Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing

Article abstract of DOI:10.1021/acs.bioconjchem.5b00430

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such

Full text of DOI:10.1021/acs.bioconjchem.5b00430

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Article
Development of anilino-maytansinoid ADCs that efficiently release cytotoxic
metabolites in cancer cells and induce high levels of bystander killing
Wayne C. Widdison, Jose F Ponte, Jennifer A Coccia, Leanne Lanieri, Yulius Setiady,
Ling Dong, Anna Skaletskaya, E. Erica Hong, Rui Wu, Qifeng Qiu, Rajeeva Singh,
Paulin Salomon, Nathan Fishkin, Luke Harris, Erin K Maloney, Yelena Kovtun, Karen
Veale, Sharon D. Wilhelm, Charlene A. Audette, Juliet A Costoplus, and Ravi V.J. Chari
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00430 • Publication Date (Web): 10 Sep 2015
Downloaded from http://pubs.acs.org on September 11, 2015
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Title: Development of anilino-maytansinoid ADCs that efficiently
release cytotoxic metabolites in cancer cells and induce high levels of
bystander killing
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Authors: Wayne C. Widdison*, Jose F. Ponte, Jennifer A. Coccia, Leanne Lanieri,
Yulius Setiady, Ling Dong, Anna Skaletskaya, E. Erica Hong, Rui Wu, Qifeng Qiu,
Rajeeva Singh, Paulin Salomon, Nathan Fishkin, Luke Harris, Erin K. Maloney, Yelena
Kovtun, Karen Veale, Sharon D. Wilhelm, Charlene A. Audette, Juliet A. Costoplus, Ravi
V. J. Chari
ABSTRACT
Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a
maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide which
in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were
prepared utilizing different dipeptides in the linkage including GlyꢀGly, LꢀValꢀLꢀCit and all four
stereoisomers of the AlaꢀAla dipeptide. The properties of AaMCs could be altered by the choice
of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a DꢀAlaꢀDꢀAla peptide
linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide
linkers. In mouse models, the antiꢀCanAg AaMC bearing a DꢀAla-L-Ala dipeptide in the linker
was shown to be more efficacious against heterogeneous HTꢀ29 xenografts than maytansinoid
ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar
tolerabilities.
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INTRODUCTION
Antibodyꢀdrug conjugates (ADCs) are prepared by conjugating a cytotoxic payload to a
monoclonal antibody (mAb) via a linker. All ADCs in the clinic bearing maytansinoid payloads,
antibodyꢀmaytansinoid conjugates (AMCs), utilize linkers that are attached to lysine residues of
the targeting antibody. The linker itself is either nonꢀcleavable or contains a cleavable disulfide
moiety. Mechanisms of how ADCs kill targeted cancer cells have been studied, and the modes
by which AMCs release metabolites to kill targeted cells are depicted in Scheme 1.1, 2 The
antibody component of the AMC binds to a cancer cell in step 1. The antibodyꢀantigen complex
is internalized in step 2, traveling through one or more preꢀlysosomal vesicles. In step 3, the
complex is degraded in a lysosome or a preꢀlysosomal vesicle until only a lysine residue of the
antibody remains, attached to the unaltered maytansinoid via the linker. The initially formed
metabolite is transported to the cytoplasm in step 4 and if the linker is nonꢀcleavable, then no
further degradation occurs. However, if the linker contains a disulfide bond a portion of the
metabolite is cleaved in step 5 to release a thiolꢀbearing maytansinoid. A portion of the thiolꢀ
bearing maytansinoid can also be Sꢀmethylated in step 6, but with different efficiencies
depending on the structure of the maytansinoid’s side chain. All of the metabolites are believed
to bind to microtubules and cause cells to arrest in G2/M phase before ultimately killing the cells
(step 7). The lysineꢀbearing metabolites are charged and it is known that a significant portion
can escape the cell (step 8a), however the lysine adduct cannot easily diffuse into and kill
neighboring bystander cells (step 9a). The thiolꢀbearing and Sꢀmethylated maytansinoid
metabolites on the other hand are not charged, and although they bind nonꢀcovalently to
microtubules, they can diffuse out (step 8b) of the targeted cells and into nearby bystander cells,
potentially killing them (step 9b). Thiolꢀbearing metabolites also have the potential to react with
proteins or disulfideꢀcontaining compounds, such as cystine, in the nonꢀreducing extracellular
environment to become less membraneꢀpermeable. Indeed the thioꢀmaytansinoid DM4 (2) is
several fold less potent than Sꢀmethylated DM4 (3) when tested in vitro.3 Also, when a thiolꢀ
bearing maytansinoid is added to buffered plasma, levels of the free thiolꢀbearing maytansinoid
decline presumably due to formation of mixed disulfides with proteins such as albumin, or with
disulfideꢀcontaining peptides. It is therefore expected that a released thioꢀmaytansinoid would
induce less bystander killing in vivo than a nonꢀthiolꢀbearing maytansinoid such as Sꢀmethylꢀ
DM4 (3).
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Scheme 1 a) Illustration of the mechanisms by which AMCs kill cells b) Control maytansinoid
conjugates used in these studies that utilize disulfide linkers and the metabolites formed from
disulfide cleavage and Sꢀmethylation.
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a)
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b)
Experiments with radiolabeled antibodies indicate that a very low proportion of an injected
tumorꢀselective antibody actually accumulates in a tumor in humans.4 Monoclonal antibodies to
tumorꢀassociated antigens have been reported to bind in vivo to cancer cells that are proximal to
blood vessels but not to penetrate far from blood vessels into the tumor mass.5 Lower molecular
weight chemotherapeutics however can penetrate more deeply into a tumor. Due to their low
molecular weight, metabolites released from a cancer cell after ADC catabolism would also be
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expected to penetrate more deeply into a tumor mass. However, only the membrane permeable
metabolites that can induce bystander killing can desirably diffuse into and kill these neighboring
cancer cells. Also ADCs with higher affinity toward an antigen are believed to have a higher
binding site barrier than ADCs with lower affinity.6ꢀ8 More of the high affinity ADCs bind to
cancer cells that are nearest to blood vessels so that even less ADC penetrates deeper into the
tumor. The cancer cells nearest to blood vessels would likely catabolize more ADC than would
be needed to kill them. If the released metabolites cannot induce bystander killing than much of
this excess killing power would not be utilized against the tumor. If the excess of released ADC
metabolites however can induce bystander killing than some of the neighboring cancer cells or
tumor stromal cells could be killed.
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In many cases, tumor stromal cells have been shown to greatly aid in tumor cell growth and
metastasis but these cells usually do not express the antigen to which an ADC is targeted.9, 10
Tumors often also contain cancer cells that do not express target antigen.11 Thus, one would
expect that ADCs with bystander killing activity would be able to kill tumor stromal cells,
antigenꢀnegative cancer cells, and even cells of the tumor neovasculature resulting in improved
antiꢀtumor activity. Indeed, tumor xenograft studies in mice have shown that AMCs that induce
bystander killing are typically several fold more efficacious than those that do not.11ꢀ13
Conjugates utilizing the auristatin MMAE payload also display bystander killing,14 which has
been cited as being important for their efficacy.
It may seem counter intuitive but maytansinoid ADCs that induce bystander killing are not
necessarily less well tolerated in humans than similar ADCs that do not induce bystander killing.
For example, the AMC mirvetuximab soravtansine (IMGN853), which contains a disulfide
linker and induces bystander killing, is dosed at approximately 6 mg/kg every three weeks in
current clinical testing.15 In contrast, the AMC adoꢀtrastuzumab emtansine (Kadcyla®),
containing a nonꢀcleavable linker and does not induce bystander killing, is dosed at 3.6 mg/kg
every three weeks in humans.16, 17 AMCs with disulfide linkers are catabolized to release Sꢀ
methylated maytansinoid, that can induce bystander killing, but reactive thioꢀmaytansinoids, and
charged lysine adducts are also released, both of which may limit bystander activity. We
therefore wished to prepare a new type of AMC that could be efficiently degraded in endocytic
compartments of cells to release only nonꢀcharged, nonꢀreactive metabolites so that even greater
bystander killing could be induced than is seen with AMCs that utilize disulfide linkers.
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Scheme 2 Depiction of the metabolism of ADCs utilizing self immolating peptideꢀanilino
linkages
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ADCs with selfꢀimmolative linkers typically incorporate an anilineꢀpeptide construct covalently
attached through a carbamate moiety on the benzylic position of the aniline so that the released
anilino compound can immolate to produce carbon dioxide and an amineꢀbearing cytotoxic
payload.18 The amineꢀbearing cytotoxic payload released by this design would be mostly in a
charged state below pH 9, Scheme 2. ADCs using pyrrolobenzodiazepine (PBD) dimer payloads
that release a highly cytotoxic anilineꢀbearing PBD dimer upon cleavage of a peptide linker have
been described.19 Anilines are atypical amines most often having pKa values of 5 or less, so the
molecules are predominantly in a nonꢀcharged state at pH values above 5.20 ADC metabolites
bearing nonꢀcharged amines, such as anilines, should be more membrane permeable and thus
induce a greater degree of bystander killing than metabolites bearing amines that are charged at
physiological pH values. Here we describe the preparation of antibody anilinoꢀmaytansinoid
conjugates (AaMCs, 8a – 8i) that can release an anilineꢀbearing maytansinoid (PAꢀMay, 9) upon
cleavage of a peptide linker (Scheme 3). We also show results on how altering the peptide
linkage affects an AaMC’s metabolism, activity, and tolerability.
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Scheme 3 Depiction of the cellular metabolism of AaMCs in a catabolic vesicle (The
stereochemistry of amino acid side chains is shown in brackets)
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RESULTS
Synthesis of antibody maytansinoid conjugates and metabolites. AaMC precursors were
prepared by the method depicted in Scheme 4. The Nꢀhydroxysuccinimide (NHS) ester of the 9ꢀ
fluorenylmethoxycarbonyl (Fmoc) protected amino acids (11a-11b) were prepared by reacting
the corresponding Fmocꢀprotected amino acids 10a-10b with Nꢀhydroxysuccinimide (NHS) in
the presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide HCl salt (EDCꢀHCl). Fmocꢀ
protected dipeptides 12a-12d were prepared by reacting the corresponding amino acid with the
respective NHSꢀactivated Fmoc protected amino acid 11a – 11b. The Fmoc protected dipeptides
were then reacted with 4ꢀamino benzyl alcohol and 2ꢀethoxyꢀ1ꢀethoxycarbonylꢀ1, 2ꢀ
dihydroquinoline (EEDQ), as described by Dubowchik et. al., to give the compounds 13a –
13f.18 Reaction of the individual peptides 13a – 13f with 1 eq. of methane sulfonyl chloride in
DMF followed by treatment with sodium bromide in the same pot gave the corresponding crude
brominated compounds which were reacted, without isolation, with DM1 to give 14a – 14f. The
Fmoc protecting groups were removed using morpholine in DMF to give 15a – 15f, which were
each then reacted with SPDB (16) to give 17a-17f . The disulfide bonds of 17a – 17f were
cleaved with 1,4ꢀdithioꢀDLꢀthreitol (DTT) to give the corresponding thioꢀmaytansinod 18a –
18f. Paraꢀanilinoꢀmaytansinoid (PAꢀMay, 9), the expected metabolite from cellular processing
of an AaMC, was prepared in two steps by reacting DM1 (18) with 4ꢀnitroꢀbenzyl bromide in
DMF to give (19) followed by iron catalyzed reduction of the nitro group (Scheme 5).
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Scheme 4 Syntheses of AaMC maytansinoids a) NHS, EDC, b) respective amino acid, c) EEDQ,
paraꢀamino benzyl alcohol, d) Methane sulfonyl chloride, NaBr, DM1, e) morpholine, f) SPDB
(16), g) DTT; The stereochemistry of amino acid side chains is shown in brackets
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Scheme 5 Synthesis of PAꢀMay (9)
AntiꢀEGFR conjugates 8a – 8f were prepared by conjugating a humanized antiꢀEGFR IgG1
antibody with the corresponding cytotoxic payloads (17a – 17f) using the Nꢀγꢀmaleimidobutyrylꢀ
oxysulfosuccinimide ester (sulfoꢀGMBS) linker (20) as shown in Scheme 6. The AaMC
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huC242-D-Ala-L-AlaꢀPAꢀMay (8g) was prepared in the same way but by conjugating 18c to the
humanized antiꢀCanAg IgG1antibody huC242. Nonꢀbinding conjugates chKTI-D-Ala-L-Alaꢀ
PAꢀMay (8h) and chKTI-L-ValꢀLꢀCitꢀPAꢀMay (8i), respectively, were prepared by conjugating
18c or 18a to a chimeric IgG1 antibody that binds to the Kunitz soybean trypsin inhibitor
(chKTI). AMCs containing DM4 (2) conjugated to antibodies utilizing disulfide linkers: antiꢀ
EGFRꢀSPDBꢀDM4 (1a), huC242ꢀsulfoꢀSPDBꢀDM4 (1b), huC242ꢀSPDBꢀDM4 (1c), chKTIꢀ
sulfoꢀSPDBꢀDM4 (1d) and antiꢀEGFRꢀsulfoꢀSPDBꢀDM4 (1e) were prepared by previously
described procedures; structural representations are shown in Scheme 1.21
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Scheme 6 Conjugation of 18a – 18f to an antibody (antiꢀEGFR, COLO 205 or chKTI), with the
stereochemistry of amino acid side chains shown in brackets.
In vitro cytotoxicity and bystander killing. Each of the antiꢀEGFR AaMCs as well as AMCs
1a and 1e, that utilize disulfide linkers, were assayed for their in vitro cytotoxicities against
several EGFR+ cell lines; IC50 values are recorded in Table 1. The AaMCs were more potent
than conjugates 1a and 1e except for antiꢀEGFR-D-Ala-D-AlaꢀPAꢀMay (8b). AntiꢀEGFR-L-
Ala-D-AlaꢀPAꢀMay (8d) was less potent than antiꢀEGFRꢀDꢀAla-L-AlaꢀPAꢀMay (8c) or antiꢀ
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EGFR AaMCs that incorporated only natural amino acids in the linker (8a, 8e, 8f). Conjugate 8c
had similar potency to antiꢀEGFR-L-Ala-L-AlaꢀPAꢀMay (8e) and antiꢀEGFR-L-Val-L-CitꢀPAꢀ
May (8a), against HSC2, PC9, H1975 and Ca9ꢀ22 cells, but 8a and 8e were more potent against
SAS and OSC19 cells. A graph of the cytotoxicities of conjugates 8a, 8c, 8e, and 1a against
H1975 cells is shown in Supplemental Figure S1a. The AaMCs were not always more potent
than AMCs that utilize disulfide linkers. For example huC242-D-Ala-L-AlaꢀPAꢀMay (8g), had a
similar cytotoxicity profile to huC242ꢀSPDBꢀDM4 (1c) against CanAgꢀpositive COLO 205
cells, while both conjugates were 100 fold less potent against CanAgꢀnegative Namalwa cells,
Supplemental Figure S1b.
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Table 1 In vitro cytotoxicities of ADCs toward EGFR+ cell lines
In vitro IC50 (nM)
ADC
HSC2
0.18
SAS
0.87
OSC19
1.39
PC9
0.18
H1975
3.36
Ca9-22
0.089
0.40
1a Anti-EGFR-SPDB-DM4
1e Anti-EGFR-sulfo-SPDB-DM4
8a Anti-EGFR-L-Val-L-Cit-PA-May
8b Anti-EGFR-D-Ala-D-Ala-PA-May
8c Anti-EGFR-D-Ala-L-Ala-PA-May
8d Anti-EGFR-L-Ala-D-Ala-PA-May
8e Anti-EGFR-L-Ala-L-Ala-PA-May
8f Anti-EGFR-Gly-Gly-PA-May
0.17
3.17
3.85
4.09
4.70
0.031
2.52
0.10
0.10
0.046
10.67
0.057
0.17
0.18
0.026
0.55
6.67
30.00
0.19
30.00
0.18
0.028
0.12
0.080
0.16
0.021
0.051
0.017
0.017
1.89
0.82
0.030
0.034
0.033
0.083
0.055
0.14
0.043
0.035
0.11
0.063
In vitro cytotoxicities were measured by WSTꢀ8 assay after a 5 day continuous exposure to the
respective conjugate.
AaMCs were also tested for their capacity to induce bystander killing via an in vitro assay as
previously described.11 The assay involves incubating an ADC with a coꢀculture of antigenꢀ
negative and antigenꢀpositive cells in various ratios, at a conjugate concentration that can
selectively kill all antigenꢀpositive cells, but is known not to kill any antigenꢀnegative cells
unless antigenꢀpositive cells are present. The ADC is internalized and processed by the antigenꢀ
positive cells releasing metabolites that can kill the antigenꢀpositive cells and any metabolites
that are membrane permeable may also diffuse into and kill neighboring antigenꢀnegative cells.
ADCs that induce the most bystander killing are those that kill antigenꢀnegative cells (bystander
cells) with the lowest amount of added antigenꢀpositive cells while keeping the ADC
concentration constant (bystander killing method 1). Bystander killing of antiꢀCanAg conjugates
was evaluated by method 1 using 5000 antigenꢀnegative Namalwa cells in the presence of
different numbers of antigenꢀpositive COLO 205 cells and 0.3 nM of either huC242-D-Ala-L-
AlaꢀPAꢀMay (8g), or huC242ꢀsulfoꢀSPDBꢀDM4 (1b), Figure 1a. AMC 1b required the addition
of 625 COLO 205 cells to induce over 50% bystander killing and 8g was approximately twice as
efficient as 1b, requiring only 250 COLO 205 cells to induce over 50% bystander killing. In a
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separate experiment huC242ꢀ SPDBꢀDM4 (1c) was found to have the same degree of bystander
killing as conjugate 1b while a conjugate utilizing a nonꢀcleavable linker displayed no bystander
killing (data not shown). In a similar bystander killing assay (method 2) a fixed number of
antigenꢀnegative cells were incubated with a fixed amount of antigenꢀpositive cells in the
presence of an ADC at a single concentration. AntiꢀEGFR AaMCs were assayed for bystander
killing via method 2 using 2000 antigenꢀnegative cells (MCF7) and 3000 antigenꢀpositive cells
(Ca9ꢀ22) in the presence of either antiꢀEGFR-L-Val-L-CitꢀPAꢀMay (8a), antiꢀEGFR-D-Ala-L-
AlaꢀPAꢀMay (8c), antiꢀEGFR-L-Ala-D-AlaꢀPAꢀMay (8d), antiꢀEGFR-L-Ala-L-AlaꢀPAꢀMay
(8e) at a concentration of 0.66 nM or with no added conjugate, Figure 1b. AaMCs 8a, 8c and 8e
displayed similar levels of bystander killing while 8d had slightly lower bystander killing. In a
separate experiment antiꢀEGFR-D-Ala-D-AlaꢀPAꢀMay (8b) was shown to have essentially no
bystander killing (data not shown). For either bystander killing method, the number of antigen
positive cells required depends on their output of metabolite, which inturn depends on their
target antigen density and ADC processing efficiency.
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Bystander Potency (anti-CanAg ADCs)
0.3 nM huC242 conjugate
2.0
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huC242-D-Ala-L-Ala-PA-May (8g) huC242-sulfo-SPDB-DM4 (1b)
Number of antigenꢀpositive COLO 205 cells
b) Bystander Potency (anti-EGFR AaMCs)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
[Ab] = 0.66 nM
Ag+ cells: Ca9-22
Ag- cells: MCF7
)
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d
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Figure 1 Bystander killing assay in which 5000 antigenꢀnegative cells (Namalwa) were mixed
with the following number of COLO 205 cells: none ■,125 ■, 250 ■, 625 ■, or 1250 ■ and the
mixtures were treated with antiꢀCanAg conjugates (1b, or 8g) at a concentration of 0.3 nM. b)
Bystander killing assay in which 2000 antigenꢀnegative cells (MCF7) were mixed with 3000
antigenꢀpositive cells (Ca9ꢀ22) and the mixtures were either treated with the indicated conjugate
at a concentration of 0.66 nM, or not treated (control). Relative cell numbers were determined
by the WSTꢀ8 assay.
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Inhibition of AaMC induced G2/M cell cycle arrest by Bafilomycin A1. Studies were
undertaken to determine if any of the AaMCs could induce cell cycle arrest in the G2/M phase if
endosomal transport to lysosomal vesicles was blocked. Bafilomycin A1 (Baf A1) selectively
inhibits VꢀATPase, a proton pump present in endosomes and lysosomes, which leads to
neutralization of the pH in these vesicles.22 The pH neutralization blocks trafficking from late
endosomes to lysosomes and lysosomal processing, but only modestly affects the rate of
internalization and recycling and does not inhibit trafficking between endosomes and transꢀ
Golgi.23 These experiments showed that G2/M phase cell arrest occurred when EGFRꢀpositive
cells were treated with cellꢀtargeting antiꢀEGFR AaMCs (8a – 8f) (Supplemental Figure S2).
Cell arrest was blocked by Baf A1 for antiꢀEGFRꢀGlyꢀGlyꢀPAꢀMay (8f) and for AaMCs 8b-8d,
which contain at least one DꢀAlanine in the linker. However the AaMCs that contained only Lꢀ
amino acids in the linkage (8a and 8e) retained between 50 ꢀ 80 % of their ability to arrest cells
in the presence of Baf A1.
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Identification of the major metabolite of AaMCs. Two experiments were conducted to
identify which metabolite(s) could be generated by catabolism of AaMCs. First antiꢀEGFR-L-
Val-L-CitꢀPAꢀMay (8a) was incubated with cathepsin B at 37 oC, followed by HPLC/MS
analysis. One major product was formed and was shown to have the same HPLC retention time
and MS profile as the synthetic standard PAꢀMay (9), Figure 2a. In a second experiment
huC242-D-Ala-L-AlaꢀPAꢀMay (8g) was incubated with COLO 205 cells in vitro for 24 h after
which the cells were pelleted, lysed and analyzed by HPLC/MS, Figure 2b. A control of
untreated COLO 205 cells was also analyzed by HPLC/MS, Figure 2c. A major HPLC peak
(retention time 26.6 – 27 min) was seen in the test sample but not in the control sample. This
new peak had the same retention time and mass spectrum as PAꢀMay 9, indicating that 9 is a
major metabolite of 8g in COLO 205 cells. The mass spectrum for 9 is shown in Supplemental
Figure S3.
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Figure 2 HPLC traces of a) Cathepsin B treated antiꢀEGFR AaMC 8a analyzed using analytical
HPLC method 1 and b) Lysate from antiꢀCanAg AaMC 8g treated COLO 205 cells (AaMC cell
extract), analyzed by analytical HPLC method 2 c) lysed COLO 205 cells that were not exposed
to any conjugate (untreated cell extract), analyzed by analytical HPLC method 2.
Quantitation of metabolite produced by anti-EGFR AaMC 8c and anti-EGFR AMC 1a.
EGFRꢀexpressing HSC2 cells were incubated with antiꢀEGFRꢀSPDBꢀDM4 (1a) or antiꢀEGFR-
D-Ala-L-AlaꢀPAꢀMay (8c) for 2 h after which cells were washed then allowed to process
internalized ADC for 24 h. Over this time frame it is known that cells remain alive and
membranes are not ruptured to release cellular contents into the cell media. The amount of
metabolite in organic extracts of cells and in unextracted media were determined using an antiꢀ
maytansinoid binding competition ELISA, Supplemental Figure S4.24 Incubations with
conjugate 8c produced approximately 25% more total metabolite than conjugate 1a. Incubations
with 8c also released approximately twice as much metabolite into the cell media as that with 1a.
HSC2 cells however retained similar amounts of metabolite from the 8c and 1a incubations.
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In vitro cytotoxicity of the anilino-maytansinoid AaMC metabolite 9. The cytotoxicity of
synthesized PAꢀMay (9) was determined on KB cells and found to be similar to SꢀmethylꢀDM4
(3), the most cytotoxic metabolite of DM4 bearing AMCs; IC50 = 7.0 x 10ꢀ11 and 6.0 x 10ꢀ11 for 9
and 3 respectively.
9
Determination of the MTD of AaMCs in mice. Pilot studies were conducted with 3 mice per
group to determine the approximate maximum tolerated dose (MTD) of selected conjugates, with
dosing based on linked maytansinoid. The MTD was considered reached for a given group when
at least one mouse of that group lost 20% or more of its preꢀtreatment weight or upon
observations of any clinical signs of distress. Each of the conjugates utilized an antiꢀEGFR
antibody that does not cross react with murine EGFR. AaMCs antiꢀEGFR-L-Val-L-CitꢀPAꢀMay
(8a) and antiꢀEGFRꢀGlyꢀGlyꢀPAꢀMay (8f) had the lowest MTDs, both approximately 900 µg/kg
based on maytansinoid dose. AaMC antiꢀEGFR-L-Ala-L-AlaꢀPAꢀMay (8e) was not tolerated at
1,400 µg/kg but the other AaMC containing alanine residues in the linkage (8b, 8c and 8d) were
tolerated at this dose (see Supplemental Figure S5). This preliminary MTD was in line with
MTDs previously determined for AMCs that utilize disulfide linkers.12 Since antiꢀEGFR-D-Ala-
L-AlaꢀPAꢀMay (8c) was the most potent of the well tolerated AaMCs, D-Ala-L-Ala was selected
as the lead dipeptide moiety for AaMCs. A second MTD study was conducted with chKTI-D-
Ala-L-AlaꢀPAꢀMay (8h) and chKTIꢀsulfoꢀSPDBꢀDM4 (1d) using a larger number of animals (9
mice per group) for comparison, Figure 3. Mice were dosed with 8h at 1000, 1250 and 1500
µg/kg and 1d was dosed at 1500 µg/kg. Neither conjugate was tolerated at 1500 µg/kg, however
the averaged weight loss was similar for the two conjugates at this dose. The MTD of 8h was
found to be between 1000 – 1250 µg/kg.
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Figure 3 Maximum tolerated dose (MTD) study of chKTI-D-Ala-L-AlaꢀPAꢀMay 8h compared
to chKTIꢀsulfoꢀSPDBꢀDM4 1d.
In vivo efficacy of AaMCs. The AaMCs antiꢀEGFR-L-Val-L-CitꢀPAꢀMay (8a), antiꢀEGFR-D-
Ala-L-AlaꢀPAꢀMay (8c), antiꢀEGFR-L-Ala-D-AlaꢀPAꢀMay (8d), and antiꢀEGFR-L-Ala-L-Alaꢀ
PAꢀMay (8e) were tested for efficacy in mice bearing H1975 xenografts, Figure 4a. H1975
xenografts displayed high homogeneous expression of EGFR, determined by
immunohistochemical analysis, Figure 4b. AntiꢀEGFRꢀSPDBꢀDM4 (1a) was also evaluated in
this study for comparison. The tumor size ratio (T/C) indicates the median tumor size of the test
group divided by the median tumor size for the control group.25 The tumor growth delay (TꢀC)
indicates the time in days that it took for the median tumor size of a test group to reach 678 mm3
minus the time in days it took for the median tumor size of the control group to reach the same
size. 678 mm3 was the maximum median tumor size achievable for all groups in this study, mice
that did not regrow tumors were not included in the (TꢀC) calculation. Most of the conjugates
were highly active at 3 mg/kg. AntiꢀEGFR-L-Ala-D-AlaꢀPAꢀMay (8d) had a T/C of 12% and
the shortest TꢀC (33 days). All other AaMCs showed similar T/C (2ꢀ4%) and TꢀC (~55 days)
values and had longer TꢀC values than the antiꢀEGFRꢀSPDBꢀDM4 (1a) (42 days). In a separate
study antiꢀEGFRꢀGlyꢀGlyꢀPAꢀMay (8f) and antiꢀEGFR-L-Val-L-CitꢀPAꢀMay (8a) showed
similar efficacy at 3 mg/kg in mice bearing H1975 xenografts (data not shown).
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a)
Homogeneous H1975 Xenograft Model
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anti-EGFR-L-Val-L-Cit-PA-May 8a
anti-EGFR-D-Ala-L-Ala-PA-May 8c
anti-EGFR-L-Ala-D-Ala-PA-May 8d
anti-EGFR-L-Ala-L-Ala-PA-May 8e
anti-EGFR-SPDB-DM4 1c
1000
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0
20
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Days Post Treatment
b)
c)
Heterogeneous HT-29 Xenograft Model
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HT29 - Median TV
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1200
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Vehicle
chKTI-D-Ala-L-Ala-PA-May 8h 7.5 mg/kg
huC242-s-SPDB-DM4 1b 7.5 mg/kg
huC242-s-SPDB-DM4 1b 5.0 mg/kg
huC242-s-SPDB-DM4 1b 2.5 mg/kg
huC242-D-Ala-L-Ala-PA-May 8g 7.5 mg/kg
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huC242-D-Ala-L-Ala-PA-May 8g 5.0 mg/kg
huC242-D-Ala-L-Ala-PA-May 8g 2.5 mg/kg
0
10 20 30 40 50 60 70
Day (post inoculation)
Animals Dosed: Day 5 Post Inoculation
Regressions
%T/C ACTIVITY
PR
ꢀ
CR
ꢀ
ꢀ
Vehicle
ꢀ
0/6
6/6
4/6
1/6
6/6
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4/6
1/6
chKTI-D-Ala-L-Ala-PA-May 8h (7.5 mg/kg)
huC242-sulfo-SPDB-DM4 1c (7.5 mg/kg)
huC242-sulfo-SPDB-DM4 1c (5.0 mg/kg)
huC242-sulfo-SPDB-DM4 1c (2.5 mg/kg)
huC242-D-Ala-L-Ala-PA-May 8g (7.5 mg/kg)
huC242-D-Ala-L-Ala-PA-May 8g (5.0 mg/kg)
huC242-D-Ala--L-Ala-PA-May 8g (2.5 mg/kg)
100%
0%
Inactive
Highly Active
Inactive
47%
72%
0%
Inactive
Highly Active
Highly Active
Active
6%
37%
d)
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Figure 4 Efficacy of AaMCs against homogeneous and heterogeneous tumor xenografts in mice
a) SCID mice (6 mice/group) bearing homogeneous H1975 NSCLC xenografts were treated
with a single intraveneous dose of PBS or 3 mg/kg of the following(○); antiꢀEGFR-L-Val-L-Citꢀ
PAꢀMay 8a (■); antiꢀEGFR-D-Ala-L-AlaꢀPAꢀMay 8c (▲); antiꢀEGFR-L-Ala-D-AlaꢀPAꢀMay
8d (▼);antiꢀEGFR-L-Ala-L-AlaꢀPAꢀMay 8e (♦); antiꢀEGFRꢀSPDBꢀDM4 1a (ꢀ) b) Staining
for EGFR on H1975 xenograft showing 2ꢀ3 Homogeneous expression c) SCID mice (6
mice/group) bearing heterogeneous HTꢀ29 xenografts that were treated with a single
intraveneous dose of PBS (○); or 7.5 mg/kg mAb-D-Ala-L-AlaꢀPAꢀMay 8h (*); 7.5 mg/kg
huC242ꢀSPDBꢀDM4 1c dashed line (■); 5.0 mg/kg huC242ꢀSPDBꢀDM4 1c dashed line (▼); 2.5
mg/kg huC242ꢀSPDBꢀDM4 1c dashed line (+);7.5 mg/kg huC242-D-Ala-L-AlaꢀPAꢀMay 8g (♦);
5.0 mg/kg huC242-D-Ala-L-AlaꢀPAꢀMay 8g (▲);2.5 mg/kg huC242-D-Ala-L-AlaꢀPAꢀMay 8g
(ꢀ). %T/C was calculated as the median tumor volume of treated groups (T) divided by the
median tumor volume of the vehicle control group (C) and activity was assigned according to the
National Cancer Institute standards (%T/C ≤ 42% = active, %T/C ≤ 1% = highly active. PR
indicates partial regression (greater than 50% reduction of tumor xenograft size) CR indicates
complete regression (tumor xenograft no longer detected). d) Staining of CanAg on HTꢀ29
Xenograft showing 3 heterogeneous expression.
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An efficacy study in mice bearing HT29 xenografts was done to assess the activity of a huC242-
D-Ala-L-AlaꢀPAꢀMay (8g) in comparison to huC242ꢀsulfoꢀSPDBꢀDM4 (1b) (Figure 4c).
Expression of CanAg antigen in HT29 xenografts is highly heterogeneous, with staining for the
antigen seen on only a small subset of HT29 cells (Figure 4d), thus providing a model to assess
bystander potency.11 Conjugates 8g and 1b were administered i.v. with dosings of 2.5 mg/kg,
5.0 mg/kg or 7.5 mg/kg. The 1b conjugate was highly active at 7.5 mg/kg with a T/C of 0% and
6/6 partial regressions (PRs) and complete regressions (CRs), but had little activity at the 2.5
mg/kg dose (T/C 72%; 0/6 CR). The 8g conjugate was active at 2.5 mg/kg (T/C = 37% with 4/6
PR and 1/6 CR) and highly active at 5 and 7.5 mg/kg (T/C 6 and 0%, 6/6 and 6/6 PR, and 4/6
and 6/6 CR, respectively). The minimally efficacious dose of 8g in this study was 2.5 mg/kg
compared to about 5.0 mg/kg for the 1b conjugate. The nonꢀtargeting AaMC 8h was inactive at
7.5 mg/kg, indicating that the efficacy of 8g was specifically targeted.
Pharmacokinetics of anti-EGFR-D-Ala-L-Ala-PA-May 8c in mice. Six mice were
administered a single 10 mg/kg i.v. dose of antiꢀEGFR-D-Ala-L-AlaꢀPAꢀMay (8c). The
concentration of the antibody component of the conjugate and the concentration of intact
maytansinoidꢀbearing conjugate from mouse blood samples were determined by ELISA as
previously described.26 The ELISA involves the capture of conjugate bearing at least one
attached maytansinoid using an antiꢀmaytansinoid antibody then the antibody component of the
conjugate is captured and detected with an enzyme labeled antiꢀFC antibody. In order to be
detected a conjugate must contain at least one covalently linked maytansinoid. The total
antibody, antibody bearing at least one maytansinoid as well as antibody with no attached
maytansinoids, is determined by capturing it with an antiꢀhuman IgG antibody then quantitated
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using an enzyme labeled antiꢀhuman IgG antibody. Results of the study are shown in Figure 5.
The PK parameters for the antibody component of the conjugate were as follows; Cmax of 190
µg/mL, T1/2 of 12.3 days, AUC0ꢀinf 25,960 h* µg/mL, Cl of 0.39 mL/h/kg and a Vss of 153.9
mL/kg. These parameters are in similar to observed PK parameters for the naked antibody in
mice, suggesting that conjugation had little effect on the PK parameters of the antibody
component of this conjugate. The PK parameters for the intact maytansinoidꢀbearing conjugate
were as follows; Cmax 225 µg/mL, T1/2 of 5 days, AUC0ꢀinf 11,969 h* µg/mL, Cl of 0.84
mL/h/kg and a Vss of 158.0 mL/kg. These parameters are in line with observed PK parameters
for typical disulfide linked DM4 conjugates, where the T1/2 for the overall clearance of the
intact conjugates were also about 5 days.27
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Figure 5 In vivo clearance rate of antiꢀEGFR-D-Ala-L-AlaꢀPAꢀMay (8c) in mice. The ꢀ graph
indicates the concentration versus time plot of the antibody component of 8c, the ■ graph
indicates the concentration versus time plot of the maytansinoid component of 8c.
Concentrations were determined by two ELISA assays which separately quantitate antibody and
conjugate that contains at least one maytansinoid.
DISCUSSION
A new class of maytansinoidꢀbased ADC has been prepared that can release an anilineꢀbearing
maytansinoid, via cleavage of a peptide linker. The metabolite PAꢀMay 9 is highly cytotoxic to
the cell in which it is formed and because it is predominantly nonꢀcharged, it can diffuse into
neighboring cells to induce greater bystander killing than is achieved with AMCs that release
charged or reactive metabolites such as AMCs with sulfoꢀSPDB linkers (Figure 1a). The type of
diꢀpeptide used in an AaMCs linker can affect the conjugates ability to induce bystander killing
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or to be inhibited by Bafilomycin A1. For example, in cases where a D-Alanine residue is linked
directly to the aniline amine (Table 1 and Figure 1b) poorer bystander killing is noted. It is
likely that a portion of these AaMCs does not undergo cleavage of the dipeptide linker, in which
case one or more charged metabolites that do not induce bystander killing, would be released.
However, a Dꢀalanine residue can be incorporated into the dipeptide of the linker without
impairing the AaMCs cytotoxicity, against most of the tested cell lines, as long as an Lꢀalanine is
attached directly to the aniline moiety. The presence of a Dꢀamino acid in either position of an
AaMCs linker or linkers having two glycines caused cytotoxicity to be blocked in the presence
of Bafilomycin A1, indicating the need for linker cleavage in lysosomes. The cytotoxicity of
AaMCs utilizing only Lꢀamino acids in the linkage however was only partially inhibited by
Bafilomycin A, indicating that the peptide linkages could be cleaved with 50% or higher
efficiency in preꢀlysosomal compartments of the tested cells. AaMCs only require the cleavage
of a single bond in their peptide linker to release the metabolite PAꢀMay (9). In contrast, AMCs
with disulfide linkers require multiple bonds of the antibody to be cleaved in order to release an
initial metabolite, which may be less efficient. Indeed we have seen that some targeted cells can
release more total metabolite from an optimized AaMC than from an AMC bearing a disulfide
linker.
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Pharmacokinetic studies indicate that the AaMC bearing the D-Ala-L-Ala dipeptide linker (8c)
has a similar clearance rate in circulation in mice as that reported for disulfideꢀlinked DM4
conjugates.12 Both types of conjugate displayed loss of maytansinoid payload from the antibody
component, but only slowly and at approximately the same rate, for an overall conjugate T1/2 of
about 5 days. Conjugates containing payloads linked to a maleimide via a thioꢀether are known
to slowly release the payload in the presence of thiols.28, 29 The half life of a maytansinoid
conjugate with a maleimide thioꢀether linkage is only about 30 h longer than that of SPDB linked
DM4 conjugates, while AMCs that utilize highly hindered disulfide linkages that are very stable
have half lives of over 9 days.12 So it a likely that a large portion of the maytansinoid released
from 8c in vivo was due to thioꢀether cleavage but some maytansinoid loss was propably also due
to cleavage of the peptide linker. AntiꢀEGFRꢀ D-Ala-L-AlaꢀPAꢀMay (8c) was better tolerated in
mice than antiꢀEGFRꢀL-Ala-L-AlaꢀPAꢀMay (8e). This may be due to differences in the stability
of the dipeptide linkages of these AaMCs during circulation in mice and will require further in
vivo PK studies.
The antiꢀEGFR AaMCs were efficacious against homogeneously expressing H1975 xenografts
and most were slightly more efficacious than antiꢀEGFRꢀSPDBꢀDM4 (1a). AntiꢀEGFRꢀLꢀAlaꢀ
DꢀAlaꢀPAꢀMay (8d) was the least efficacious of the tested AaMCs, and this AaMC showed
lower cytotoxicity and lower bystander killing than AaMCs bearing an Lꢀamino acid attached to
the aniline. As previously stated this could be due to less efficient cleavage of the peptide linker
of the conjugate resulting in the release of less total metabolite. Also partial cleavage of the
peptide linker of 8d would give a positively charged metabolite that still contained the Dꢀalanine
residue of the linker, which would presumably have poor bystander killing. If a portion of the
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conjugate did not undergo cleavage of the peptide linker, then metabolites bearing a lysine
residue or a larger peptide fragment derived from the antibody would be released. These lysine
or peptide bearing metabolites would not be expected to induce bystander killing and their ability
to diffuse from the catabolic vesicle into the cytoplasm of the cell might be impaired. When the
antigen is expressed in a heterogeneous manner, such as in HTꢀ29 xenografts, the AaMC,
huC242ꢀDꢀAlaꢀLꢀAlaꢀPAꢀMay (8g) was substantially more efficacious than the corresponding
AMC bearing a disulfide linker (huC242ꢀsulfoꢀSPDBꢀDM4 1b). In vivo efficacy studies using
xenografts composed of a mixture of antigenꢀpositive and antigenꢀnegative cells in various ratios
have been reported.30 Similar mixed xenograft efficacy studies would be of value in future work
to more exactly determine the percent of antigen positive cells required for in vivo efficacy
against heterogenous xenogtrafts.
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Efficacy studies on heterogeneous xenografts may better mimic the treatment of tumors in
patients for several reasons. Antigen expression in human tumors often varies and may not
always be as homogeneous as EGFR expression in H1975 xenografts. Reports also indicate that
mouse stroma secretes mouse cytokines and growth factors that in many situations do not aid the
survival and promotion of metastasis of human cancer cells as effectively as those secreted from
human stroma.31ꢀ33 Many types of human cancer cells however secrete growth factors and
cytokines. 34, 35 So nonꢀtargeted antigenꢀnegative cancer cells in a heterogeneous xenograft might
take on some of the role human tumor stroma plays in aiding cancer cells to survive. AaMCs are
a promising new class of maytansinoid conjugate, which may be well suited for killing a
patient’s cancerous cells as well as the tumor stromal cells that support cancer cell survival. The
studies described herein should also be of use in the design of ADCs that utilize peptide linkages
to nonꢀmaytansinoid payloads.
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EXPERIMENTAL
All reagents were obtained from Chem Impex, Bachem, or SigmaꢀAldrich unless otherwise
stated. All synthetic reactions were conducted under an argon atmosphere with magnetic stirring
unless otherwise stated. Proton magnetic resonance (1H NMR) and carbon magnetic resonance
spectra (13C NMR) were obtained on a Bruker Avance 400 spectrometer operating at 400 MHz
and 100 MHz respectively. In cases where 13C NMRs were taken using DMSOꢀd6 as the
solvent, a small amount of CDCl3 was also added. The NMR chemical shifts are reported in δ
values relative to the utilized NMR solvent. Centrifugations whether under vacuum or without
vacuum were performed on a Thermo electron SPD SpeedVac equipped with a UVS800DDA
universal vacuum system. Lyophilizations were performed using a VerTis Benchtop K. Flash
chromatography was performed on an Agilent Intelliflash system. HPLC purifications were
performed using Gilson 334 pumps in series with a Gilson UV/Visꢀ156 dual wavelength detector
and a Gilson FC204 fraction collector. High resolution mass spectra were obtained on a Thermo
Fisher QꢀExactive instrument. Analytical HPLC/MS data was obtained on an Agilent 1100
HPLC in series with a Bruker esquire 3000 ion trap MS operating in alternating positive/negative
ion mode or on an Agilent 1260 HPLC in tandem with an Agilent 6120 single quadrupole mass
spectrometer. Fmoc-L-Val-L-CitꢀOH and Fmoc-L-Val-L-CitꢀPAꢀOH were prepared as described
by Dubowchik et. al.18 Conjugates containing DM4 linked to an antibody via the SPDB or sulfoꢀ
SPDB linkers were prepared as described previously with maytansinoid/antibody ratios (MAR)
as follows, 1a MAR = 4.0, 1b MAR = 4.1, 1c MAR = 3.9, 1d MAR = 3.8, 1e MAR = 3.9 each of
the conjugates contained less than 2% aggregate and endotoxin units (EU) were below 0.2
EU/mg.21 Enzymeꢀlinked immunosorbent assay (ELISA) methods for the determination of
relative antibody and maytansinoid amounts in a given conjugate were performed as previously
described.26 Samples for HPLC/MS analysis of AaMC metabolites formed by incubating 8g
with COLO 205 cells were performed as described by Erickson et. al.1
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Preparative C18 HPLC method 1. Column 250 x 29 cm C18 packed in a load and lock system
(available from Agilent); Flow rate 40 mL/min; Eluting with deionized water containing 0.2%
formic acid with 5% acetonitrile for 5 min then a linear gradient from 5% ꢀ 95% acetonitrile for
the next 30 min and maintaining 95% acetonitrile for 5 min with 220 nm and 252 nm detection.
If required, particulates were removed from samples by centrifugation prior to purification.
HPLC analytical method 1. A HISEP 25 cm x 4.6 mm, 5 µm, Supelco column was eluted at
room temperature, flow rate 0.7 mL/min with 100 mM ammonium acetate pH 7.0 buffer and a
gradient of acetonitrile as follows: linear 25 – 40% 0 – 25 min; linear 40 – 100% 25 – 27 min.
After which the column was maintained at 100% acetonitrile for 5 min then reequilibrated in
starting conditions.
HPLC analytical method 2. A C8 150 x 2.1 mm 100, 5 µm, Kromasil column was eluted at
room temperature, flow rate of 0.2 mL/min with deionized water containing 0.1% formic acid
and with a gradient of acetonitrile as follows: linear 15 ꢀ 32% 0ꢀ15 min; linear 32 ꢀ 48% 15 – 25
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min; linear 48 ꢀ58% 25 – 35 min; linear 58ꢀ90% 35ꢀ45 min; linear 90ꢀ15% 45 – 50 min with 9
min reꢀequilibration between runs. Detection by uv/vis 220 – 350 nm and by mass spectroscopy
in alternating positive and negative ion modes.
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Preparation of AaMC precursors and the AaMC metabolite PA-May 9.
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4-Nitro-PA-May (20): To a 57 mM solution of DM1 (211 mg, 0.286 mmol) in DMSO was
added 1ꢀ(bromomethyl)ꢀ4ꢀnitrobenzene (74.1 mg, 0.343 mmol), then DBU (47.9 mg, 0.314
mmol) at room temperature. After 16 h the mixture was purified on an intelliflash system with a
100 g C18 cartridge, using deionized water with 0.1% formic acid and gradient of acetonitrile 30
% ꢀ 90% over 30 min, at a flow rate of 60 mL/min. The product fractions were combined, frozen
and lyophilized to give 109 mg, (47% yield) as an offꢀwhite solid. 1H NMR (400 MHz,
Chloroformꢀd) δ 0.77 (s, 3H), 1.15 – 1.26 (m, 2H), 1.26 – 1.32 (m, 3H), 1.45 (td, J = 10.1, 5.4
Hz, 2H), 1.56 (s, 1H), 1.63 (s, 3H), 2.16 (dd, J = 14.5, 3.1 Hz, 2H), 2.43 – 2.78 (m, 6H), 2.81 (s,
3H), 2.96 – 3.04 (m, 2H), 3.16 (s, 3H), 3.35 (s, 3H), 3.44 – 3.54 (m, 2H), 3.74 (d, J = 3.1 Hz,
2H), 4.00 (s, 3H), 4.21 – 4.31 (m, 1H), 4.76 (dd, J = 12.0, 3.1 Hz, 1H), 5.34 (q, J = 6.8 Hz, 1H),
5.62 (dd, J = 15.3, 9.0 Hz, 1H), 6.20 (s, 1H), 6.35 – 6.46 (m, 1H), 6.55 (d, J = 1.7 Hz, 1H), 6.65
(d, J = 11.1 Hz, 1H), 6.78 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 8.08 (d, J = 8.7 Hz, 2H).
HRMS (M + H)+ calcd. 873.3142, found 873.3124.
PA-May (9): To a 0.05 M solution of 4ꢀNitroꢀPAꢀMay (109 mg, 0.125 mmol) in 3:1
tetrahydrofuran:methanol (40 mL) was added a 5.5 M aqueous solution of ammonium chloride
(66.8 mg, 1.248 mmol) and powdered iron (34.8 mg, 0.624 mmol) at 50 ºC. After 16 h the
reaction was cooled to room temperature, diluted with 10:1 tetrahydrofuran/methanol
(~equivalent volume) and vacuum filtered through Celite filter aid. The greenishꢀyellow filtrate
was rotary evaporated under vacuum. The yellow solid was redissolved in 4:1 acetonitrile:water
(20 mL) and purified on an intelliflash system with a 100 g flash C18 cartridge, using deionized
water with 0.1% formic acid and gradient of acetonitrile 20% ꢀ 95% over 30 min, at a flow rate
of 60 mL/min. The product fractions were frozen and lyophilized to give 32.5 mg (27% yield)
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of desired product as a white solid. H NMR (400 MHz, chloroformꢀd) δ 0.78 (s, 3H), 1.17 –
1.26 (m, 2H), 1.29 (d, J = 6.6 Hz, 3H), 1.40 – 1.51 (m, 2H), 1.52 – 1.60 (m, 1H), 1.63 (s, 6H),
2.16 (dd, J = 14.4, 3.1 Hz, 2H), 2.32 – 2.45 (m, 1H), 2.49ꢀ2.68 (m, 4H), 2.76 (s, 3H), 3.01 (d, J =
9.6 Hz, 1H), 3.08 (d, J = 12.6 Hz, 1H), 3.14 (s, 3H), 3.35 (s, 3H), 3.49 (d, J = 9.0 Hz, 1H), 3.57
(s, 2H), 3.62 (d, J = 12.7 Hz, 1H), 3.99 (s, 3H), 4.20 – 4.32 (m, 1H), 4.76 (dd, J = 12.0, 3.1 Hz,
1H), 5.35 (q, J = 6.8 Hz, 1H), 5.64 (dd, J = 15.3, 9.0 Hz, 1H), 6.24 (s, 1H), 6.42 (dd, J = 15.4,
11.1 Hz, 1H), 6.54 – 6.63 (m, 3H), 6.70 (d, J = 11.1 Hz, 1H), 6.82 (d, J = 1.8 Hz, 1H), 6.99 (d, J
= 8.2 Hz, 2H). HRMS (M + H)+ calcd. 843.3400, found 843.3388.
General Procedure for the preparation of Fmoc protected dipeptides Fmoc-D-Ala-D-Ala-
OH (12b) and Fmoc-D-Ala-L-Ala-OH (12c ). Fmoc-D-AlaꢀOH (3.0 g, 9.6 mmol), N-
hydroxysuccinimide (6.4 g, 12.1 mmol) and EDC (1.8 g, 9.6 mmol) were taken up in
dichloromethane (50 mL) at room temperature. After 2 h the solution was washed with 0.25 M
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HCl (2 x 50 mL) then with saturated aqueous sodium chloride (40 mL). The organic layer was
dried over anhydrous sodium sulfate then vacuum filtered and solvent was remove from the
filtrate under vacuum. Residue was taken up in 1,2ꢀdimethoxyethane (45 mL) to which was
added a solution of Lꢀalanine or Dꢀalanine (2.3 g, 25 mmol) in sodium bicarbonate (1.94 g, 23
mmol) dissolved in deionized water (45 mL) and dimethoxyethane (10 mL) followed after 2 min
by addition of dimethoxyethane (35 mL). After 2 h the mixture was vacuum filtered and the
filtrate was purified on a 450 g C18 cartridge eluting at 80 mL/min with deionized water
containing 0.1% formic acid and a gradient of acetonitrile (5 to 95% over 35 min). Fractions
containing desired compound were pooled, frozen and lyophilized.
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Fmoc-D-Ala-D-Ala-OH (12b). White solid (41% yield). 1H NMR (400 MHz, DMSOꢀd6) δ
12.52 (s, 1H), 8.12 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 7.2 Hz, 2H), 7.49 (d,
J = 7.8 Hz, 1H), 7.42 (td, J = 7.5,1.1 Hz, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 4.30 – 4.16 (m, 4H),
4.09 (q, J = 7.3 Hz, 1H), 1.27 (d, J = 7.3 Hz, 3H), 1.22 (d, J = 7.1 Hz, 3H). HRMS (M + Na)+
calcd. 405.1427, found 405.1417.
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Fmoc-D-Ala-L-Ala-OH (12c ). White solid (38 % yield). H NMR (400 MHz, DMSOꢀd6) δ
1.23 (d, J = 7.3 Hz, 3H), 1.26 (d, J = 7.2 Hz, 3H), 4.05 – 4.16 (m, 1H), 4.24 (dq, J = 13.2, 7.2
Hz, 4H), 7.33 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.73 (t, J =
7.2 Hz, 2H), 7.88 (d, J = 7.5 Hz, 2H), 8.10 (d, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO) δ
17.47, 18.54, 46.64, 47.47, 49.86, 65.64, 120.05, 125.27, 125.32, 127.05, 127.60, 140.68, 143.78,
143.89, 155.56, 172.13, 173.93. HRMS (M + Na)+ calcd. 405.1427, found 405.1411.
Fmoc-L-Ala-D-Ala-OH (12d). Fmoc-L-AlaꢀONHS (3.0 g, 7.35 mmol) was taken up in 1,2ꢀ
dimethoxyethane (45 mL) to which was added a solution of H-D-AlaꢀOH (2.5 g, 26 mmol) in
sodium bicarbonate (2.0 g, 23.2 mmol) dissolved in deionized water (47 mL) and
dimethoxyethane (11 mL) followed after 2 min by adding dimethoxyethane (37 mL). After 2 h
the mixture was vacuum filtered and the filtrate was purified on a 450 g C18 cartridge eluting at
80 mL/min with deionized water and an acetonitrile gradient of acetonitrile (5 to 95% over 35
min). Fractions containing desired compound were pooled, frozen and lyophilized to give 1.3 g,
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(46 % yield) of product as a white solid. H NMR (400 MHz, DMSOꢀd6) δ 1.24 (d, J = 7.2 Hz,
3H), 1.27 (d, J = 7.3 Hz, 3H), 4.14 (t, J = 7.5 Hz, 1H), 4.19 – 4.35 (m, 3H), 7.32 (t, J = 7.5 Hz,
2H), 7.41 (t, J = 7.5 Hz, 2H), 7.48 (d, J = 8.1 Hz, 1H), 7.74 (t, J = 7.5 Hz, 2H), 7.87 (d, J = 7.5
Hz, 2H), 8.13 (t, J = 8.2 Hz, 1H), 11.97 – 13.16 (m, 1H). 13C NMR (101 MHz, DMSO) δ
174.02, 172.25, 155.64, 143.95, 143.83, 140.75, 127.67, 127.11, 125.39, 125.34, 120.12, 65.71,
49.93, 47.53, 46.70, 18.62, 17.51. HRMS (M + Na)+ calcd. 405.1427, found 405.1409.
General Procedure for the preparation of 13b – 13f. To a 20 mL capacity flask was added 4ꢀ
aminoꢀbenzylalcohol (0.25 g, 3.0 mmol), one of the Fmoc protected dipeptides (12b-12d) or
FmocꢀGlyꢀGlyꢀOH (0.65 mmol), and EEDQ (0.32 g, 1.3 mmol) followed by 2:1
dichloromethane:methanol (10 mL). After 16 h, solvent was evaporated under vacuum and
residue was vigorously triturated with diethyl ether (40 mL). The suspension was vacuum
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filtered while continuously scrapping the filter paper. The filter cake was washed with diethyl
ether (10 mL) and dried under vacuum.
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Fmoc-D-Ala-D-Ala-PAB-OH (13b). White solid (56 % yield). H NMR (400 MHz, DMSOꢀ
d6) δ 1.24 (d, J = 7.0 Hz, 3H), 1.31 (d, J = 7.0 Hz, 3H), 4.11 (q, J = 7.1 Hz, 1H), 4.19 – 4.33 (m,
3H), 4.36 – 4.51 (m, 4H), 5.10 (t, J = 5.6 Hz, 1H), 7.23 (d, J = 8.1 Hz, 2H), 7.33 (t, J = 7.4 Hz,
2H), 7.42 (t, J = 7.4 Hz, 2H), 7.55 (dd, J = 7.8, 4.9 Hz, 3H), 7.72 (t, J = 8.1 Hz, 2H), 7.89 (d, J =
7.5 Hz, 2H), 8.10 (d, J = 7.2 Hz, 1H), 9.88 (s, 1H). HRMS (M + Na)+ calcd. 510.2005, found
510.1985.
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Fmoc-D-Ala-L-Ala-PAB-OH (13c). White solid (64 % yield). 1H NMR (400 MHz, DMSOꢀd6)
δ 1.27 (d, J = 7.1 Hz, 3H), 1.34 (d, J = 7.0 Hz, 3H), 4.14 (t, J = 7.0 Hz, 1H), 4.22 (t, J = 6.9 Hz,
1H), 4.31 (d, J = 6.2 Hz, 2H), 4.46 (q, J = 4.4, 3.2 Hz, 3H), 5.14 (d, J = 5.4 Hz, 1H), 7.25 (d, J =
8.3 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.40 (td, J = 7.4, 4.6 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.64
– 7.77 (m, 3H), 7.87 (dd, J = 7.5, 2.8 Hz, 2H), 8.33 (d, J = 7.5 Hz, 1H), 9.75 (s, 1H). 13C NMR
(101 MHz, DMSO) δ 172.53, 70.86, 156.00, 143.87, 143.74, 140.74, 137.64, 137.40, 127.65,
127.09, 126.89, 125.29, 125.24, 120.10, 119.12, 65.77, 62.65, 50.25, 48.95, 46.68, 18.17, 17.93.
HRMS (M + Na)+ calcd. 510.2005, found 510.1987.
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Fmoc-L-Ala-D-Ala-PAB-OH (13d). White solid (48 % yield). H NMR (400 MHz, DMSOꢀd6)
δ 1.26 (d, J = 7.0 Hz, 3H), 1.32 (d, J = 7.0 Hz, 3H), 4.13 (p, J = 7.3 Hz, 1H), 4.22 (q, J = 6.7, 4.7
Hz, 1H), 4.30 (d, J = 6.2 Hz, 2H), 4.45 (d, J = 5.0 Hz, 3H), 5.13 (t, J = 5.7 Hz, 1H), 7.24 (d, J =
8.2 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.40 (td, J = 7.5, 4.9 Hz, 2H), 7.60 (d, J = 7.9 Hz, 2H), 7.63
– 7.75 (m, 3H), 7.87 (dd, J = 7.5, 3.0 Hz, 2H), 8.31 (d, J = 7.6 Hz, 1H), 9.73 (s, 1H). HRMS (M
+ Na)+ calcd. 510.2005, found 510.1986.
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Fmoc-L-Ala-L-Ala-PAB-OH (13e). White solid (75 % yield). H NMR (400 MHz, DMSOꢀd6)
δ 1.24 (d, J = 7.1 Hz, 3H), 1.31 (d, J = 7.0 Hz, 3H), 4.11 (q, J = 7.1 Hz, 1H), 4.17 – 4.32 (m,
3H), 4.42 (dd, J = 11.9, 6.3 Hz, 3H), 5.10 (t, J = 5.7 Hz, 1H), 7.24 (d, J = 8.3 Hz, 2H), 7.33 (t, J
= 7.4 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.55 (dd, J = 7.7, 4.2 Hz, 3H), 7.72 (t, J = 8.2 Hz, 2H),
7.89 (d, J = 7.5 Hz, 2H), 8.11 (d, J = 7.3 Hz, 1H), 9.88 (s, 1H). 13C NMR (101 MHz, DMSO) δ
171.02, 170.57, 170.53, 168.09, 155.24, 151.21, 143.85, 143.75, 141.25, 141.23, 140.68, 139.39,
138.30, 137.59, 133.23, 132.56, 128.93, 128.89, 128.45, 127.60, 127.25, 127.05, 125.25, 125.13,
121.35, 120.07, 119.99, 119.02, 117.11, 113.90, 109.69, 88.19, 79.97, 78.62, 77.62, 73.14, 66.74,
59.99, 56.53, 56.09, 51.64, 49.99, 49.96, 48.96, 46.64, 45.40, 37.68, 36.33, 35.16, 34.86, 33.32,
31.91, 29.68, 25.97, 18.14, 15.01, 14.39, 13.04, 11.35. HRMS (M + Na)+ calcd. 510.2005, found
510.1989.
Fmoc-Gly-Gly-PAB-OH (13f). White solid (56 % yield). 1H NMR (400 MHz, DMSOꢀd6) δ
3.71 (d, J = 6.0 Hz, 2H), 3.92 (d, J = 5.8 Hz, 2H), 4.24 (t, J = 7.0 Hz, 1H), 4.32 (d, J = 7.1 Hz,
2H), 4.44 (s, 2H), 5.11 (s, 1H), 7.25 (d, J = 8.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.5
Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.66 (t, J = 6.1 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.88 (d, J =
7.5 Hz, 2H), 8.25 (q, J = 8.5, 5.9 Hz, 1H), 9.82 (s, 1H). 13C NMR (101 MHz, DMSO) δ 40.88,
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41.80, 44.86, 60.83, 64.01, 117.14, 118.28, 123.43, 125.14, 125.27, 125.82, 135.56, 135.68,
138.92, 142.02, 154.83, 165.62, 167.77. HRMS (M + Na)+ calcd. 510.2005, found 510.1992.
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General Procedure for the preparation of compounds (14a – 14f). To a solution of one of the
compounds 13a – 13f (0.24 mmol) in anhydrous DMF (700 ꢁL) dichloromethane was added
DIPEA (56 ꢁL, 0.32 mmol) and methanesulfonyl chloride (20 ꢁL, 0.26 mmol). After 1 h sodium
bromide (0.82 mg, 0.80 mmol) was added then after 90 min 1:1 dichloromethane:DMF (2 mL)
was added and the mixture was centrifuged in a speedvac for 5 min without vacuum. The
supernatent was transferred to a 10 mL flask to which was added DM1 (118 mg, 0.16 mmol).
After 45 min the sample was purified by preparative HPLC method 1. Fractions containing pure
desired product were combined in a 200 mL flask, frozen in a dry ice acetone bath then
lyophilized.
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Fmoc-L-Val-L-Cit-PA-May (14a). White solid (32 % yield). 1H NMR (400 MHz, CDCl3) 0.81
(s, 3H), 0.98 – 0.86 (m, 6H), 1.19 (d, J = 6.1 Hz, 3H), 1.23 (d, J = 6.6 Hz, 3H), 1.56 – 1.29 (m,
5H), 1.63 (s, 3H), 1.84 – 1.72 (m, 1H), 2.12 – 1.98 (m, 2H), 2.44 (dd, J = 14.9, 7.4 Hz, 1H), 2.57
– 2.50 (m, 3H), 2.59 (s, 2H), 2.70 – 2.63 (m, 1H), 2.73 (s, 3H), 2.85 (d, J = 9.5 Hz, 1H), 3.07 –
2.95 (m, 1H), 3.17ꢀ3.08(m, 5H), 3.31 (s, 3H), 3.54 – 3.34 (m, 4H), 3.67 – 3.55 (m, 2H), 3.97 (s,
3H), 4.01 (d, J = 6.7 Hz, 1H), 4.14 (t, J = 11.4 Hz, 1H), 4.32 – 4.21 (m, 2H), 4.41 – 4.32 (m,
1H), 4.63 – 4.45 (m, 2H), 5.46 – 5.33 (m, 2H), 5.63 (dd, J = 14.9, 9.1 Hz, 1H), 6.09 – 5.90 (m,
1H), 6.68 – 6.47 (m, 3H), 6.80 (s, 1H), 7.14 – 7.07 (m, 2H), 7.38ꢀ7.30 (m, 3H), 7.46 – 7.38 (m,
2H), 7.53 (d, J = 8.1 Hz, 2H), 7.74 (t, J = 7.4 Hz, 2H), 7.86 (d, J = 7.4 Hz, 2H), 8.10 (d, J = 7.4
Hz, 1H), δ 10.00 (s, 1H). HRMS (M + Na)+ calcd. 1343.5441; Found 1343.5378
Fmoc-D-Ala-D-Ala-PA-May (14b). White solid (41 % yield). 1H NMR (400 MHz, DMSOꢀd6)
δ 0.76 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.21 – 1.26 (m, 6H), 1.31 (dd, J
= 7.3, 3.0 Hz, 5H), 1.44 (dd, J = 15.3, 9.9 Hz, 2H), 1.57 (s, 3H), 2.03 (dd, J = 14.7, 2.4 Hz, 1H),
2.37 (ddd, J = 16.0, 7.6, 3.5 Hz, 1H), 2.67 (s, 3H), 2.78 (d, J = 9.7 Hz, 1H), 3.07 (s, 3H), 3.13 (d,
J = 12.5 Hz, 1H), 3.24 (s, 3H), 3.37 (d, J = 12.4 Hz, 1H), 3.47 (d, J = 9.0 Hz, 1H), 3.55 – 3.66
(m, 2H), 3.93 (s, 3H), 4.02 – 4.14 (m, 3H), 4.19 – 4.30 (m, 4H), 4.42 (d, J = 9.4 Hz, 3H), 4.52
(dd, J = 11.9, 2.8 Hz, 1H), 5.30 (q, J = 6.7 Hz, 1H), 5.56 (dd, J = 13.7, 9.0 Hz, 1H), 6.49 – 6.60
(m, 3H), 6.87 (s, 1H), 7.09 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H),
7.33 (t, J = 7.5 Hz, 3H), 7.41 (t, J = 7.6 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.54 (dd, J = 8.0, 2.9
Hz, 3H), 7.72 (t, J = 8.1 Hz, 2H), 7.89 (d, J = 7.6 Hz, 2H), 8.10 (t, J = 6.4 Hz, 1H), 9.89 (d, J =
11.6 Hz, 1H). HRMS (M + Na)+ calcd 1229.4649, 1229.4584.
Fmoc-D-Ala-L-Ala-PA-May (14c). White solid (35 % yield). 1H NMR (400 MHz, DMSOꢀd6)
δ 0.72 (d, J = 6.5 Hz, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H), 1.24 (dd, J = 7.0,
3.7 Hz, 3H), 1.29 – 1.33 (m, 3H), 1.38 – 1.46 (m, 2H), 1.56 (d, J = 2.5 Hz, 3H), 2.02 (d, J = 14.5
Hz, 1H), 2.32 – 2.41 (m, 1H), 2.59 – 2.64 (m, 1H), 2.67 (s, 3H), 2.78 (d, J = 9.6 Hz, 1H), 3.06 (d,
J = 1.4 Hz, 2H), 3.11 (s, 1H), 3.19 (s, 1H), 3.24 (s, 1H), 3.32 (s, 5H), 3.43 (dd, J = 12.3, 9.1 Hz,
1H), 3.55 – 3.64 (m, 2H), 3.93 (s, 3H), 4.02 – 4.14 (m, 2H), 4.21 (t, J = 6.2 Hz, 1H), 4.24 – 4.29
(m, 2H), 4.42 (t, J = 6.8 Hz, 1H), 4.51 (dd, J = 11.9, 2.5 Hz, 1H), 5.29 (q, J = 6.7 Hz, 1H), 5.54
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(ddd, J = 13.7, 8.9, 3.9 Hz, 1H), 5.91 (s, 1H), 6.45 – 6.52 (m, 2H), 6.56 (d, J = 11.1 Hz, 1H),
6.86 (d, J = 10.8 Hz, 1H), 7.07 (dd, J = 8.3, 2.6 Hz, 2H), 7.14 (dd, J = 8.1, 1.7 Hz, 1H), 7.32 (td,
J = 7.5, 5.7, 3.9 Hz, 3H), 7.39 (q, J = 7.1 Hz, 3H), 7.49 (dd, J = 8.3, 5.3 Hz, 2H), 7.62 (d, J = 6.9
Hz, 1H), 7.70 (td, J = 12.1, 10.2, 5.1 Hz, 2H), 7.87 (dd, J = 7.5, 3.5 Hz, 2H), 8.27 – 8.32 (m,
1H), 9.75 (d, J = 11.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 172.48, 172.43, 171.09, 170.89,
170.53, 168.08, 155.87, 155.22, 151.21, 143.84, 143.79, 143.71, 143.64, 141.23, 140.67, 138.34,
137.42, 133.35, 132.54, 129.40, 128.89, 128.85, 128.42, 127.61, 127.05, 125.26, 125.20, 121.53,
120.08, 119.15, 117.08, 113.91, 88.16, 82.95, 79.95, 77.60, 73.13, 66.72, 65.69, 59.98, 56.53,
56.08, 51.62, 50.10, 48.89, 46.60, 45.38, 37.67, 36.33, 35.15, 34.84, 33.27, 31.89, 29.66, 25.95,
18.12, 18.02, 14.99, 14.39, 13.04, 11.32. HRMS (M + Na)+ 1229.4649, found 1229.4615.
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Fmoc-L-Ala-D-Ala-PA-May (14d). White solid (33 % yield). 1H NMR (400 MHz, DMSOꢀd6)
δ 0.76 (s, 3H), 1.12 (d, J = 6.5 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 7.2 Hz, 3H), 1.31
(dd, J = 6.8, 2.4 Hz, 3H), 1.40 – 1.49 (m, 2H), 1.58 (s, 3H), 2.03 (dd, J = 14.5, 2.8 Hz, 1H), 2.37
(ddd, J = 16.3, 8.2, 5.8 Hz, 1H), 2.59 – 2.65 (m, 1H), 2.67 (s, 3H), 2.69 ꢀ 2.76 (m, 1H), 2.78 (d, J
= 9.7 Hz, 1H), 3.07 (s, 3H), 3.13 (d, J = 12.7 Hz, 1H), 3.24 (d, J = 3.1 Hz, 3H), 3.47 (dd, J = 9.0,
3.1 Hz, 1H), 3.55 – 3.65 (m, 2H), 3.93 (d, J = 2.4 Hz, 3H), 4.03 – 4.12 (m, 2H), 4.22 (d, J = 6.8
Hz, 1H), 4.27 (d, J = 8.1 Hz, 1H), 4.41 (q, J = 7.1, 6.6 Hz, 1H), 4.52 (dd, J = 11.9, 2.8 Hz, 1H),
5.30 (q, J = 6.8 Hz, 1H), 5.55 (dd, J = 13.6, 9.1 Hz, 1H), 5.93 (s, 1H), 6.49 – 6.59 (m, 3H), 6.87
(s, 1H), 7.07 – 7.12 (m, 2H), 7.15 (d, J = 1.8 Hz, 1H), 7.30 – 7.37 (m, 2H), 7.41 (t, J = 7.5 Hz,
2H), 7.47 (dd, J = 8.5, 2.7 Hz, 2H), 7.55 (d, J = 7.5 Hz, 1H), 7.72 (t, J = 8.1 Hz, 2H), 7.84 (d, J =
7.4 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 8.14 (d, J = 7.2 Hz, 1H), 9.94 (s, 1H). 13C NMR (101
MHz, DMSO) δ 172.47, 170.89, 170.52, 168.06, 155.22, 151.21, 143.82, 143.63, 141.25, 141.20,
140.66, 138.38, 133.29, 132.51, 128.86, 127.59, 127.03, 125.26, 125.19, 121.50, 120.04, 119.15,
117.08, 113.86, 88.17, 79.94, 77.61, 77.57, 73.12, 66.71, 65.72, 59.95, 56.51, 56.00, 51.60,
50.12, 48.98, 46.60, 37.67, 36.32, 35.13, 34.85, 33.26, 31.87, 29.64, 25.90, 18.07, 17.99, 14.98,
14.39, 13.03, 11.33. HRMS (M + Na)+ 1229.4649, found 1229.4622.
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Fmoc-L-Ala-L-Ala-PA-May (14e). White solid (43% yield). H NMR (400 MHz, DMSOꢀd6) δ
0.76 (s, 3H), 1.12 (d, J = 6.5 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 7.2 Hz, 3H), 1.31
(dd, J = 6.8, 2.4 Hz, 3H), 1.40 – 1.49 (m, 2H), 1.58 (s, 3H), 2.03 (dd, J = 14.5, 2.8 Hz, 1H), 2.37
(ddd, J = 16.3, 8.2, 5.8 Hz, 1H), 2.59 – 2.65 (m, 1H), 2.67 (s, 3H), 2.69 ꢀ 2.76 (m, 1H), 2.78 (d, J
= 9.7 Hz, 1H), 3.07 (s, 3H), 3.13 (d, J = 12.7 Hz, 1H), 3.24 (d, J = 3.1 Hz, 3H), 3.47 (dd, J = 9.0,
3.1 Hz, 1H), 3.55 – 3.65 (m, 2H), 3.93 (d, J = 2.4 Hz, 3H), 4.03 – 4.12 (m, 2H), 4.22 (d, J = 6.8
Hz, 1H), 4.27 (d, J = 8.1 Hz, 1H), 4.41 (q, J = 7.1, 6.6 Hz, 1H), 4.52 (dd, J = 11.9, 2.8 Hz, 1H),
5.30 (q, J = 6.8 Hz, 1H), 5.55 (dd, J = 13.6, 9.1 Hz, 1H), 5.93 (s, 1H), 6.49 – 6.59 (m, 3H), 6.87
(s, 1H), 7.07 – 7.12 (m, 2H), 7.15 (d, J = 1.8 Hz, 1H), 7.30 – 7.37 (m, 2H), 7.41 (t, J = 7.5 Hz,
2H), 7.47 (dd, J = 8.5, 2.7 Hz, 2H), 7.55 (d, J = 7.5 Hz, 1H), 7.72 (t, J = 8.1 Hz, 2H), 7.84 (d, J =
7.4 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 8.14 (d, J = 7.2 Hz, 1H), 9.94 (s, 1H). HRMS (M + Na)+
1229.4649, found 1229.4614.
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Fmoc-Gly-Gly-PA-May (14f). White solid (38 % yield). 1H NMR (400 MHz, DMSOꢀd6) δ
0.74 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.23 (d, J = 13.0 Hz, 2H), 1.43
(dd, J = 14.3, 7.7 Hz, 2H), 1.58 (s, 3H), 2.06 (dd, J = 20.2, 8.5 Hz, 1H), 2.30 – 2.45 (m, 1H),
2.69 (d, J = 7.9 Hz, 5H), 2.79 (d, J = 9.7 Hz, 2H), 3.00 – 3.18 (m, 4H), 3.25 (d, J = 10.5 Hz, 4H),
3.61 (q, J = 13.5 Hz, 2H), 3.70 (d, J = 5.8 Hz, 2H), 3.83 – 4.04 (m, 5H), 4.07 (t, J = 11.4 Hz,
1H), 4.25 (d, J = 6.5 Hz, 1H), 4.30 (d, J = 7.0 Hz, 2H), 4.40 – 4.62 (m, 1H), 5.30 (q, J = 6.8 Hz,
1H), 5.55 (dd, J = 14.2, 9.2 Hz, 1H), 5.93 (s, 1H), 6.42 – 6.68 (m, 3H), 6.82 – 6.95 (m, 1H), 7.04
– 7.20 (m, 3H), 7.32 (dd, J = 14.9, 7.5 Hz, 2H), 7.38 – 7.52 (m, 4H), 7.65 (t, J = 5.8 Hz, 1H),
7.72 (d, J = 7.4 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.15 – 8.37 (m, 1H). 13C NMR (101 MHz,
DMSO) δ 11.34, 13.05, 14.40, 15.01, 25.98, 29.68, 31.91, 33.31, 34.85, 35.16, 36.34, 37.68,
42.67, 43.54, 45.39, 46.63, 51.63, 56.08, 56.54, 59.99, 65.80, 66.74, 73.15, 77.61, 79.97, 88.18,
113.91, 117.10, 119.04, 120.09, 121.56, 125.09, 125.23, 127.07, 127.62, 128.42, 128.94, 132.58,
133.27, 137.41, 138.39, 140.70, 141.25, 143.81, 151.23, 155.24, 156.61, 167.47, 168.10, 169.57,
170.54, 170.57. HRMS (M + Na)+ calcd. 1229.4649, found 1229.4584.
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General Procedure for the preparation of compounds (15a – 15f). One of the compounds
(14a – 14f) (0.10 mmol) was dissolved in anhydrous DMF (2 mL) to which excess morpholine
(250 ꢁL) was added. After 45 min the reaction was purified by preparative HPLC method 1.
Fractions containing the desired product were combined in a 200 mL flask, frozen in a dry ice
acetone bath then lyophilized.
H2N-L-Val-L-Cit-PA-May (15a). Thick colorless oil (72% yield). 1H NMR (400 MHz, DMSOꢀ
d6) δ 0.76 (s, 3H), 0.95 (s, 3H), 0.97 (s, 3H), 1.14 (d, J = 6.3 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H),
1.24 – 1.55 (m, 5H), 1.58 (s, 3H), 1.60 – 1.71 (m, 1H), 1.71 – 1.84 (m, 1H), 1.99 (d, J = 11.8 Hz,
1H), 2.05 – 2.18 (m, 1H), 2.30 – 2.43 (m, 1H), 2.58 – 2.66 (m, 1H), 2.67 (s, 3H), 2.80 (d, J = 9.6
Hz, 1H), 2.96 – 3.06 (m, 1H), 3.07 (s, 3H), 3.27 (s, 3H), 3.36 – 3.78 (m, 2H), 3.93 (s, 3H), 4.05 –
4.16 (m, 1H), 4.49 – 4.64 (m, 2H), 5.32 (dd, J = 13.4, 6.6 Hz, 1H), 5.58 (dd, J = 15.0, 9.1 Hz,
1H), 6.43 – 6.63 (m, 3H), 6.71 (s, 1H), 7.06 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 8.07 –
8.18 (m, 2H), 8.69 (d, J = 7.8 Hz, 1H), 10.10 (s, 1H). HRMS (M + Na )+ calcd. 1121.4761;
found 1121.4717.
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H2N-D-Ala-D-Ala-PA-May (15b). Thick colorless oil (76 % yield). H NMR (400 MHz,
DMSOꢀd6) δ 0.77 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 6H), 1.24 (d, J = 13.1
Hz, 1H), 1.31 (d, J = 6.9 Hz, 3H), 1.45 (tt, J = 10.1, 2.6 Hz, 2H), 1.58 (s, 3H), 2.03 (dd, J = 14.5,
2.9 Hz, 1H), 2.31 – 2.42 (m, 2H), 2.55 – 2.65 (m, 3H), 2.67 (s, 3H), 2.69 – 2.76 (m, 1H), 2.78 (d,
J = 9.7 Hz, 1H), 3.07 (s, 3H), 3.13 (d, J = 12.5 Hz, 1H), 3.25 (s, 3H), 3.34 – 3.37 (m, 2H), 3.48
(d, J = 9.1 Hz, 1H), 3.55 – 3.65 (m, 2H), 3.69 (t, J = 4.6 Hz, 1H), 3.94 (s, 3H), 4.02 – 4.10 (m,
1H), 4.39 – 4.48 (m, 1H), 4.52 (dd, J = 12.0, 2.8 Hz, 1H), 5.30 (q, J = 6.8 Hz, 1H), 5.51 – 5.59
(m, 1H), 5.92 (s, 1H), 6.49 – 6.54 (m, 2H), 6.56 (s, 1H), 6.88 (s, 1H), 7.08 – 7.13 (m, 2H), 7.16
(d, J = 1.8 Hz, 1H), 7.27 – 7.42 (m, 1H), 7.43 – 7.49 (m, 2H), 7.70 (d, J = 7.4 Hz, 0.5H), 7.86 (d,
J = 7.6 Hz, 0.5H), 8.19 (s, 1H), 10.02 (s, 1H). HRMS (M + Na)+ calcd. 1007.3962, found
1007.3958.
H2N-D-Ala-L-Ala-PA-May (15c). Thick colorless oil (84 % yield). 1H NMR (400 MHz,
DMSOꢀd6) δ 0.76 (s, 3H), 1.06 – 1.24 (m, 7H), 1.24 – 1.49 (m, 5H), 1.58 (s, 3H), 1.99 (d, J =
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14.2 Hz, 1H), 2.36 (dd, J = 18.3, 9.7 Hz, 1H), 2.58 – 2.74 (m, 4H), 2.80 (dd, J = 9.6, 4.3 Hz,
1H), 3.07 (d, J = 3.4 Hz, 3H), 3.27 (d, J = 4.6 Hz, 4H), 3.50 – 3.67 (m, 3H), 3.94 (d, J = 3.9 Hz,
3H), 4.09 (t, J = 11.1 Hz, 1H), 4.45 (d, J = 7.1 Hz, 1H), 4.49 – 4.62 (m, 1H), 5.33 (t, J = 6.7 Hz,
1H), 5.57 (dd, J = 14.9, 8.9 Hz, 1H), 5.90 (s, 1H), 6.34 – 6.67 (m, 3H), 6.66 – 6.78 (m, 1H), 7.07
(d, J = 8.0 Hz, 2H), 7.46 (dt, J = 8.5, 2.8 Hz, 2H), 8.28 (d, J = 37.9 Hz, 2H), 9.95 (d, J = 6.9 Hz,
1H). HRMS (M + H)+ calcd. 985.4148, found 985.4134.
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H2N-L-Ala-D-Ala-PA-May (15d). Thick colorless oil (75 % yield). 1H NMR (400 MHz,
DMSOꢀd6) δ 0.76 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.21 (d, J = 6.9 Hz,
3H), 1.32 (d, J = 7.0 Hz, 3H), 1.38 – 1.50 (m, 2H), 1.58 (s, 3H), 2.03 (dd, J = 14.4, 2.9 Hz, 1H),
2.38 (ddd, J = 16.0, 8.1, 5.9 Hz, 1H), 2.54 (d, J = 2.4 Hz, 1H), 2.58 – 2.8 (m, 1H), 2.67 (s, 3H),
2.79 (d, J = 9.6 Hz, 1H), 3.07 (s, 3H), 3.14 (d, J = 12.3 Hz, 1H), 3.25 (s, 3H), 3.38 (d, J = 12.4
Hz, 1H), 3.48 (d, J = 8.9 Hz, 1H), 3.48 – 3.60 (m, 2H), 3.60 (d, J = 7.5 Hz, 1H), 3.94 (s, 3H),
4.01 – 4.12 (m, 1H), 4.13ꢀ4.38 (m, 2H), 4.43 (d, J = 6.4 Hz, 1H), 4.52 (dd, J = 11.9, 2.8 Hz, 1H),
5.30 (q, J = 6.7 Hz, 1H), 5.56 (dd, J = 13.5, 9.2 Hz, 1H), 6.48 – 6.61 (m, 3H), 6.89 (s, 1H), 7.10
(d, J = 8.4 Hz, 2H), 7.16 (d, J = 1.9 Hz, 1H), 7.49 (dd, J = 8.6, 2.5 Hz, 2H), 8.43 (s, 1H), 8.45 –
8.55 (m, 1H), 10.14 (s, 1H). 13C NMR (101 MHz, DMSO) δ 173.48, 171.02, 170.58, 170.55,
168.11, 155.27, 151.27, 141.26, 138.32, 137.57, 133.33, 132.58, 128.91, 128.47, 125.15, 121.59,
119.24, 117.14, 113.90, 88.22, 79.99, 77.63, 73.17, 66.77, 66.27, 60.00, 56.55, 56.11, 51.66,
49.39, 48.96, 45.44, 44.99, 37.71, 36.36, 35.17, 34.88, 33.36, 31.92, 29.70, 25.97, 20.05, 18.48,
15.03, 14.40, 13.06, 11.38. HRMS (M + H)+ calcd. 985.4148, found 985.4131.
H2N-L-Ala-L-Ala-PA-May (15e). Thick colorless oil (81% yield). 1H NMR (400 MHz,
DMSOꢀd6) δ 0.77 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 12.2
Hz, 1H), 1.28 (d, J = 6.9 Hz, 3H), 1.33 (d, J = 7.0 Hz, 3H), 1.40 – 1.50 (m, 2H), 1.58 (s, 3H),
2.03 (dd, J = 14.5, 2.8 Hz, 1H), 2.37 (ddd, J = 16.1, 8.1, 5.8 Hz, 1H), 2.54 (d, J = 2.3 Hz, 1H),
2.62 (dd, J = 13.8, 6.1 Hz, 1H), 2.67 (s, 3H), 2.68 – 2.75 (m, 1H), 2.78 (d, J = 9.8 Hz, 1H), 2.86
(t, J = 4.8 Hz, 0H), 3.06 (s, 3H), 3.14 (d, J = 12.6 Hz, 1H), 3.25 (s, 3H), 3.39 (d, J = 12.5 Hz,
1H), 3.48 (d, J = 9.1 Hz, 1H), 3.60 (d, J = 7.6 Hz, 2H), 3.63 – 3.71 (m, 1H), 3.93 (s, 3H), 4.02 –
4.10 (m, 1H), 4.45 (t, J = 6.5 Hz, 1H), 4.51 (dd, J = 12.1, 2.8 Hz, 1H), 5.30 (q, J = 6.8 Hz, 1H),
5.51 – 5.59 (m, 1H), 6.50 – 6.59 (m, 3H), 6.88 (s, 1H), 7.10 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 1.8
Hz, 1H), 7.46 – 7.50 (m, 2H), 8.29 (s, 2H), 8.62 (d, J = 7.3 Hz, 1H), 10.13 (s, 1H). HRMS (M +
Na)+ calcd. 985.4148, found 985.4137.
H2N-Gly-Gly-PA-May (15f). Thick colorless oil (80 % yield). 1H NMR (400 MHz, DMSOꢀd6)
δ 0.76 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 1.16 (d, J = 6.8 Hz, 4H), 1.24 (d, J = 12.8 Hz, 1H), 1.39
– 1.51 (m, 2H), 1.58 (s, 3H), 2.03 (d, J = 12.1 Hz, 1H), 2.33 – 2.44 (m, 1H), 2.59 – 2.66 (m, 1H),
2.67 (s, 3H), 2.69 – 2.75 (m, 1H), 2.78 (d, J = 9.7 Hz, 2H), 3.07 (s, 3H), 3.16 (d, J = 12.7 Hz,
1H), 3.24 (s, 3H), 3.48 (d, J = 9.0 Hz, 1H), 3.60 (d, J = 7.9 Hz, 1H), 3.64 (s, 1H), 3.94 (s, 3H),
4.01 (d, J = 5.6 Hz, 2H), 4.02 – 4.11 (m, 2H), 4.51 (dd, J = 12.0, 2.5 Hz, 1H), 5.30 (q, J = 6.7
Hz, 1H), 5.55 (dd, J = 12.9, 10.1 Hz, 1H), 5.92 (s, 1H), 6.50 – 6.60 (m, 4H), 6.88 (s, 1H), 7.10
(d, J = 8.4 Hz, 2H), 7.19 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 8.10 – 8.33 (m, 1H), 8.80 (t, J = 5.6
29
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