Cyclic enediyne-amino acid chimeras as new aminopeptidase N inhibitors

Article abstract of DOI:10.1007/s00726-012-1292-0

Enediyne-peptide conjugates were designed with the aim to inhibit aminopeptidase N, a widespread ectoenzyme with a variety of functions, like protein digestion, inactivation of cytokines in the immune system and endogenous opioid peptides in the central nervous system. Enediyne moiety was embedded within the 12-membered ring with hydrophobic amino acid alanine, valine, leucine or phenylalanine used as carriers. Aromatic part of the enediyne bridging unit and the amino acid side chains were considered as pharmacophores for the binding to the aminopeptidase N (APN) active site. Additionally, the fused enediyne-amino acid ''heads'' were bound through a flexible linker to the L-lysine, an amino group donor. The synthesis included building the aromatic enediyne core at the C-terminal of amino acids and subsequent intramolecular N-alkylation. APN inhibition test revealed that the alanine-based derivative 9a inhibits the APN with IC₅₀ of 34 ± 11 lM. Enediyne-alanine conjugate 12 missing the flexible linker was much less effective in the APN inhibition. These results show that enediynefused amino acids have potential as new pharmacophores in the design of APN inhibitors.

Full text of DOI:10.1007/s00726-012-1292-0

Amino Acids
DOI 10.1007/s00726-012-1292-0
ORIGINAL ARTICLE
Cyclic enediyne–amino acid chimeras as new aminopeptidase
N inhibitors
•
Matija Gredicak Marija Abramic
•
ˇ
´
´
Ivanka Jeric
Received: 18 November 2011 / Accepted: 3 April 2012
Ó Springer-Verlag 2012
Abstract Enediyne–peptide conjugates were designed
with the aim to inhibit aminopeptidase N, a widespread
ectoenzyme with a variety of functions, like protein
digestion, inactivation of cytokines in the immune system
and endogenous opioid peptides in the central nervous
system. Enediyne moiety was embedded within the
12-membered ring with hydrophobic amino acid alanine,
valine, leucine or phenylalanine used as carriers. Aromatic
part of the enediyne bridging unit and the amino acid side
chains were considered as pharmacophores for the binding
to the aminopeptidase N (APN) active site. Additionally,
the fused enediyne–amino acid ‘‘heads’’ were bound
through a flexible linker to the L-lysine, an amino group
donor. The synthesis included building the aromatic
enediyne core at the C-terminal of amino acids and sub-
sequent intramolecular N-alkylation. APN inhibition test
revealed that the alanine-based derivative 9a inhibits the
APN with IC50 of 34 11 lM. Enediyne–alanine conju-
gate 12 missing the flexible linker was much less effective
in the APN inhibition. These results show that enediyne-
fused amino acids have potential as new pharmacophores
in the design of APN inhibitors.
Introduction
Aminopeptidases catalyze the hydrolysis of the N-terminal
peptide bond in peptides and proteins. They are widely
distributed in many tissues and cells in animals, plants,
bacteria and viruses and have wide substrate specificity
(Taylor 1993). Aminopeptidase N (APN; CD13; membrane
alanyl aminopeptidase; EC 3.4.11.2) is a zinc-containing
metalloenzyme involved in cleavage of neutral or basic
amino acids (Ala[Phe[Leu[Arg) from almost all unsub-
stituted oligopeptides and from amide or aryl amide
derivatives of amino acids (Turner 2004). APN is a wide-
spread ectoenzyme, particularly abundant in the brush
border membranes of small intestine, kidney and placenta,
but its expression range includes also fibroblasts and epi-
thelial cells in the liver, brain and lung, endothelial cells in
blood vessels and synaptic membranes (Turner 2004). This
transmembrane metallopeptidase has a variety of functions,
depending on the cell type and tissue environment. It is
involved in the final stages of protein and peptide digestion
in intestine, in terminating the actions of certain bioactive
peptides and inactivation of cytokines in the immune sys-
tem. Within the hematopoietic system, APN, also named
the thirteenth ‘‘cluster of differentiation’’ antigen or CD13,
is accepted as a marker for normal and malignant cells of
the myeloid lineage, but has been detected as well on the
lymphocytes, following cell activation, inflammation and
malignant transformation (Gabrilovac et al. 2005). In
addition to being identified as tumor marker of the hema-
topoietic system, accumulated data suggest that APN plays
a pivotal role in solid tumor invasion and angiogenesis
(Petrovic et al. 2004; Fukasawa et al. 2006). In tumor
vasculature, APN is overexpressed in the endothelium and
promotes angiogenesis. Thus, APN inhibition is expected
to be useful for cancer treatment. APN is also involved in
Keywords Aminopeptidase N ꢀ Enediyne–peptide
conjugate ꢀ Enzyme inhibitors ꢀ Amino acids
ˇ
´
´
M. Gredicak ꢀ M. Abramic ꢀ I. Jeric (&)
Division of Organic Chemistry and Biochemistry,
ˇ
´
ˇ
Ruder Boskovic Institute, Bijenicka cesta 54,
¯
HR-10002 Zagreb, Croatia
e-mail: ijeric@irb.hr
123

ˇ
M. Gredicak et al.
the inactivation of endogenous opioid peptides, the most
important analgesia medium in the central nervous system
(Chen et al. 1998). Recently, it has been reported that
synthetic dual inhibitors of APN and another cell-surface
metallopeptidase, neprilysin, can block the degradation of
enkephalins and, hence, produce potent physiological
analgetic responses (Noble and Roques 2007).
and the observed DNA damage governs scientists to search
for new targets and propose proteins as the most probable
ones (Nicolaou et al. 1993; Fouad et al. 2005; Dutta et al.
2009). Three independent classes of photoactivated
enediynes have been designed to target albumin, histone and
estrogen receptor and showed correlation between affinity
and protein degrading activity (Fouad et al. 2005). Anti-
bacterial activity of cyclic enediyne–phendioxy conjugates
has also been revealed (Joshi et al. 2007). Conjugation of
enediyne chromophore with amino acids or peptides was
shown to induce novel thermally initiated cyclization–
The crystal structure of APN from Escherichia coli was
first reported in 2006 (Addlagatta et al. 2006; Ito et al.
2006), but the complete crystal structure of human APN
has not been determined so far. The active site of APN was
determined by X-ray crystallographic studies on the com-
plex of the enzyme and bestatin (also known under the
tradename Ubenimex), an aminopeptidase inhibitor cur-
rently in the clinical trials for the treatment of acute
myelocytic leukemia (Hirayama et al. 2003). Studies
revealed that the active site comprises Zn2? ion as a cat-
alytic center of APN, two hydrophobic binding pockets (S1
and S10) in the vicinity of the Zn2?, and the binding site for
the a-amino group of the ligand (bestatin) formed by three
glutamic acid residues. Key pharmacophores have been
outlined for the potential inhibitor to possess: carbonyl
oxygen and hydroxamate or similar group for Zn2? coor-
dination and two hydrophobic groups for the interaction
with S1 and S10 pockets (S1 pocket prefers aromatic rings
rather than aliphatic chains). In addition, free a-amino
group or a cation that may interact with the glutamic acid
residues adjacent to S1 pocket is also advisable (Zhang and
Xu 2008). Since the appearance of bestatin, numerous
inhibitors have been prepared according to afore-men-
tioned guidelines as a transition state analogs (Zhang et al.
2011). They mostly belong to peptidomimetics or peptide
mimics—the strategy of design used to overcome draw-
backs of natural peptides, such as low bioavailability,
proteolytic degradation and rapid elimination. The most
prominent APN inhibitors are derivatives of bestatin (Gao
et al. 2011; Rao et al. 2011; Luan et al. 2011), phosphinic
acid (Grzywa and Oleksyszyn 2008; Grzywa et al. 2010),
L-iso-glutamine (Li et al. 2009), gallic acid (Zhang and Xu
2008), sulfonyl pyrrolidine (Cheng et al. 2008), amino-
benzosuberone derivatives (Maiereanu et al. 2011) and
compounds containing hydroxamate group (Lee et al.
2005; Flipo et al. 2007).
ˇ
elimination pathway (Gredicak et al. 2010), but also to
inhibit certain enzymes, such as topoisomerase I (Lin et al.
2001), proprotein convertase (Basak et al. 2009), chymo-
trypsin (Dutta et al. 2009) and tyrosine phosphatase
(Chandra et al. 2011). In this work, we have designed and
synthesized fused enediyne–amino acid ‘‘heads’’, attached
them to the L-lysine through a flexible linker and tested for
the APN inhibitor activity.
Materials and methods
General
Optical rotations were measured at 20 °C with an Optical
Activity LTD automatic AA-10 Polarimeter (some com-
pounds are too colored for an unambiguous determination
of optical rotation). Reactions were monitored by TLC on
Silica Gel 60 F254 plates (Merck; Darmstadt, Germany)
using detection with ninhydrin. Column chromatography
was performed on Silica Gel (Merck, 0.040–0.063). RP
HPLC analysis was performed on HPLC system coupled
with UV detector; C-18 semipreparative (250 9 8 mm, ID
5 lm) column at flow rate of 1 mL/min, or analytical
(150 9 4.5 mm, ID 5 lm) column at flow rate of 0.5
mL/min was used under isocratic conditions using different
concentration of MeOH in 0.1 % aqueous TFA. UV
detection was performed at 254 nm. NMR spectra were
recorded on 600 and 300 MHz spectrometers, operating at
150. 92 or 75.47 MHz for 13C and 600.13 or 300.13 MHz
for 1H nuclei. TMS was used as an internal standard.
Spectra were assigned based on 2D homonuclear (COSY)
and heteronuclear (HMQC and HMBC) spectra. Mass
spectrometry measurements were performed on HPLC
system coupled with triple quadrupole mass spectrometer,
operating in positive electrospray ionization (ESI) mode.
Spectra were recorded from a 10 lg/mL compound solu-
tion in 50 % MeOH/0.1 % FA by injection of 3 lL into the
ion source of the instrument by autosampler, at the flow
rate of 0.2 mL/min (mobile phase 50 % MeOH/0.1 % FA).
HRMS analysis was performed on MALDI-TOF mass
spectrometer operating in reflectron mode. Mass spectra
Here, we propose structurally distinct group of com-
pounds as potential APN inhibitors. These compounds are
based on the enediyne chromophore, a key moiety of the
enediyne anticancer antibiotics (Nicolaou and Dai 1991).
Enediyne group (Z-1,5-diyn-3-hexene) is known to
undergo the Bergman cyclization (Jones and Bergman
1972) that results in the cleavage of double stranded DNA.
However, some recent findings raise questions about DNA
as single biological target of enediyne compounds. Gen-
erally, poor correlation between their antitumor activity
123

Cyclic enediyne–amino acid chimeras
were acquired by accumulating three spectra after 400 laser
shots per spectrum. Calibrant and analyte spectra were
obtained in positive ion mode. Calibration type was inter-
nal with calibrants produced by matrix ionization (mono-
meric, dimeric and trimeric CHCA), with azithromycin and
angiotensin II dissolved in a-cyano-4-hydroxycinnamic
acid matrix in the mass range m/z 190.0499 to 749.5157 or
1046.5417. Accurately measured spectra were internally
calibrated and elemental analysis was performed on Data
Explorer v. 4.9 Software with mass accuracy better than 5
ppm. Samples were prepared by mixing 1 lL of analyte
methanol solution with 5 lL of saturated (10 mg/mL)
solution of a-cyano-4-hydroxycinnamic acid (a-CHCA)
and internal calibrants (0.1 mg/mL) dissolved in 50 %
acetonitrile/0.1 % TFA. MM2 calculations were performed
by a modified version of Allinger’s MM2 force field,
integrated into the ChemBioOffice 2008 programme.
Synthesis of amino acid derivatives 1a–d has been
analyzed at the same time and visualization was accom-
plished using ninhydrin (2 % in ethanol). Additionally,
samples were analyzed by HPLC–MS. Mobile phase was
0.1 % FA in water (solvent A) and 0.1 % FA in MeOH
(solvent B). Gradient was applied as follows: 0 min 100 %
A (0 % B); 0–12 min 100 % A (0 % B) – 90 % B (10 %
A); 12–15 min 90 % B (10 % A); 15–17 min 90 % B
(10 % A) – 100 % A (0 % B). Aliquots (25 lL) of the
reaction mixture were diluted with 100 lL of 0.1 % FA in
water and 5 lL were injected on the column (Zorbax XDB
C18, 3.5 lm, 4.6975 mm) at the flow rate of 0.5 mL/min.
Synthesis of amino acid derivatives 2
CuI (0.161 mmol), Pd(PPh3)4 (0.016 mmol), piperidine
(1.608 mmol) and 1,2-diiodobenzene (2.412 mmol) were
dissolved in dry THF under argon and stirred at room
temperature for 30 min. A premixed solution of 1 (1.608
mmol) and piperidine (1.608 mmol) in dry THF under
argon was added dropwise into the initial reaction mixture.
The solution was stirred at room temperature for 3 h. The
solvent was evaporated and the product extracted with
EtOAc, washed with brine and water and purified by flash
chromatography.
ˇ
described previously (Gredicak et al. 2008).
APN activity and inhibition assay
The enzyme activity was assayed using L-Leu-2-naph-
thylamide (Leu-2NA) as the substrate according to the
´
described procedure (Abramic and Vitale 1992). The
enzyme (porcine kidney microsomal aminopeptidase, EC
3.4.11.2, Sigma), in concentration of 1.58 9 10-10 M, was
incubated with the selected compound (6, 9 or 10) for
10 min at 23 °C in 50 mM Na-PO4 buffer pH 7.4, followed
by 5 min at 37 °C, in the total volume of 935 lL. The
enzyme reaction was then initiated by the addition of
65 lL of 1.36 mM solution of Leu-2NA. After 10-min
incubation at 37 °C, the reaction was stopped by adding
0.2 mL of Fast Blue B salt (1.5 mg/mL) solution con-
taining 10 % Tween 80 in 2M sodium acetate buffer pH
4.2. The absorbance at 530 nm was measured after
15 min. The release of the reaction product 2-naphthyl-
amine was followed also continuously, by measuring its
fluorescence at 25 °C by Perkin-Elmer luminescence
spectrometer LS 50, using excitation and emission
(S)-N-(3-(2-iodophenyl)prop-2-ynyl)-2-(2-nitrophenylsulfon-
amido)propanamide (2a) Yield: 41 %. Brown oil. Rf 0.51
(EtOAc–petrol ether 3:2). Mr 513.31. 13C NMR (CDCl3):
d = 19.0 (b Ala), 30.4 (H1 propargyl), 53.5 (a Ala), 85.9,
87.9 (C2,3 propargyl), 100.9 (C2 iodophenyl), 125.7 (C3
oNbs), 128.8 (C1 iodophenyl), 127.8, 129.7, 131.1, 132.7,
133.1, 134.1 (C4,5,6 oNbs, C4,5,6 iodophenyl), 138.7 (C3
1
iodophenyl), 147.9 (C2 oNbs), 170.3 (CO Ala). H NMR
3
(CDCl3): d = 1.39 (d, 3H, b Ala, Ja,b = 7.2 Hz), 4.05
3
(q, 1H, a Ala, Ja,b = 7.2 Hz), 4.21–4.27 (m, 2H, H1
propargyl), 5.97 (br s, 1H, NHa), 6.70 (br t, 1H, NHamide),
6.98–7.06, 7.28–7.34, 7.39–7.44 (m, 4H, H3,4,5,6 iodo-
phenyl), 7.69–7.76 (m, 2H, H4,5 oNbs), 7.81–7.91 (m, 1H,
H6 oNbs), 8.11–8.17 (m, 1H, H3 oNbs).
´
wavelength of 332 and 420 nm, respectively (Abramic
(S)-N-(3-(2-iodophenyl)prop-2-ynyl)-3-methyl-2-(2-nitroph-
enylsulfonamido)butanamide (2b) Yield: 67 %. Brown
oil. Rf 0.40 (petrol ether–EtOAc 2:1). Mr 541.36. 13C NMR
(CDCl3): d = 16.8, 18.7 (cc0 Val), 29.7 (CH2 propargyl),
30.5 (b Val), 62.9 (a Val), 85.3 (C2 propargyl), 87.4 (C3
propargyl), 100.2 (C2 iodophenyl), 125.1 (C3 oNbs), 128.4
(C1 iodophenyl), 127.4, 129.2, 130.2, 132.2, 132.4, 133.4
(C4,5,6 iodophenyl, C4,5,6 oNbs), 132.9 (C1 oNbs), 138.2
(C3 iodophenyl), 169.1 (CO Val). 1H NMR (CDCl3):
et al. 2004).
To check if potential inhibitors are hydrolyzed by APN,
the incubation of selected compound was performed with
the enzyme for 60 min in 0.025 M Na-PO4 buffer pH 7.4,
at 37 °C. The concentration of APN in reaction mixture of
100 lL was 1.58 9 10-9 M, and of the compounds 9a and
10, it was 5 and 25 mM, respectively. Control samples did
not contain the enzyme. Aliquots (5 lL) of the reaction
mixture were analyzed by thin-layer chromatography
(TLC) on glass plates precoated with silica gel 60 F254
(Merck, Darmstadt, Germany) using mixture of solvents
EtOH–H2O–NH4OH (60:25:15, v/v). L-Lysine was
3
d = 0.86, 0.96 (d, 6H, cc0 Val, Jb,c = 6.9 Hz), 2.23–2.29
(m, 1H, b Val), 4.10–4.15 (m, 1H, a Val), 4.18 (t, 2H, CH2
3
3
propargyl, JH,NH = 5.0 Hz), 6.03 (d, 1H, NHa, Ja,NH
7.2 Hz), 6.50 (br t, 1H, NHamide), 6.99–7.04 (m, 1H, H4
=
123

ˇ
M. Gredicak et al.
(S)-N-(3-(2-(3-hydroxyprop-1-ynyl)phenyl)prop-2-ynyl)-2-
(2-nitrophenylsulfonamido)propanamide (3a) Yield: 75 %.
Yellow oil. Rf 0.40 (EtOAc–petrol ether 2:1). [a]D -40.0°
(c 1.0 MeOH). Mr 441.46. 13C NMR (CDCl3): d = 19.1
(b Ala), 30.5 (C1 propargyl), 51.8 (C10 propargyl), 53.6
(a Ala), 82.4, 84.3 (C2,3 propargyl), 88.7 (C30 propargyl),
92.1 (C20 propargyl), 125.5, 126.0 (C1,2 enediyne aromatic
ring), 125.8 (C3 oNbs), 128.3, 128.4, 131.2, 131.4, 132.4,
133.3, 134.2 (C4,5,6 oNbs, C3,4,5,6 enediyne aromatic
ring), 133.5 (C1 oNbs), 147.9 (C2 oNbs), 171.2 (CO Ala).
1H NMR (CDCl3): d = 1.36 (d, 3H, b Ala, 3Ja,b = 7.0 Hz),
4.02–4.31 (m, 3H, H1 propargyl, a Ala), 4.58 (s, 2H,
H10 propargyl), 7.16 (br t, 1H, NHamide), 7.24–7.49
(m, 4H, H3,4,5,6 enediyne aromatic ring), 7.60–7.67
(m, 2H, H4,5 oNbs), 7.81–7.87 (H6 oNbs), 8.08–8.15
(H3 oNbs).
iodophenyl), 7.28–7.32, 7.38–7.41 (m, 2H, H5,6 iodo-
phenyl), 7.64–7.77 (m, 3H, H3 iodophenyl, H4,5 oNbs),
7.83–7.86 (m, 1H, H6 oNbs), 8.08–8.11 (m, 1H, H3
oNbs).
(S)-N-(3-(2-iodophenyl)prop-2-ynyl)-4-methyl-2-(2-nitroph-
enylsulfonamido)pentanamide (2c) Yield: 41 %. Brown
oil. Rf 0.51 (petrol ether–EtOAc 1:1). Mr 555.39. 13C NMR
(CDCl3): d = 20.6, 22.4 (dd0 Leu), 23.8 (c Leu), 29.7 (C1
propargyl), 41.4 (b Leu), 56.1 (a Leu), 85.3, 87.4 (C2,3
propargyl), 100.3 (C2 iodophenyl), 125.1 (C3 oNbs), 128.4
(C1 iodophenyl), 127.4, 129.2, 130.4, 132.2, 132.4, 133.4
(C4,5,6 iodophenyl, C4,5,6 oNbs), 132.8 (C1 oNbs),
138.3 (C3 iodophenyl), 147.2 (C2 oNbs), 169.9 (CO Leu).
1H NMR (CDCl3): d = 0.75, 0.89 (d, 6H, dd0 Leu,
3Jc,d = 6.4 Hz), 1.59–1.70 (m, 3H, b,c Leu), 3.95–4.01 (m,
1H, a Leu), 4.11–4.20 (m, 2H, H1 propargyl), 5.99 (br s,
1H, NHa), 6.58 (br t, 1H, NHamide), 6.99–7.03, 7.28–7.32,
7.39–7.42 (m, 4H, H3,4,5,6 iodophenyl), 7.64–7.72 (m,
2H, H4,5 oNbs), 7.82–7.87 (m, 1H, H6 oNbs), 8.11–8.16
(m, 1H, H3 oNbs).
(S)-N-(3-(2-(3-hydroxyprop-1-ynyl)phenyl)prop-2-ynyl)-3-
methyl-2-(2-nitrophenylsulfonamido)-butanamide
(3b)
Yield: 73 %. Yellow oil. Rf 0.47 (EtOAc–petrol ether 2:1).
[a]D -63.0° (c 1.0 MeOH). Mr 469.51. 13C NMR (CD3OD):
d = 15.7, 16.6 (cc0 Val), 21.2 (b Val), 29.9 (C1 propargyl),
48.4 (C10 propargyl), 61.3 (a Val), 79.2 (C3 propargyl), 81.1
(C2 propargyl), 86.6 (C30 propargyl), 90.3 (C20 propargyl),
123.3 (C3 oNbs), 123.5, 124.0 (C1,2 enediyne aromatic
ring), 126.3, 126.5, 128.7, 130.0, 130.2, 130.8, 132.1
(C3,4,5,6 enediyne aromatic ring, C4,5,6 oNbs), 131.9 (C1
oNbs), 146.3 (C2 oNbs), 169.3 (CO Val). 1H NMR
(S)-N-(3-(2-iodophenyl)prop-2-ynyl)-2-(2-nitrophenylsulfo-
namido)-3-phenylpropanamide (2d) Yield: 53 %. Brown
oil. Rf 0.67 (petrol ether–EtOAc 2:1). Mr 589.40. 13C NMR
(CDCl3): d = 30.4 (C1 propargyl), 38.5 (b Phe), 59.7 (a
Phe), 86.0 (C3 propargyl), 88.2 (C2 propargyl), 101.2 (C2
iodophenyl), 126.2 (C6 oNbs), 127.5, 128.0, 128.9, 129.3,
129.9 (d,e,f Phe, C4,5 iodophenyl), 129.2 (C1 iodophenyl),
131.1 (C3 oNbs), 133.0 (C1 oNbs), 132.9, 133.3, 133.9
(C4,5 oNbs, C6 iodophenyl), 135.2 (c Phe), 138.9 (C3
3
(CD3OD): d = 0.95, 0.98 (d, 6H, cc0 Val, Jb,c = 6.7 Hz),
3
1.94–2.04 (m, 1H, b Val), 3.73 (d, 1H, a Val, Ja,b
=
7.4 Hz), 3.92, 3.99 (d, 2H, H1 propargyl, 2JH,H = 17.5 Hz),
4.45 (s, 2H, H10 propargyl), 7.29–7.46 (m, 4H, H3,4,5,6
enediyne aromatic ring), 7.58–7.74 (m, 2H, H4,5 oNbs),
7.76–7.80 (m, 1H, H6 oNbs), 8.05–8.09 (m, 1H, H3 oNbs).
0
1
iodophenyl), 147.3 (C2 oNbs), 169.9 (CO Phe). H NMR
2
0
(CDCl3): d = 2.95, 3.26 (dd, 2H, b Phe, Jb,b = 14.1 Hz,
3
Ja,b = 5.2 Hz, 3Ja,b = 8.9 Hz), 4.15–4.18 (m, 1H, a Phe),
4.24, 4.36 (dd, 2H, H1 propargyl, JNH,H1 = 5.7 Hz,
0
3
3
2
JNH,H1 = 4.9 Hz, JH1,1 = 17.7 Hz), 6.83 (br t, 1H,
0
0
(S)-N-(3-(2-(3-hydroxyprop-1-ynyl)phenyl)prop-2-ynyl)-4-
(3c)
methyl-2-(2-nitrophenylsulfonamido)pentanamide
NHa), 6.99–7.04 (m, 5H, d,e,f Phe), 7.28–7.32 (m, 2H,
H4,5 iodophenyl), 7.41–7.44, 7.62–7.68 (m, 3H, H4,5
oNbs, H6 iodophenyl), 7.74–7.76 (m, 1H, H6 oNbs),
7.82–7.85 (m, 1H, H3 iodophenyl), 7.95–7.98 (m, 1H, H3
oNbs).
Yield: 63 %. Yellow oil. Rf 0.22 (EtOAc–petrol ether 1:1).
[a]D -61.0° (c 1.0 MeOH). Mr 483.54. 13C NMR
(CD3OD): d = 18.7, 20.4 (dd0 Leu), 22.6 (c Leu), 27.4 (C1
propargyl), 40.3 (b Leu), 48.4 (C10 propargyl), 54.2 (a
Leu), 79.3 (C3 propargyl), 81.1 (C2 propargyl), 86.7 (C30
propargyl), 90.3 (C20 propargyl), 123.2 (C3 oNbs), 123.6,
123.9 (C1,2 enediyne aromatic ring), 126.3, 126.5, 128.7,
130.0, 130.2, 130.8, 132.2 (C3,4,5,6 enediyne aromatic
ring, C4,5,6 oNbs), 131.8 (C1 oNbs), 146.3 (C2 oNbs),
Synthesis of amino acid derivatives 3
Compound 2 (1.294 mmol), Pd(PPh3)4 (0.013 mmol), CuI
(0.129 mmol) and piperidine (2.588 mmol) were dissolved
in dry THF under argon and stirred at room temperature for
30 min. Propargyl alcohol (3.883 mmol) was added and the
reaction mixture was stirred at room temperature overnight.
The solvent was evaporated and the product extracted with
EtOAc, washed with brine and water and purified by flash
chromatography.
1
170.4 (CO Leu). H NMR (CD3OD): d = 0.87, 0.94 (d,
3
6H, dd0 Leu, Jc,d = 6.8 Hz), 1.43–1.82 (m, 3H, b,c Leu),
3.99 (s, 2H, H1 propargyl), 4.02–4.09 (m, 1H, a Leu), 4.47
(s, 2H, H10 propargyl), 7.27–7.49 (m, 4H, H3,4,5,6 ened-
iyne aromatic ring), 7.59–7.82 (m, 3H, H4,5,6 oNbs),
8.05–8.13 (m, 1H, H3 oNbs).
123

Cyclic enediyne–amino acid chimeras
(S)-N-(3-(2-(3-hydroxyprop-1-ynyl)phenyl)prop-2-ynyl)-2-
(2-nitrophenylsulfonamido)-3-phenylpropanamide
(S)-N-(3-(2-(3-bromoprop-1-ynyl)phenyl)prop-2-ynyl)-3-
methyl-2-(2-nitrophenylsulfonamido) butanamide (4b)
Yield: 82 %. Colorless oil. Rf 0.44 (EtOAc–petrol ether
1:1). [a]D -69.0° (c 1.0 MeOH). Mr 532.41. 13C NMR
(CDCl3): d = 15.81 (C10 propargyl), 17.56, 19.41 (cc0
Val), 30.39 (C1 propargyl), 31.38 (b Val), 63.64 (a Val),
82.22, 85.49 (C3,30 propargyl), 88.46, 88.52 (C2,20 prop-
argyl), 125.06, 125.48 (C1,2 enediyne aromatic ring),
125.75 (C3 oNbs), 128.53, 128.84, 130.92, 132.13, 132.23,
133.12, 134.05 (C4,5,6 oNbs, C3,4,5,6 enediyne aromatic
ring), 133.66 (C1 oNbs), 147.96 (C2 oNbs), 169.85 (CO
(3d)
Yield: 61 %. Brown oil. Rf 0.38 (EtOAc–petrol ether 1:1).
Mr 517.55. 13C NMR (CDCl3): d = 30.4 (C1 propargyl),
38.4 (b Phe), 51.5 (C10 propargyl), 59.6 (a Phe), 82.1 (C30
propargyl), 84.0 (C3 propargyl), 88.5 (C2 propargyl), 92.1
(C20 propargyl), 125.3, 125.9 (C1,2 enediyne aromatic
ring), 125.8 (C6 oNbs), 127.2, 128.0, 128.2, 128.5, 129.2
(d,e,f Phe, C4,5 enediyne aromatic ring), 130.8 (C3 oNbs),
131.3, 131.4 (C4,5 oNbs), 133.0 (C1 oNbs), 133.1, 133.6
(C3,6 enediyne aromatic ring), 135.2 (c Phe), 147.1 (C2
1
1
oNbs), 170.3 (CO Phe). H NMR (CDCl3): d = 2.95, 3.20
Val). H NMR (CDCl3): d = 0.88, 0.96 (d, 6H, cc0 Val,
3
3
2
(dd, 2H, b Phe, Ja,b = 5.3 Hz, Ja,b = 8.5 Hz, Jb,b
14.0 Hz), 4.10, 4.34 (dd, 2H, H1 propargyl, JH,H
=
3Jb,c = 6.7 Hz), 2.20–2.27 (m, 1H, b Val), 3.79–3.83 (m,
0
0
2
0
=
1H,
3
a
Val), 4.15, 4.20 (dd, 2H, H1 propargyl,
3
3
17.6 Hz, JNH,H = 6.2 Hz, JNH,H = 4.8 Hz), 4.18–4.25
(m, 1H, a Phe), 4.57 (s, 2H, H10 propargyl), 6.96–7.06 (m,
5H, d,e,f Phe), 7.18 (br t, 1H, NHamide), 7.24–7.30 (m, 2H,
H4, 5 enediyne aromatic ring), 7.36–7.43 (m, 2H, H3,6
enediyne aromatic ring), 7.55–7.61 (m, 2H, H4,5
oNbs), 7.70–7.75 (m, 1H, H6 oNbs), 7.91–7.96 (m, 1H, H3
oNbs).
JNH,H = 5.6 Hz, 2JH,H = 17.8 Hz), 4.25, 4.28 (d, 2H, H10
0
0
2
0
propargyl,
JH,H = 14.1 Hz), 6.07 (d, 1H, NHa,
3JNH,a = 8.3 Hz), 6.50 (br t, 1H, NHamide), 7.28–7.31 (m,
2H, H4,5 enediyne aromatic ring), 7.38–7.45 (m, 2H, H3,6
enediyne aromatic ring), 7.59–7.67 (m, 2H, H4,5 oNbs),
7.81–7.84 (m, 1H, H6 oNbs), 8.08–8.11 (m, 1H, H3 oNbs).
(S)-N-(3-(2-(3-bromoprop-1-ynyl)phenyl)prop-2-ynyl)-4-
methyl-2-(2-nitrophenylsulfonamido)pentanamide
(4c)
Synthesis of amino acid derivatives 4
Yield: 82 %. Colorless oil. Rf 0.57 (EtOAc–petrol ether
1:1). [a]D -70.0° (c 1.0 MeOH). Mr 545.45. 13C NMR
(CDCl3): d = 15.7 (C10 propargyl), 21.1, 23.0 (dd0 Leu),
24.3 (c Leu), 30.2 (C1 propargyl), 41.9 (b Leu), 56.6 (a
Leu), 82.0, 82.9 (C3,30 propargyl), 85.3, 88.3 (C2,20
propargyl), 124.8, 125.3 (C1,2 enediyne aromatic ring),
125.6 (C3 oNbs), 128.4, 128.7, 130.9, 131.9, 132.0, 133.0,
133.9 (C4,5,6 oNbs, C3,4,5,6 enediyne aromatic ring),
Compound 3 (0.917 mmol) was dissolved in dry DCM and
PBr3 (1.834 mmol) was added. The reaction mixture was
stirred for 2 h at room temperature. The reaction was
quenched with an ice cold 10 % NaHCO3 solution. The
solvent was evaporated and the product extracted with
EtOAc, washed with brine and water and purified by flash
chromatography.
1
133.3 (C1 oNbs), 147.7 (C2 oNbs), 170.5 (CO Leu). H
NMR (CDCl3): d = 0.76, 0.89 (d, 6H, dd0 Leu,
3Jc,d = 6.1 Hz), 1.58–1.76 (m, 3H, b,c Leu), 3.96–4.05 (m,
(S)-N-(3-(2-(3-bromoprop-1-ynyl)phenyl)prop-2-ynyl)-2-(2-
nitrophenylsulfonamido)propanamide (4a) Yield: 79 %.
Yellow oil. Rf 0.60 (EtOAc–petrol ether 2:1). [a]D -27.0°
(c 1, MeOH). Mr 504.35. 13C NMR (CDCl3): d = 15.1 (C10
propargyl), 18.6 (b Ala), 29.9 (C1 propargyl), 53.0 (a Ala),
81.6 (C3 propargyl), 83.4 (C2 propargyl), 84.8 (C30 prop-
argyl), 87.8 (C20 propargyl), 124.6, 124.7 (C1,2 enediyne
aromatic ring), 125.2 (C3 oNbs), 127.8, 128.1 (C4,5
enediyne aromatic ring), 130.5 (C6 oNbs), 131.4. 131.5
(C3,6 enediyne aromatic ring), 132.6 (C4 oNbs), 132.7 (C1
oNbs), 133.6 (C5 oNbs), 147.3 (C2 oNbs), 169.8 (CO
1H, a Leu), 4.13–4.20 (m, 2H, H1 propargyl), 4.25, 4.31 (d,
2
2H, H10 propargyl, JH,H = 14.2 Hz), 6.01 (d, 1H, NHa,
0
3Ja,NH = 7.7 Hz), 6.55 (t, 1H, NHamide, JH,NH = 4.9 Hz),
3
7.25–7.33, 7.38–7.47 (m, 4H, H3,4,5,6 enediyne aromatic
ring), 7.58–7.70 (m, 2H, H4,5 oNbs), 7.81–7.87 (m, 1H, H6
oNbs), 8.10–8.16 (m, 1H, H3 oNbs).
(S)-N-(3-(2-(3-bromoprop-1-ynyl)phenyl)prop-2-ynyl)-2-
(2-nitrophenylsulfonamido)-3-phenylpropanamide
(4d)
Yield: 78 %. Yellow oil. Rf 0.53 (EtOAc–petrol ether 1:1).
[a]D -3.0° (c 0.5, MeOH). Mr 580.45. 13C NMR (CDCl3):
d = 15.1 (C10 propargyl), 29.9 (C1 propargyl), 38.0 (b
Phe), 59.2 (a Phe), 81.5 (C10 propargyl), 84.7 (C3 prop-
argyl), 87.8, 87.9 (C2,20 propargyl), 124.4, 124.8 (C1,2
enediyne aromatic ring), 125.4 (C6 oNbs), 126.8, 127.8,
128.1, 128.2, 128.6, 130.4, 131.5, 132.6, 133.2 (d,e,f Phe,
C4,5 oNbs, C3,4,5,6 enediyne aromatic ring), 131.5 (C3
oNbs), 132.3 (C1 oNbs), 138.2 (c Phe), 146.6 (C2 oNbs),
1
3
Ala). H NMR (CDCl3): d = 1.39 (d, 3H, b Ala, Ja,b
=
7.2 Hz), 4.06 (m, 1H, a Ala), 4.24, 4.28 (dd, 2H, H1
3
2
0
propargyl, JH,NH = 4.8 Hz, JH,H = 17.8 Hz), 4.26 (s,
2H, H10 propargyl), 5.98 (br d, 1H, NHa), 6.64 (br t, 1H,
NHamide), 7.27–7.31 (m, 2H, H4,5 enediyne aromatic ring),
7.40–7.45 (m, 2H, H3,6 enediyne aromatic ring), 7.66–7.73
(m, 2H, H4,5 oNbs), 7.85–7.88 (H6 oNbs), 8.11–8.15
(H3 oNbs).
123

ˇ
M. Gredicak et al.
1
169.2 (CO Phe). H NMR (CDCl3): d = 2.96, 3.25 (dd,
aromatic ring), 7.31–7.35 (m, 2H, H4,5 oNbs), 7.49–7.51
(m, 2H, H3,6 oNbs).
2
3
0
2H, b Phe, Jb,b = 14.6 Hz, Ja,b = 5.1 Hz), 4.17–4.20
(m, 1H, a Phe), 4.23, 4.37 (dd, 2H, H1 propargyl,
JH,H = 17.8 Hz, JNH,H = 5.9 Hz), 4.25 (s, 2H, H10
propargyl), 6.10 (d, 1H, NHa, 3Ja,NH = 6.2 Hz), 6.79 (br t,
1H, NHamide), 6.98–7.06 (m, 5H, d,e,f Phe), 7.26–7.31 (m,
2H, H4,5 enediyne aromatic ring), 7.40–7.45 (m, 2H, H3,6
enediyne aromatic ring), 7.59–7.65 (m, 2H, H4,5 oNbs),
7.72–7.75 (m, 1H, H6 oNbs), 7.95–7.98 (m, 1H, H3 oNbs).
2
3
0
(5S)-5-(2-methylpropyl)-4-(2-nitrophenylsulfonyl)-benz-4,
7-diazacyclododeca-1,9-diyn-6-one (5c) Yield: 43 %.
Colorless oil. Rf 0.55 (EtOAc–petrol ether 1:1). [a]D
?67.0° (c 1.0 MeOH). Mr 465.52. 13C NMR (CDCl3):
d = 22.1, 22.7 (dd0 Leu), 24.4 (c Leu), 30.4 (C1 propar-
gyl), 34.6 (C10 propargyl), 37.6 (b Leu), 56.9 (a Leu), 82.4,
83.1 (C3,30 propargyl), 88.3, 90.3 (C2,20 propargyl), 123.9
(C3 oNbs), 125.6, 126.7 (C1,2 enediyne aromatic ring),
127.7, 128.2, 129.1, 130.9, 131.6, 132.3, 134.1 (C4,5,6
oNbs, C3,4,5,6 enediyne aromatic ring), 132.7 (C1 oNbs),
147.7 (C2 oNbs), 168.5 (CO Leu). 1H NMR (CDCl3):
d = 0.79, 0.81 (d, 6H, dd0 Leu, 3Jc,d = 2.1 Hz), 1.24–1.35,
Synthesis of amino acids derivatives 5
Compound 4 (0.079 mmol) was dissolved in 10 mL DMF
and added dropwise via a syringe pump into the solution
of K2CO3 (0.158 mmol) in 10 mL DMF (flow rate 0.49
mL/h). The solvent was evaporated and the product
extracted with EtOAc, washed with brine and water and
purified by flash chromatography.
1.99–2.10 (m, 2H, bb0 Leu), 1.42–1.54 (m, 1H, c Leu),
2
3.62, 4.58 (dd, 2H, H1 propargyl, JH,H = 17.5 Hz,
0
3
3
0
JH,NH = 4.5 Hz, JH ,NH = 8.1 Hz), 4.31–4.36 (m, 1H, a
2
Leu), 4.34, 4.74 (d, 2H, H10 propargyl, JH,H = 18.7 Hz),
7.15–7.45, 7.58–7.64 (m, 7H, H3,4,5,6 enediyne aromatic
ring, H4,5,6 oNbs), 8.18–8.26 (m, 1H, H3 oNbs).
0
(5S)-5-methyl-4-(2-nitrophenylsulfonyl)-benz-4,7-diaza-
cyclododeca-1,9-diyn-6-one (5a) Yield: 33 %. Colorless
oil. Rf 0.62 (EtOAc–petrol ether 3:1). [a]D ?55.0° (c 0.5
MeOH). Mr 423.44. 13C NMR (CDCl3): d = 13.4 (b Ala),
30.0 (C1 propargyl), 33.7 (C10 propargyl), 53.9 (a Ala),
82.4, 82.6 (C2,20 propargyl), 87.2, 90.2 (C3,30 propargyl),
123.8 (C3 oNbs), 124.9, 126.3 (C1,2 enediyne aromatic
ring), 127.2, 127.7 (C4,5 enediyne aromatic ring), 128.4
(C6 oNbs), 130.4 (C4 oNbs), 131.3, 131.5 (C3,6 enediyne
aromatic ring), 132.0 (C1 oNbs), 133.7 (C5 oNbs), 168.6
(CO Ala). 1H NMR (CDCl3): d = 1.34 (d, 3H, b Ala,
(5S)-5-benzyl-4-(2-nitrophenylsulfonyl)-benz-4,7-diazacycl-
ododeca-1,9-diyn-6-one (5d) Yield: 28 %. Colorless oil.
Rf 0.49 (EtOAc–petrol ether 1:1). [a]D ?60.0° (c 0.5
MeOH). Mr 499.54. 13C NMR (CDCl3): d = 30.8 (C1
propargyl), 35.1, 35.1 (C10 propargyl, b Phe), 60.6 (a Phe),
82.9, 83.2 (C2,20 propargyl), 88.4, 90.5 (C3,30 propargyl),
124.6 (C3 oNbs), 125.8, 127.1 (C1,2 enediyne aromatic
ring), 127.0, 127.9, 128.3, 128.6, 129.2, 129.6, 131.2,
132.0, 132.2, 134.1 (d,e,f Phe, C4,5,6 oNbs, C3,4,5,6
enediyne aromatic ring), 133.0 (C1 oNbs), 136.2 (c Phe),
3Ja,b = 7.0 Hz), 3.71, 4.65 (d, 2H, H10 propargyl,
0
JH,H = 18.5 Hz), 4.30, 4.71 (dd, 2H, H1 propargyl,
3
JH,NH = 4.1 Hz, JH ,NH = 8.6 Hz, JH,H = 17.5 Hz),
2
3
2
147.8 (C2 oNbs), 168.2 (CO Phe). 1H NMR (CDCl3):
0
0
3
3
Phe, Ja,b = 6.8 Hz,
4.53 (q, 1H, Ja,b = 7.0 Hz), 7.20–7.35 (m, 4H, H3,4,5,6
enediyne aromatic ring), 7.56–7.70 (m, 3H, H4,5,6 oNbs),
8.18–8.21 (m, 1H, H3 oNbs).
d = 2.89, 3.39 (dd, 2H,
b
Ja,b = 7.9 Hz, Jb,b = 14.1 Hz), 3.60, 3.63 (dd, 2H, H1
3
2
0
0
2
propargyl, JH,H = 17.5 Hz, JNH,H = 4.2 Hz, JNH,H
3
3
0
0
=
=
2
13.0 Hz), 4.51, 4.83 (d, 2H, H10 propargyl, JH,H
0
(5S)-5-isopropyl-4-(2-nitrophenylsulfonyl)-benz-4,7-diaza-
cyclododeca-1,9-diyn-6-one (5b) Yield: 55 %. Colorless
oil. Rf 0.44 (EtOAc–petrol ether 2:1). [a]D ?89.0° (c 0.5
MeOH). Mr 451.49. 13C NMR (CDCl3): d = 18.4, 19.6 (cc0
Val), 26.5 (b Val), 30.5 (C1 propargyl), 35.0 (C10 prop-
argyl), 65.3 (a Val), 82.0, 83.7 (C2,20 propargyl), 88.7, 90.2
(C3,30 propargyl), 123.5 (C3 oNbs), 126.1, 126.8 (C1,2
enediyne aromatic ring), 127.9, 128.3 (C4,5 enediyne
aromatic ring), 129.4 (C6 oNbs), 131.1, 131.4, 132.4, 133.9
(C3,6 enediyne aromatic ring, C4,5 oNbs), 133.3 (C1
oNbs), 147.8 (C2 oNbs), 168.5 (CO Val). 1H NMR
18.6 Hz), 4.55–4.60 (m, 1H, a Phe), 7.01–7.10 (m, 5H,
d,e,f Phe), 7.20–7.25, 7.33–7.36, 7.41–7.44, 7.53–7.59
(m, 7H, H4,5,6 oNbs, H3,4,5,6 enediyne aromatic ring),
8.00–8.04 (m, 1H, H3 oNbs).
Synthesis of amino acid derivatives 6
Compound 5 (0.026 mmol), K2CO3 (0.052 mmol) and
PhSH (0.052 mmol) were dissolved in dry DMF and stirred
for 1 h at room temperature. The solvent was evaporated
and the product extracted with EtOAc, washed with brine
and water and purified by flash chromatography.
3
(CDCl3): d = 0.80, 0.93 (d, 6H, cc0 Val, Jb,c = 6.9 Hz),
2.34–2.44 (m, 1H, b Val), 3.55, 3.90 (d, 2H, H10 propargyl,
2
0
JH,H = 17.4 Hz), 4.33, 4.76 (d, 2H, H1 propargyl,
(5S)-5-methylbenz-4,7-diazacyclododeca-1,9-diyn-6-one
(6a) Yield: 67 %. Colorless oil. Rf 0.13 (EtOAc–petrol
ether 3:1). [a]D ?33.0° (c 0.25 MeOH). Mr 238.28. 13C
2
3
0
JH,H = 18.3 Hz), 4.54 (dd, 1H, a Val, JNH,a = 9.8 Hz,
3Ja,b = 7.5 Hz), 7.18–7.25 (m, 4H, H3,4,5,6 enediyne
123

Cyclic enediyne–amino acid chimeras
NMR (CDCl3): d = 20.7 (b Ala), 30.1 (C1 propargyl),
40.1 (C10 propargyl), 60.9 (a Ala), 82.4, 84.1 (C2,20
propargyl), 90.8, 92.6 (C3,30 propargyl), 124.7, 125.6 (C1,2
enediyne aromatic ring), 127.6, 127.7, 128.7, 131.0
(C3,4,5,6 enediyne aromatic ring), 176.4 (CO Ala). 1H
129.2, 131.2 (d,e,f Phe, C3,4,5,6 enediyne aromatic ring),
137.5 (c Phe), 175.4 (CO Phe). 1H NMR (CDCl3):
3
3
0
d = 2.65, 3.32 (dd, 2H, b Phe, Ja,b = 4.1 Hz, Ja,b
=
2
10.4 Hz, Jb,b = 14.2 Hz), 3.36, 3.88 (d, 2H, H1 propar-
0
2
3
0
gyl, JH,H = 17.4 Hz), 3.39 (dd, 1H, a Phe, Ja,b
=
3
3
4.1 Hz, Ja,b = 10.4 Hz), 3.85, 4.62 (dd, 2H, H10 propar-
0
NMR (CDCl3): d = 1.37 (d, 3H, b Ala, Ja,b = 7.2 Hz),
3
2
3
3.23 (q, 1H, a Ala, Ja,b = 7.1 Hz), 3.67, 4.04 (d, 2H, H10
gyl,
JH,H = 17.6 Hz, 3JNH,H = 3.1 Hz,
9.5 Hz), 7.19–7.35 (m, 9H, H3,4,5,6 enediyne aromatic
JNH,H
0
0
=
2
propargyl, JH,H = 17.4 Hz), 3.88, 4.56 (dd, 2H, H1
0
3
propargyl, JH,NH = 3.5 Hz, JH ,NH = 9.3 Hz, JH,H
3
2
0
0
=
ring, d,e,f Phe), 7.65–7.70 (m, 1H, NHa).
17.6 Hz), 7.17–7.37 (m, 4H, H3,4,5,6 enediyne aromatic
ring).
Synthesis of Boc-L-Lys(Boc)-d-Gly-OH (7)
(5S)-5-isopropylbenz-4,7-diazacyclododeca-1,9-diyn-6-one
(6b) Yield: 67 %. Colorless oil. Rf 0.44 (EtOAc–petrol
ether 2:1). [a]D ?64.0° (c 0.5 EtOAc). Mr 266.34. 13C
NMR (CDCl3): d = 18.1, 19.8 (cc0 Val), 30.4 (C1 prop-
argyl), 31.8 (b Val), 41.1 (C10 propargyl), 71.2 (a Val),
82.5, 84.1 (C2,20 propargyl), 91.5, 92.8 (C3,30 propargyl),
125.0, 128.0 (C1,2 enediyne aromatic ring), 127.8, 127.9,
129.0, 131.3 (C3,4,5,6 enediyne aromatic ring), 175.5 (CO
Boc-L-Lys(Boc)-OH (200 mg, 0.057 mmol) and NMM
(76 lL, 0.069 mmol) were dissolved in 2 mL dry DMF
and cooled down to 0 °C. Isobutyl chloroformate (91 lL,
0.069 mmol) was added and the reaction was stirred for
10 min at 0 °C. 5-aminopentoic acid (81 mg, 0.069 mmol)
was added and the solution was stirred for 30 min at 0 °C
and then at room temperature overnight. The solvent was
evaporated and the product extracted with EtOAc, washed
with brine and water and purified by flash chromatography
in petrol ether–EtOAc–AcOH 10:10:0.5.
1
Val). H NMR (CDCl3): d = 0.96, 1.05 (d, 6H, cc0 Val,
3Jb,c = 7.1 Hz), 2.12–2.18 (m, 1H, b Val), 2.96 (d, 1H, a
3
Val, Ja,b = 5.0 Hz), 3.59, 4.12 (d, 2H, H10 propargyl,
Yield: 236 mg (92 %). Colorless oil. Rf 0.42 (petrol
ether-EtOAc–AcOH 10:10:0,5). [a]D = -7.5° (c 1.0
MeOH). Mr = 445.55. 13C NMR (DMSO-d6): d = 21.9 (b
d-Gly); 22.7 (c Lys), 28.1, 28.2 (CH3 Boca, CH3 Boce),
28.5 (b Lys), 29.2 (c d-Gly), 31. 8 (d Lys), 33.4 (a d-Gly),
38.0 (d d-Gly), 39.1 (e Lys), 54.3 (a Lys), 77.3, 77.9 (C
Boca, C Boce), 155.2, 155.5 (CO Boca, CO Boce), 171.9
(CO Lys), 174.4 (CO d-Gly). 1H NMR (DMSO-d6):
d = 1.36, 1.37 (s, 18H, CH3 Boca, CH3 Boce), 1.29–1.50
(m, 10H, b,c d-Gly, b,c,d Lys), 2.18 (t, 2H, a d-Gly,
3Ja,b = 7.2 Hz), 2.83–2.89 (m, 2H, e Lys), 2.97–3.09 (m,
2H, d d-Gly), 3.77, 3.85 (m, 1H, a Lys), 6.65 (d, 1H,
2
0
JH,H = 17.5 Hz), 3.98, 4.56 (dd, 2H, H1 propargyl,
3
3
2
JH,NH = 3.8 Hz, JH ,NH = 9.0 Hz, JH,H = 17.7 Hz),
0
0
7.23–7.26 (m, 2H, H4,5 enediyne aromatic ring),
7.29–7.32, 7.37–7.39 (m, 2H, H3,6 enediyne aromatic
ring).
(5S)-5-(2-methylpropyl)-benz-4,7-diazacyclododeca-1,9-
diyn-6-one (6c) Yield: 61 %. Colorless oil. Rf 0.52
(EtOAc–petrol ether 1:1). [a]D ?72.0° (c 0.5 MeOH). Mr
13
280.16. C NMR (CDCl3): d = 21.9, 23.1 (dd0 Leu), 25.1
(c Leu), 30.2 (C1 propargyl), 40.4 (b Leu), 43.6 (C10
propargyl), 63.9 (a Leu), 82.4, 83.9 (C3,30 propargyl), 91.0,
92.6 (C2,20 propargyl), 124.8, 127.8 (C1,2 enediyne aro-
matic ring), 127.6, 127.7, 128.7, 131.1 (C3,4,5,6 enediyne
aromatic ring), 176.5 (CO Leu). 1H NMR (CDCl3):
3
NHaLys, JNH,a = 7.9 Hz), 6.69 (br t, 1H, NHeLys), 7.71 (t,
3
1H, NHd-Gly, JNH,d = 6.0 Hz).
Synthesis of amino acid derivatives 8
3
d = 0.94, 0.96 (d, 6H, dd0 Leu, Jc,d = 6.6 Hz), 1.37–1.48
(m, 2H, c Leu), 1.57–1.77 (m, 2H, b Leu), 3.09–3.19 (m,
Compound 7 (0.094 mmol) and NMM (0.094 mmol) were
dissolved in 2 mL of dry DMF and cooled down to 0 °C.
Isobutyl chloroformate (0.057 mmol) was added and the
reaction mixture was stirred for 10 min at 0 °C. Compound
6 (0.047 mmol) was added and the solution was stirred for
30 min at 0 °C and then at room temperature overnight.
The solvent was evaporated and the product purified by
flash chromatography.
2
0
1H, a Leu), 3.61, 4.05 (d, 2H, H1 propargyl, JH,H
=
2
17.1 Hz), 3.89, 4.51 (dd, 2H, H10 propargyl, JH,H
=
0
3
3
0
17.6 Hz, JH,NH = 3.5 Hz, JH ,NH = 9.4 Hz), 7.17–7.36
(m, 4H, H3,4,5,6 enediyne aromatic ring), 7.54–7.63 (m,
1H, NHa).
(5S)-5-benzylbenz-4,7-diazacyclododeca-1,9-diyn-6-one (6d)
Yield: 68 %. Colorless oil. Rf 0.44 (petrol ether–EtOAc
1:1). [a]D ?46.0° (c 0.5 MeOH). Mr 314.38. 13C NMR
(CDCl3): d = 30.4 (C1 propargyl), 40.3, 40.8 (C10 prop-
argyl, b Phe), 66.8 (a Phe), 82.8, 84.5 (C2,20 propargyl),
90.9, 92.9 (C3,30 propargyl), 125.0, 128.0 (C1,2 enediyne
aromatic ring), 127.3, 127.9, 127.9, 128.5, 128.9, 129.1,
(5S)-5-methyl-4-[N-tert-butyloxycarbonyl-L-lysyl(N-tert-
butyloxycarbonyl)-d-glycyl]-benz-4,7-diazacyclododeca-
1,9-diyn-6-one (8a) Yield: 32 %. Colorless oil. Rf 0.28
(EtOAc–petrol ether 3:1). Mr 665.82. 13C NMR (CDCl3):
d = 13.4 (b Ala), 21.9 (b d-Gly), 22.9 (c Lys), (CH3 Boca,
123

ˇ
M. Gredicak et al.
CH3 Boce), 28.7 (b Lys), 29.8 (c d-Gly), 30.4 (C1 prop-
argyl), 32.3 (d Lys), 33.1 (C10 propargyl), 33.4 (a d-Gly),
39.1 (d d-Gly), 40.2 (e Lys), 50.3 (a Ala), 54.7 (a Lys),
79.1, 80.0 (C Boca, C Boce), 82.0, 82.4 (C2,20 propargyl),
88.4, 90.9 (C3,30 propargyl), 125.9, 126.3 (C1,2 enediyne
aromatic ring), 127.7, 128.3, 129.4, 130.8 (C3,4,5,6 ened-
iyne aromatic ring), 155.8, 156.2 (CO Boca, CO Boce),
propargyl), 126.1, 126.3 (C1,2 enediyne aromatic ring),
127.6, 128.2, 129.4, 130.7 (C3,4,5,6 enediyne aromatic
ring), 171.9, 175.2, 176.7 (CO Leu, CO d-Gly, CO Lys). 1H
NMR (CDCl3): 0.86, 0.95 (d, 6H, dd0 Leu, 3Jc,d = 6.7 Hz),
1.43, 1.44 (s, 18H, CH3 Boca, CH3 Boce), 1.46–1.82 (m,
11H, b,c Leu, b,c Lys, b,c d-Gly), 2.26–2.57, 2.64–2.74
(m, 4H, a d-Gly, d Lys), 3.05–3.24 (m, 4H, d d-Gly, e Lys),
1
3
170.6, 172.15, 174.9 (CO Ala, CO Lys, CO d-Gly). H
3.68, 4.59 (d, 2H, H1 propargyl, JH,NH = 4.4 Hz,
3
3
2
0
0
NMR (CDCl3): d = 1.35 (d, 3H, b Ala, Ja,b = 7.1 Hz),
JH ,NH = 8.4 Hz, JH,H = 17.6 Hz), 3.98 (br s, 1H, a
1.42, 1.43 (s, 18H, CH3 Boca, CH3 Boce), 1.48–1.85 (m,
8H, b,c d-Gly, b,c Lys), 2.43–2.73 (m, 4H, a d-Gly, d Lys),
3.04–3.14 (m, 2H, d d-Gly), 3.16–3.24 (m, 2H, e Lys),
Lys), 4.23–4.32 (m, 2H, H10 propargyl), 5.12 (br s, 1H,
NHaLys), 5.24 (t, 1H, a Leu, 3Ja,b = 7.3 Hz), 6.31 (br s, 1H,
NHeLys), 6.87 (br s, 1H, NHamide), 7.20–7.36 (m, 4H,
H3,4,5,6 enediyne aromatic ring).
3
3.69, 4.59 (dd, 2H, H1 propargyl, JH,NH = 4.5 Hz,
3
2
0
0
JH ,NH = 8.1 Hz, JH,H = 17.7 Hz), 3.94–4.06 (m, 1H, a
2
Lys), 4.27, 4.35 (d, 2H, H10 propargyl, JH,H = 18,9 Hz),
4.66–4.75 (m, 1H, NHdGly), 5.21 (d, 1H, NHaLys,
3Ja,NH = 8,1 Hz), 5.33 (q, 1H, a Ala, 3Ja,b = 7.1 Hz), 6.44
(br s, 1H, NHeLys), 7.0 (br s, 1H, NHamide), 7.18–7.39 (m,
4H, H3,4,5,6 enediyne aromatic rings).
Synthesis of amino acid derivatives 9
0
Compound 8 was dissolved in 1 mL TFA–H2O 9:1 and
stirred for 1 h at room temperature. Solvent was evaporated
and the product was purified on HPLC. Residual TFA was
removed by flushed through a silicagel filled column (20 %
NH3 in EtOAc was used as an eleunt).
(5S)-5-isopropyl-4-[N-tert-butyloxycarbonyl-L-lysyl(N-tert-
butyloxycarbonyl)-d-glycyl]-benz-4,7-diazacyclododeca-1,
9-diyn-6-one (8b) Yield: 29 %. Colorless oil. Rf 0.37
(EtOAc–petrol ether 3:1). Mr 693.87. 13C NMR (CDCl3):
d = 18.2, 19.7 (cc0 Val), 21.7 (b d-Gly), 22.7 (c Lys), 28.3,
28.4 (CH3 Boca, CH3 Boce), 28.8 (b Lys), 29.4 (c d-Gly),
29.7 (C1 propargyl), 30.2 (b Val), 31.9 (d Lys), 32.7 (C10
propargyl), 33.9 (a d-Gly), 37.1 (d d-Gly), 38.8 (e Lys),
54.5 (a Lys), 61.3 (a Val), 78.5, 79.1 (C Boca, C Boce),
81.7, 82.5 (C2,20 propargyl), 88.7, 90.8 (C3,30 propargyl),
125.6, 126.2 (C1,2 enediyne aromatic ring), 127.6, 128.2,
129.5, 130.7 (C3,4,5,6 enediyne aromatic ring), 171.9,
172.4, 173.2 (CO Val, CO d-Gly, CO Lys). 1H NMR
(5S)-5-methyl-4-(L-lysyl-d-glycyl)-benz-4,7-diazacyclodod-
eca-1,9-diyn-6-one (9a) Yield: 43 %. RT 10.7 min (40 %
MeOH in 0.1 % TFA). Purity 98.7 %. HRMS (MALDI):
m/z [M?H]? calcd for C26H35N5O3, 466.2813; found:
1
466.2816. Mr 465.59. H NMR (DMSO): d = 1.27–1.68
(m, 11H, b Ala, b,c d-Gly, b,c Lys), 2.71–2.78 (m, 4H, d
Lys, a d-Gly), 3.30 (a Lys, d d-Gly—under the HOD signal
from DMSO), 3.61–3.68 (m, 2H, e Lys), 3.94 (s, 2H, H1
2
propargyl), 4.26, 4.52 (d, 2H, H10 propargyl, JH,H
=
0
17.6 Hz), 4.36–4.45 (m, 1H, a Lys), 4.80 (q, 1H, a Ala,
3Ja,b = 7.4 Hz), 7.36–7.51 (m, 4H, H3,4,5,6 enediyne
aromatic ring).
3
(CDCl3): d = 0.81, 1.01 (d, 6H, cc0 Val, Jb,c = 7,1 Hz,
3Jb,c = 6.4 Hz), 1.45, 1.46 (s, 18H, CH3 Boca, CH3 Boce),
1.49–1.85 (m, 8H, b,c d-Gly, b,c Lys), 2.29–2.79 (m, 6H, a
d-Gly, d Lys, b Val), 3.07–3.26 (m, 4H, d d-Gly, e Lys),
3.63–3.76, 4.55–4.81 (m, 2H, H1 propargyl, a Val),
3.94–4.07 (m, 1H, a Lys), 4.27–4.35 (br s, 2H, H10 prop-
argyl), 5.14 (br s, 1H, NHaLys), 6.34 (br s, 1H, NHeLys),
6.81 (br s, 1H, 1H, NHamide), 7.22–7.75 (m, 4H, H3,4,5,6
enediyne aromatic ring).
(5S)-5-isopropyl-4-(L-lysyl-d-glycyl)-benz-4,7-diazacyclod-
odeca-1,9-diyn-6-one (9b) Yield: 30 %. RT 14.3 min
(40 % MeOH in 0.1 % TFA). Purity 96.9 %. HRMS
(MALDI): m/z [M?H]? calcd for C28H39N5O3, 494.3125;
found: 494.3128. Mr 493.64. 1H NMR (CD3OD):
d = 0.86–0.93 (m, 6H, cc0 Val), 1.41–1.65 (m, 8H, b,c d-
Gly, b,c Lys), 2.24–2.36 (m, 6H, a d-Gly, d Lys, b Val),
3.17–3.19 (m, 1H, a Lys), 3.28–3.35, 3.40–3.43 (m, 4H, d
d-Gly, e Lys), 3.62–3.66, 4.53–4.55 (m, 2H, H1 propargyl),
4.04–4.08 (m, 1H, a Val), 4.19–4.22 (m, 2H, H10 propar-
gyl), 7.60–7.63 (m, 2H, H4,5 enediyne aromatic ring),
7.70–7.73 (m, 2H, H3,6 enediyne aromatic ring).
(5S)-5-isobutyl-4-[N-tert-butyloxycarbonyl-L-lysyl(N-tert-
butyloxycarbonyl)-d-glycyl]-benz-4,7-diazacyclododeca-
1,9-diyn-6-one (8c) Yield: 31 %. Colorless oil. Rf 0.47
(EtOAc–petrol ether 3:1). Mr 707.90. 13C NMR (CDCl3):
d = 21.7 (b d-Gly), 22.2, 22.9 (dd0 Leu), 22.7 (c Lys), 24.8
(c Leu), 28.3, 28.4 (CH3 Boca, CH3 Boce), 28.8 (b Lys),
29.4 (c d-Gly), 30.2 (C1 propargyl), 31.9 (d Lys), 32.8 (C10
propargyl), 33.5 (a d-Gly), 37.1 (d d-Gly), 38.8 (e Lys),
39.9 (b Leu), 52.9, 54.6 (a Lys, a Leu), 79.2, 79.3 (C Boca,
C Boce), 81.9, 82.5 (C2,20 propargyl), 88.4, 90.9 (C3,30
(5S)-5-isobutyl-4-(L-lysyl-d-glycyl)-benz-4,7-diazacyclodo-
deca-1,9-diyn-6-one (9c) Yield: 33 %. RT 13.3 min
(43.5 % MeOH in 0.1 % TFA). Purity 97 %. HRMS
(MALDI): m/z [M?H]? calcd for C29H41N5O3, 508.3282;
found: 508.3288. Mr 507.67. 1H NMR (CD3OD): d = 0.92,
123

Cyclic enediyne–amino acid chimeras
3
0.96 (d, 6H, dd0 Leu, Jc,d = 6.7 Hz), 1.35–1.90 (m, 11H,
Synthesis of (5S)-5-methyl-4-(L-lysyl)-benz-4,7-
diazacyclododeca-1,9-diyn-6-one (12)
b,c Leu, b,c Lys, b,c d-Gly), 2.25–2.34 (m, 4H, a d-Gly, d
Lys), 2.91–2.97 (m, 1H, a Lys), 3.13–3.26 (m, 4H, e Lys, d
d-Gly), 3.78, 4.46 (d, 2H, H1 propargyl, 2JH,H = 17.6 Hz),
0
Compound 11 (16 mg, 0.028 mmol) was dissolved in 1 mL
TFA–H2O 9:1 and stirred for 1h at room temperature.
Solvent was evaporated and the product was purified on
HPLC in 25 % MeOH. Isolated product was dissolved in
20 % NH3 in EtOAc and flushed through silicagel filled
column.
4.19–4.23 (m, 1H, a Leu), 4.42 (s, 2H, H10 propargyl),
7.24–7.40 (m, 4H, H3,4,5,6 enediyne aromatic ring).
Synthesis of H–L-Lys-d-Gly-OH (10)
Yield: 87 %. Colorless oil. RT 33.7 min (25 % MeOH in
0.1 % TFA). Purity 96.6 %. HRMS (MALDI): m/z
[M?H]? calcd for C21H27N4O2, 367.4645; found:
367.4640. Mr 366.46. 13C NMR (CD3OD): d = 11.9 (b
Ala), 21.7 (c Lys), 26.4 (d Lys), 29.9, 30.6, 33.2 (C1,10
propargyl, b Lys), 38.6 (e Lys), 50.9, 51.4 (a Ala, a Lys),
81.9, 83.2 (C2,20 propargyl), 88.2, 90.4 (C3,30 propargyl),
124.8, 125.4 (C1,2 enediyne aromatic ring), 128.6, 129.2,
129.7, 131.3 (C3,4,5,6 enediyne aromatic ring), 170.8,
171.4 (CO Ala, CO Lys). 1H NMR (CD3OD): d = 1.29 (d,
Compound 7 (20 mg, 0.045 mmol) was dissolved in 1 mL
TFA–H2O 9:1 and stirred for 1h at room temperature.
Solvent was evaporated and the product is purified by
HPLC. Residual TFA was removed on a Dowex 1 9 2 200
(Ac) column (water was used as an eluent).
Yield: 36 %. RT 8.2 min (35 % MeOH in 0.1 % TFA).
Purity 98.2 %. HRMS (MALDI): m/z [M?H]? calcd for
C11H23N3O3, 246.3259; found: 246.3257. Mr 245.32. 13C
NMR (CD3OD): d = 23.1 (b d-Gly), 24.4 (c Lys), 28.3 (d
Lys), 30.0 (c d-Gly), 32.6 (a d-Gly), 36.9 (b Lys), 40.4,
3
1
3H, b Ala, Ja,b = 6.7 Hz), 1.58–1.72, 2.37–2.62 (m, 6H,
40.5 (d d-Gly, e Lys), 54.6 (a Lys). H NMR (CD3OD):
3
b,c,d Lys), 2.94 (t, 1H, a Lys, Ja,b = 7.1 Hz), 3.84, 4.29
d = 1.43–1.51, 1.53–1.66, 1.67–1.74, 1.80–1.92 (m, 10H,
(d, 2H, H1 propargyl, 2JH,H = 18.1 Hz), 4.41, 4.58 (d, 2H,
b,c d-Gly, b,c,d Lys), 2.24 (t, 2H, a d-Gly, 3Ja,b = 7.4 Hz),
0
2
3
H10 propargyl, JH,H = 19.6 Hz), 5.29 (q, 1H, a Ala,
0
2.94 (t, 2H, d Lys, Jc,d = 7.5 Hz), 3.29–3.32 (m, 2H, d
3Ja,b = 6.7 Hz), 7.28–7.50 (m, 4H, H3,4,5,6 enediyne
aromatic ring).
3
d-Gly), 3.80 (t, 1H, a Lys, Ja,b = 6.4 Hz).
Synthesis of (5S)-5-methyl-4-[N-tert-butyloxycarbonyl-L-
lysyl(N-tert-butyloxycarbonyl)]-benz-4,7-diazacyclodo-
deca-1,9-diyn-6-one (11)
Results and discussion
Design of enediyne-based APN inhibitors
Boc-L-Lys(Boc)-OH (23 mg, 0.066 mmol) and NMM
(16 lL, 0.132 mmol) were dissolved in dry DMF and
cooled down to 0 °C. Isobutyl chloroformate (10 lL,
0.073) was added and the reaction mixture was stirred for
10 min at 0 °C. Compound 6a (10 mg, 0.055 mmol) was
added and the solution was stirred for 30 min at 0 °C and
then at room temperature overnight. The solvent was
evaporated and the product purified by flash chromatog-
raphy in EtOAc–petrol ether 2:1.
Crystal structure of the APN-bestatin complex revealed
extended conformation of the inhibitor with pharmaco-
phores occupying S1 and S10 pocket (phenyl and isobutyl
unit, respectively) placed away from each other (Ito et al.
2006). Having in mind this conformation, we designed
enediyne–peptide chimeras 9 with general structure pre-
sented in Fig. 1A. Enediyne moiety is embedded within the
12-membered ring, and can be considered as amino acid
N?C bridging unit. Having in mind affinity of APN
(Ala[Phe[Leu[Arg), hydrophobic amino acids alanine,
valine, leucine and phenylalanine were used as carriers of
the enediyne moiety. Secondary amino group is bound
through a flexible linker to the lysine residue. Binding to
the S1 pocket is targeted with the aromatic ring of the
enediyne moiety, while hydrophobic amino acid side chain
aims the S10 pocket. Rough MM2 calculations show that
the distance between the enediyne aromatic ring and the
Yield: 26 %. Colorless oil. Rf 0.43 (EtOAc–petrol ether
2:1). [a]D ?54.0° (c 1.0 MeOH). Mr 566.69. 13C NMR
(CDCl3): d = 15.5 (b Ala), 23.1 (c Lys), 29.6 (d Lys),
28.3, 28.5 (CH3 Boca, CH3 Boce), 30.2 (C1 propargyl),
33.2 (C10 propargyl), 32.4 (b Lys), 40.1 (e Lys), 50.4, 54.4
(a Lys, a Ala), 81.1, 81.2 (C Boca, C Boce), 82.1, 83.1
(C2,20 propargyl), 89.3, 90.8 (C3,30 propargyl), 125.8, 126.
2 (C1,2 enediyne aromatic ring), 127.9, 128.4, 129.6, 130.7
(C3,4,5,6 enediyne aromatic ring), 155.6, 156.9 (CO
Boca, CO Boce), 168.8, 170.3 (CO Lys, CO Ala). 1H NMR
(CDCl3): d = 1.39–1.47 (m, 21H, b Ala, CH3 Boca,
CH3 Boce), 1.54–1.89 (m, 6H, b,c,d Lys), 3.01–3.18 (m,
2H, e Lys), 3.57–5.38 (m, 6H, a Lys, a Ala, H1,10 prop-
argyl), 7.13–7.39 (m, 4H, H3,4,5,6 enediyne aromatic
ring).
˚
amino acid side chains is around 8 A (derivative 9c,
Fig. 1B), while the distance between two bestatin chro-
˚
mophores in the active site of APN is 8.4 A (Fig. 1C).
These calculations also pointed two carbonyl groups
towards the same direction aiming to coordinate Zn2? ion.
123

ˇ
M. Gredicak et al.
Fig. 1 A Chemical structures
of designed peptide-based
enediyne derivatives;
B optimized structure of 9c
obtained by the MM2
calculation; C bestatin
(A)
NH2
O
conformation in the APN active
site (Ito et al. 2006)
H
N
N
H2N
N
H
O
O
R
(B)
9
a
:-CH3 (Ala)
b:-CH(CH3)2 (Val)
R=
8Å
c:-CH2CH(CH3)2 (Leu)
d:-CH2C6H5 (Phe)
(C)
8.4 Å
Synthesis
The rigidity of the enediyne-containing ring ensures
extended-like conformation, but requires introduction of
free NH2 group for the interaction with the glutamic resi-
due in the vicinity of S1 pocket; we opted for the L-lysine
as an amino group donor. It has already been shown that
presence of carbonyl group capable of Zn2? coordination,
as well as the free amino group in L-lysine-derived com-
pounds, contributes to the APN inhibition (Wang et al.
2008). Additional flexible linker was placed between
enediyne ‘‘head’’ and the lysine residue thus to enable
closer interaction of the lysine amino group with acidic
binding site of the APN.
The key step in the synthesis of 12-membered enediyne
rings 6 is intramolecular N-alkylation of C-terminal mod-
ified amino acids Ala, Val, Leu and Phe (Scheme 1).
ˇ
Amino acid derivatives 1 (Gredicak et al. 2008) were
submitted to two consecutive Sonogashira couplings (So-
nogashira et al. 1975) that result in moderate yields of 2
and 3. When aliphatic reactant(s) are involved, the Sono-
gashira coupling must be performed under rigorous reac-
tion conditions and generally results in lower yields
ˇ
´
(Gredicak and Jeric 2009). The typical procedure includes
halogen arene and terminal alkyne mixed with piperidine,
Pd(PPh3)4 and CuI in THF under the argon atmosphere.
Compound 4 was obtained in satisfying yields as a result of
the SN2 substitution, utilizing PBr3 in THF. The closing of
the 12-membered ring 5 was performed by intramolecular
N-alkylation reaction in DMF with K2CO3. It is worth
noting that we tried to obtain 5 directly from 3 using
Mitsunobu reaction conditions (Mitsunobu and Yamada
1967), but only traces of 5 were obtained. The last step was
the removal of the oNbs protection group with PhSH and
K2CO3.
Known feature of enediyne-related compounds is ther-
mally or photochemically triggered Bergman cyclization
producing diradical species and leading to the DNA
cleavage (Smith and Nicolaou 1996) or protein degradation
(Fouad et al. 2005). The 12-membered ring comprising
enediyne moiety is expected to be stable toward the
Bergman cyclization owing to the large distance between
terminal acetylene carbon atoms (cd distance). It was
˚
shown that cyclic enediynes cd = 3.2–3.3 A undergo
cyclization at room temperature (10-membered and some
˚
11-membered rings), while enediynes with cd [ 3.7 A
require elevated temperatures for the cyclization (Nicolaou
Stability of prepared macrocycles 6 toward the Bergman
cyclization was tested at this stage by the temperature-
dependent FT-IR spectroscopy, an established method for
simple and rapid monitoring of thermally induced physical
et al. 1992). MM2 calculations showed that the cd dis-
˚
tances in compounds 9 are around 4.2 A; therefore, the
Bergman cyclization is not expected under the conditions
applied in this work.
ˇ
and chemical rearrangements (Gredicak et al. 2010).
123

Cyclic enediyne–amino acid chimeras
Scheme 1 (i) 1,2-diiodobenzene (1.2 eqv.), piperidine (2 eqv.),
Pd(PPh3)4 (0.02 eqv.), CuI (0.1 eqv.) THF; (ii) propargyl alcohol (3
eqv.), piperidine (2 eqv.), Pd(PPh3)4 (0.02 eqv.), CuI (0.1 eqv.) THF;
(iii) PBr3 (2 eqv.), THF; (iv) K2CO3 (2 eqv.), DMF; (v) PhSH (2 eqv.),
K2CO3 (2 eqv.), DMF
APN inhibition test
Compounds were heated up to 240 °C without any changes
observed in FT-IR spectra, proving stability of the
12-membered enediyne rings.
Enediyne macrocycles 6a–d and dipeptide mimic 10 were
submitted to the APN inhibition experiments to see whe-
ther two structural elements alone can inhibit the enzyme.
The inhibition experiments with derivatives 6 revealed that
only valine-based macrocycle 6b caused 30 % inhibition at
150 lM concentration. Dipeptide 10 was not inhibitory at
200 lM concentration and at 1 mM it lowered the enzyme
activity only moderately (25.3 7.5 %). The inhibition
experiments conducted with enediyne conjugates 9 showed
diverging results. The most prominent inhibitor was the
alanine-based derivative 9a, manifesting an IC50 value at
33.7 11.4 lM concentration. Valine-based derivative
9b, in spite of causing some inhibition as a tailless mac-
rocycle 6b, did not show any inhibition whatsoever, even at
400 lM concentration, and similar result was obtained
with the leucine-based derivative 9c. Clearly, conjugation
of the alanine-related enediyne ‘‘head’’ with L-lysine
through a flexible linker fulfilled basic structural and con-
formational requirements for the APN inhibition. In addi-
tion, we examined the role of flexible linker in positioning
of 9a into the APN active site, and prepared compound 12
With enediyne macrocycles 6 in hands, final deriva-
tives 9 with incorporated L-lysine have been prepared
(Scheme 2). Boc-protected L-lysine was coupled with
5-aminopentoic acid by a mixed anhydride method to
provide 7. The same method was applied for the coupling
of 7 with 6 to give 8 in moderate yields, while 8d could
not be obtained. Although the obtained yields are con-
sidered satisfactory with regard to secondary amine as a
participant, several other coupling methods were tried,
focusing on 6d. The BOP/HOBt, HATU and DCC/HOSu
methods provided lower yields of the compounds 8,
while 8d could not be obtained by any method used,
most likely due to the sterical hindrances of the phen-
ylalanine aromatic ring over nitrogen. Final compounds 9
were obtained by Boc deprotection in acidic conditions.
Following the same procedure, compound 10 was pre-
pared from 7. The reason for the synthesis of 10 is to
exclude that eventually observed inhibition stems only
from the ‘‘dipeptide’’ part (L-lysine ? flexible linker) of
the molecule.
Scheme 2 (i) ClOCOiBu (1.2
eqv.), NMM (2 eqv.),
5-aminopentoic acid (1.2 eqv.);
(ii) 6 (1.2 eqv.), ClOCOiBu (1.2
eqv.), NMM (2 eqv.); (iii) TFA–
H2O 9:1 (v/v)
123

ˇ
M. Gredicak et al.
and provide interactions with a-amino group and charged
groups of the Lys and Arg side chains. This combination of
hydrophobic and hydrophilic binding surfaces accounts for
the ability of enzyme to cleave Lys, Arg, Phe, and Tyr.
Drag et al. have shown, by examining a library of 61
natural and unnatural amino acids substrates, that the most
favored amino acids in mammalian APN synthetic (fluo-
rogenic) substrates have rather large hydrophobic side
chains, indicating an open S1 pocket (Drag et al. 2010).
These authors revealed a large and hydrophobic character
for the S1 pocket of mammalian (human, pig and rat) APN
(Drag et al. 2010).
160
140
120
100
80
60
40
20
Considering structure of enediyne–peptide conjugates
presented in this work, S1 pocket can accommodate either
hydrophobic aromatic part of enediyne ‘‘heads’’ or hydro-
carbon part of the lysine side chain. Obviously, the
hydrocarbon linker contributes to the binding into the APN
active site, since the compound 12 was much less potent,
compared to the 9a. It can be speculated that presence of
linker allowed interaction of free NH2 group with glutamic
acid side chain of the APN active site. Alternatively, the e-
amino group of the lysine side chain could contact with
polar top of the S1 subsite (Addlagatta et al. 2008). Since
we did not observe the hydrolytic cleavage of Lys residue,
upon APN incubation with 9a, we speculate that the
enzyme interaction with the e-amino group of the lysine
side chain, and accommodation of enediyne ‘‘head’’ in S1
subsite, is more likely.
0
0
1
2
3
4
5
6
7
8
9
10 11
t (min)
Fig. 2 Hydrolysis rate plots of Leu-2NA cleavage by APN in the
absence (filled square) or in the presence (filled circle) of compound
9a
(Scheme 2) with L-lysine directly bound to the secondary
amine of the alanine-related enediyne ‘‘head’’. Linkerless
derivative 12 was found to be far less active towards APN
than the corresponding 9a, with IC50 value above 1 mM.
To verify if the inhibition by 9a is maintained over the
APN assay period (10 min), the enzyme activity was fol-
lowed continuously (Fig. 2). The activity decline was
observed in the presence of 9a from the beginning till the
end of measurement of enzyme reaction.
Finally, it was important to compare our results with
other known APN inhibitors. Different classes of com-
pounds have been prepared and tested as potential inhibi-
tors of the APN, but also of some other zinc-dependent
metallopeptidases, like leucine aminopeptidase (Drag et al.
2005) and matrix metalloproteinases-2 (MMP-2) (Wang
et al. 2008). Peptidomimetics containing 3-galloylamido-
N0-substituted-2,6-piperidinedione-N-acetamide, showed
high inhibitory activity against MMP-2 and low activity
against APN, except two derivatives with IC50 values of
3.1 and 5.2 lM (Li et al. 2007). A series of peptidomimetic
L-iso-glutamine derivatives exhibited selective inhibition
against APN with IC50 values in lM range (Li et al. 2009).
A broad comparison study using phosphinate inhibitors
yielded a potent APN inhibitor with an IC50 about 60 nM
(Grzywa et al. 2010). Amino-benzosuberone derivatives, a
low molecular weight, nonpeptidic compounds are among
the most active APN inhibitors found so far, with inhibitory
constant in picomolar range (Maiereanu et al. 2011).
Regarding derivatives of L-lysine, synthesized compounds
were inferior APN inhibitors compared with MMP-2 and
only 1 compound out of 20 showed inhibition activity
comparable with bestatin (Wang et al. 2008). Other com-
pounds were less active (IC50 values higher than 100 lM)
and fall into the same range as compounds considered here.
Since lysine residue could be recognized at the P1
position of the APN substrates, susceptibility of the most
active enediyne macrocycle 9a and the dipeptide mimic 10
to the hydrolysis by the APN was examined. Results
obtained by the TLC and the HPLC–MS analysis revealed
that both compounds are stable toward the enzymatic
hydrolysis. Moreover, presence of only 9a in the reaction
mixture incubated with enzyme confirms that structural
properties of the enediyne conjugate and not chemical
reaction of the enediyne moiety are responsible for the
observed activity. These findings are in accordance with
previously published data on the recognition role of
enediyne-related compounds as enzyme inhibitors (Lin
et al. 2001; Dutta et al. 2009; Chandra et al. 2011).
Addlagatta et al. (2008) investigated the structural basis
for the unusual specificity of E. coli APN. This enzyme
cleaves adjacent to the nonpolar amino acids Phe and Tyr
but also next to the polar residues Lys and Arg. They
determined the structure of the APN in complex with dif-
ferent amino acids (Addlagatta et al. 2008). The authors
found that hydrophobic walls of the S1 pocket accommo-
date nonpolar or largely nonpolar side chains of Phe and
Tyr, but also hydrocarbon part of the polar Lys and Arg
side chains. The bottom and top of the S1 pocket are polar
123

Cyclic enediyne–amino acid chimeras
enkephalin-degrading enzymes: a class of central analgesics.
Proc Natl Acad Sci USA 95:12028–12033
Cheng XC, Wang Q, Fang H, Tang W, Xu WF (2008) Design,
synthesis and evaluation of novel sulfonyl pyrrolidinederivatives
as matrix metalloproteinase inhibitors. Bioorg Med Chem
16:5398–5404
Preliminary results on small number of enediyne–pep-
tide conjugates presented in this work, point toward
enediyne–amino acid chimeras as new pharmacophore for
the APN S1/S10 pockets and identified enediyne macro-
cycle 9a as the most promising candidate for further studies
(IC50 value 33.7 11.4 lM). To approach efficiency of
the most active APN inhibitors, in the second generation of
enediyne–peptide conjugates, emphasis will be given to the
modification of linker length and introduction of better
zinc-binding moieties.
Drag M, Grembecka J, Pawełczak M, Kafarski
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Acknowledgments This research has been supported by the Cro-
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