues, the use of Nle at R₁(10m−10o′) in combination

with Tyr or Trp at R₂provided the most potent ERAP2 and

IRAP inhibitors. Among them, 10n, with IC₅₀= 755 nM for

ERAP2, is the most selective inhibitor against ERAP1 (>130-

*

NI, no inhibition observed up to 100 μM; >100 indicates that a

α

limited inhibition is observed in the 50−100 μM range. Data taken

from ref 23.

three enzymes, hTyr in combination with Trp displayed low-

micromolar inhibition for IRAP (4q and 4r). In particular, 4q

proved to be the most selective IRAP inhibitor against both

ERAP1 and ERAP2 (IC₅₀> 100 μM). The combination of

R₁Nle (4s−4v) or Arg (4w−4y) with R₂Tyr or Trp

provided the most potent inhibitors. Compound 4u is the most

potent inhibitor of ERAP1 (IC₅₀= 0.92 μM) and IRAP (IC₅₀

=

105 nM), followed by its corresponding carboxylic acid 4u′

(IC₅₀= 296 nM for IRAP). It is interesting to note that 4s and

4t are both signiﬁcantly selective inhibitors of IRAP (IC₅₀

=

1.2−1.3 μM) with respect to ERAP1 (IC₅₀> 100 μM). As

predicted, Arg in the R₁position provided two more

submicromolar inhibitors, 4x and 4y, for both ERAP2 and

IRAP, with 4x (IC₅₀= 518 nM) displaying a ∼20-fold

selectivity with respect to ERAP1 (IC₅₀= 9.6 μM).

Concerning the in vitro evaluation of the 1,4-disubstituted

DABA derivatives (Table 2), our results indicate a signiﬁcant

structural eﬀect from the orientation of the two amino acid

substituents on their inhibitory eﬀect, especially regarding

ERAP1. A striking example is inactive compound 10a, which is

the regioisomer of the initial ERAP1 hit, 4a (IC₅₀= 2.0 μM). In

fold), and 10o′ is the most potent inhibitor of ERAP2 (IC₅₀

=

237 nM). The corresponding benzyl ester of the latter, 10o,

displayed a higher potency for IRAP (IC₅₀= 438 nM) with

respect to ERAP2 (IC₅₀= 709 nM). Finally, 10q with R₁Arg,

the para-isomer of potent ERAP2/IRAP inhibitor 4x, provided

an equipotent inhibitor of the three enzymes (IC₅₀= 1.4−2.6

μM).

Overall, the in vitro evaluation of the designed DABA

derivatives revealed several potent inhibitors for the three M1

aminopeptidases. ERAP1 appears to be the most challenging

target in terms of inhibitor potency and selectivity, with 4u

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(IC₅₀= 0.92 μM) and 10j′ (IC₅₀= 0.80 μM) being the most

eﬀective inhibitors and 10h being the most selective with

respect to ERAP2 (∼20-fold). On the other hand, our

optimization eﬀorts resulted in signiﬁcantly more potent

inhibitors of ERAP2 (10o′, IC₅₀= 237 nM) and IRAP (4u,

IC₅₀= 105 nM). In addition, we identiﬁed two remarkably

selective inhibitors, 10n, displaying IC₅₀= 755 nM for ERAP2

and IC₅₀> 100 μM for ERAP1, and 4s, with IC₅₀= 1.3 μM for

IRAP and IC₅₀> 100 μM for both ERAP1 and ERAP2.

Conclusively, the inhibitory potency of the synthesized

compounds revealed that Nle as the R₁and Trp as the R₂

substituents provided the most potent inhibitors for the three

aminopeptidases. It is possible that the linear side chain of Nle

is easily accommodated within the S1 pocket and facilitates the

proper orientation of the inhibitors in both topologies of the

DABA derivatives. The indole ring of Trp is most probably

mediating key aromatic interactions at the S-primed subsites in

addition to potential hydrogen-bonding interactions. Similar

observations were drawn from the crystallographic structure of

ERAP1 in complex with a phosphinic transition state analogue

tripeptide comprising a Trp residue, which was found to be

stacked with catalytic Y438.²¹

Structure−Activity Relationships. Recent crystallo-

graphic studies revealed that ERAP1 can adopt at least two

distinct conformational states that involve large interdomain

motions, a closed, active-form (Figure 1a) and an open, inactive

conformation that exposes a large internal peptide-binding

cavity to the solvent.²⁵Although ERAP2 has been crystallized

in the closed conformation only, the absence of substrate access

into the active site in the published structure suggests that

ERAP2 should be able to adopt open conformations as well.²⁶

These two states diﬀer in the organization of key active site

residues and the adjacent S1 speciﬁcity pocket, which is

disordered in the open state, making inhibitor design a

challenging task.¹⁴Speciﬁcally, the conserved ERAP1 residue

F433 that comprises the bottom of the S1 pocket as well as the

catalytic Y438 are both disordered or dislocated in the open-

state crystal structure.²⁷

It is therefore plausible that inhibitors that target these

aminopeptidases may form initial interactions inside the active

site at diﬀerent conﬁgurations than those expected by analyzing

the closed crystallographic structures. This may be exempliﬁed

by a lack of a strong zinc binding group serving as anchor point.

As a result, molecular docking calculations of the synthesized

compounds reveal multiple putative binding poses inside the

active sites of all three aminopeptidases. Still, the most potent

inhibitors display a common binding motif among the top-

ranked predicted conformations, where the carbonyl group of

R₁is bound to zinc so that the R₁side chain can be

accommodated within the S1 pocket (Figure 1c). In such

conformation, the α-amino acid terminal amine displays salt

bridge interactions with two conserved glutamates, E183/200/

295 and E320/337/431 (residue numbering of ERAP1/

ERAP2/IRAP, respectively), and the free aniline could provide

an additional hydrogen bond with the catalytic E354/371/465

residue. It is therefore possible that only the best inhibitors

bind in the expected conformation through an induced-ﬁt

mechanism that promotes correct folding of the APA active

site.

Figure 2. Close-up view of the active site from molecular models of

(a) ERAP1−10j′, (b) ERAP2−10n, and (c) IRAP−4u complexes. The

inhibitors are shown with carbon atoms in orange, and the key

interacting residues, with cyan carbons. Potential hydrogen bonds are

indicated with dashed lines, and the surface representations illustrate

the three potential subsites of the enzymes.

residue that belongs to the C-terminal domain IV. The indole

ring of 10j′ could interact with the H873 residue of domain IV,

whereas its terminal carboxylic acid might form salt bridges

with K308 and K403. Considering the bound conformation of

the most selective inhibitor of ERAP2 (>100-fold with respect

to ERAP1), 10n is predicted to bind in a similar orientation as

that for 10j′. However, the phenolic ring in 10n could be

accommodated between the aromatic side chains of Y892 and

W363 so that a hydrogen bond is formed between its

hydroxylate group and R345 of ERAP2 (Figure 2b). The

nonconserved W363 residue of ERAP2 corresponds to G346 of

ERAP1 and therefore such an interaction might not be possible

between ERAP1 and 10n. The most potent IRAP inhibitor, 4u,

is predicted to interact mostly with conserved residues and thus

displays low-micromolar aﬃnity for ERAP1 and ERAP2 as well.

The high binding aﬃnity of 4u for IRAP (105 nM) can be

attributed to a potential stacking interaction of the indole

moiety with Y961 and the two hydrogen bonds with S546

(Figure 2c). The terminal benzyl group of 4u is predicted to

provide minor contacts; therefore, the corresponding depro-

tected analogue, 4u′, displayed a marginally lower binding

aﬃnity for IRAP (296 nM).

Biological Evaluation of Inhibitors. To assess the

biological eﬃcacy of this group of inhibitors we utilized two

specialized cellular assays that relate to the function of the

human innate and adaptive immune responses. It has been

recently demonstrated that macrophages respond to the

inﬂammatory modulator interferon-γ in combination with

lipopolysaccharide (LPS), which is a component of the outer

membrane of Gram-negative bacteria, by secreting ERAP1 in a

The docked conformation of the most potent ERAP1

inhibitor, 10j′, displays the side chain of its hTyr stacked above

F433 inside the S1 pocket and, at the same time, forms a

hydrogen bond with the backbone NH of S869 (Figure 2a), a

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Figure 3. (a) Eﬀect of interferon-γ and LPS on the phagocytic activity of RAW264.7 macrophages as followed by the internalization of ﬂuorescent

latex beads. (b) Fluorescence enhancement due to macrophage activation. (c, d) Reduction of ﬂuorescence enhancement due to inhibition of

phagocytosis by the ERAP1 inhibitors and the calculated ED₅₀values.

TLR-dependent manner.^10,11The aminopeptidase activity of

the secreted ERAP1 induces the enhancement of phagocytosis

by macrophages, which can be followed by the internalization

of ﬂuorescently labeled latex beads (Figure 3a). To evaluate the

eﬀect of the inhibitors on this process, we measured the total

cell ﬂuorescence in stimulated and unstimulated cells. Total

ﬂuorescence enhancement upon macrophage stimulation was

in the range of 100−150%, which is a suﬃcient signal change to

detect inhibitory eﬀects (Figure 3b). Adding compounds 24 h

premeasurement revealed a dose dependent reduction in

phagocytosis with calculated ED₅₀values (Figure 3c,d) that

correspond well to the in vitro IC₅₀values calculated for

ERAP1 using recombinant protein, suggesting that the

biological eﬀect was indeed mediated through the inhibition

of ERAP1.

continued inhibitor internalization into phagosomes and

endosomes.

To date, only a series of phosphinic pseudopeptide transition

state analogues has been reported as potent inhibitors of all

three APAs,²¹albeit with low selectivity among the three

enzymes. Speciﬁcally, the most potent inhibitor displayed IC₅₀

values of 33, 11, and 30 nM for ERAP1, ERAP2, and IRAP,

respectively. On the basis of the observation that peptide

inhibitors of IRAP can enhance cognitive functions, peptidomi-

metic analogues of angiotensin IV have been designed using a

wide range of synthetic approaches.²⁸⁻³⁰Among the most

potent angiotensin IV mimetics reported, a series of macro-

cyclic compounds attained inhibition constants as low as 1.8

nM for IRAP.³⁰With regard to small molecule inhibitors of

IRAP, a series of benzopyran compounds that had been

identiﬁed through a virtual screening approach using a

homology model of IRAP³¹led to the development of the

ﬁrst lead compound with K_i= 30 nM through a medicinal

chemistry campaign.³²Recently, a series of arylsulfonamides

was identiﬁed as being moderate inhibitors of IRAP, with the

most potent exhibiting IC₅₀= 1.1 μM.³³These IRAP inhibitors,

however, have either not been tested for their ability to inhibit

ERAP1 and ERAP2 or have been tested and found not to be

active.³¹As a result, the 3,4-diaminobenzoic acid derivatives

described here appear to constitute not only an alternative

potent chemical tool for controlling APA activity but also one

with very good selectivity properties.

Furthermore, we evaluated the eﬀectiveness of the most

potent IRAP inhibitor, 4u, on blocking cross-presentation by

bone marrow-derived DCs (BMDCs). To assess the speciﬁcity

of the inhibitor, we utilized BMDCs from both wild-type and

IRAP knockout mice. We ﬁrst conﬁrmed that 4u is able to

inhibit mouse IRAP using mouse recombinant enzyme and

discovered that this compound is 2-fold more eﬀective for

mouse IRAP (IC_50,mouse= 52 nM, Supporting Information

Figure S1) compared to that for human IRAP (IC_50,human= 105

nM). Mouse BMDCs were ﬁrst exposed to yeast cells

expressing surface ovalbumin and then added to CD8⁺

T

cells from the lymph nodes of transgenic mice expressing a T

cell receptor speciﬁc for the peptide ovalbumin247−254. T cell

activation by the cross-presented epitope was read out as IL-2

secretion (Figure 4a). The same experiment was performed in

the presence of increasing doses of 4u (Figure 4b). In the

presence of the inhibitor, cross-presentation was reduced in a

dose-dependent manner with an ED₅₀of 6.5 nM. No eﬀect was

seen when we used BMDCs from IRAP knockout mice,

indicating that the eﬀect is speciﬁc for the IRAP-dependent

cross-presentation pathway. The surprisingly high eﬀectiveness

of the inhibitor in this setting may be related to the

compartmentalization of the cross-presentation process in

which high local concentrations may be obtained due to

CONCLUSIONS

■

In summary, we have investigated the ability of 3,4-

diaminobenzoic acid derivatives to act as inhibitors of three

critical aminopeptidases involved in the human innate and

adaptive immune response, ERAP1, ERAP2, and IRAP. Our

analysis revealed potent inhibitors for ERAP2 and IRAP as well

as excellent selectivity proﬁles for all three enzymes.

Furthermore, our analysis conﬁrmed previously hypothesized

diﬃculties in the rational design of inhibitors for this class of

enzymes based on existing crystal structures, especially for

ERAP1, possibly due to the conformational heterogeneity of

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commercial suppliers and used without further puriﬁcation. Reactions

requiring anhydrous conditions were carried out in ﬂame-dried

(vacuum <0.5 Torr) glassware, using anhydrous, freshly distilled

solvents and under an Ar atmosphere. All reactions were stirred with

Teﬂon-coated magnetic stir bars, and temperatures were measured

externally. Yields refer to chromatographically and spectroscopically

(¹H NMR) homogeneous materials. Reactions were monitored by

thin-layer chromatography (TLC) carried out on 0.25 mm silica gel

plates (60 F254, E. Merck). Silica gel (60, particle size: 0.040−0.063

mm, E. Merck) was used for ﬂash column chromatography. NMR

spectra were recorded on a Bruker Avance DRX-500 instrument at 298

K using residual undeuterated solvent for ¹H NMR [δ_H= 7.26

(CHCl₃), 2.50 (DMSO-d₅), and 3.31 (MeOH-d₃) ppm] and ¹³C

deuterated solvent for ¹³C NMR [δ_C= 77.16 (CDCl₃), 39.52 (DMSO-

d₅), and 49.00 (MeOH-d₄) ppm] as an internal reference. The

following abbreviations were used to designate the multiplicities: s,

singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad.

Quantitation of the ﬁnal compounds was achieved using an internal

standard of 2,5-dimethylfuran (DMFu, 0.1 mM in MeOD).³⁷High-

resolution mass spectra (HRMS) were measured on Agilent 6224

Accurate Mass TOF LC/MS at the Faculty of Chemistry and

Chemical Technology, University of Ljubljana. Compounds were

puriﬁed by reversed-phase HPLC on a C18 chromolith column

(Merck) using a 0−50% (v/v) acetonitrile gradient in water containing

0.05% TFA while following the absorbance at 257 nm. The purity of

all ﬁnal compounds employed in the biological assays was greater than

95%, and the structures of the synthesized compounds were

determined by ¹H NMR, ¹³C NMR, and high-resolution mass

spectroscopy (HRMS).

General Methods for the Synthesis of the Inhibitors. Amino

Acid Coupling (Method A). A mixture of 1 (1.0 equiv), the desired

N-Boc protected amino acid (1.1 equiv), HBTU (2.0 equiv), and

DIEA (3.0 equiv) in anhydrous DMF (0.25 mM) was stirred under an

Ar atmosphere at ambient temperature for 4 h. Similarly, a mixture of

the carboxylic acid derivative of 2 or 7 (1.0 equiv), the desired O-Me

or O-Bn protected amino acid (1.5 equiv), HBTU (3.0 equiv), and

DIEA (4.0 equiv) in anhydrous DMF (0.25 mM) was stirred under an

Ar atmosphere at ambient temperature for 8−12 h. For the

preparation of the 1,4-substituted compounds, a mixture of 5 or 8

(1.0 equiv), the N-Boc protected amino acid (2.0 equiv), HATU (3.0

equiv), and DBU (4.0 equiv) in anhydrous DMF (0.25 mM) was

stirred under an Ar atmosphere at ambient temperature for 24−36 h.

The reaction mixture was then diluted with EtOAc and washed

sequentially with aqueous HCl 1.0 N, sat. NaHCO₃, and sat. NaCl.

Consequently, the solvent was evaporated under reduced pressure to

provide the crude product, which was puriﬁed using a gradient of

EtOAc in hexanes to yield the desired products in yields of 45−92%.

Saponiﬁcation of Methyl Esters (Method B). The desired

compound was dissolved in a mixture of 1:1 dioxane/aqueous LiOH

1.0 M (20 equiv) under rigorous stirring. After 3−6 h (monitored by

TLC) at ambient temperature, the mixture was diluted with aqueous

sat. NaCl and the organic solvent was evaporated. The remaining

aqueous solution (pH 12) was washed with EtOAc and then acidiﬁed

with HCl 1.0 N until no further precipitation could be observed (pH

2). The aqueous phase was then extracted three times with EtOAc,

and the combined organic extracts were dried with MgSO₄, ﬁltered,

and washed with EtOAc. After evaporating the solvents and drying

under high vacuum, the product was obtained at yields of 87−95% and

purity > 95% (NMR).

Figure 4. (a) Murine BMDCs from either wild-type or IRAP knockout

mice were exposed to several ratios of ovalbumin-expressing yeast cells

and subsequently to CD8⁺T cells puriﬁed from lymph nodes of OT-I

mice. T cell activation was evaluated by measuring the IL-2

concentration in the culture supernatant. (b) The same experiment

was repeated in the presence of increasing concentrations of 4u. Note

that titration of the inhibitor aﬀected only wild-type, not IRAP

knockout, cells and reduced the response of the wild-type cells to the

levels of the IRAP knockout cells, indicating complete inhibition of the

IRAP-dependent cross-presentation pathway.

this enzyme that greatly aﬀects the folding of the S1 speciﬁcity

pocket. Our best compounds were found to be active in

specialized cellular assays related to their biological functions of

the targeted enzymes in regulation of the adaptive and innate

immune responses. We conclude that this class of compounds

may ﬁnd important applications in the regulation of

inﬂammatory human immune responses for the treatment of

a variety of major human diseases, varying from viral infections

to cancer and autoimmunity. More speciﬁcally, ERAP1

inhibitors such as 4a may be useful for reducing inﬂammation

by controlling macrophage activation¹¹and selective ERAP2

inhibitors like 10n may be useful for controlling autoimmune

responses brought about by high ERAP2 expression, such as

that seen in birdshot chorioretinopathy,³⁴or for enhancing

immune responses toward immune-evading cancer cells.³⁵

Finally, potent IRAP inhibitors such as 4u or 4u′ may ﬁnd

uses in controlling inﬂammatory autoimmune diseases or as

cognitive enhancers.^36,32

General Procedure for the Deprotection of O-Bn and N-Cbz

Groups (Method C). The desired compound (0.1 mmol) was

dissolved in MeOH (4 mL, 0.025 mM), and the solution was degassed

under an Ar atmosphere. A catalytic amount of activated Pd/C 10%

was added and degassed under Ar, followed by the introduction of H₂

gas. The mixture was stirred under H₂at ambient temperature for 1−2

h (monitored by TLC) and then ﬁltered through Celite and washed

with MeOH. The resulting product was acquired in yields of 90−95%.

General Procedure for the Deprotection of N-Boc and O-tBu

Groups (Method D). The desired compound (0.1 mmol) was

dissolved in a mixture of CH₂Cl₂/TFA (2:1 mL, 0.033 mM) and

EXPERIMENTAL SECTION

Materials and General Procedures. Unless otherwise noted, all

solvents and reagents for organic synthesis were obtained from

■

G

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stirred at ambient temperature for 20−40 min (monitored by TLC).

Consequently, the solvents were evaporated, and the product was

treated with HCl 1.0 N (0.5 mL). The solvents were evaporated using

toluene (2 × 2 mL) to azeotrop the remaining aqueous solution and

dried under high vacuum overnight to yield the hydrochloric salt of the

amine in yields of 95−99%.

130.2, 129.3, 128.5, 126.9, 125.9, 121.6, 117.0, 117.0, 56.5, 52.8, 37.8.

HRMS (ESI): m/z calcd for C₁₇H₁₉N₃O₄+ H⁺[M + H⁺], 330.1448;

found, 330.1444.

(S)-4-Amino-3-(2-amino-3-(4-hydroxyphenyl)propanamido)-

benzoic Acid (3b′).

Methyl 4-Amino-3-((tert-butoxycarbonyl)amino)benzoate

(5). To a stirred solution of 1 (500 mg, 3.0 mmol) and Gu·HCl (45

mg, 0.45 mmol) in 5 mL of EtOH was added (Boc)₂O (650 mg, 3.0

mmol), and the mixture was heated at 45 °C for 40 min. Subsequently,

ethanol was evaporated under reduced pressure, and the reaction

mixture was redissolved in EtOAc and washed sequentially with

aqueous HCl 1.0 N, sat. NaHCO₃, and sat. NaCl. The organic layer

was evaporated under reduced pressure to provide the crude product,

which was puriﬁed using a gradient of 35−65% EtOAc in hexanes to

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (br.d, 1H), 7.81 (s, 1H),

7.25−7.21 (m, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H),

4.33−4.25 (m, 1H), 3.30−3.24 (m, 1H), 3.16−3.08 (m, 1H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 179.0, 169.6, 158.5, 140.5, 131.7,

131.7, 130.6, 129.5, 126.9, 125.8, 121.4, 121.4, 117.0, 117.0, 56.5, 37.9.

HRMS (ESI): m/z calcd for C₁₆H₁₇N₃O₄+ H⁺[M + H⁺], 316.1292;

found, 316.1298.

1

yield the N-Boc protected amine 5 (665 mg, 2.5 mmol, 83%). H

NMR (500.13 MHz, CDCl₃): δ 7.83 (s, 1H), 7.56 (dd, J = 8.4, 1.7 Hz,

1H), 6.76 (s, 1H), 6.58 (d, J = 8.4 Hz, 1H), 4.39 (s, 2H), 3.74, (s, 3H),

1.41 (s, 9H). ¹³C NMR (125.76 MHz, CDCl₃): δ 167.0, 154.2, 145.8,

128.3, 127.6, 122.7, 119.4, 115.5, 80.5, 51.5, 28.1, 28.1, 28.1.

Saponiﬁcation of methyl ester of 5 using method B yielded 7, as

reported in the literature.³⁸

(S)-Methyl 4-Amino-3-(2-amino-4-(4-(benzyloxy)phenyl)-

butanamido)benzoate (3c).

(S)-Methyl 4-Amino-3-(2-amino-3-(4-(benzyloxy)phenyl)-

propanamido)benzoate (3a).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.95 (s, 1H), 7.90 (dd, J = 8.4,

1.8 Hz, 1H), 7.42−7.37 (m, 2H), 7.37−7.32 (m, 2H), 7.32−7.25 (m,

2H), 7.20 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 5.02 (s, 2H),

4.33−4.26 (m, 1H), 3.87 (s, 3H), 2.84−2.74 (m, 2H), 2.42−2.33 (m,

1H), 2.32−2.24 (m, 1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ

169.9, 167.3, 158.9, 138.8, 133.5, 130.4, 130.4, 130.1, 130.1, 129.4,

129.4, 128.9, 128.8, 128.5, 128.5, 127.9, 127.3, 122.1, 116.2, 116.2,

71.1, 55.0, 52.8, 34.5, 31.3. HRMS (ESI): m/z calcd for C₂₅H₂₇N₃O₄+

H⁺[M + H⁺], 434.2074; found, 434.2079.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.82 (d, J = 8.4 Hz, 1H), 7.65

(s, 1H), 7.47−7.42 (m, 2H), 7.38−7.32 (m, 2H), 7.31−7.25 (m, 3H),

7.10−7.06 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H), 5.16−5.08 (m, 2H),

4.32−4.25 (m, 1H), 3.85 (s, 3H), 3.25−3.17 (m, 2H). ¹³C NMR

(125.76 MHz, MeOH-d₄): δ 171.9, 169.4, 160.1, 147.1, 138.6, 131.8,

131.8, 131.8, 130.5, 130.5,129.6, 129.5, 129.5, 128.9, 128.6, 127.5,

127.5, 119.3, 116.7, 116.7, 71.1, 56.4, 52.6, 38.0. HRMS (ESI): m/z

calcd for C₂₄H₂₅N₃O₄+ H⁺[M + H⁺], 420.1918; found, 420.1922.

(S)-4-Amino-3-(2-amino-3-(4-(benzyloxy)phenyl)-

propanamido)benzoic Acid (3a′).

(S)-Methyl 4-Amino-3-(2-amino-4-(4-hydroxyphenyl)-

butanamido)benzoate (3d).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.93 (s, 1H), 7.86 (d, J = 8.4

Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 6.73 (d, J

= 8.3 Hz, 2H), 4.30−4.22 (m, 1H), 3.88 (s, 3H), 2.80−2.68 (m, 2H),

2.41−2.28 (m, 1H), 2.28−2.20 (m, 1H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 169.9, 167.6, 157.1, 140.4, 131.9, 130.3, 130.3, 130.2,

129.1, 126.7, 125.8, 121.0, 116.5, 116.5, 54.9, 52.7, 34.8, 31.3. HRMS

(ESI): m/z calcd for C₁₈H₂₁N₃O₄+ H⁺[M + H⁺], 344.1605; found,

344.1607.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (d, J = 8.3 Hz, 1H), 7.71

(s, 1H), 7.46−7.41 (m, 2H), 7.38−7.32 (m, 2H), 7.31−7.25 (m, 3H),

7.23 (dd, J = 8.3, 2.5 Hz, 1H), 7.03 (d, J = 8.2 Hz, 2H), 5.18−5.03 (m,

2H), 4.34−4.28 (m, 1H), 3.28−3.23 (m, 1H), 3.22−3.16 (m, 1H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 169.5, 168.5, 160.0, 139.6, 138.6,

131.8, 131.8, 131.8, 130.6, 129.6, 129.5, 129.5, 128.9, 128.6, 128.6,

127.5, 127.0, 121.2, 116.7, 116.7, 71.0, 56.3, 37.8. HRMS (ESI): m/z

calcd for C₂₃H₂₃N₃O₄+ H⁺[M + H⁺], 406.1761; found, 406.1767.

(S)-Methyl 4-Amino-3-(2-amino-3-(4-hydroxyphenyl)-

propanamido)benzoate (3b).

(S)-Methyl 4-Amino-3-(2-amino-4-methylpentanamido)-

benzoate (3e).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.94 (s, 1H), 7.88 (d, J = 8.0,

1H), 7.21 (d, J = 8.0 Hz, 1H), 4.25−4.15 (m, 1H), 3.88 (s, 3H), 1.97−

1.76 (m, 3H), 1.11−1.05 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 170.6, 167.6, 140.6, 130.4, 129.2, 126.7, 125.7, 121.0, 53.5, 52.7,

41.5, 25.6, 23.3, 21.9. HRMS (ESI): m/z calcd for C₁₄H₂₁N₃O₃+ H⁺

[M + H⁺], 280.1656; found, 280.1659.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (br.d, 1H), 7.71 (s, 1H),

7.28−7.23 (m, 1H), 7.18 (d, J = 8.3 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H),

4.33−4.27 (m, 1H), 3.91 (s, 3H), 3.26−3.11 (m, 2H). ¹³C NMR

(125.76 MHz, MeOH-d₄): δ 174.0, 169.7, 167.3, 158.5, 131.7, 131.7,

H

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(S)-4-Amino-3-(2-amino-4-methylpentanamido)benzoic

Acid (3e′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.73 (s, 1H), 7.60 (dd, J = 8.4,

1.9 Hz, 1H), 7.30−7.14 (m, 5H), 6.84 (d, J = 8.4 Hz, 1H), 4.64−4.56

(m, 1H), 3.91−3.83 (m, 1H), 3.72 (s, 3H), 3.02−2.89 (m, 2H), 2.86−

2.72 (m, 2H), 2.28−2.19 (m, 1H), 2.14−1.97 (m, 2H), 1.94−1.68 (m,

3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.9, 173.5, 169.9,

148.1, 142.1, 129.6, 129.5, 129.4, 128.3, 127.7, 127.3, 123.2, 122.9,

122.4, 116.6, 55.6, 53.6, 52.8, 40.3, 36.6, 32.7, 29.3, 25.3. HRMS (ESI):

m/z calcd for C₂₃H₃₁N₅O₄+ H⁺[M + H⁺], 442.2449; found,

442.2450.

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.08−7.88 (m, 2H), 7.42−7.35

(m, 1H), 4.27−4.19 (m, 1H), 2.01−1.75 (m, 3H), 1.11−1.05 (m, 6H).

¹³C NMR (125.76 MHz, MeOH-d₄): δ 170.7, 168.2, 136.5, 130.4,

129.4, 129.1, 123.0, 115.4, 53.5, 41.4, 25.6, 23.3, 21.9. HRMS (ESI):

m/z calcd for C₁₃H₁₉N₃O₃+ H⁺[M + H⁺], 266.1499; found,

266.1500.

( S ) - 5 - A m i n o - 2 - ( 4 - a m i n o - 3 - ( ( S ) - 2 - a m i n o - 4 -

phenylbutanamido)benzamido)pentanoic Acid (4d′).

(S)-Methyl 4-Amino-3-(2-aminohexanamido)benzoate (3f).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.68 (s, 1H), 7.56 (dd, J = 8.4,

1.9 Hz, 1H), 7.32−7.09 (m, 5H), 6.81 (d, J = 8.4 Hz, 1H), 4.49−4.40

(m, 1H), 3.90−3.75 (m, 1H), 2.88−2.72 (m, 2H), 2.32 (s, 1H), 2.26−

2.20 (m, 1H), 2.17−2.13 (m, 1H), 2.12−2.03 (m, 1H), 2.00−1.84 (m,

4H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.4, 173.3, 169.4,

147.8, 142.1, 129.6, 129.5, 129.4, 128.1, 127.5, 127.3, 123.7, 123.1,

122.5, 116.7, 55.6, 51.4, 42.8, 36.7, 32.7, 28.9, 22.5. HRMS (ESI): m/z

calcd for C₂₂H₂₉N₅O₄+ H⁺[M + H⁺], 428.2292; found, 428.2299.

( S ) - 6 - A m i n o - 2 - ( 4 - a m i n o - 3 - ( ( S ) - 2 - a m i n o - 3 -

phenylpropanamido)benzamido)hexanoic Acid (4e).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.99 (d, J = 1.9 Hz, 1H), 7.90

(dd, J = 8.4, 1.9 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 4.34−4.16 (m, 1H),

3.89 (s, 3H), 2.15−1.94 (m, 2H), 1.61−1.36 (m, 4H), 1.09−0.87 (m,

3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 170.4, 167.1, 136.2,

130.0, 129.1, 128.8, 123.3, 115.3, 55.1, 52.9, 32.2, 28.1, 23.4, 14.0.

HRMS (ESI): m/z calcd for C₁₄H₂₁N₃O₃+ H⁺[M + H⁺], 280.1656;

found, 280.1655.

(S)-Methyl 4-Amino-3-(2-amino-5-guanidinopentanamido)-

benzoate (3g).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.11 (s, 1H), 7.97 (d, J = 8.3

Hz, 1H), 7.53−7.46 (m, 1H), 4.40−4.28 (m, 1H), 3.92 (s, 3H), 3.37−

3.32 (m, 2H), 2.27−2.20 (m, 1H), 2.16−2.07 (m, 1H), 1.91−1.80 (m,

2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 170.0, 166.9, 158.6,

144.8, 130.2, 129.9, 128.7, 124.3, 124.2, 54.6, 53.0, 41.8, 29.6, 25.7.

HRMS (ESI): m/z calcd for C₁₄H₂₂N₆O₃+ H⁺[M + H⁺], 323.1826;

found, 323.1818.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.92 (d, J = 8.3 Hz, 1H), 7.69

(s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.47−7.34 (m, 5H), 4.64−4.55 (m,

1H), 4.53−4.46 (m, 1H), 3.51−3.43(m, 1H), 3.29−3.21 (m, 1H),

3.00−2.93 (m, 2H), 2.12−1.88 (m, 2H), 1.83−1.70 (m, 2H), 1.68−

1.50 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 175.0, 173.0,

169.8, 168.3, 135.4, 130.8, 130.7, 130.7, 130.3, 130.3, 129.1, 128.1,

127.1, 127.0, 125.0, 56.4, 54.2, 40.5, 38.4, 31.8, 28.1, 24.2. HRMS

(ESI): m/z calcd for C₂₂H₂₉N₅O₄+ H⁺[M + H⁺], 428.2292; found,

428.2289.

(S)-4-Amino-3-(2-amino-5-guanidinopentanamido)benzoic

Acid (3g′).

( S ) - 6 - A m i n o - 2 - ( 4 - a m i n o - 3 - ( ( S ) - 2 - a m i n o - 3 - ( 4 -

hydroxyphenyl)propanamido)benzamido)hexanoic Acid (4f).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.12 (br.s, 1H), 8.04−7.97 (m,

1H), 7.60−7.47 (m, 1H), 4.40−4.28 (m, 1H), 3.38−3.32 (m, 2H),

2.29−2.20 (m, 1H), 2.15−2.06 (m, 1H), 1.90−1.80 (m, 2H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 170.0, 167.8, 158.6, 131.7, 130.6,

130.1, 128.8, 124.7, 124.4, 54.6, 41.8, 29.6, 25.7. HRMS (ESI): m/z

calcd for C₁₃H₂₀N₆O₃+ H⁺[M + H⁺], 309.1670; found, 309.1677.

(S)-Methyl 5-Amino-2-(4-amino-3-((S)-2-amino-4-

phenylbutanamido)benzamido) pentanoate (4d).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.92 (d, J = 8.3 Hz, 1H), 7.82

(s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.1 Hz, 2H), 6.83 (d, J =

8.1 Hz, 2H), 4.62−4.55 (m, 1H), 4.49−4.38 (m, 1H), 3.45−3.37 (m,

1H), 3.19−3.10 (m, 1H), 3.02−2.91 (m, 2H), 2.09−1.90 (m, 2H),

1.85−1.70 (m, 2H), 1.67−1.48 (m, 2H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 175.1, 170.0, 168.3, 158.3, 141.3, 131.8, 131.8, 129.6,

129.5, 128.1, 127.0, 125.9, 125.3, 117.0, 117.0, 56.6, 54.3, 40.5, 37.6,

31.7, 28.1, 24.2. HRMS (ESI): m/z calcd for C₂₂H₂₉N₅O₄+ H⁺[M +

H⁺], 444.2241; found, 444.2251.

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(S)-Methyl 6-Amino-2-(4-amino-3-((S)-2-amino-4-(4-

hydroxyphenyl)butanamido) benzamido)hexanoate (4g).

1.45 (m, 2H), 1.11−1.06 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 174.0, 170.8, 168.7, 151.2, 132.0, 128.6, 128.3, 127.3, 122.7,

54.2, 53.5, 52.8, 41.5, 40.5, 31.7, 28.0, 25.6, 24.2, 23.4, 21.9. HRMS

(ESI): m/z calcd for C₂₀H₃₃N₅O₄+ H⁺[M + H⁺], 408.2605; found,

408.2606.

( S ) - 6 - A m i n o - 2 - ( 4 - a m i n o - 3 - ( ( S ) - 2 - a m i n o - 4 -

methylpentanamido)benzamido)hexanoic Acid (4j′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.99−7.85 (m, 2H), 7.54−7.46

(m, 1H), 7.13 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 4.64−4.54

(m, 1H), 4.38−4.32 (m, 1H), 3.74 (s, 3H), 3.00−2.90 (m, 2H), 2.83−

2.69 (m, 2H), 2.44−2.33 (m, 1H), 2.32−2.22 (m, 1H), 2.06−1.87 (m,

2H), 1.80−1.68 (m, 2H), 1.64−1.46 (m, 2H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 173.9, 170.2, 168.5, 157.0, 134.1, 133.1, 132.0,

130.4, 130.4, 130.1, 128.2, 127.0, 124.2, 116.4, 116.4, 55.1, 54.3, 52.9,

40.5, 34.8, 31.6, 31.4, 28.0, 24.1. HRMS (ESI): m/z calcd for

C₂₄H₃₃N₅O₅+ H⁺[M + H⁺], 472.2554; found, 472.2554.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.96−7.85 (m, 2H), 7.46 (d, J =

8.2 Hz, 1H), 4.65−4.53 (m, 1H), 4.34−4.21 (m, 1H), 2.12−1.68 (m,

9H), 1.64−1.48 (m, 2H), 1.14−1.01 (m, 6H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 175.1, 170.8, 168.5, 151.2, 129.5, 128.2, 127.2,

123.7, 123.5, 54.1, 53.6, 41.4, 40.6, 31.9, 28.1, 25.6, 24.2, 23.4, 21.9.

HRMS (ESI): m/z calcd for C₁₉H₃₁N₅O₄+ H⁺[M + H⁺], 394.2449;

found, 394.2455.

(S)-Methyl 6-Amino-2-(4-amino-3-((S)-2-amino-4-(4-

(benzyloxy)phenyl)butanamido) benzamido)hexanoate (4h).

( S ) - M e t h y l 6 - A m i n o - 2 - ( 4 - a m i n o - 3 - ( ( S ) - 2 -

aminohexanamido)benzamido)hexanoate (4k).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.05−7.93 (m, 2H), 7.67−7.57

(m, 1H), 7.50−7.19 (m, 7H), 7.00−6.88 (m, 2H), 5.04 (s, 2H), 4.66−

4.56 (m, 1H), 4.46−4.31 (m, 1H), 3.82−3.68 (m, 3H), 3.02−2.90 (m,

2H), 2.88−2.77 (m, 2H), 2.52−2.24 (m, 2H), 2.10−1.88 (m, 2H),

1.82−1.68 (m, 2H), 1.67−1.46 (m, 2H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 173.8, 170.3, 168.2, 158.9, 138.8, 135.9, 133.5, 131.6,

130.5, 130.5, 129.5, 129.5, 128.8, 128.8, 128.5, 128.5, 128.1, 126.9,

125.6, 116.2, 116.2, 71.0, 55.0, 54.4, 52.9, 40.5, 34.6, 31.6, 31.4, 28.0,

24.2. HRMS (ESI): m/z calcd for C₃₁H₃₉N₅O₅+ H⁺[M + H⁺],

562.3024; found, 562.3026.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.92 (s, 1H), 7.88 (d, J = 8.4

Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.63−4.57 (m, 1H), 4.28−4.22 (m,

1H), 3.76 (s, 3H), 2.99−2.92 (m, 2H), 2.15−2.06 (m, 1H), 2.05−1.96

(m, 2H), 1.95−1.88 (m, 1H), 1.78−1.68 (m, 2H), 1.62−1.43 (m, 6H),

1.03−0.97 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.9,

170.5, 168.6, 134.3, 133.2, 129.5, 128.2, 127.1, 123.6, 55.1, 54.3, 52.9,

40.5, 32.3, 31.6, 28.2, 28.0, 24.2, 23.4, 14.1. HRMS (ESI): m/z calcd

for C₂₀H₃₃N₅O₄+ H⁺[M + H⁺], 408.2605; found, 408.2603.

(S)-Methyl 6-Amino-2-(4-amino-3-((S)-2-amino-5-

guanidinopentanamido)benzamido)hexanoate (4l).

(S)-Methyl 6-Amino-2-(4-amino-3-((S)-2-amino-3-

(benzyloxy)propanamido)benzamido)hexanoate (4i).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.95−7.83 (m, 2H), 7.48 (m,

1H), 7.45−7.23 (m, 5H), 4.74−4.64 (m, 2H), 4.64−4.56 (m, 1H),

4.55−4.50 (m, 1H), 4.11−4.02 (m, 2H), 3.79−3.68 (m, 3H), 2.99−

2.90 (m, 2H), 2.07−1.86 (m, 2H), 1.82−1.68 (m, 2H), 1.64−1.48 (m,

2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.9, 168.5, 167.9,

138.4, 133.7, 129.6, 129.6, 129.2, 129.2, 129.2, 128.2, 127.2, 123.9,

123.8, 115.8, 74.6, 68.8, 55.2, 54.3, 52.9, 40.5, 31.7, 28.0, 24.2. HRMS

(ESI): m/z calcd for C₂₄H₃₃N₅O₅+ H⁺[M + H⁺], 472.2554; found,

472.2550.

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.00 (s, 1H), 7.88 (d, J = 8.2

Hz, 1H), 7.47−7.38 (m, 1H), 4.63−4.56 (m, 1H), 4.35−4.29 (m, 1H),

3.75 (s, 3H), 3.37−3.32 (m, 2H), 2.99−2.91 (m, 2H), 2.17−2.07 (m,

2H), 2.05−1.90 (m, 2H), 1.90−1.81 (m, 2H), 1.79−1.68 (m, 2H),

1.64−1.49 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.0,

171.5, 169.9, 168.6, 158.6, 129.1, 128.3, 127.2, 123.4, 123.3, 54.7, 54.4,

52.9, 41.9, 40.7, 31.8, 29.8, 28.2, 25.8, 24.3. HRMS (ESI): m/z calcd

for C₂₀H₃₄N₈O₄+ H⁺[M + H⁺], 451.2776; found, 451.2777.

(S)-Benzyl 2-(4-Amino-3-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido)benzamido)-3-methylbutanoate (4m).

(S)-Methyl 6-Amino-2-(4-amino-3-((S)-2-amino-4-

methylpentanamido)benzamido) hexanoate (4j).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.94−7.80 (m, 2H), 7.42−7.35

(m, 1H), 4.63−4.57 (m, 1H), 4.28−4.22 (m, 1H), 3.78−3.70 (m, 3H),

2.98−2.90 (m, 2H), 2.09−1.79 (m, 5H), 1.78−1.69 (m, 2H), 1.64−

J

DOI: 10.1021/jm501867s

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.78 (d, J = 8.2 Hz, 1H), 7.69

(s, 1H), 7.41−7.29 (m, 6H), 7.21 (d, J = 8.3 Hz, 2H), 6.83 (d, J = 8.3

Hz, 2H), 5.26−5.21 (m, 1H), 5.19−5.14 (m, 1H), 4.51−4.46 (m, 1H),

4.39−4.33 (m, 1H), 3.36−3.32 (m, 1H), 3.16−3.08 (m, 1H), 2.32−

2.22 (m, 1H), 1.06−0.94 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.0, 169.9, 169.1, 167.3, 158.5, 137.2, 131.8, 131.8, 131.8,

129.6, 129.5, 129.5, 129.4, 129.4, 128.2, 127.3, 125.8, 122.9, 117.0,

117.0, 117.0, 67.9, 60.5, 56.5, 37.8, 31.7, 19.6, 19.2. HRMS (ESI): m/z

calcd for C₂₈H₃₂N₄O₅+ H⁺[M + H⁺], 505.2445; found, 505.2447.

(S)-2-(4-Amino-3-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido)benzamido)-3-methylbutanoic Acid (4m′).

117.1, 117.1, 68.6, 60.3, 56.7, 52.8, 37.4, 20.1. HRMS (ESI): m/z calcd

for C₂₁H₂₆N₄O₆+ H⁺[M + H⁺], 431.1925; found, 431.1931.

(S)-Benzyl 2-(4-Amino-3-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido)benzamido)-3-(1H-indol-3-yl)propanoate (4p).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.55 (d, J = 7.9 Hz, 1H), 7.49

(s, 1H), 7.46−7.41 (m, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.30−7.25 (m,

3H), 7.19−7.14 (m, 2H), 7.14−7.05 (m, 3H), 7.03−6.97 (m, 2H),

6.78−6.71 (m, 3H), 5.07 (s, 2H), 4.93−4.87 (m, 1H), 3.82−3.76 (m,

1H), 3.43−3.37 (m, 1H), 3.35−3.33 (m, 1H), 3.05−2.97 (m, 1H),

2.95−2.86 (m, 1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 175.3,

173.8, 169.8, 157.5, 148.0, 138.1, 137.1, 131.6, 131.6, 129.5, 129.2,

129.2, 128.9, 128.8, 128.3, 127.7, 124.5, 123.1, 122.5, 122.2, 120.0,

119.2, 117.2, 116.7, 116.6, 116.6, 116.2, 112.5, 110.8, 68.2, 57.8, 55.6,

41.5, 28.6. HRMS (ESI): m/z calcd for C₃₄H₃₃N₅O₅+ H⁺[M + H⁺],

592.2554; found, 592.2564.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.86 (d, J = 8.3 Hz, 1H), 7.74

(s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.3 Hz, 2H), 6.83 (d, J =

8.3 Hz, 2H), 4.52−4.46 (m, 1H), 4.43−4.37 (m, 1H), 3.40−3.33 (m,

1H), 3.18−3.07 (m, 1H), 2.35−2.24 (m, 1H), 1.09−1.01 (m, 6H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 174.8, 169.9, 168.7, 167.1, 158.4,

135.4, 131.8, 131.8, 130.4, 128.1, 127.1, 125.8, 124.6, 117.0, 117.0,

60.1, 56.6, 37.7, 31.7, 19.7, 18.9. HRMS (ESI): m/z calcd for

C₂₁H₂₆N₄O₅+ H⁺[M + H⁺], 415.1976; found, 415.1982.

(S)-Methyl 2-(4-Amino-3-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido) benzamido)-3-(4-hydroxyphenyl)propanoate

(4n).

(S)-2-(4-Amino-3-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido)benzamido)-3-(1H-indol-3-yl)propanoic Acid

(4p′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.71−7.50 (m, 3H), 7.39−7.29

(m, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.15 (s, 1H), 7.11−7.02 (m, 1H),

7.02−6.63 (m, 1H), 6.82 (d, J = 8.4 Hz, 2H), 4.95−4.90 (m, 1H),

4.42−4.30 (m, 1H), 3.55−3.40 (m, 1H), 3.40−3.32 (m, 1H), 3.25−

3.15 (m, 1H), 3.14−3.04 (m, 1H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 175.1, 169.9, 168.2, 158.4, 138.1, 133.9, 131.7, 131.7, 129.5,

128.8, 127.9, 127.0, 125.8, 124.5, 123.6, 122.4, 122.1, 119.9, 119.3,

117.1, 117.1, 112.4, 111.1, 56.5, 55.5, 37.8, 28.3. HRMS (ESI): m/z

calcd for C₂₇H₂₇N₅O₅+ H⁺[M + H⁺], 502.2085; found, 502.2080.

(S)-Benzyl 2-(4-Amino-3-((S)-2-amino-4-(4-(benzyloxy)-

phenyl)butanamido) benzamido)-3-(1H-indol-3-yl)propanoate

(4q).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.70 (d, J = 8.3 Hz, 1H), 7.63

(s, 1H), 7.34 (dd, J = 8.4, 3.1 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.07

(d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H),

4.80−4.74 (m, 1H), 4.38−4.32 (m, 1H), 3.72 (s, 3H), 3.23−3.15 (m,

2H), 3.14−3.07 (m, 1H), 3.06−2.99 (m, 1H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 173.7, 169.9, 168.4, 158.5, 157.4, 132.9, 131.8,

131.8, 131.2, 131.2, 129.0, 128.9, 128.0, 127.2, 125.9, 123.1, 117.1,

117.1, 116.9, 116.3, 116.3, 56.6, 56.2, 52.8, 37.8, 37.4. HRMS (ESI):

m/z calcd for C₂₆H₂₈N₄O₆+ H⁺[M + H⁺], 493.2082; found,

493.2081.

(2S,3R)-Methyl 2-(4-Amino-3-((S)-2-amino-3-(4-

hydroxyphenyl)propanamido)benzamido)-3-hydroxybuta-

noate (4o).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.75−7.63 (m, 2H), 7.55−7.48

(m, 1H), 7.41−7.11 (m, 14H), 7.07−6.86 (m, 5H), 5.06 (br.d, 2H),

4.99 (br.d, 2H), 4.95−4.89 (m, 1H), 4.38−4.22 (m, 1H), 3.49−3.35

(m, 1H), 3.36−3.30 (m, 1H), 2.87−2.67 (m, 2H), 2.41−2.31 (m, 1H),

2.29−2.19 (m, 1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.4,

170.1, 168.4, 158.9, 138.7, 138.0, 137.0, 134.9, 133.5, 132.8, 130.4,

130.4, 129.4, 129.4, 129.2, 129.2, 129.1, 129.1, 128.9, 128.8, 128.8,

128.7, 128.5, 128.5, 128.1, 127.0, 124.6, 123.2, 122.5, 119.9, 119.1,

116.2, 116.2, 112.4, 110.7, 71.0, 68.0, 55.9, 54.9, 34.6, 31.4, 28.3.

HRMS (ESI): m/z calcd for C₄₂H₄₁N₅O₅+ H⁺[M + H⁺], 696.3180;

found, 696.3174.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.80 (d, J = 8.4 Hz, 1H), 7.69

(s, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 8.3 Hz, 2H), 6.84 (d, J =

8.3 Hz, 2H), 4.69−4.65 (m, 1H), 4.43−4.37 (m, 1H), 4.33−4.27 (m,

1H), 3.78 (s, 3H), 3.29−3.25 (m, 1H) 3.15−3.09 (m, 1H), 1.27−1.23

(m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 169.6, 169.2, 165.1,

158.5, 158.2, 131.7, 131.7, 128.3, 127.5, 125.9, 120.6, 120.5, 120.4,

K

DOI: 10.1021/jm501867s

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(S)-2-(4-Amino-3-((S)-2-amino-4-(4-hydroxyphenyl)-

butanamido)benzamido)-3-(1H-indol-3-yl)propanoic Acid (4r).

1.04−0.93 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.7,

173.5, 170.7, 168.0, 157.4, 135.4, 131.2, 131.2, 131.0, 129.0, 127.9,

126.8, 125.0, 116.3, 116.3, 56.3, 55.1, 52.8, 37.3, 32.2, 28.1, 23.4, 14.1.

HRMS (ESI): m/z calcd for C₂₃H₃₀N₄O₅+ H⁺[M + H⁺], 443.2289;

found, 443.2291.

(S)-Benzyl 2-(4-Amino-3-((S)-2-aminohexanamido)-

benzamido)-3-(1H-indol-3-yl)propanoate (4u).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.65 (s, 1H), 7.59−7.39 (m,

3H), 7.29 (br.d, 1H), 7.14−6.91 (m, 5H), 6.74 (br.d, 2H), 4.71−4.49

(m, 1H), 4.30−4.04 (m, 1H), 3.46−3.38 (m, 1H), 3.30−3.28 (m, 1H),

2.76−2.64 (m, 2H), 2.32−2.22 (m, 1H), 2.23−2.13 (m, 1H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 175.3, 169.8, 168.9, 157.1, 141.2,

138.0, 132.0, 130.3, 130.3, 128.4, 128.2, 127.6, 127.3, 125.5, 124.4,

122.4, 120.0, 119.8, 119.2, 116.5, 116.5, 112.3, 111.1, 55.3, 54.9, 35.0,

31.4, 28.3. HRMS (ESI): m/z calcd for C₂₈H₂₉N₅O₅+ H⁺[M + H⁺],

516.2241; found, 516.2248.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.78 (s, 1H), 7.70 (d, J = 7.8

Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.40−7.30 (m, 2H), 7.30−7.25 (m,

3H), 7.22−7.16 (m, 2H), 7.11−7.06 (m, 1H), 7.04 (s, 1H), 7.01−6.95

(m, 1H), 5.09 (s, 2H), 4.24−4.16 (m, 1H), 3.48−3.38 (m, 1H), 3.38−

3.32 (m, 2H), 2.12−2.04 (m, 1H), 2.03−1.93 (m, 1H), 1.56−1.39 (m,

4H), 1.02−0.94 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ

173.4, 170.4, 168.3, 138.1, 137.0, 133.3, 129.5, 129.5, 129.2, 129.2,

129.1, 129.1, 128.7, 128.0, 127.0, 124.5, 123.5, 123.4, 122.5, 119.9,

119.1, 112.4, 110.8, 68.0, 55.9, 55.1, 32.3, 28.3, 28.1, 23.4, 14.0. HRMS

(ESI): m/z calcd for C₃₁H₃₅N₅O₄+ H⁺[M + H⁺], 542.2762; found,

542.2759.

(S)-Benzyl 2-(4-Amino-3-((S)-2-aminohexanamido)-

benzamido)-3-methylbutanoate (4s).

(S)-2-(4-Amino-3-((S)-2-aminohexanamido)benzamido)-3-

(1H-indol-3-yl)propanoic Acid (4u′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.85−7.77 (m, 2H), 7.40−7.29

(m, 6H), 5.27−5.13 (m, 2H), 4.50−4.46 (m, 1H), 4.24−4.18 (m, 1H),

2.31−2.22 (m, 1H), 2.15−2.04 (m, 1H), 2.03−1.93 (m, 1H), 1.57−

1.42 (m, 4H), 1.04−0.95 (m, 9H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.0, 170.4, 168.9, 151.1, 137.2, 129.6, 129.6, 129.5, 129.4,

128.6, 128.2, 128.2, 127.2, 127.2, 122.8, 67.9, 60.5, 55.1, 32.3, 31.6,

28.1, 23.4, 19.6, 19.2, 14.1. HRMS (ESI): m/z calcd for C₂₅H₃₄N₄O₄+

H⁺[M + H⁺], 455.2653; found, 455.2653.

(S)-2-(4-Amino-3-((S)-2-aminohexanamido)benzamido)-3-

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.76 (s, 1H), 7.70−7.64 (m,

1H), 7.58 (d, J = 7.8 Hz, 1H), 7.41−7.36 (m, 1H), 7.32 (d, J = 7.8 Hz,

1H), 7.15 (s, 1H), 7.09−7.03 (m, 1H), 7.00−6.93 (m, 1H), 4.93−4.90

(m, 1H), 4.26−4.18 (m, 1H), 3.53−3.44 (m, 1H), 3.37−3.32 (m, 1H),

2.14−2.04 (m, 1H), 2.02−1.94 (m, 1H), 1.55−1.40 (m, 4H), 1.02−

0.92 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 175.1, 170.5,

168.2, 138.0, 134.3, 133.2, 129.9, 128.8, 127.9, 126.9, 124.5, 124.0,

122.4, 119.8, 119.2, 112.3, 111.2, 55.5, 55.1, 32.2, 28.2, 28.1, 23.4, 14.1.

HRMS (ESI): m/z calcd for C₂₄H₂₉N₅O₄+ H⁺[M + H⁺], 452.2292;

found, 452.2302.

methylbutanoic Acid (4s′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.91−7.86 (m, 2H), 7.56 (br.d,

1H), 4.50−4.46 (m, 1H), 4.28−4.22 (m, 1H), 2.34−2.25 (m, 1H),

2.16−2.06 (m, 1H), 2.03−1.96 (m, 1H), 1.58−1.42 (m, 4H), 1.08−

1.02 (m, 6H), 1.02−0.96 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 178.5, 171.0, 169.0, 147.6, 127.9, 127.3, 124.2, 122.3, 116.8,

61.7, 55.2, 33.1, 33.0, 28.3, 23.5, 20.2, 18.7, 14.1. HRMS (ESI): m/z

calcd for C₁₈H₂₈N₄O₄+ H⁺[M + H⁺], 365.2183; found, 365.2177.

(S)-Methyl 2-(4-Amino-3-((S)-2-aminohexanamido)-

benzamido)-3-(4-hydroxyphenyl)propanoate (4t).

(S)-Methyl 2-(4-Amino-3-((S)-2-aminohexanamido)-

benzamido)-5-guanidinopentanoate (4v).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.00 (s, 1H), 7.98−7.94 (d, J =

7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 4.65−4.58 (m, 1H), 4.33−4.27

(m, 1H), 3.75 (s, 3H), 2.17−1.92 (m, 4H), 1.85−1.62 (m, 3H), 1.60−

1.40 (m, 5H), 1.04−0.92 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.6, 170.6, 168.2, 158.6, 135.8, 131.5, 130.6, 128.1, 126.9,

125.6, 55.1, 54.2, 53.0, 42.0, 32.1, 29.2, 28.1, 26.6, 23.4, 14.1. HRMS

(ESI): m/z calcd for C₂₀H₃₃N₇O₄+ H⁺[M + H⁺], 436.2667; found,

436.2670.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.83−7.72 (m, 2H), 7.51 (d, J =

8.3 Hz, 1H), 7.13−7.02 (m, 2H), 6.74−6.65 (m, 2H), 4.81−4.74 (m,

1H), 4.26−4.19 (m, 1H), 3.72 (s, 3H), 3.26−3.16 (m, 1H), 3.08−2.96

(m, 1H), 2.15−2.05 (m, 1H), 2.05−1.95 (m, 1H), 1.57−1.41 (m, 4H),

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(S)-2-(4-Amino-3-((S)-2-amino-5-guanidinopentanamido)-

benzamido)-3-methylbutanoic Acid (4w).

(S)-Methyl 3-Amino-4-(2-amino-3-(4-(benzyloxy)phenyl)-

propanamido)benzoate (6b).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90−7.85 (m, 1H), 7.79 (dd, J

= 8.3, 1.7 Hz, 1H), 7.45−7.40 (m, 2H), 7.38−7.32 (m, 2H), 7.31−7.24

(m, 4H), 7.02 (d, J = 8.5 Hz, 3H), 5.18−4.99 (m, 2H), 4.45−4.27 (m,

1H), 3.95−3.85 (m, 3H), 3.37−3.32 (m, 1H), 3.20−3.10 (m, 1H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 169.5, 167.0, 159.9, 143.4, 138.6,

133.1, 131.8, 131.8, 130.5, 129.5, 129.5, 128.9, 128.5, 128.4, 127.8,

127.5, 126.9, 124.3, 116.6, 116.6, 70.9, 56.4, 52.9, 37.7. HRMS (ESI):

m/z calcd for C₂₄H₂₅N₃O₄+ H⁺[M + H⁺], 420.1845; found,

420.1843.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.91 (s, 1H), 7.83 (d, J = 8.3

Hz, 1H), 7.44−7.37 (m, 1H), 4.50−4.45 (m, 1H), 4.33−4.27 (m, 1H),

3.36−3.32 (m, 2H), 2.35−2.05 (m, 3H), 1.89−1.80 (m, 2H), 1.08−

1.01 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.9, 169.9,

169.1, 169.0, 158.7, 128.3, 128.1, 127.2, 122.5, 122.3, 60.0, 54.6, 41.8,

31.7, 29.7, 25.6, 19.7, 19.6. HRMS (ESI): m/z calcd for C₁₈H₂₉N₇O₄+

H⁺[M + H⁺], 408.2354; found, 408.2354.

( S ) - M e t h y l 2 - ( 4 - A m i n o - 3 - ( ( S ) - 2 - a m i n o - 5 -

guanidinopentanamido)benzamido)-3-(4-hydroxyphenyl)-

propanoate (4x).

(S)-Methyl 3-Amino-4-(2-amino-3-(4-hydroxyphenyl)-

propanamido)benzoate (6c).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.63 (s, 1H), 7.50 (d, J = 8.2

Hz, 1H), 7.30−7.16 (m, 3H), 6.81 (d, J = 8.1 Hz, 2H), 4.40−4.30 (m,

1H), 3.95−3.85 (m, 3H), 3.30−3.07 (m, 2H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 169.2, 167.8, 158.4, 143.4, 131.8, 131.8, 130.3,

126.8, 125.9, 123.7, 121.5, 121.3, 116.9, 116.9, 56.4, 52.7, 37.9. HRMS

(ESI): m/z calcd for C₁₇H₁₉N₃O₄+ H⁺[M + H⁺], 330.1448; found,

330.1457.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (s, 1H), 7.76 (br.d, 1H),

7.60−7.47 (m, 1H), 7.08 (br.d, 2H), 6.70 (br.d, 2H), 4.82−4.74 (m,

1H), 4.39−4.29 (m, 1H), 3.72 (s, 3H), 3.40−3.27 (m, 2H), 3.26−3.14

(m, 1H), 3.13−2.97 (m, 1H), 2.25−2.06 (m, 2H), 1.92−1.76 (m, 2H).

¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.6, 170.0, 167.9, 158.6,

157.3, 151.2, 135.8, 131.2, 131.2, 129.0, 127.9, 126.8, 126.6, 125.3,

116.3, 116.3, 56.3, 54.6, 52.8, 41.8, 37.3, 29.6, 25.6. HRMS (ESI): m/z

calcd for C₂₃H₃₁N₇O₅+ H⁺[M + H⁺], 486.2459; found, 486.2466.

(S)-2-(4-Amino-3-((S)-2-amino-5-guanidinopentanamido)-

benzamido)-3-(1H-indol-3-yl)propanoic Acid (4y).

(S)-Methyl 3-Amino-4-(2-amino-4-methylpentanamido)-

benzoate (6d).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.15−8.04 (m, 2H), 7.62 (dd, J

= 8.5, 3.2 Hz, 1H), 4.33−4.25 (m, 1H), 3.94 (s, 3H), 2.00−1.76 (m,

3H), 1.12−1.04 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ

170.8, 166.5, 135.4, 130.8, 130.8, 127.2, 126.4, 115.9, 53.7, 53.1, 41.3,

25.5, 23.4, 21.8. HRMS (ESI): m/z calcd for C₁₄H₂₁N₃O₃+ H⁺[M +

H⁺], 280.1656; found, 280.1650.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.83−7.55 (m, 2H), 7.39−7.21

(m, 2H), 7.20−6.88 (m, 4H), 4.32−4.24 (m, 1H), 3.93−3.81 (m, 1H),

3.69−3.49 (m, 2H), 3.34−3.40 (m, 2H), 2.17−2.00 (m, 2H), 1.88−

1.76 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.1, 170.5,

169.7, 168.6, 158.6, 138.0, 128.0, 127.2, 125.9, 124.5, 122.5, 121.6,

120.1, 119.8, 119.6, 119.1, 112.4, 110.9, 55.6, 54.5, 41.8, 29.7, 28.3,

25.6. HRMS (ESI): m/z calcd for C₂₄H₃₀N₈O₄+ H⁺[M + H⁺],

495.2463; found, 495.2470.

(S)-Methyl 3-Amino-4-(2-aminohexanoyl)benzoate (6e).

(S)-Methyl 3-Amino-4-(2-amino-4-phenylbutanamido)-

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.10 (s, 1H), 8.04 (d, J = 8.4

Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 4.31−4.25 (m, 1H), 3.94 (s, 3H),

2.16−1.96 (m, 2H), 1.58−1.39 (m, 4H), 1.02−0.94 (m, 3H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 170.4, 166.5, 143.4, 135.3, 130.6,

128.0, 127.0, 126.2, 55.2, 53.1, 32.2, 28.1, 23.4, 14.1. HRMS (ESI): m/

z calcd for C₁₄H₂₁N₃O₃+ H⁺[M + H⁺], 280.1656; found, 280.1663.

(S)-Methyl 3-Amino-4-(2-amino-5-guanidinopentanamido)-

benzoate (6f).

benzoate (6a).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.17−8.06 (m, 2H), 7.67−7.58

(m, 1H), 7.35−7.27 (m, 4H), 7.25−7.18 (m, 1H), 4.45−4.36 (m, 1H),

3.94 (s, 3H), 2.90−2.80 (m, 2H), 2.48−2.37 (m, 1H), 2.36−2.26 (m,

1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 170.1, 166.4, 141.2,

135.6, 131.2, 131.1, 130.7, 129.7, 129.6, 129.4, 127.6, 127.1, 126.6,

126.5, 55.2, 53.1, 34.5, 32.3. HRMS (ESI): m/z calcd for C₁₈H₂₁N₃O₃

+ H⁺[M + H⁺], 328.1656; found, 328.1661.

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¹H NMR (500.13 MHz, MeOH-d₄): δ 8.13 (s, 1H), 8.08 (dd, J = 8.4,

1.8 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 4.40−4.33 (m, 1H), 3.94 (s,

3H), 3.29−3.24 (m, 2H), 2.25−2.16 (m, 1H), 2.13−2.04 (m, 1H),

1.87−1.78 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 167.9,

166.7, 158.3, 145.0, 131.6, 130.3, 126.8, 121.1, 118.6, 53.5, 52.8, 41.8,

29.3, 25.4. HRMS (ESI): m/z calcd for C₁₄H₂₂N₆O₃+ H⁺[M + H⁺],

323.1826; found, 323.1822.

(m, 1H), 6.99−6.94 (m, 1H), 6.92−6.87 (m, 1H), 4.92−4.85 (m, 1H),

3.53−3.40 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 175.4,

168.7, 138.1, 131.9, 128.9, 128.1, 126.6, 124.4, 124.1, 122.4, 119.8,

119.3, 118.1, 117.5, 112.3, 111.2, 55.3, 28.3. HRMS (ESI): m/z calcd

for C₁₈H₁₈N₄O₃+ H⁺[M + H⁺], 339.1452; found, 339.1447.

(S)-Methyl 2-(3,4-Diaminobenzamido)-5-guanidinopenta-

noate (9d).

(S)-Methyl 6-Amino-2-(3,4-diaminobenzamido)hexanoate

(9a).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.77 (d, J = 1.9 Hz, 1H), 7.72

(dd, J = 8.5, 1.9 Hz 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.62−4.53 (m, 1H),

3.73 (s, 3H), 3.28−3.18 (m, 2H), 2.10−1.85 (m, 2H), 1.84−1.63 (m,

2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.0, 169.1, 158.6,

143.6, 128.8, 125.2, 124.6, 119.7, 118.4, 53.9, 52.8, 42.0, 29.4, 26.6.

HRMS (ESI): m/z calcd for C₁₄H₂₂N₆O₃+ H⁺[M + H⁺], 323.1826;

found, 323.1821.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.80 (s, 1H), 7.75−7.70 (m,

1H), 7.04 (d, J = 8.5 Hz, 1H), 4.60−4.52 (m, 1H), 3.74 (s, 3H), 2.99−

2.91 (m, 2H), 2.06−1.87 (m, 2H), 1.73 (m, 2H), 1.63−1.45 (m, 2H).

¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.1, 169.0, 142.9, 128.9,

125.8, 124.7, 120.1, 118.8, 54.2, 52.8, 40.5, 31.6, 28.0, 24.2. HRMS

(ESI): m/z calcd for C₁₄H₂₂N₄O₃+ H⁺[M + H⁺], 295.1765; found,

295.1765.

(S)-Methyl 6-Amino-2-(3-amino-4-((S)-2-amino-4-

phenylbutanamido)benzamido)hexanoate (10a).

(S)-Methyl 2-(3,4-Diaminobenzamido)-3-(4-hydroxyphenyl)-

propanoate (9b).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.68 (s, 1H), 7.60 (d, J = 8.3

Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 6.70 (d, J

= 8.4 Hz, 2H), 4.76−4.68 (m, 1H), 3.70 (s, 3H), 3.21−3.12 (m, 1H),

3.07−2.97 (m, 1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.8,

168.7, 157.3, 142.5, 131.2, 131.2, 129.0, 128.7, 126.1, 124.6, 120.3,

119.0, 116.3, 116.3, 56.2, 52.7, 37.3. HRMS (ESI): m/z calcd for

C₁₇H₁₉N₃O₄+ H⁺[M + H⁺], 330.1448; found, 330.1447.

(S)-Benzyl 2-(3,4-Diaminobenzamido)-3-(1H-indol-3-yl)-

propanoate (9c).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.05 (s, 1H), 8.00 (br.d, 1H),

7.62 (br.d, 1H), 7.33−7.16 (m, 5H), 4.64−4.56 (m, 1H), 4.46−4.40

(m, 1H), 3.75 (s, 3H), 3.03−2.91 (m, 2H), 2.92−2.84 (m, 2H), 2.52−

2.40 (m, 1H), 2.38−2.28 (m, 1H), 2.08−1.80 (m, 2H), 1.82−1.70 (m,

2H), 1.66−1.46 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ

173.8, 170.1, 168.0, 141.3, 134.4, 129.7, 129.6, 129.6, 129.4, 129.4,

129.2, 127.6, 127.5, 127.1, 125.1, 55.2, 54.4, 52.9, 40.5, 34.4, 32.3, 31.6,

28.0, 24.2. HRMS (ESI): m/z calcd for C₂₄H₃₃N₅O₄+ H⁺[M + H⁺],

456.2605; found, 456.2596.

( S ) - B e n z y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 4 -

phenylbutanamido)benzamido)-3-methylbutanoate (10b).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.63 (s, 1H), 7.58−7.46 (m,

2H), 7.38−7.23 (m, 4H), 7.20−7.11 (m, 2H), 7.11−6.88 (m, 4H),

5.09 (s, 2H), 4.92−4.85 (m, 1H), 3.47−3.34 (m, 2H). ¹³C NMR

(125.76 MHz, MeOH-d₄): δ 173.6, 168.8, 152.0, 143.4, 138.1, 137.1,

132.1, 129.5, 129.2, 129.2, 128.6, 126.9, 125.4, 125.4, 124.5, 122.5,

119.9, 119.2, 118.3, 117.6, 112.4, 110.8, 68.0, 55.8, 28.3. HRMS (ESI):

m/z calcd for C₂₅H₂₄N₄O₃+ H⁺[M + H⁺], 429.1921; found,

429.1930.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (s, 1H), 7.85 (d, J = 8.3

Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.40−7.25 (m, 9H), 7.23−7.15 (m,

1H), 5.25−5.20 (m, 1H), 5.18−5.13 (m, 1H), 4.52−4.47 (m, 1H),

4.44−4.38 (m, 1H), 2.90−2.83 (m, 2H), 2.48−2.38 (m, 1H), 2.37−

2.23 (m, 2H), 1.04−0.96 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 172.9, 170.0, 168.6, 141.3, 137.1, 134.6, 133.6, 130.2, 130.1,

129.8, 129.6, 129.5, 129.3, 128.8, 128.6, 128.1, 127.6, 127.4, 127.0,

126.9, 124.5, 67.9, 60.3, 55.1, 32.3, 31.8, 31.3, 19.6, 19.3. HRMS (ESI):

m/z calcd for C₂₉H₃₄N₄O₄+ H⁺[M + H⁺], 503.2653; found,

503.2649.

(S)-2-(3,4-Diaminobenzamido)-3-(1H-indol-3-yl)propanoic

Acid (9c′).

(S)-2-(3-Amino-4-((S)-2-amino-4-phenylbutanamido)-

benzamido)-3-methylbutanoic Acid (10b′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.63−7.53 (m, 2H), 7.49 (dd, J

= 8.5, 2.0 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.12 (s, 1H), 7.09−7.04

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¹H NMR (500.13 MHz, MeOH-d₄): δ 7.61 (s, 1H), 7.54 (d, J = 8.2

Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.36−7.13 (m, 5H), 4.51−4.44 (m,

1H), 4.36−4.30 (m, 1H), 2.87−2.78 (m, 2H), 2.44−2.20 (m, 3H),

1.05 (d, J = 6.4 Hz, 6H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 174.8,

169.5, 169.4, 141.3, 135.7, 134.7, 130.2, 129.7, 129.7, 129.4, 129.4,

127.5, 126.8, 123.1, 121.0, 59.9, 55.0, 34.7, 32.2, 31.7, 19.7, 18.9.

HRMS (ESI): m/z calcd for C₂₂H₂₈N₄O₄+ H⁺[M + H⁺], 413.2183;

found, 413.2185.

134.3, 129.7, 129.6, 129.4, 127.6, 127.5, 127.1, 125.1, 124.8, 55.1, 54.2,

53.0, 41.9, 34.5, 32.3, 29.3, 26.6. HRMS (ESI): m/z calcd for

C₂₄H₃₃N₇O₄+ H⁺[M + H⁺], 484.2667; found, 484.2660.

(S)-Methyl 2-(3-Amino-4-((S)-2-amino-3-(4-(benzyloxy)-

phenyl)propanamido) benzamido)-3-(4-hydroxyphenyl)-

propanoate (10f).

( S ) - M e t h y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 4 -

phenylbutanamido)benzamido)-3-(4-hydroxyphenyl)-

propanoate (10c).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.74 (s, 1H), 7.69−7.65 (m,

1H), 7.47−7.41 (m, 2H), 7.39−7.34 (m, 2H), 7.31−7.26 (m, 3H),

7.24−7.20 (m, 1H), 7.09−7.00 (m, 4H), 6.73−6.68 (m, 2H), 5.13−

5.10 (m, 2H), 4.82−4.77 (m, 1H), 4.42−4.36 (m, 1H), 3.74−3.71 (m,

3H), 3.37−3.32 (m, 1H), 3.24−3.14 (m, 2H), 3.08−2.97 (m, 1H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 173.6, 169.6, 168.0, 165.8, 159.9,

157.4, 138.6, 135.8, 134.8, 131.8, 131.8, 131.2, 131.2, 129.9, 129.6,

128.9, 128.9, 128.5, 127.4, 127.0, 127.0, 123.7, 121.7, 116.8, 116.8,

116.3, 116.3, 71.0, 56.5, 56.2, 52.8, 37.7, 37.4. HRMS (ESI): m/z calcd

for C₃₃H₃₄N₄O₆+ H⁺[M + H⁺], 583.2511; found, 583.2504.

(S)-Methyl 2-(3-Amino-4-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido) benzamido)-3-(4-hydroxyphenyl)propanoate

(10g).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.89 (s, 1H), 7.84 (d, J = 8.3

Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.31−7.28 (m, 4H), 7.24−7.15 (m,

1H), 7.08 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 4.82−4.76 (m,

1H), 4.43−4.32 (m, 1H), 3.72 (s, 3H), 3.24−3.15 (m, 1H), 3.08−3.00

(m 1H), 2.91−2.81 (m, 2H), 2.49−2.37 (m, 1H), 2.36−2.24 (m, 1H).

¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.6, 170.1, 167.6, 157.4,

141.2, 134.6, 134.5, 131.2, 131.2, 129.7, 129.7, 129.4, 129.4, 129.1,

128.9, 127.5, 127.1, 126.8, 125.2, 116.3, 116.3, 56.3, 55.1, 52.8, 37.3,

34.4, 32.3. HRMS (ESI): m/z calcd for C₂₇H₃₀N₄O₅+ H⁺[M + H⁺],

491.2289; found, 491.2298.

( S ) - B e n z y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 4 -

phenylbutanamido)benzamido)-3-(1H-indol-3-yl)propanoate

(10d).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.79 (br.s, 1H), 7.74 (br.d, 1H),

7.27 (br.d, 1H), 7.19 (d, J = 8.3 Hz, 2H), 7.07 (br.d, 2H), 6.82 (d, J =

8.3 Hz, 2H), 6.69 (br.d, 2H), 4.82−4.76 (m, 1H), 4.41−4.35 (m, 1H),

3.73 (s, 3H), 3.28−3.09 (m, 3H), 3.06−2.99 (m, 1H). ¹³C NMR

(125.76 MHz, MeOH-d₄): δ 173.6, 169.8, 167.8, 158.4, 157.4, 134.8,

133.4, 131.8, 131.8, 131.2, 131.2, 128.9, 127.8, 127.7, 127.1, 125.8,

124.2, 117.0, 117.0, 116.3, 116.3, 56.6, 56.3, 52.8, 37.7, 37.4. HRMS

(ESI): m/z calcd for C₂₆H₂₈N₄O₆+ H⁺[M + H⁺], 493.2082; found,

493.2082.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.75 (s, 1H), 7.66 (dd, J = 8.4,

1.7 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.36−

7.15 (m, 11H), 7.12−6.96 (m, 3H), 5.10 (s, 2H), 4.98−4.92 (m, 1H),

4.40−4.32 (m, 1H), 3.50−3.40 (m, 1H), 3.38−3.32 (m, 1H), 2.90−

2.77 (m, 2H), 2.45−2.35 (m, 1H), 2.34−2.21 (m, 1H). ¹³C NMR

(125.76 MHz, MeOH-d₄): δ 173.4, 169.9, 168.2, 141.2, 138.1, 137.0,

134.5, 132.8, 130.3, 129.7, 129.7, 129.5, 129.5, 129.4, 129.4, 129.2,

129.2, 128.7, 127.6, 126.9, 126.7, 126.7, 124.5, 123.6, 122.5, 119.9,

119.1, 112.4, 110.7, 68.1, 55.9, 55.1, 34.5, 32.2, 28.3. HRMS (ESI): m/

z calcd for C₃₅H₃₅N₅O₄+ H⁺[M + H⁺], 590.2762; found, 590.2759.

( S ) - M e t h y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 4 -

phenylbutanamido)benzamido)-5-guanidinopentanoate

(10e).

(S)-Benzyl 2-(3-Amino-4-((S)-2-amino-3-(4-(benzyloxy)-

phenyl)propanamido)benzamido)-3-(1H-indol-3-yl)-

propanoate (10h).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.72 (s, 1H), 7.61 (br.d, 1H),

7.55 (d, J = 7.9 Hz, 1H), 7.46−7.39 (m, 2H), 7.38−7.32 (m, 3H),

7.31−7.23 (m, 6H), 7.22−7.16 (m, 3H), 7.11−6.95 (m, 5H), 5.14−

5.06 (m, 4H), 4.98−4.92 (m, 1H), 4.38−4.33 (m, 1H), 3.49−3.40 (m,

1H), 3.38−3.32 (m, 2H), 3.20−3.10 (m, 1H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 173.3, 169.7, 167.8, 159.9, 138.6, 138.0, 137.0,

134.6, 131.8, 131.8, 131.6, 129.5, 129.5, 129.4, 129.4, 129.2, 129.2,

128.9, 128.7, 128.5, 128.5, 128.3, 128.1, 127.4, 127.0, 124.6, 124.6,

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.07−7.91 (m, 2H), 7.58 (br.d,

1H), 7.34−7.16 (m, 5H), 4.67−4.58 (m, 1H), 4.43−4.35 (m, 1H),

3.75 (s, 3H), 3.29−3.20 (m, 2H), 2.91−2.81 (m, 2H), 2.47−2.24 (m,

2H), 2.13−1.89 (m, 2H), 1.89−1.66 (m, 2H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 173.7, 171.9, 170.1, 168.1, 158.6, 154.2, 141.3,

O

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Journal of Medicinal Chemistry

Article

122.5, 119.9, 119.1, 116.6, 116.6, 116.5, 112.5, 110.7, 70.9, 68.1, 56.5,

55.9, 37.6, 28.3. HRMS (ESI): m/z calcd for C₄₁H₃₉N₅O₅+ H⁺[M +

H⁺], 682.3024; found, 682.3020.

(S)-2-(3-Amino-4-((S)-2-amino-3-(4-hydroxyphenyl)-

propanamido)benzamido)-3-(1H-indol-3-yl)propanoic Acid

(10h′).

(S)-2-(3-Amino-4-((S)-2-amino-4-(4-hydroxyphenyl)-

butanamido)benzamido)-3-(1H-indol-3-yl)propanoic Acid

(10j′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.90 (s, 1H), 7.84 (br.d, 1H),

7.58−7.50 (m, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.14−6.96 (m, 4H),

6.76−6.69 (m, 3H), 4.93−4.91 (m, 1H), 4.40−4.31 (m, 1H), 3.38−

3.32 (m, 2H), 2.81−2.70 (m, 2H), 2.43−2.32 (m, 1H), 2.31−2.20 (m,

1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 175.0, 170.3, 167.6,

157.1, 138.1, 134.7, 131.9, 130.4, 130.4, 129.5, 127.1, 125.5, 125.4,

124.5, 122.5, 122.4, 119.9, 119.2, 119.1, 116.4, 116.4, 112.4, 112.4,

55.7, 55.1, 34.7, 31.4, 28.2. HRMS (ESI): m/z calcd for C₂₈H₂₉N₅O₅+

H⁺[M + H⁺], 516.2241; found, 516.2249.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.58 (br.d, 1H), 7.46−6.90 (m,

9H), 6.84−6.76 (m, 2H), 4.92−4.88 (m, 1H), 4.31−4.21 (m, 1H),

3.50−3.42 (m, 1H), 3.25−3.07 (m, 3H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 175.3, 169.8, 168.7, 158.4, 143.1, 138.1, 134.4, 131.8,

129.5, 128.9, 128.5, 126.7, 126.0, 124.4, 122.4, 119.9, 119.3, 117.5,

116.9, 116.9, 116.7, 112.3, 111.1, 56.3, 55.2, 38.1, 28.3. HRMS (ESI):

m/z calcd for C₂₇H₂₇N₅O₅+ H⁺[M + H⁺], 502.2085; found,

502.2088.

( S ) - B e n z y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 4 -

methylpentanamido)benzamido)-3-(1H-indol-3-yl)propanoate

(10k).

(S)-Methyl 6-Amino-2-(3-amino-4-((S)-2-amino-3-(4-

hydroxyphenyl)propanamido)benzamido)hexanoate (10i).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.78 (s, 1H), 7.72 (br.d, 1H),

7.54 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.36−7.25 (m, 4H),

7.22−7.16 (m, 2H), 7.11−6.96 (m, 3H), 5.13−5.06 (s, 2H), 4.98−

4.92 (m, 1H), 4.30−4.21 (m, 1H), 3.48−3.40 (m, 1H), 3.39−3.33 (m,

1H), 1.96−1.76 (m, 3H), 1.11−1.04 (br.s, 6H). ¹³C NMR (125.76

MHz, MeOH-d₄): δ 173.3, 170.7, 168.1, 138.1, 137.1, 134.8, 133.2,

129.8, 129.5, 129.4, 129.3, 129.2, 128.8, 128.2, 127.3, 127.0, 124.5,

124.0, 122.5, 119.9, 119.2, 112.4, 110.8, 68.1, 55.9, 53.7, 41.4, 28.3,

25.5, 23.4, 21.9. HRMS (ESI): m/z calcd for C₃₁H₃₅N₅O₄+ H⁺[M +

H⁺], 542.2762; found, 542.2761.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.86 (s, 1H), 7.79 (br.d, 1H),

7.30 (br.d, 1H), 7.20 (br.d, 2H), 6.81 (br.d, 2H), 4.63−4.55 (m, 1H),

4.43−4.36 (m, 1H), 3.75 (s, 3H), 3.38−3.32 (m, 1H), 3.23−3.10 (m,

1H), 3.00−2.90 (m, 2H), 2.11−1.86 (m, 2H), 1.81−1.67 (m, 2H),

1.59−1.50 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.9,

169.6, 168.5, 158.3, 134.4, 132.6, 131.8, 131.8, 130.8, 127.1, 126.7,

125.8, 123.4, 116.9, 116.9, 56.5, 54.4, 52.9, 40.6, 37.7, 31.6, 28.0, 24.2.

HRMS (ESI): m/z calcd for C₂₃H₃₁N₅O₅+ H⁺[M + H⁺], 458.2398;

found, 458.2407.

(S)-Benzyl 2-(3-Amino-4-((S)-2-amino-4-(4-(benzyloxy)-

phenyl)butanamido) benzamido)-3-(1H-indol-3-yl)propanoate

(10j).

(S)-Methyl 6-Amino-2-(3-amino-4-((S)-2-amino-4-

methylpentanamido)benzamido)hexanoate (10l).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.62 (br.s, 1H), 7.58−7.49 (m,

2H), 7.44−7.23 (m, 10H), 7.23−6.89 (m, 9H), 5.12−5.01 (m, 4H),

4.98−4.90 (m, 1H), 4.31−4.23 (m, 1H), 3.48−3.39 (m, 1H), 3.27−

3.20 (m, 1H), 2.82−2.71 (m, 2H), 2.38−2.29 (m, 1H), 2.28−2.19 (m,

1H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.4, 169.7, 168.6,

159.0, 138.8, 138.1, 137.1, 134.6, 133.5, 132.3, 131.3, 130.4, 130.4,

130.4, 129.5, 129.5, 129.3, 129.2, 128.8, 128.8, 128.5, 126.8, 124.5,

124.5, 122.5, 122.4, 122.0, 119.9, 119.2, 119.2, 116.3, 116.3, 112.4,

110.8, 109.7, 71.1, 68.1, 55.9, 55.0, 34.7, 31.3, 28.3. HRMS (ESI): m/z

calcd for C₄₂H₄₁N₅O₅+ H⁺[M + H⁺], 696.3175; found, 696.3180.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.99−7.93 (m, 2H), 7.60 (d, J =

8.4 Hz, 1H), 4.65−4.57 (m, 1H), 4.32−4.23 (m, 1H), 3.76 (s, 3H),

3.00−2.90 (m, 2H), 2.11−1.81 (m, 5H), 1.79−1.68 (m, 2H), 1.67−

1.46 (m, 2H), 1.13−1.04 (m, 6H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.9, 170.8, 168.2, 134.6, 133.9, 128.4, 128.3, 127.1, 124.6,

54.4, 53.6, 52.9, 41.4, 40.5, 31.7, 28.1, 25.5, 24.2, 23.4, 21.8. HRMS

(ESI): m/z calcd for C₂₀H₃₃N₅O₄+ H⁺[M + H⁺], 408.2605; found,

408.2604.

P

DOI: 10.1021/jm501867s

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Journal of Medicinal Chemistry

Article

(S)-Benzyl 2-(3-Amino-4-((S)-2-aminohexanamido)-

benzamido)-3-methylbutanoate (10m).

129.5, 129.5, 129.3, 129.2, 128.8, 127.1, 126.8, 124.5, 123.9, 122.5,

119.9, 119.2, 112.4, 110.8, 110.7, 68.1, 55.9, 55.2, 32.3, 28.3, 28.1, 23.4,

14.1. HRMS (ESI): m/z calcd for C₃₁H₃₅N₅O₄+ H⁺[M + H⁺],

542.2762; found, 542.2760.

(S)-2-(3-Amino-4-((S)-2-aminohexanamido)benzamido)-3-

(1H-indol-3-yl)propanoic Acid (10o′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.92−7.68 (m, 2H), 7.58 (d, J =

8.4 Hz, 1H), 7.42−7.28 (m, 5H), 5.26−5.14 (m, 2H), 4.52−4.47 (m,

1H), 4.27−4.22 (m, 1H), 2.35−2.20 (m, 1H), 2.14−2.06 (m, 1H),

2.05−1.96 (m, 1H), 1.56−1.41 (m, 4H), 1.05−0.94 (m, 9H). ¹³C

NMR (125.76 MHz, MeOH-d₄): δ 172.9, 170,4, 168.5, 137.2, 134.8,

133.9, 129.6, 129.6, 129.5, 129.5, 129.4, 129.4, 128.4, 126.9, 124.7,

67.9, 60.5, 55.2, 32.3, 31.6, 28.1, 23.4, 19.6, 19.2, 14.1. HRMS (ESI):

m/z calcd for C₂₅H₃₄N₄O₄+ H⁺[M + H⁺], 455.2653; found,

455.2653.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.81 (s, 1H), 7.75 (br.d, 1H),

7.58 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.0 Hz,

1H), 7.14 (s, 1H), 7.10−7.04 (m, 1H), 7.02−6.94 (m, 1H), 4.95−4.91

(m, 1H), 4.26−4.18 (m, 1H), 3.56−3.39 (m, 2H), 2.10−1.90 (m, 2H),

1.56−1.36 (m, 4H), 1.03−0.92 (m, 3H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 170.4, 167.8, 159.6, 138.1, 133.8, 128.3, 128.1, 126.9,

124.5, 124.4, 122.5, 122.4, 119.9, 119.8, 119.2, 119.1, 112.3, 61.3, 55.2,

32.3, 28.2, 28.1, 23.4, 14.1. HRMS (ESI): m/z calcd for C₂₄H₂₉N₅O₄+

H⁺[M + H⁺], 452.2292; found, 452.2297.

(S)-2-(3-Amino-4-((S)-2-aminohexanamido)benzamido)-3-

methylbutanoic Acid (10m′).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.99−7.92 (m, 2H), 7.60 (d, J =

7.9 Hz, 1H), 4.51−4.46 (m, 1H), 4.29−4.22 (m, 1H), 2.34−2.24 (m,

1H), 2.15−2.06 (m, 1H), 2.05−1.95 (m, 1H), 1.58−1.35 (m, 4H),

1.08−1.03 (m, 6H), 1.02−0.96 (m, 3H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 174.7, 170.5, 168.2, 135.0, 134.3, 129.0, 127.3, 127.0,

125.1, 60.1, 55.2, 32.2, 31.6, 28.1, 23.4, 19.7, 18.9, 14.1. HRMS (ESI):

m/z calcd for C₁₈H₂₈N₄O₄+ H⁺[M + H⁺], 365.2183; found,

365.2177.

( S ) - M e t h y l 6 - A m i n o - 2 - ( 3 - a m i n o - 4 - ( ( S ) - 2 -

aminohexanamido)benzamido)hexanoate (10p).

(S)-Methyl 2-(3-Amino-4-((S)-2-aminohexanamido)-

benzamido)-3-(4-hydroxyphenyl)propanoate (10n).

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.07−7.94 (m, 2H), 7.62 (d, J =

8.3 Hz, 1H), 4.63−4.57 (m, 1H), 4.32−4.22 (m, 1H), 3.75 (s, 3H),

3.00−2.90 (m, 2H), 2.17−1.90 (m, 4H), 1.91−1.66 (m, 2H), 1.65−

1.32 (m, 6H), 1.06−0.83 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.9, 170.4, 168.1, 134.4, 134.3, 128.9, 127.5, 127.0, 125.0,

55.2, 54.4, 52.9, 40.5, 32.2, 31.6, 28.1, 28.0, 24.2, 23.4, 14.1. HRMS

(ESI): m/z calcd for C₂₀H₃₃N₅O₄+ H⁺[M + H⁺], 408.2605; found,

408.2608.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.86−7.76 (m, 2H), 7.56 (br.d,

1H), 7.11−7.04 (m, 2H), 6.72−6.67 (m, 2H), 4.82−4.75 (m, 1H),

4.27−4.18 (m, 1H), 3.73 (s, 3H), 3.24−3.16 (m, 1H), 3.07−2.97 (m,

1H), 2.15−2.04 (m, 1H), 2.02−1.93 (m, 1H), 1.58−1.42 (m, 4H),

1.03−0.94 (m, 3H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.6,

170.4, 167.8, 157.4, 134.7, 134.0, 131.2, 131.2, 128.9, 128.4, 126.9,

124.7, 116.3, 116.3, 116.2, 56.3, 55.2, 52.8, 37.4, 32.2, 28.1, 23.4, 14.1.

HRMS (ESI): m/z calcd for C₂₃H₃₀N₄O₅+ H⁺[M + H⁺], 443.2289;

found, 443.2288.

( S ) - M e t h y l 2 - ( 3 - A m i n o - 4 - ( ( S ) - 2 - a m i n o - 5 -

guanidinopentanamido)benzamido)-3-(4-hydroxyphenyl)-

propanoate (10q).

(S)-Benzyl 2-(3-Amino-4-((S)-2-aminohexanamido)-

benzamido)-3-(1H-indol-3-yl)propanoate (10o).

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.95−7.82 (m, 2H), 7.67 (d, J =

8.4 Hz, 1H), 7.09 (d, J = 8.0 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H), 4.82−

4.74 (m, 1H), 4.40−4.28 (m, 1H), 3.73 (s, 3H), 3.38−3.32 (m, 2H),

3.23−3.15 (m, 1H), 3.09−2.99 (m, 1H), 2.30−2.17 (m, 1H), 2.16−

2.05 (m, 1H), 1.95−1.76 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-

d₄): δ 173.6, 169.9, 167.6, 158.6, 157.4, 134.7, 134.6, 131.2, 131.2,

129.4, 128.9, 127.1, 126.4, 125.4, 116.3, 116.3, 56.3, 54.7, 52.8, 41.8,

37.3, 29.6, 25.7. HRMS (ESI): m/z calcd for C₂₃H₃₁N₇O₅+ H⁺[M +

H⁺], 486.2459; found, 484.2458.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.77 (s, 1H), 7.70 (br.d, 1H),

7.55 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.36−7.26 (m, 4H),

7.23−7.16 (m, 2H), 7.12−6.96 (m, 3H), 5.11 (s, 2H), 4.98−4.92 (m,

1H), 4.25−4.18 (m, 1H), 3.48−3.40 (m, 1H), 3.37−3.32 (m, 1H),

1.55−1.37 (m, 6H), 1.02−0.94 (m, 3H). ¹³C NMR (125.76 MHz,

MeOH-d₄): δ 173.4, 170.3, 168.1, 138.1, 137.1, 134.6, 134.5, 133.1,

Q

DOI: 10.1021/jm501867s

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Journal of Medicinal Chemistry

Article

(S)-2-(3-Amino-4-((S)-2-amino-5-guanidinopentanamido)-

benzamido)-3-(1H-indol-3-yl) propanoic Acid (10r).

where Y is the enzymatic activity and X is the inhibitor concentration.

Phagocytosis Assay. Murine macrophage cell line RAW264.7

cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal

bovine serum. For assays, cells were transferred to and cultured in a

96-well black culture plate (2 × 10⁴cells/well) for 24 h at 37 °C, 5%

CO₂. The next day, they were washed twice with cold phosphate-

buﬀered saline (PBS) and activated with IFN-γ (100 IU/mL) and LPS

(1 μg/mL) in the presence or absence of various inhibitor

concentrations. Phagocytosis was assessed by measuring the amount

of uptake of latex beads coated with FITC-labeled rabbit IgG into cells

using a phagocytosis assay kit (FITC) (Cayman Chemical, Ann Arbor,

MI) according to the instruction manual. In brief, cells with or without

various stimulants were treated with the beads and cultured in RPMI

1640 without FBS (1% FBS) for 24 h at 37 °C, 5% CO₂. Twenty-four

hours after stimulation, cells were washed twice with cold phosphate-

buﬀered saline (PBS). The uptake of the beads into cells was

calculated by measuring ﬂuorescence intensity in a TECAN inﬁnite

M200 microplate ﬂuorescence reader using an excitation of 485 nm

and an emission of 535 nm. The cell supernatant from treated cells was

used for measuring aminopeptidase activity. The enzymatic activity of

ERAP1 was determined with ^L-leucine-7-amido-4-methyl coumarin (L-

AMC; Sigma). The reaction mixture containing 100 μM Leu-AMC

and 50 μL of culture medium in 50 mM HEPES pH 7.0, 100 mM

NaCl. The amount of 7-amino-4-methylcoumarin released was

measured by a TECAN inﬁnite M200 microplate ﬂuorescence reader

at an excitation wavelength of 380 nm and an emission wavelength of

460 nm. For calculation of the ED₅₀values, experimental data were ﬁt

to the equation described in the Enzymatic Assay section using the

GraphPad Prism software.

¹H NMR (500.13 MHz, MeOH-d₄): δ 7.80 (s, 1H), 7.74 (d, J = 8.1

Hz, 1H), 7.63−7.56 (m, 2H), 7.32 (d, J = 8.1 Hz, 1H), 7.14 (s, 1H),

7.10−7.05 (m, 1H), 7.01−6.94 (m, 1H), 4.97−4.89 (m, 1H), 4.37−

4.26 (m, 1H), 3.37−3.32 (m, 2H), 3.25−3.20 (m, 2H), 2.28−2.03 (m,

2H), 1.91−1.79 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ

175.0, 169.9, 167.8, 158.7, 138.1, 133.8, 128.06, 126.9, 124.6, 124.4,

122.5, 122.4, 119.9, 119.2, 119.1, 112.4, 112.3, 111.2, 64.9, 54.7, 41.8,

29.7, 28.3, 25.7. HRMS (ESI): m/z calcd for C₂₄H₃₀N₈O₄+ H⁺[M +

H⁺], 495.2463; found, 495.2468.

(S)-Methyl 6-Amino-2-(3-amino-4-((S)-2-amino-5-

guanidinopentanamido)benzamido)hexanoate (10s).

Mice. Previously described IRAP knockout mice on an Sv129

background obtained from S. Keller were backcrossed up to 10 times

to C57BL/6 mice obtained from Janvier (St. Quentin-Fallavier,

France). Control mice were mixed-background mice bred in our

facility. RAG1-deﬁcient OT-1 T cell receptor transgenic mice were

obtained from Taconic (Germantown, NY) and bred in our animal

facility. Animal experimentation was approved by the Comite

¹H NMR (500.13 MHz, MeOH-d₄): δ 8.02−7.94 (m, 2H), 7.70 (br.d,

1H), 4.65−4.58 (m, 1H), 4.37−4.30 (m, 1H), 3.76 (s, 3H), 3.37−3.32

(m, 2H), 2.99−2.91 (m, 2H), 2.14−1.90 (m, 4H), 1.89−1.69 (m, 4H),

1.65−1.48 (m, 2H). ¹³C NMR (125.76 MHz, MeOH-d₄): δ 173.9,

169.9, 168.1, 158.8, 134.5, 134.1, 128.6, 127.1, 126.9, 124.9, 54.7, 54.4,

52.9, 41.9, 40.6, 31.7, 29.7, 28.1, 25.7, 24.1. HRMS (ESI): m/z calcd

for C₂₀H₃₄N₈O₄+ H⁺[M + H⁺], 451.2776; found, 451.2778.

Protein Expression and Puriﬁcation. The expression and

puriﬁcation of recombinant human endoplasmic reticulum amino-

peptidase 1 (ERAP1), endoplasmic reticulum aminopeptidase 2

(ERAP2), and insulin-regulated aminopeptidase (IRAP) have been

described before.^23,24Recombinant baculovirus containing each

ERAP1 variant was produced in sf9 cells according to the

manufacturer’s instructions (Bac-to-Bac baculovirus expression system,

Invitrogen). Recombinant proteins were expressed in Hi5 cells after

infection with the appropriate recombinant baculovirus and puriﬁed as

previously described.²⁴Proteins were aliquoted in and stored in a

buﬀer containing 10 mM Hepes, pH 7, 100 mM NaCl, and 10%

glycerol at −80 °C, until needed.

Enzymatic Assays. The enzymatic activity of the enzymes was

calculated by following the time-dependent increase in ﬂuorescence at

460 nm (excitation was at 380 nm) of the ﬂuorigenic substrates ^L-

leucine-7-amido-4-methyl coumarin (L-AMC; Sigma) for ERAP1 and

IRAP and ^L-arginyl-7-amido-4-methyl coumarin (R-AMC; Sigma) for

ERAP2. All measurements were performed on a TECAN inﬁnite

M200 microplate ﬂuorescence reader. For evaluation of the eﬀect of

the compound on activity, 30 nM ERAP1, 6 nM ERAP2, or 6 nM

IRAP was added to each well along with 50 μM substrate and varied

concentrations of compound. In all cases, the enzyme concentration

used was signiﬁcantly less than the calculated IC₅₀values for the

inhibitors in order to avoid ligand depletion artifacts. The reaction was

followed for 5−10 min, and activity was calculated by measuring the

slope of the time course. For calculation of the in vitro IC₅₀values,

experimental data were ﬁt to the following equation using the

GraphPad Prism software package

́

d’et

P2.LS.156.10).

́ ́

hique pour l’experimentation animale Paris Descartes (no.

In Vitro Cross-Presentation Assays. Murine BMDCs were

generated in vitro by culturing progenitor cells extruded from large

bones for 7 days in complete medium (IMDM medium completed

with 10% FCS, 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL

streptomycin, 50 mM β-mercaptoethanol) supplemented with J558

supernatant containing 20 μg/mL GM-CSF. On day 6, BMDCs were

seeded at 50 000 cells/well into a 96-well ﬂat bottom culture plate with

increasing concentrations of inhibitor 4u. On day 7, cells were washed

twice with PBS and incubated with serial dilutions of yeast cells

expressing ovalbumin on their cell surface in complete medium

(prepared as described in ref 39), in the presence of the same

concentration of 4u as that used before. After 6 h, the cells were

washed twice with PBS, and CD8⁺T cells puriﬁed from lymph nodes

of OT-I mice were added to the culture for 20 h at a ratio T/BMDCs

of 1.5:1, again in the presence of inhibitor 4u. To assess T cell

activation, the IL-2 concentration in supernatants was measured by

sandwich ELISA using Nunc Maxisorp plates, streptavidin/horse

radish peroxidase (Thermo Scientiﬁc), and OptEIA TMB substrate

(BD Biosciences). As negative controls, an aliquot of BMDCs were

ﬁxed with 0.04% glutaraldehyde (as described in ref 39) prior to the

addition of antigen, and the signal obtained was set as the background

and subtracted from all OD values. Results represent the means of

duplicate wells.

Computational Methods. The crystallographic structures of

ERAP1 (PDB ID: 2YD0),⁴⁰ERAP2 (PDB ID: 3SE6),²⁴and IRAP

(PDB ID: 4PJ6)⁴¹were used without any further reﬁnement. Polar

hydrogen atoms were added and Gasteiger charges were applied using

AutoDock Tools 1.5.6.⁴²The initial conformations of the inhibitors

were generated from SMILES representations using the program

Omega 2.4,⁴³and then Gasteiger charges were applied. The search

space was deﬁned by a grid box centered next to the catalytic zinc and

comprised 81 × 81 × 81 grid points of 0.375 Å spacing. For each

₅₀−X)×Hill Slope)

Y = bottom + (top − bottom)/(1 + 10^{((Log IC}

)

R

DOI: 10.1021/jm501867s

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

complex, 100 docking rounds were calculated with AutoDock 4.2

using the Lamarckian genetic algorithm with the default parameters of

AutoDock 3.⁴⁴The maximum number of energy evaluations was set to

10 × 10⁶, and the docked conformations were clustered using a

tolerance of 2.0 Å. Visual examination of the complexes and rendering

of the ﬁgures were performed with VMD 1.9.⁴⁵Calculations were

carried out using Intel Xeon workstations running Linux 2.6.32

kernels.

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ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat.

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ASSOCIATED CONTENT

■

S

* Supporting Information

¹H and ¹³C NMR spectra of biologically active compounds 4a,

4u, 4x, and 10n; titration curve of 4u with recombinant mouse

IRAP. This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Authors

*(D.V.) E-mail: vourloumis@chem.demokritos.gr. Phone:

(+30)2106503624. Fax: (+30)2106511766.

*(E.S.) E-mail: stratos@rrp.demokritos.gr. Phone: (+30)

2106503918. Fax: (+30)2106503918.

Author Contributions

A.P. performed the computational design, synthesis, and

characterization of the compounds; E.Z. prepared the

recombinant enzymes and performed the in vitro and

phagocytosis assays measurements and HPLC analysis; S.T.

assisted in the synthesis of the compounds; F.-X.M. and P.v.E.

designed and performed the cross-presentation assay; G.S and

D.C.M. helped to establish the macrophage activation assay;

E.A.T. co-supervised the project along with D.V. and E.S., who

conceived the experiments and analyzed the results. All authors

contributed to the preparation of the manuscript and have

approved its ﬁnal version.

(9) Alvarez-Navarro, C.; Lopez de Castro, J. A. ERAP1 structure,

function and pathogenetic role in ankylosing spondylitis and other

MHC-associated diseases. Mol. Immunol. 2014, 57, 12−21.

(10) Goto, Y.; Ogawa, K.; Nakamura, T. J.; Hattori, A.; Tsujimoto,

M. TLR-mediated secretion of endoplasmic reticulum aminopeptidase

1 from macrophages. J. Immunol. 2014, 192, 4443−52.

(11) Goto, Y.; Ogawa, K.; Hattori, A.; Tsujimoto, M. Secretion of

endoplasmic reticulum aminopeptidase 1 is involved in the activation

of macrophages induced by lipopolysaccharide and interferon-gamma.

J. Biol. Chem. 2011, 286, 21906−14.

(12) Aldhamen, Y. A.; Seregin, S. S.; Rastall, D. P.; Aylsworth, C. F.;

Pepelyayeva, Y.; Busuito, C. J.; Godbehere-Roosa, S.; Kim, S.;

Amalfitano, A. Endoplasmic reticulum aminopeptidase-1 functions

regulate key aspects of the innate immune response. PLoS One 2013,

8, e69539.

Notes

The authors declare no competing ﬁnancial interest.

(13) Aldhamen, Y. A.; Pepelyayeva, Y.; Rastall, D. P. W.; Seregin, S.

S.; Zervoudi, E.; Koumantou, D.; Aylsworth, C. F.; Quiroga, D.;

Godbehere, S.; Georgiadis, D.; Stratikos, E.; Amalfitano, A. Auto-

immune disease-associated variants of extracellular endoplasmic

reticulum aminopeptidase 1 induce altered innate immune responses

by human immune cells. J. Innate Immun. 2015, Jan 14. DOI:10.1159/

000368899.

(14) Stratikos, E. Regulating adaptive immune responses using small

molecule modulators of aminopeptidases that process antigenic

peptides. Curr. Opin. Chem. Biol. 2014, 23C, 1−7.

(15) York, I. A.; Brehm, M. A.; Zendzian, S.; Towne, C. F.; Rock, K.

L. Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC

class I-presented peptides in vivo and plays an important role in

immunodominance. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9202−7.

(16) Hammer, G. E.; Gonzalez, F.; James, E.; Nolla, H.; Shastri, N. In

the absence of aminopeptidase ERAAP, MHC class I molecules

present many unstable and highly immunogenic peptides. Nat.

Immunol. 2007, 8, 101−8.

(17) Rastall, D. P.; Aldhamen, Y. A.; Seregin, S. S.; Godbehere, S.;

Amalfitano, A. ERAP1 functions override the intrinsic selection of

specific antigens as immunodominant peptides, thereby altering the

potency of antigen-specific cytolytic and effector memory T-cell

responses. Int. Immunol. 2014, 26, 685−95.

ACKNOWLEDGMENTS

■

This research was ﬁnanced by the European Union (European

Social Fund) and Greek national funds through the Operational

Program “Education and Lifelong Learning” of the National

Strategic Reference Framework: Research Funding Program of

the General Secretariat for Research & Technology (grant nos.

LS7-2199 and ERC-14). F.-X.M. was supported by a grant from

INSERM (poste d’accueil). Work in the laboratory of P.v.E.

́

was supported by the Fondation pour la Recherche Medicale

(grant no. DEQ20130326539).

ABBREVIATIONS USED

■

DABA, 3,4-diaminobenzoic acid; hPhe, L-homophenylalanine;

hTyr, ^L-homotyrosine; Nle, L-norleucine; TFA, triﬂuoroacetic

acid; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-

uronium hexaﬂuorophosphate; HATU, 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate; DIEA, N,N-diisopropylethylamine; DBU,

1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-dimethylforma-

mide

(18) Cifaldi, L.; Lo Monaco, E.; Forloni, M.; Giorda, E.; Lorenzi, S.;

Petrini, S.; Tremante, E.; Pende, D.; Locatelli, F.; Giacomini, P.; Fruci,

D. Natural killer cells efficiently reject lymphoma silenced for the

endoplasmic reticulum aminopeptidase associated with antigen

processing. Cancer Res. 2011, 71, 1597−606.

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3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of m1 aminopeptidases with immune-regulating properties

DOI: 10.1021/jm501867s

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