Synthesis, antimycobacterial activity and influence on mycobacterial InhA and PknB of 12-membered cyclodepsipeptides

Article abstract of DOI:10.1016/j.bmc.2018.04.045

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at μM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 μg/ml) and did not show any significant antiproliferative effect (HUVEC > 5 μg/ml; K-562 > 5 μg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.

Full text of DOI:10.1016/j.bmc.2018.04.045

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antimycobacterial activity and influence on mycobacterial
InhA and PknB of 12-membered cyclodepsipeptides
Katja Laqua a, Marcel Klemm a, Melissa Richard-Greenblatt b, Adrian Richter a, Linda Liebe a,
Tingting Huang c, Shuangjun Lin c, Ana Guardia d, Esther Pérez-Herran d, Lluís Ballell d, Yossef Av-Gay b,
Peter Imming a,
⇑
a Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany
b Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, British Columbia V6H 3Z6, Canada
c State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao
Tong University, Shanghai 200030, PR China
d Diseases of the Developing World, Alternative Discovery & Development, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have
antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series
of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within
the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis
and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were
inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB).
PknB inhibitors were active at mM concentration against mycobacteria while inducers were inactive. PknB
regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the ser-
ies against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the
parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 mg/
ml) and did not show any significant antiproliferative effect (HUVEC > 5 mg/ml; K-562 > 5 mg/ml). Within
the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identi-
fied, but the data suggest interaction with PknB or other kinases may partly cause the activity.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 3 March 2018
Revised 16 April 2018
Accepted 20 April 2018
Available online xxxx
Keywords:
Cyclodepsipeptides
Antimycobacterial agent
PknB
InhA
Tuberculosis
1. Introduction
cordycommunin, enniatins, griselimycins, nocardithiocin, pyrido-
mycin, sansanmycins, trichoderins and wollamides.3–9 For many
Tuberculosis (TB) remains the primary cause of death due to a
single infectious agent worldwide. An additional challenge in the
global control of TB is the emergence and spread of drug resistant
strains. The increasing prevalence of multi (MDR) and extensively
(XDR) Mycobacterium tuberculosis (Mtb) strains necessitate the
development of antitubercular drugs with a distinct mode of action
or novel mode of bindings to a known target. Bedaquiline and dela-
manid follow these criteria and were recently incorporated into
current treatment plans for TB, the first since the 1970s. However,
the FDA has restricted their use to patients that have failed stan-
dard treatment. To date, only a limited number of compounds with
novel modes of action are in clinical trials.1,2
of them, the molecular target was not identified yet. There is hope
that they inhibit Mtb growth through hitherto untapped inhibition
mechanisms. We focused on the synthesis of a (cyclo)depsipeptide
medium-sized ring system with twelve atoms, comparing activity
with their open-chain precursors. The ring system was devised fol-
lowing the long-known7 pyridomycin structure.
2. Results and discussion
2.1. Cyclodepsipeptidic structures
Peptides, depsipeptides and cyclo(depsi)peptides have recently
shown early antitubercular promise, among them calpinactam,
The 12-membered ring system of 22a/b, 23a/b, 24 DS1 as well
as 31 and 38 (Schemes 1–3) was synthesized starting from
benzaldehyde (1) and methyl 4-nitrobutyrate (2). A nitro aldol
condensation afforded the (E)-nitrostyrene of 1-methoxy-4-nitro-
5-phenylpent-4-en-1-ol as the main product (3) (Scheme 1).
⇑
Corresponding author.
E-mail address: peter.imming@pharmazie.uni-halle.de (P. Imming).
https://doi.org/10.1016/j.bmc.2018.04.045
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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2
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
O
O
O
O2N
H
O
i
+
O
NO2
1
2
3 (73 %)
O
O
iii
OH
ii
O
+
NH2
HN
NO2
4 (93 %)
6 (20 %)
5 (70 %)
O
iv
O
v
OH
+
Cl-
NH3
7 (69 %)
O
O
Cl-
+
NH3
8b (80 %)
Scheme 1. Synthesis of the 4-amino-5-phenylpentanoic acid followed by benzylation. Reaction conditions: i) n-butylamine, toluene, 140 °C, 6 h; ii) NaBH4, dioxan, EtOH, 90
min; iii) Pd/C, H2, MeOH, 20 bar, RT, 24 h, c ꢀ 0.12 M; iv) 25% HCl, reflux, 18 h; v) SOCl2, BnOH, 0 °C ? reflux, 4 h.
t-Boc
OH
t-Boc
O
HN
HN
O
+H3N
Cl-
Bn
H
HO
HO
N
Bn
i
O
+
O
R1
O
O
R1
9
10 : R1 = H (98 %)
11 : R1 = Bn (87 %)
8a : R1 = H (55 %)
8b : R1 = Bn
t-Boc
R2
HN
O
H
ii
R4
O
N
R3
O
O
O
O
R1
R2
Bn
O
13a : R1 = R2 = H; R3 = R4 = Bn (70 %)
O
13b : R1 = H; R2 = CH3; R3 = R4 = Bn (58 %)
14a : R1 = R3 = R4 = Bn; R2 = H (48 %)
14b : R1 = R3 = R4 = Bn; R2 = CH3 (63 %)
OH
12a : R2 = H (63 %)
12b : R2 = CH3
iii
15a : R1 = R3 = R2 = R4 = H (97 %)
15b : R1 = R3 = R4 = H; R2 = CH3 (93 %)
16a : R1 = Bn; R2 = R3 = R4 = H (78 %)
16b : R1 = Bn; R2 = CH3; R3 = R4 = H (79 %)
17 : R1 = Bn; R2 = R3 = CH3; R4 = H (86 %)
Scheme 2. Chain prolongation to the desired depsipeptidic intermediates. Reaction conditions: i) PyBOP, DIPEA, DCM, RT, 16 h; ii) DIPEA, DCM, ꢁ20 °C, 15 min, PyBOP, RT, 5–
18 h; iii) Pd/C, H2, MeOH or i-prop, 5 bar, RT, 24 h.
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
N
R3
t-Boc
NH
O
O
NH
O
O
ii
i
15a/b;
16a/b
R1
NH
O
O
O
R1
NH
O
O
O
O
9
9
R3
R2
R2
18a : R1 = R2 = H (20 %)
22a : R1 = R2 = R3 = H (42 %)
N
CO2H
18b : R1 = H; R2 = CH3 (26 %)
19 DS1: R1 = Bn; R2 = H (20 %)
19 DS2: R1 = Bn; R2 = H (21 %)
20 DS1: R1 = Bn; R2 = CH3 (27 %)
20 DS2: R1 = Bn; R2 = CH3 (17 %)
22b : R1 = R2 = H; R3 = OH (5 %)
23a : R1 = R3 = H; R2 = CH3 (48 %)
23b : R1 = H; R2 = CH3; R3 = OH (28 %)
24 DS1: R1 = Bn; R2 = H; R3 = OH (49 %)
21a : R3 = H
21b : R3 = OH
Scheme 3. Final ring closure and subsequent coupling with picolinic acid and 3-hydroxypicolinic acid. Reaction conditions: i) DIPEA, DCM, ꢁ20 °C, 15 min, PyBOP, RT,
overnight or 3 d; ii) 1. TFA/DCM, 0 °C, 5 min; 2. DIPEA, PyBOP, DMF, RT, 4 d.
N-Butylamine in toluene at 140 °C gave 3 without the concomi-
tant formation of a Henry reaction intermediate, viz. the 5-meth-
oxy-2-nitro-1-phenylpentane-1,5-diol.10 Direct reduction of 3 to
afford 5 and 6 in ethanolic or methanolic solutions caused unde-
sired side products, especially the addition of ethanol or methanol
to the carbon-carbon double bond,11 or the formation of an oxime.
Further hydrogenolysis of the oxime failed. Therefore the reduction
of 3 was performed in two steps, starting with the reduction of the
carbon-carbon double bond with sodium borohydride, followed by
catalytic hydrogenolysis of the nitro group. The hydrogenolysis of 3
to give 4 needed addition of 3 to a suspension of sodium borohy-
dride in a mixture of dioxane and ethanol while maintaining low
concentration of 3 in the reaction medium.12 The use of a mixture
of protic and aprotic solvents (ethanol and dioxane) afforded excel-
lent yields. Dioxane proved to be the best solvent for dissolving
nitrostyrenes (3) and precipitation of the simultaneously formed
19 DS2, and 20 DS1 and 20 DS2 (Schemes 2 and 3). Ring-closures
needed to be performed at concentrations below 1 mM to prevent
the formation of oligomers and polymers as intermolecular side
products. The diastereomeric mixtures of 19 and 20 were sepa-
rated to give the corresponding diastereomers (19 DS1 and 19
DS2; 20 DS1 and 20 DS2). The configuration of the stereochemical
center at C-9 was assigned later by stereoconservative synthesis
(see 3.4 below).
With the synthesis of the scaffold established, we introduced a
few exocyclic variations. The t-Boc group had deliberately been
chosen to allow for this. Quite a few cyclodepsipeptidic antibiotics
bear 3-hydroxypicolinic acid (3-HPA) exocyclically coupled to
threonine, suggesting it may have importance for antimicrobial
activity.17–21
Consequently, the last step in the synthetic sequence consisted
in the removal of the t-Boc group and sequential attachment of
picolinic acid (21a) and 3-hydroxypicolinic acid (21b) to yield
22a/b, 23a/b and 24 DS1 (Scheme 3). Further substitution of 19
DS2 with 3-hydroxypicolinic acid failed; however, 19 DS1 yielded
24 DS1. The yield total of the acylations varied considerably which
was due to the fact that in some cases, the deacylation took longer,
favoring competitive reactions leading to unidentified, probably
oligomeric side products.
For the antimycobacterial evaluation, compound 17 was
included as an open-chain methyl ester analogue of compounds
19 and 20. We decided to include this motif so as to compare directly
how the t-Boc protected ring systems 19 and 20 and the open-chain
system 17 performed in antimycobacterial tests. The esterification
was accomplished by catalytic hydrogenolysis of the depsipeptide
14b to give 17 as a diastereomeric mixture in excellent yield.
nitronate salt.12 Hydrogenolysis of
4 afforded 4-amino-5-
phenylpentanoic acid (6) and the corresponding 5-benzylpyrro-
lidin-2-one (5). With isopropanol instead of methanol, ester forma-
tion was avoided and the lactam 5 obtained quantitatively.
Reactant concentration proved to be a simple but crucial parame-
ter towards formation of 6 (see Experimental Part for details).
Hydrolysis of 5 afforded 7.13 The amine 6 and its hydrochloride 7
were converted to the benzyl ester 8b.14 Compounds 6, 7 and 8b
were prepared and used as racemic mixtures.
The
5-oxo-1-phenylpentan-2-aminium chloride (8b) were coupled
with N-Boc- -Thr-OH (9) to give the dipeptides 10 and 11
c
-aminobutyric acid benzyl ester14 (8a) and 5-(benzyloxy)-
L
(Scheme 2). Benzotriazol-1-yl-oxytripyrrolidinophosphonium hex-
afluorophosphate (PyBOP) in dichloromethane proved to be the
best coupling reagent even for this amidation step.15 Subsequently,
10 and 11 were coupled with glycolic acid derivatives, again using
PyBOP. Compound 10 was esterified with benzyloxy acetic acid
(12a) to obtain 13a and with the commercially available benzyloxy
propionic acid (12b) to form compound 13b. The depsipeptide 11
was esterified with 12a to afford 14a in good yield as well as with
12b to yield compound 14b. Benzyloxy acetic acid (12a) was syn-
thesized following a literature procedure.16 The resulting dep-
sipeptides (13a/b, 14a/b) were hydrogenated which led to the
cleavage of both benzyl protecting groups to give 15a/b and 16a/
b, followed by ring-closure with PyBOP to give 18a/b, 19 DS1 and
2.1.1. Cyclodepsipeptides with exocyclic alkyl side chains
With variation at position 6 accessible and exemplified by a few
target molecules, we further varied the twelve-membered ring sys-
tem and its periphery, working from and with readily accessible
building blocks (Scheme 4): (1) The ester group at position C-12
was exchanged for an amide moiety. (2) Pregabalin as an easily
accessible c-aminobutyric acid derived building block was incor-
porated. (3) An isobutyl group was put in position C-2. Variations
(1) and (2) addressed the left and right hand ‘‘corners” (the way
the formulae are depicted here). The synthesis was initiated by
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4
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
O
O
i
H
N
H
+
O
N
O
t-Boc
OH
H2N
Bn
t-Boc
N
H
Bn
O
O
25
26 (quanꢀtaꢀve)
27 (97 %)
t-Boc
HO
NH
O
ii
H
N
O
N
H
R1
O
O
t-Boc
HO
28 : R1 = Bn (90 %)
29 : R1 = H (quanꢀtaꢀve)
NH
iii
OH
O
N
9
t-Boc
NH
O
O
O
O
NH
O
iv
v
NH
O
O
NH
O
O
N
H
COOH
N
H
N
21a
30
31 (16 %)
Scheme 4. Synthesis of cyclodepsipeptides 30 and 31. Reaction conditions: i) DIPEA, PyBOP, DCM, RT, 5 h; ii) 1. TFA/DCM, RT, 1 h; 2. DIPEA, PyBOP, DCM, RT, 3 h; iii) Pd/C, H2,
MeOH, 5 bar, RT, 6 h; iv) DCM, ꢁ20 °C, DIPEA, PyBOP, 15 min, RT, 5 h; v) 1. TFA/DCM, RT, 5 min; 2. DIPEA, PyBOP, DMF, 37 °C, 4 d.
coupling t-Boc-pregabalin (25) to the benzyl ester of
L-isoleucine
2.2. Open-chain depsipeptidic structures
(26) to yield compound 27 (Scheme 4). The benzyl ester of
L
-isoleu-
cine was formed quantitatively.22 Following removal of the t-Boc
A ring system may be of importance for the binding of a ligand
to its target, viz. if conformational and configurational constraints
imposed by the ring create a molecular shape that will ideally fit
into the binding site. However, the concomitant loss of flexibility
may reduce affinity, especially in cases of mutually induced fit.
Higher flexibility could also lead to (favourable) high koff values.24
Since little is known about the binding modes and kinetics of
(cyclo)depsipeptide-target interaction, we included open-chain
analogues in our study (Scheme 6). The tripeptides 13a and 13b,
intermediates for depsipeptide synthesis, were converted into
picolinic (39, 43), 3-hydroxypicolinic (40, 44), salicylic (42, 46)
and 3-fluoropyridine-2-carboxylic acid derivatives (41, 45).
protecting group, the amine intermediate was coupled directly
with N-Boc- -Thr-OH (9). The resulting tripeptide 28 was smoothly
L
deprotected via catalytic hydrogenolysis to give 29 quantitatively.
The ring closure to the depsipeptide 30 needed both DIPEA as aux-
iliary base and PyBOP as coupling reagent. Compound 30 was fur-
ther substituted with picolinic acid to yield 31.
2.1.2. Cyclodepsipeptides with an endocyclic phenyl group
The derivatives described so far did not bear any planar group
endocyclically. A double bond or a phenyl group may result in
highly constrained and/or planar cycles. To introduce more rigidity
in the 12-membered ring system, we again used a readily accessi-
ble building block (a salicylic acid derivative) to give a benzo-
2.3. Stereoselective synthesis of compound 19 DS1
annulated macrocycle. Scheme
5 delineates the synthesis of
cyclodepsipeptides 37 and 38.
The synthetic pathway described above had yielded, after chro-
matographic separation, the diastereomers 19 DS1, 19 DS2 and 24
DS1. However, the absolute configurations could not be assigned
via X-ray diffractometry because the compounds did not form
suitable crystals. Circular dichroism (CD) spectra indeed gave
mirror images for the individual members of pairs of opposite
enantiomers. However, the simulation of the CD spectra of the
compounds 19 DS1, DS2 and 24 DS1 did not yield unequivocal
results. Consequently, the stereoselective synthesis of compound
19 seemed to be the best approach. For the synthesis of a single
diastereomer of compound 19, the building block 8b was prepared
The benzyl ester of b-alanin23 (32) was coupled with 9 using
DCC as coupling reagent. Again using DCC in a Steglich esterifica-
tion, the dipeptide 33 was coupled with benzyloxybenzoic acid
(34) (In this case, PyBOP did not yield the desired products.).
Subsequent hydrogenolysis of 35 afforded 36 quantitatively. For
the final cyclization, compound 36 was diluted in DCM to a con-
centration of less than 1 mM. The depsipeptide 37 did form, but
in low yield. Following removal of the t-Boc protecting group, the
intermediate free amine was directly acylated with 3-hydroxypi-
colinic acid (21b), yielding the 12-membered ring 38 (53%).
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
t-Boc
t-Boc
HO
NH
NH
O
H
N
HO
O
OH
Bn
O
i
Cl- +H3N
O
Bn
+
O
O
O
33 (46 %)
32 (90 %)
9
O
O
R1
O
N
O
ii
H
HN
O
t-Boc
O
R1
OH
35 : R1 = Bn (61 %)
36 : R1 = H (43 %)
iii
O
N
OH
Bn
34
t-Boc
HN
HN
O
O
O
iv
v
NH
O
O
O
NH
O
O
OH
O
O
O
N
COOH
37 (15 %)
21b
38 (53 %)
Scheme 5. Synthesis of a 12-membered ring system with endocyclic phenyl group. Reaction conditions: i) DCC, DCM, RT, 5 h; ii) DMAP, DCC, DCM, RT, overnight; iii) Pd/C, H2,
i-prop, 5 bar, RT, 5 h; iv) DIPEA, DCM, ꢁ20 °C, 30 min, PyBOP, RT, 5 d; v) 1. TFA/DCM, 0 °C, 30 min; 2. DIPEA, PyBOP, DMF, RT, 3 d.
X
R2
t-Boc
R1
HN
O
R1
HN
O
O
H
N
H
Bn
O
Bn
Bn
O
N
Bn
O
O
O
O
i
O
O
O
O
13a : R1 = H
13b : R1 = CH3
39-46
39: X = N; R1 = H; R2 = H (45 %)
40: X = N; R1 = H; R2 = OH (16 %)
41: X = N; R1 = H; R2 = F (26 %)
42: X = C; R1 = H; R2 = OH (6 %)
43: X = N; R1 = CH3; R2 = H (52 %)
44: X = N; R1 = CH3; R2 = OH (50 %)
45: X = N; R1 = CH3; R2 = F (48 %)
46: X = C; R1 = CH3; R2 = OH (20 %)
X
R2
COOH
21a : X = N; R2 = H
21b : X = N; R2 = OH
21c : X = N; R2 = F
21d : X = C; R2 = OH
Scheme 6. Synthesis of several open-chain derivatives. Reaction conditions: i) 1. TFA/DCM, 0 °C, 1 h; 2. PyBOP, DCM, DIPEA, RT, overnight.
following a stereoconservative synthetic route.25–27 The starting
materials N-Boc- -Phe-OH (47) and Meldrum’s acid (48) were com-
mercially available and the synthetic route is illustrated in
Scheme 7.
First, Meldrum’s acid (47) was acylated with N-Boc- -Phe-OH
L
L
(52) using EDC and DMAP in DCM at 0 °C. Reduction of the ketone
(49) with sodium borohydride yielded compound 50. Subsequent
refluxing of 50 in toluene for 4 h afforded lactam 51 quantitatively
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
t-Boc
t-Boc
O
O
t-Boc
O
O
O
NH
NH
NH
i
ii
+
O
O
O
O
O
O
O
OH
O
O
50 (24 %)
49 (quanꢀtaꢀve)
47
48
NH3+CF3COO-
O
t-Boc
t-Boc
t-Boc
NH
NH
N
OBn
iii
iv
v
vi
OH
OBn
O
O
O
54 (quanꢀtaꢀve)
52 (71 %)
53 (68 %)
51 (quanꢀtaꢀve)
Scheme 7. Stereoselective synthesis of the building block (2R)-5-(benzyloxy)-5-oxo-1-phenylpentan-2-aminium trifluoroacetate (54). Reaction conditions: i) EDC-HCl,
DMAP, DCM, 0 °C ? RT, 16 h; ii) NaBH4, DCM, CH3COOH, 0 °C, 12 h; iii) toluene, reflux, 4 h; iv) acetone, 1 M NaOH, 30 min; v) BnBr, Na2CO3, DMF, 0 °C, 12 h; vi) TFA/DCM, RT,
1 h.
t-Boc
t-Boc
OH
NH3+CF3COO-
HN
O
HN
H
OBn
i
N
+
OBn
O
OH
O
OH
O
55
56 (63 %)
9
t-Boc
HN
O
H
ii
O
N
BnO
OBn
O
O
57 (92 %)
O
BnO
OH
12a
t-Boc
t-Boc
HN
O
HN
O
O
H
O
N
iv
iii
HO
OH
NH
O
O
O
O
O
58 (85 %)
63 = 19 DS1 (64 %)
Scheme 8. Preparation of the enantiopure ring system 63 (= 19 DS1). Reaction conditions: i) PyBOP, DIPEA, DCM, RT, 16 h; ii) DMAP, EDC-HCl, DCM, 0 °C, 2 h, RT, 16 h; iii) Pd/
C, H2, i-PrOH, 4 bar, RT, 24 h; iv) DIPEA, DCM, ꢁ20 °C, 15 min, PyBOP, RT, 3 d.
that could be used without further purification. Hydrolysis of 51
led to the desired -amino acid 52 as a white solid with a yield
of 71%. The amino acid 52 was converted directly to the benzyl
ester 53 using benzyl bromide and sodium carbonate. Final
removal of the t-Boc protecting group was carried out with
trifluoroacetic acid in DCM yielding the salt 54.
The
c
-aminocarboxylate 55 was coupled with N-Boc- -Thr-OH
L
c
(9) to give peptide 56 (Scheme 8). The subsequent esterification
of 56 with 12a was optimized via replacement of PyBOP by a com-
bination of EDC-HCl and DMAP simplifying the reaction conditions
and increasing the yield to 92% for compound 57 compared to 48%
for 14a. The depsipeptide 57 was hydrogenated, cleaving both
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
Fig. 1. Circular dichroism spectra of diastereomers 19 DS1 and 19 DS2 and of 63, the product of the stereoselective synthesis.
Table 1
2.5. In vitro MIC determination against M. tuberculosis and smegmatis
Antimycobacterial activity determined by minimal inhibitory concentration of
depsipeptides.
For the active compounds of the agar diffusion assay, the mini-
mal inhibitory concentration (MIC) values were determined using
M. smegmatis and M. tuberculosis as test organisms (Table 1).
Compound 19 DS1 was the most active diastereomer with a
MIC value of 3.1 mg/ml against M. smegmatis and 1.7 mg/ml against
M. tuberculosis. Its activity suggests that a lipophilic side-chain on
N-6 conveys antimycobacterial activity while a side-chain incorpo-
rating a heterocyclic system is not an absolute requirement in this
position. In comparison to 19 DS1, its diastereomer 19 DS2 showed
lower activity (MICM. smeg. = 12.5 mg/ml; MICM. tb. = 3.5 mg/ml). The
diastereomers vary at the stereochemical centre at C-9 only. Com-
pound 24 DS1, containing the exocyclic 3-hydroxypicolinyl residue
typical of several antibacterial compounds (see 3.1, above), showed
better activity against M. smegmatis (MIC = 6.3 mg/ml) and M. tuber-
culosis (MIC = 3.6 mg/ml) than 19 DS2 but with lower activity than
the non-acylated depsipeptide 19 DS1. Though compound 20 had
been inactive in the agar diffusion assay, the MIC for M. tuberculosis
was determined and found to be high (56.1 mg/ml).
Since we used phenotypic screens with isolated mycobacteria,
activity at this point is a summary effect of cell penetration,
ligand-target binding and possible metabolism of the test com-
pounds by the mycobacteria. Structure-activity conclusions have
to take all factors into account. In contrast to the protected open-
chain tripeptide 17, the direct precursors of the depsipeptides 19
and 20 (16a and 16b) possessed polar functional groups such as a
free carboxylic acid. However, no inhibitory activity was observed.
With the given very limited number of active and inactive ana-
logues, structure-activity relationships (SAR) can only be derived in
a very tentative way. However, the following conclusions appear to
be justified and useful to guide further studies:
1.) Since the (hydroxy)picolinic acid side chain did not improve
activity in this small series, simply a lipophilic acyl substituent
may be sufficient in this position. 2.) All open-chain derivatives
were inactive, so a 12-membered ring system is obviously the
starting point of choice for further activity improvement. 3.) The
open-chain molecules possess increased polarity (except 17) which
usually leads to poorer membrane permeability compared to their
ring-closed analogues. Thus, sufficient lipophilicity is important.
4.) The introduction of an endocyclic phenyl moiety to increase
planarity and reduce conformational flexibility of the ring system
resulted in no activity, indicating that either a particular topology
or some flexibility, perhaps induced fit, is essential. 5.) Replace-
ment (38) or displacement (31) of the ring ester group did not
induce activity. This could be caused by either loss of essential
hydrogen bonds to the target or different conformations of the
cyclopeptide, indicating a specific target rather than unspecific
interference with mycobacterial membrane integrity. 6.) The lack
Compound
M. smegmatis SG987*)
M. tuberculosis H37Rv
mg/ml
mM
mg/ml
mM
19 DS1
19 DS2
20 DS1
20 DS2
24 DS1
Isoniazid
3.1
7.2
1.7
4
12.5
n.d.
n.d.
6.3
28.8
n.d.
n.d.
13.7
29.2
3.5
8
56.1
56.1
3.6
125
125
8
430
0.04
0.31
*)
n.d. = not determined; The initial read-out of the MIC against M. smegmatis was
carried out by fluorescence after a 48 h-incubation.
benzyl protecting groups (58), followed by the final macrolac-
tonization with PyBOP. The desired ring system 63 which proved
to be 19 DS1 was obtained with 64% yield.
The CD spectrum of 19 DS1 was characterised by a negative
Cotton effect (CE) at around 215 nm, whereas 19 DS2 displayed a
positive CE at around 219 nm (Fig. 1). The final product of the
stereoselective synthesis 63 showed the same CD profile as 19
DS1. So both possess the same configuration at all positions, and
19 DS1 has R configuration at C-9.
2.4. Diffusion assay determination of antibacterial activity
For the evaluation of antimycobacterial and antimicrobial activ-
ities, the depsipeptides (19 DS1, 19 DS2, 20 DS1, 20 DS2, 22a/b,
23a/b, 24 DS1, 31, 37, 38) and open-chain analogues (17, 39–46)
were subjected to a standard agar diffusion antimicrobial sensitiv-
ity test (agar diffusion assay).28,29 Mycobacterium smegmatis,
Mycobacterium vaccae and Mycobacterium aurum, Gram-positive
and Gram-negative bacteria (Escherichia coli and Bacillus subtilis)
and the yeast Sporobolomyces salmonicolor were used for antimi-
crobial evaluations. The activity was expressed as diameter of zone
of inhibition in comparison to zone of inhibition of positive con-
trols ciprofloxacin and amphotericin B. None of the compounds
showed activity against the non-mycobacterial Gram-positive
and Gram-negative bacteria and yeast (data not shown).
In contrast, compounds 19 DS1, 19 DS2 and 24 DS1 showed
good antimycobacterial activity against M. smegmatis and M. vac-
cae. The incorporation of a phenylmethylene moiety at C-9 gave
rise to active compounds against different mycobacterial strains.
In contrast, the addition of one methyl group at position C-2 had
a dramatic negative effect on antimycobacterial activity. Both the
ring system 20 and its open-chain precursor 17 showed no activity.
The large effect of a small structural change suggests that some
specific binding is necessary for activity rather than a more general
molecular interaction.
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8
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 2
Minimal inhibitory concentrations [mM] of cyclodepsipeptides for a Mycobacterium bovis overexpressing InhA.
Compound
M. tuberculosis H37Rv
M. bovis BCG:pATB45 (WT)a
M. bovis BCG:pATB45::inhA (OE)
Ratio (MIC of BCG:pATB45::inhA/MIC of BCG:pATB45)
19 DS1
19 DS2
24 DS1
Isoniazid
4
8
8
0.31
7.8
7.8
1.0
2.0
1.0
15.6
62.5
62.5
0.8
125
62.5
12.5
a
The BCG:pATB45 strain represents the control strain with an incorporation of the promoter region for inhA overexpression but without the enzyme gene InhA included. In
contrast, the mutant BCG:pATP::inhA overexpressor strain includes the promoter region as well as InhA.
of a benzyl group at position C-9 caused inactivity. 7.) The active
diastereomers bearing the benzyl motif at C-9 showed different
MICs, suggesting a crucial impact of the relative configuration at
position C-9 on antimycobacterial activity. 8.) The introduction of
a single methyl group had negative effects on the inhibition of
mycobacterial growth. With a ring size of twelve, bordering on
medium-sized rings with pronounced transannular interaction,
even a small methyl group may alter the ring conformation consid-
erably, leading to marked changes in activity. The fact that the
introduction of just a methyl group indeed had this consequence,
together with entries 6 and 7 is a strong indication of a specific
molecular mechanism of action meriting further investigation.
may contribute to the observed bacteriostatic effect of these com-
pounds. Interesting, an earlier study identified Streptomyces 85E
aerial hyphae formation to be sensitive to inhibition by protein
kinase inhibitors.33 Using this strain in an assay to identify novel
kinase inhibitors, the authors discovered pyridomycin as one of
several compounds able to inhibit aerial hyphae formation. Similar
to Streptomyces, mycobacteria are also known to contain eukary-
otic-like protein kinases34 and pyridomycin was also found to
interfere with the growth of various mycobacterial species.31,33
Consequently, we hypothesized that Mtb eukaryotic-like serine/
threonine protein kinases (STPKs) could be potential targets of
the three cyclodepsipeptides.
STPK regulation of Mtb growth occurs mainly through the
kinases PknA and PknB.35 Recent studies of the PknB homolog in
Streptomyces (AfsK) was found to regulate polar growth and
hyphal branching.36 As Waters et al.33 had previously observed
pyridomycin to inhibit hyphae formation, it became of interest to
investigate Mtb PknB as a target of cyclodepsipeptides. Using an
in vitro kinase assay, we monitored the influence of several dep-
sipeptides on PknB autophosphorylation as subsequent transpho-
rylation of substrates is dependent on PknB autophosphorylation
(Fig. 2).37–39 Interestingly, the three compounds that displayed
the greatest antimycobacterial activity in our MIC assays all
demonstrated an inhibitory effect on PknB activity (Fig. 2A). Both
19 DS1 and 19 DS2 decreased PknB autophosphorylation by
approximately 60% at the highest concentration tested (4.8 mM)
whereas 24 DS1 only achieved a 35% reduction in activity. As
observed in the MIC assay, 20 DS1 had minimal and tripeptide
17 had no effect on bacterial growth. These results are in accor-
dance with our findings that PknB autophosphorylation was unaf-
fected by tripeptide 17 at the tested concentrations; however, 20
DS1 showed an approximate 60% increase in autophosphorylation
activity at 4.8 mM. Pyridomycin was additionally examined in the
assay, and as expected had no influence PknB autophosphorylation
activity. The findings suggest that inhibition of PknB by cyclodep-
sipeptides is detrimental to mycobacterial growth meanwhile
enhanced autophosphorylation activity has no effect under the
assay conditions.
2.6. InhA-overexpressor assay
In a preliminary search for molecular targets, we first investi-
gated InhA, an enoyl-acyl carrier protein reductase that is involved
in Mtb cell wall biosynthesis, as it had previously been shown to
be the target of a related cyclodepsipeptide.31 To assess InhA as a
direct molecular target of our active compounds, we evaluated
whether overexpression of the inhA gene caused an increase in resis-
tance to the compounds in wild-type M. bovis BCG. Therefore, the
ratio of the MIC values against BCG:pATB45 (wild-type, WT) and
BCG:pATP::inhA (overexpressor, OE) was determined and a MIC
shift (OE/WT) would strongly suggest InhA as the molecular target.
Overexpression of InhA demonstrated little (19 DS2) to no impact
(19 DS1 and 24 DS1) on the MIC values (Table 2) of our three leading
antimycobacterial compounds. However, the antituberculosis drug
isoniazid, that is known to target InhA32, as expected, showed an
approximate 16 fold increase in M. bovis BCG resistance suggesting
that InhA is an unlikely target of the tested cyclodepsipeptides.
2.7. PknB autophosphorylation activity is modulated by depsipeptides
in vitro
As the results from the InhA overexpressor assay indicated that
InhA is unlikely to be the direct molecular target for 19 DS1, 19
DS2, or 24 DS1, we further explored alternative mechanisms that
Fig. 2. Influence of depsipeptides on PknB autophosphorylation activity. A, In vitro kinase assay monitoring the dose dependent effect of several depsipeptides on PknB
autophosphorylation activity. M. tuberculosis PknB purified as GST fusion from E. coli was incubated with each depsipeptide for 30 min prior to the addition of 10 mCi [
32P]
c
-
ATP for another 30 min. Reactions were separated by 12% SDS-PAGE and stained with Coomassie Blue or silver stain followed by autoradiography. B, Graphical representation
of the percent change in PknB autophosphorylation activity at varying concentrations (0–4.8 mM) of depsipeptides with antimycobacterial activity. Error bars represent the
mean S.E. of three independent experiments.
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
The MICs of 19 DS1, 19 DS2 and 24 DS1 did not directly trans-
late to the PknB inhibitory concentrations as observed in the kinase
assay. Generally, inhibitory concentrations for enzymatic assays
are lower than MICs due to the direct interaction of the compound
with its target and the absence of a complex cell system. However,
inhibition of PknB phosphorylation was not directly correlated to
the determined MICs. Lower MICs compared to PknB IC50 values
were previously observed for the PknB inhibitor IMB-YH-8 which
was found to have an IC50 of 20 mM which significantly greater than
that of its MIC for Mtb (1.14 mM).40 These and our results might be
explained by the essentiality of PknB in Mtb growth both in vitro
and in vivo, and the observation that stringent regulation of PknB
expression is further necessary for cell growth. Depletion or over-
expression of PknB alters cell morphology eventually leading to
cell death.41,42 Further analysis of this PknB conditional knock-
out mutant transformed with plasmids expressing the kinase-dead
PknB-K40M mutant demonstrated an inability to recover growth,
underlining the importance of PknB kinase activity in mediating
cell survival. Accordingly, it is plausible that despite an incomplete
loss of PknB autophosphorylation activity, 19 DS1, 19 DS2 and 24
DS1 could induce a bacteriocidal effect at the concentrations
observed in the MIC assays.42,43
Since tight regulation of PknB activity is essential for the survival
of Mtb, it could also be rationalized that the observed increase in
PknB autophosphorylation by 20 DS1 would also have a bacterioci-
dal effect. However, a pknB mutant expressing approx. 4.5-fold more
PknB than wildtype Mtb in aerated culture demonstrated similar
growth kinetics.43 As the addition of 4.8 mM of 20 DS1 in our kinase
assay was only able to induce an approximate 2-fold increase in
PknB autophosphorylation it is unlikely that the compound would
have any impact on Mtb growth at these concentrations. The dose
dependent increase of PknB autophosphorylation suggests that
higher concentrations of 20 DS1 could significantly enhance kinase
activity and result in killing at the concentrations observed in the
MIC. Future studies are needed to support the notion that higher
concentrations (125 mM) of 20 DS1 can further alter PknB autophos-
phorylation and catalytic activity.
comparison of Mtb PknB with eukaryotic kinases was found to
have <30% sequence similarity.48 Despite limited homology, we
performed a preliminary analysis of the cytotoxic and antiprolifer-
ative effects of 19 DS1, 19 DS2 and 24 DS1. All tested compounds
did not exhibit any cytotoxic effects on HeLa cells or antiprolifera-
tive activity on HUVEC or K-562 cells up to a concentration of CC50
and GI50 > 5 mg/ml (data not shown).
To further confirm the absence of any cytotoxic effects or
antiproliferative activities at higher concentrations for lead com-
pound 19 DS1, additional tests were performed. No cytotoxic effect
on HeLa cells or antiproliferative activity on HUVEC or K-562 cells
up to a concentration of CC50 and GI50 > 50 mg/ml was observed.
Therefore at the concentrations tested, 19 DS1, 19 DS2 and 24
DS1 appear to be more specific for mycobacterial STPKs and may
have limited off-target effects on human cells in vitro.
3. Conclusion
We established a synthetic pathway towards new (cyclo)dep-
sipeptidic structures. Within the series studied, the 12-membered
ring system was favored for antimycobacterial activity. We identi-
fied a number of compounds which were able to prevent the growth
of Mycobacterium smegmatis and Mycobacterium tuberculosis in the
micromolar range. Unlike pyridomycin and isoniazid, they did not
inhibit the mycobacterial reductase InhA. While the search for their
target is going on, we explored if they impact the autophosphoryla-
tion activity of PknB. We indeed observe the same cyclodepsipep-
tides that exhibited antimycobacterial activity to also inhibit PknB
autophosphorylation. Further investigation of this finding may shed
more light on the equivocal correlation of PknB and antimycobacte-
rial growth inhibition because potent PknB inhibitors were found to
impair40 and not impair47 mycobacterial growth. Findings from this
study identify a novel class of cyclodepsipeptides that may hold pro-
mise for the management of drug resistant and latent TB.
4. Experimental section
Although mild overexpression of PknB does not affect Mtb
growth in aerated culture, Ortega et al.43 discovered that this
mutant exhibited a 10-fold decrease in survival under hypoxic con-
ditions. These findings are of particular interest in light of our
observations that 20 DS1 is able to enhance PknB autophoshphory-
lation. Several studies have demonstrated that hypoxia is able to
arrest Mtb growth in vitro43,44 and that Mtb dormancy is associated
with the oxygen limited environment of the granuloma.45,46 It is
bacteria in this dormant-like, non-replicating state that are respon-
sible for Mtb persistence and reactivation of the disease. As there
are currently a limited number of antimycobacterial therapies that
are able to target this subpopulation of Mtb, MIC assays examining
20 DS1 under hypoxic conditions should be further assessed.
Despite the observation that specific cyclodepsipeptides are
able to modulate the level of autophosphorylation of PknB, further
studies are warranted to support PknB as a target of these com-
pounds. Furthermore, if these compounds are indeed kinase inhibi-
tors, studies regarding specificity for other Mtb STPKs as well as
eukaryote kinases are necessary. Previous in vitro studies examin-
ing PknB inhibitors discovered cross-reactivity with other Mtb
STPKs40,47 and if also true for our cyclodepsipeptides, this may also
contribute to the increased sensitivity of Mtb to 19 DS1, 19 DS2
and 24 DS1 in the MIC assay.
4.1. Biological assays
4.1.1. Antimicrobial inhibition assay
For screening of antimycobacterial and antimicrobacterial
activities, the following test organisms were used: Mycobacterium
vaccae IMET 10670 (200 ml), Mycobacterium aurum SB66 (100 ml),
Mycobacterium smegmatis SG987 (100 ml), Bacillus subtilis ATCC
6633 (100 ml), Escherichia coli SG 458 (100 ml), and Sporobolomyces
salmonicolor 549 (400 ml). The agar diffusion test was used for qual-
itative determination. General methods were described in litera-
ture.49,50 The test organisms (appropriate volume of test
organisms is noted above, McFarland standard no. 0.5 Biomerieux)
were suspended in a 34 ml melted agar medium (Müller-Hinton-
Bouillon) and poured into petri dishes. The test substances were
dissolved in DMSO and diluted with methanol in a ratio of 1:9.
After cooling, 9-mm holes were cut out and 50 ml of the test com-
pounds (concentration of 100 mg/ml) were filled into the holes. The
positive controls used were ciprofloxacin (concentration of 5 mg/
ml), for antimicrobacterial and antimycobacterial tests, and
amphotericin B (concentration of 10 mg/ml) for Sporobolomyces
salmonicolor 549 strain. The bacterial strains were incubated at
37 °C overnight and the yeast at 30 °C overnight. The diameter of
zone of inhibition was evaluated after 24 h.
2.8. Cytotoxicity and antiproliferative effects
4.1.2. Mycobacterium smegmatis inhibition assay
For quantitative conclusions the MIC values were determined
by using a standard microbroth dilution method and the resazurin
microtiter assay.51,52
Mtb STPKs were first described as ‘‘eukaryotic-like” based on
the presence of conserved residues and motifs, known as Hank’s
subdomains, found in the kinase domain of eukaryotes.34 However,
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10
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
The inoculum of M. smegmatis SG987 was adjusted to McFar-
medium. Isoniazid was used as the positive control and DMSO as
blank control. The inoculum (over-expressing strain BCG:
pATB45::InhA, control strain BCG:pATB45) was standardized to
approximately 1 ꢂ 105 CFU/ml in Middlebrook 7H9 broth. 100 ml
of the inoculum was added to the entire, except for the blank con-
trols (columns H7–H12). All plates were placed in a sealed box to
prevent drying out of the peripheral wells, and they were incu-
bated at 37 °C without shaking for 6 days. A resazurin solution
was prepared by dissolving one tablet of resazurin (Resazurin
Tablets for Milk Testing; ref 330884Y, VWR International Ltd.) in
30 ml of sterile phosphate-buffered saline (PBS). Of this solution,
land standard no. 0.5 (Biomerieux) and subsequently standardized
to approximately 1 ꢂ 106 CFU/ml (final concentration of the inocu-
lum was 5 ꢂ 105 CFU/ml). Stock solutions of the test compounds
(1 mg/ml DMSO) were diluted with methanol (initial concentra-
tion: 400 lg/ml). To each well of the 96-well microtiter plates
50 ml Mueller-Hinton broths were added as well as 50 ml of the
drug solutions (row 1, column A–E). Ciprofloxacin and isoniazid
were used as positive controls. 50 ml of the reference compounds
(row 1, column F, final concentration: 100 mg/ml) and 50 ml of the
solvent (row 1–12, column G) were added. Column H was reserved
for growth control. Twelve twofold-dilutions of the test compound
solutions were prepared (from row 1 to 12) starting at 100 mg/ml
(row 1) and ending in 0.05 mg/ml (row 12) as final concentration.
25 ll was added to each well. Fluorescence was measured (Spec-
tramax M5, Molecular Devices, excitation 530 nm, emission 590
nm) after 48 h, to determine the MIC values. For each compound,
the average value of the duplicate samples was calculated.
Sequences of the primers used in this manuscript:
Additionally, 50 ll of inoculum were added to each vial of the
96-well microtiter plate. Evaluation of the inhibition assay was
performed optically after incubation for 48 h and after 72 h
through adding resazurin. 30 ml of a resazurin solution (0.01% in
aq. dest.) were added to each well and the plates incubated for
another 24 h at 37 °C. Fluorescence was detected after 72 h with
a Nephelocan Ascent 1.4 automatic plate reader (Labsystems, Van-
taa, Finland) at k = 630 nm.
PCR 1: (Primer colony 1) 50-AATCCAAAGTTCAAACGAGGGG-30
PCR 2: (Primer colony 2) 50-CCACCACCCGATAAGAGAAAGG-30
4.1.5. PknB autophosphorylation assay
The kinase reaction for measuring the in vitro autophosphoryla-
tion of PknB was prepared in a mixture containing 6.5 ml of PknB
(1.2 mM of PknB for 24 DS1 and 2.4 mM of PknB for 17, 19 DS1,
19 DS2, 20 DS1 and 20 DS2), 6.5 ml of the appropriate concentra-
tion of the cyclodepsipeptides (0.24 mM, 1.2 mM, 2.4 mM, 4.8 mM
and 12 mM), Tris-HCl at pH 7.4 (20 mM), MgCl2 (5 mM), MnCl2 (5
mM) and DTT (1 mM). Pure PknB (2.4 mM) was used as blank con-
trol. The mixture was kept on ice for 30 min for incubation. The
Results are summarized in Table 3.
4.1.3. Mycobacterium tuberculosis inhibition assay
The measurement of the MIC for each tested compound was
performed in sterile 384-well white microtiter plates TC surface
(353988 BC Falcon). To each well 250 nl of the test compound
(10 mM) in neat DMSO (Sigma D8418) was added. Isoniazid was
reactions were initiated through the addition of 10 mCi of [c-
32P]
used as the positive control (0.1 mg/ml). Then 5
l
l of neat DMSO
ATP and incubated at room temperature for 30 min. The kinase
reactions were terminated by the addition of SDS-PAGE sample
loading buffer followed by boiling at 95 °C for 5 min. After separa-
tion of the samples by 12% SDS-PAGE the samples were stained
using Coomassie Blue or silver stain prior to gel drying and visual-
ization by autoradiography. The kinase’s respective bands were
excised from dried gels and the radioactive protein (resp. PknB
activity) was quantified by using a scintillation counter (Beckman).
All reactions were performed in triplicates.
was used as the growth and blank controls (column 6). The inocu-
lum of M. tuberculosis H37Rv was standardized to approximately 1
ꢂ 107 CFU/ml (OD600 = 0.125) and diluted 1 in 100 (105 CFU/ml) in
Middlebrook 7H9 broth (Difco 27310) supplied with 10% ADC (Bec-
ton Dickinson 262710) and 0.025% Tyloxapol (Sigma T8761), to
produce the final inoculum. Then 25 ll of this inoculum was added
to the entire plate, except for the blank controls. Each plate was
sealed with ParafilmTM to prevent evaporation and the plates were
incubated at 37 °C for 7 days. After the incubation period, 10 ml of
reconstituted BacTiter-GloTM Microbial Cell viability Assay (Pro-
mega G8231) reagent was added to each well and incubated for
further 30 min at room temperature. Luminescence was measured
(Spectramax M5, Molecular Devices, integration time 250 ms) and
the effect of a given compound was calculated as% inhibition at sin-
gle shot. MIC determinations for each test compound were per-
formed in triplicate.
4.1.6. Cytotoxicity and antiproliferative effects
General methods for the determination of the cytotoxicity and
the antiproliferative effects were described in literature.53 Cells
of HUVEC (ATCC CRL-1730), K-562 (DSM ACC 10), and HeLa
(DSM ACC 57) were cultured in DMEM (CAMBREX 12-614F), RPMI
1640 (CAMBREX 12-167F), and RPMI 1640 (CAMBREX 12-167F),
respectively. All cells were grown in the appropriate cell culture
medium supplemented with 10 ml/l ultraglutamine 1 (CAMBREX
17-605E/U1), 500 ml/l gentamicin sulfate (CAMBREX 17-518Z),
and 10% heat inactivated fetal bovine serum (PAA A15-144) at
37 °C in high density polyethylene flasks (NUNC 156340). For the
cytotoxic assay, HeLa cells were pre-incubated for 48 h without
the test compounds. The dilutions of the compounds were carried
out carefully on the subconfluent monolayers of HeLa cells after
4.1.4. InhA overexpressor assay
The measurement of the MIC for each tested compound was
performed in 96-well flat-bottomed, polystyrene microtiter plates.
Ten 2-fold drug dilutions in neat DMSO starting at the appropriate
concentration were performed from column 1 to 12. Then, 5
l
l of
these drug solutions were added to 95
ll of Middlebrook 7H9
Table 3
Inhibition of M. smegmatis growth (agar diffusion assay).
Compd
Conc. [
l
g/ml]
Solvent
Diameter of zone of inhibition (ZoI) [mm]
M. smegatis
M. vaccae
M. aurum
SG987
10670
SB66
19 DS1
19 DS2
24 DS1
20 DS1
20 DS2
100
100
100
100
100
DMSO/MeOH
DMSO/MeOH
DMSO/MeOH
DMSO/MeOH
DMSO/MeOH
21(p)
17p
17p
0
24/31(p)
20/26(p)
21/29(p)
0
0
0
0
0
0
0
0
p: a few colonies in wider ZoI.
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
11
the preincubation time. For the antiproliferative assay, the test
substances were dissolved in DMSO before being diluted in DMEM.
The adherent cells were harvested at the logarithmic growth phase
after soft trypsinization, using 0.25% trypsin in PBS containing
0.02% EDTA (Biochrom KG L2163; Biochrom, Berlin, Germany).
For each experiment approx. 10,000 cells were seeded with 0.1
ml culture medium per well of the 96-well microplates (NUNC
167008). The cells (HUVEC, K-562 and HeLa) were incubated with
dilutions of the compounds for 72 h at 37 °C in a humidity atmo-
sphere and 5% CO2. To estimate the influence of chemical com-
pounds on cell proliferation of K-562, the numbers of viable cells
present in multi-well plates via CellTiter-Blue1 assay with resa-
zurin was determined. The signal from the CellTiter-Blue1 reagent
is proportional to the number of viable cells. The adherent HUVEC
and HeLa cells were fixed by glutaraldehyde and stained with a
0.05% solution of methylene blue for 15 min. After gently washing,
the stain was eluted with 0.2 ml of 0.33 M HCl in the wells. The
optical densities (k = 660 nm) were measured in SUNRISE micro-
plate reader (TECAN, Switzerland) and the evaluation of the GI50
and the CC50 values were performed with the software Magellan
(TECAN) (Fig. 3).
Geduran SI 60 (0.063–0.200 mm) silica gel. During hydrogenolyses,
the reaction mixtures were purged with argon before adding Pd/C
before displacing the argon with hydrogen. Again, before removing
the catalyst by filtration, the hydrogen containing solution was
purged with argon.
5.2. Materials
All reagents and solvents used are commercially available. Most
chemicals were purchased from Sigma–Aldrich or VWR. Methanol
(MeOH) and dichloromethane (DCM) were distilled and dried over
activated molecular sieve 4 Å. Dimethylformamide (DMF) and 1,4-
dioxane were dried over activated molecular sieve 4 Å. Chloroform
(CHCl3), methyl tert-butyl ether (MTBE), n-butylamine and N,N-
diisopropylethylamine (DIPEA) were distilled before use. For
hydrogenolysis processes, 96% ethanol (EtOH) was used as an
undenatured solvent and 99% 2-propanol. Pyridomycin was iso-
lated from Streptomyces pyridomyceticus following a published
procedure.27 Compounds 8a,14 12a,16 18a,54 22b,54 31,55 32,23
49,26 50,25 51,25 52,25 and 5356 were prepared following the litera-
ture; their identity was ascertained spectroscopically.
5. Syntheses
5.3. Instrumentation
5.1. General experimental methods
Electrospray ionization mass spectra (ESI-MS) were obtained
using a LCQ-Classic spectrometer of Thermo Finnigan. High resolu-
tion electrospray mass spectra (ESI HR-MS) were recorded with a
Bruker Apex III 70e Fouriertransform ion cyclone resonance spec-
trometer. Positive and negative ionization was performed with a
capillary temperature of 220 °C, a voltage of 4.5 kV, and a scanning
range of 50–2000. All melting points were determined on an elec-
trically heated Boëtius microscope hot stage and are uncorrected.
Organic solvents were concentrated using a Büchi rotary evap-
orator at 38–50 °C under reduced pressure. Analytical thin layer
chromatography (TLC) was performed on ALUGRAMÒ SIL G/UV254
plates (0.20 mm, 60 Å). Visualization was done under a 254 nm
and 365 nm UV light source and iodine vapor. Column chromatog-
raphy was performed on Geduran SI 60 (0.040–0.063 mm) and
Fig. 3. Results of the antiproliferative and cytotoxic analysis for lead compound 19 DS1 on HeLa cells or HUVEC or K-562 cells up to a concentration of CC50 and GI50 > 50 mg/
ml.
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12
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Elemental analyses were measured on a LECO CHNS-932 instru-
A solution of compound 12a (1.41 g, 8.5 mmol) in 4 ml of DCM
was flushed with argon. DIPEA (2.17 ml, 12.8 mmol) was added
and the temperature cooled to ꢁ20 °C in acetone/dry ice bath.
Compound 10 (3.35 g, 8.5 mmol) was dissolved in 10 ml of DCM
and added to the solution. The resulting mixture was stirred at
ꢁ20 °C for 15 min. A solution (kept under argon) of PyBOP (4.42
g, 8.5 mmol) in 4 ml of DCM was injected to the above mixture
with further stirring at ambient temperature for 5 h. The organic
phase was washed with 1 M NaHSO4 (3ꢂ), brine (1ꢂ), 5% NaHCO3
(3ꢂ). The organic layer was dried over MgSO4 and the solvent
removed in vacuo. The yellow residue was purified by flash chro-
matography (CHCl3) to result in compound 13a as colorless syrup
(70%, 3.26 g).
ment. Data are given as percentages and as averages for duplicate
determinations. Hydrogenolysis was performed in autoclaves of
Parr Instruments, Instrumental Company, series 5500 or series
4790.
1H and 13C NMR were recorded at 25 °C on VNMRS 400 MHz
(Agilent Technologies) with 400 MHz operating frequency for 1H
NMR and 100 MHz operating frequency for 13C NMR and INOVA
500 MHz (Varian) spectrometer with 500 MHz operating fre-
quency for 1H NMR and 125 MHz operating frequency for 13C
NMR. Coupling constants are reported in hertz and the chemical
shifts in parts per million (ppm). The data for the 1H NMR are doc-
umented as follows: chemical shift (d, ppm), coupling constant (J,
Hz), integration and multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, qui = quintet, br = broad).
Rf (CHCl3/MeOH 9:1): 0.69; 1H NMR: (CDCl3) d [ppm]: 1.25 (d,
3H, J = 6.25 Hz, –CH3), 1.41 (s, 9H, t-Boc), 1.72–1.79 (m, 2H,
–CH2–CH2–CH2–), 2.32 (m, 2H, –CH2–COO–), 3.11–3.27 (m, 2H,
–NH–CH2–), 4.04 (d, 2H, J = 1.24 Hz, –O–CH2–COO–), 4.27–4.28
(bd, 1H, –CH–CO–NH–), 4.54 (d, 2H, J = 1.24 Hz, –O–CH2–Ar), 5.06
(s, 2H, –COO–CH2–Ar), 5.41–5.45 (m, 1H, CH3–CH–O–), 5.49 (d,
1H, J = 8.7 Hz, –NH), 6.83 (bt, 1H, –NH), 7.26–7.31 (m, 10H, Ar–
H); 13C NMR: (CDCl3) d [ppm]: 16.2, 24.4, 28.3, 31.9, 39.0, 57.6,
66.4, 67.3, 70.7, 73.3, 80.5, 127.99, 128.07, 128.2, 128.3, 128.5,
128.6, 135.8, 137.0, 155.7, 168.8, 169.0, 173.0; ESI-MS: calcd.
542.6206, found 565.04 [M+Na+]+; Anal. calcd. for C29H38N2O8: C
64.19, H 7.06, N 5.16; Found C 63.44, H 7.17, N 4.90.
Circular dichroism was measured on a Jasco J-815-Spectrometer
with a scan speed 50–100 nm/min and bandwidth 1.0 nm. Samples
were dissolved in methanol.
5.3.1. Benzyl 4-(2-{[(tert-butoxy)carbonyl]amino}-3-
hydroxybutanamido)butanoate (10)
t-Boc
HN
O
H
N
HO
5.3.3. Benzyl 4-[(2S,3R)-3-{[(2R)-2-(benzyloxy)propanoyl]oxy}-2-
{[(tert-butoxy)carbonyl]amino}butanamido]butanoate (4b)
O
O
t-Boc
HN
O
H
N
DIPEA (13.0 ml, 76.45 mmol), Boc-
L
-Thr-OH (9) (5.54 g, 25.3
O
mmol) and PyBOP (14.87 g, 28.6 mmol) were mixed together.
Compound 8a (4.89 g, 21.3 mmol) was added and the mixture
was dissolved in 360 ml DCM. The solution was stirred for 16 h
at ambient temperature. The solvent was removed in vacuo and
the residue was taken up with EtOAc. The organic layer was
washed with 1 M KHSO4 (3ꢂ), once with water, 5% NaHCO3 and
brine (3ꢂ). After drying over MgSO4, the organic solvent was evap-
orated under reduced pressure. The light brown residue was puri-
fied by flash chromatography with CHCl3 to give a yellow viscous
product (98%, 8.28 g).
O
O
O
O
(R)-(+)-2-Benzyloxypropionic acid (12b) (458 mg, 2.54 mmol)
was dissolved in 1.2 ml of DCM and DIPEA (648 ml, 3.81 mmol)
was added. The mixture was cooled to ꢁ20 °C under an argon
atmosphere. Compound 10 (1 g, 2.54 mmol) (dissolved in 2.4 ml
DCM) was added to the reaction mixture and the temperature
was kept at ꢁ20 °C. The reaction mixture was stirred for 15 min
at ꢁ20 °C. After addition of PyBOP (1.32 g, 2.54 mmol), the reaction
mixture was allowed to stir overnight with gradual warming to RT.
The mixture was diluted with DCM and washed with 1 M NaHSO4,
5% NaHCO3 and brine. The organic phase was dried over MgSO4
and the solvent removed in vacuo. Subsequent flash chromatogra-
phy with CHCl3 and heptane/EtOAc 7:3 delivered the product (13b)
as white solid (58%, 815 mg).
Rf (CHCl3/MeOH 9:1): 0.6–0.65; 1H NMR (CDCl3) d [ppm]: 1.16
(d, 3H, J = 6.3 Hz, –CH3), 1.44 (s, 9H, t-Boc), 1.82–1.85 (m, 2H,
–CH2–CH2–CH2–), 2.4 (t, 2H, J = 7.3 Hz, –CH2–COO–), 3.2–3.36 (m,
2H, –NH–CH2–), 3.98 (d, 2H, J = 6.9 Hz, –CH–CO–NH–), 4.34 (dq,
1H, J = 6.3/2.1 Hz, CH3–CH–CH–), 5.1 (s, 2H, –COO–CH2–Ar), 5.5
(d, 1H, –NH), 6.78 (bs, 1H, –NH), 7.31–7.37 (m, 10H, Ar–H); 13C
NMR (CDCl3) d [ppm]: 18.3, 24.5, 28.3, 31.5, 38.7, 57.9, 66.4, 66.6,
80.4, 128.2, 128.3, 128.6, 135.8, 156.6, 171.8, 172.9; ESI-MS: calcd
394.462, found 417.06 [M+Na]+; Anal. calcd. for C20H30N2O6: C
60.9; H 7.67; N 7.1; Found: C 60.43, H 7.80, N 7.10.
Rf (CHCl3/MeOH 9:1): 0.86; MP: 104 °C; 1H NMR: (CDCl3) d
[ppm]: 1.2 (d, 3H, J = 6.6 Hz, –CH3), 1.32 (d, 3H, J = 6.6 Hz, –CH3),
1.38 (s, 9H, t-Boc), 1.75 (qui, 2H, J = 7.1 Hz, –CH2–CH2–CH2–), 2.33
(t, 2H, J = 7.3 Hz, –CH2–COO–), 3.1 (m, 1H, –NH–CHH–), 3.26 (m,
1H, –NH–CHH–), 4.03 (q, 1H, J = 6.8 Hz, –O–CH–CH3–), 4.22 (bd,
1H, –NH–CH–CH–), 4.43 (d, 1H, J = 11.6 Hz, –CHH–Ar–), 4.61 (d,
1H, J = 11.7 Hz, –CHH–Ar–), 5.10 (s, 2H, –COO–CH2–Ar–), 5.33 (bd,
1H, NH), 5.45 (m, 1H, CH3–CH–CH–), 6.41 (m, 1H, –NH), 7.31–7.36
(m, 10H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 16.2, 18.6, 24.4, 28.2,
31.5, 38.9, 57.6, 66.4, 70.4, 71.8, 74.2, 80.5, 127.7, 127.9, 128.2,
128.3, 128.48, 128.54, 135.8, 137.4, 155.7, 168.8, 171.8, 172.9; ESI-
MS: calcd. 556.6472, found: 579.14 [M+Na]+; Anal. calcd. for
5.3.2. Benzyl 4-(3-{[2-(benzyloxy)acetyl]oxy}-2-{[(tert-
butoxy)carbonyl]amino}butanamido)butanoate (13a)
t-Boc
HN
O
H
N
O
O
O
O
O
C30H40N2O8: C 64.73, H 7.24, N 5.03; Found C 64.89, H 7.07, N 5.00.
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
13
5.3.4. Methyl-4-(E)-4-nitro-5-phenylpent-4-enoat (3)
5.3.6. 4-Carboxy-1-phenylbutan-2-aminium chloride (7)
O
OH
O
+
Cl-
NH3
H
O
NO2
Compound 4 (560 mg, 2.4 mmol, 0.12 M) was dissolved in 20
ml of MeOH (HPLC grade) and the catalyst Pd over charcoal was
added. To prevent the concomitant formation of the corresponding
lactam, a concentration of c ꢀ 0.12 M has to be ensured. The sus-
pension was transferred to an autoclave and pressurized with H2
(20 bar). The final heterogeneous solution was stirred at RT for
24 h. After completion of the reaction, the catalyst was removed
by filtration through CeliteÒ and the filtrate was concentrated in
vacuo. A small amount of acetone was added to the residual syrup
and the resulting crystals were collected by filtration. The white
solid contained valerianic acid (6) (20%, 95.3 mg) and the filtrate,
the corresponding lactam (5), was a red syrup (quantitative). The
lactam was used for the next step without further purification.27
In a round bottom flask, compound 5 (2.58 g, 14.7 mmol) was
mixed with 98 ml of 25% aq. HCl and heated at 110 °C for 18 h. The
cooled solution was washed with Et2O (3ꢂ) and the acidic water
layer was evaporated under reduced pressure to give a light brown
solid. The residue was recrystallized from acetone and the resulting
solid filtered (69%, 2.32 g, calculated as HCl). (To avoid esterification,
ethanol or methanol should not be used for recrystallization.)
Rf (CHCl3/MeOH 9:1): 0.07; MP: 148–150 °C; 1H NMR: (D2O) d
[ppm]: 1.88–1.93 (m, 2H, –CH2–CH2–), 2.48 (t, 2H, J = 7.5 Hz, –
CH2–COO–), 2.83 (dd, 1H, J = 14.3/8.1 Hz, –CHH–CH–), 3.00 (dd, 1H,
J = 14.3/6.2 Hz, –CHH–CH–), 3.52–3.57 (m, 1H, –CH–NH3–), 7.24–
7.37 (m, 5H, Ar–H); 13C NMR: (D2O) d [ppm]: 27.1; 29.8; 37.9;
52.4; 127.6; 129.1; 129.4; 135.5; 176.9; ESI-MS: calcd. 193.2423,
found 193.99 [M+H+]+; Anal. calcd. for C11H16ClNO2: C 57.52, H
7.02, Cl 15.43, N 6.1; Found: C 57.31, H 6.80, Cl 15.76, N 6.09.57,58
The solution of benzaldehyde (1) (1.64 ml, 16.2 mmol), methyl
4-nitrobutyric acid (2) (2 ml, 16.2 mmol) and n-butylamine (81
ml, 0.05 äq.) in 9 ml of toluene was refluxed for 6 h with a Dean-
Stark trap. The reaction mixture has to reach a temperature of
140 °C. The dark brown residue was taken up with EtOAc and 1
M KHSO4. The aq. layer was extracted with EtOAc (3ꢂ) and the
combined organic layers were washed with brine (1ꢂ), dried over
MgSO4 and concentrated in vacuo to yield a dark brown syrup.
After flash chromatography with heptane/EtOAc (7:3) a light yel-
low syrup was obtained (73%, 2.77 g).
Rf (CHCl3/MeOH 9:1): 0.79; Rf (heptane/EtOAc 7:3): 0.48; 1H
NMR: (CDCl3) 2.67 (t, 2H, J = 7.82 Hz, –CH2–COO–), 3.19 (t, 2H, J
= 7.63 Hz, –C–CH2–), 3.67 (s, 3H, –CH3), 7.44–7.46 (m, 5H, Ar–H),
8.11 (s, 1H, –CH–NO2–); ESI-MS: calcd. 235.2359, found 254.08
[M+H2O+H]+; Anal. calcd. for C12H13NO4: C 61.27, H 5.57, N 5.95;
Found C 61.57, H 5.62, N 6.16.
5.3.5. Methyl 4-nitro-5-phenylpentanoate (4)
O
O
NO2
5.3.7. 5-(Benzyloxy)-5-oxo-1-phenylpentan-2-aminium chloride (8b)
Sodium borohydride (919.8 mg, 24.3 mmol, 2.16 eq.) was sus-
pended in a mixture of 25 ml of dioxan und 8 ml of EtOH. Com-
pound 3 (2.64 g, 11.23 mmol) was dissolved in 25 ml of dioxan
and then added dropwise to the suspension over a period of 45
min. The temperature was kept at 30 °C. After stirring for 45 min
at RT the mixture was diluted with ice/water. An excess of sodium
borohydride was decomposed by the dropwise addition of acetic
acid (50%). After an extraction with CHCl3 (3ꢂ), the combined
organic phases were washed with water (3ꢂ) and brine (1ꢂ). The
organic layer was dried over MgSO4 and concentrated under
reduced pressure to result in a yellow oil. If additional purification
is required, the product can be chromatographed via flash chro-
matography with CHCl3 (93.4%, 2.49 g).
O
O
+
Cl-
NH3
In a two necked flask armed with a dropping funnel and a reflux
condenser, the compound 6 (308 mg, 1.59 mmol) and 7 (1.64 g,
7.11 mmol) were suspended in benzyl alcohol (11.14 ml, 107.65
mmol). While stirring and ice cooling, thionyl chloride (4.5 ml,
62 mmol) was carefully added dropwise (gas evolution). The mix-
ture was heated under reflux for another 4 h. A yellow slurry
formed. After cooling to RT, Et2O was added. This leads to the pre-
cipitation of white crystals. They were collected by filtration and
recrystallized from an amount of Et2O/EtOH (1:1) that led to the
formation of a milky suspension. The mixture was refrigerated
overnight and the resulting white solid was collected by filtration
and dried in vacuo (80%, 2.21 g, calculated as HCl).
Rf (CHCl3/MeOH 9:1): 0.8; 1H NMR: (CDCl3) d [ppm]: 1.99–2.07
(m, 2H, –CH2–COO–), 2.09–2.17 (m, 1H, –CHH–CH2–), 2.23–2.28
(m, 1H, –CHH–CH2–), 2.95 (dd, 1H, J = 14.28/5.48 Hz, Ar–CHH–
CH–), 3.13 (dd, 1H, J = 14.28/8.8 Hz, Ar–CHH–CH–), 3.51 (s, 3H, –
CH3), 4.70–4.77 (m, 1H, –CH–NO2), 7.06–7.20 (m, 5H, Ar–H); 13C
NMR: (CDCl3) d [ppm]: 28.3, 29.9, 40.0, 51.9, 88.7, 127.5, 128.9,
128.9, 135.1, 172.2; ESI-MS: calcd. 237.2518, found 260.20 [M
+Na]+; Anal. calcd. for C12H15NO4: C 60.75, H 6.37, N 5.9; Found C
60.91, H 6.53, N 5.76.
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

14
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Rf (CHCl3/MeOH/TEA 89:10:1): 0.65; MP: 161–163 °C; 1H NMR:
under an argon atmosphere. After stirring for 15 min, PyBOP
(913 mg, 1.755 mmol) was dissolved in 15 ml of DCM and then
added dropwise to the solution above. The mixture stirred for 18
h with gradual warming to RT. The reaction mixture was diluted
with DCM and the organic layer washed with 1 M NaHSO4, 5%
NaHCO3 and brine. The organic layer was dried over MgSO4 and
removed in vacuo. Purification by flash chromatography (MTBE)
yielded the product as a white solid which was an inseparable
diastereomeric mixture (48%, 408 mg).
(CDCl3) d [ppm]: 2.01–2.06 (m, 2H, –CH2–CH2–), 2.55–2.69 (m, 2H,
–CH2–CH2–COO–CH2–), 2.93 (dd, 1H, J = 13.7/8.60 Hz, Ar–CHH–
CH–), 3.23 (dd, 1H, J = 13.7/5.5 Hz, Ar–CHH–CH–), 3.56–3.61 (m,
1H, –CH–NH2), 5.02 (s, 2H, –COO–CH2–Ar), 7.2–7.33 (m, 10H, Ar–
H), 8.53 (bs, 3H, –NH); 13C NMR: (D2O) d [ppm]: 27.1, 29.9, 37.9,
52.2, 67.2, 127.5, 128.4, 128.7, 128.83, 129.08, 129.3, 135.47,
135.50, 174.5; ESI-MS: calcd. 283.36, found 284.10 [M+H]+; Anal.
calcd. for C18H22ClNO2: C 67.6, H 6.93, Cl 11.09, N 4.38; Found: C
67.1, H 6.792, N 4.312.
Rf (MTBE): 0.81; MP: 90–100 °C; 1H NMR: (CDCl3) d [ppm]:
diastereomeric mixture (1:1): 1.05 (d, 1.4H, J = 6.2 Hz, –CH3), 1.23
(d, 1.2H, J = 6.5 Hz, –CH3), 1.45 (2s, 9H, t-Boc), 1.55–1.65 (m, 1H,
–CHH–CH2–), 1.83–1.9 (m, 1H, –CHH–CH2–), 2.26–2.35 (m, 1H,
–CH2–CHH–COO–), 2.38 (t, 1H, J = 7.5 Hz, –CH2–CHH–COO–), 2.64
(dd, 0.4H, J = 13.7/7.1 Hz, –CH–CHH–Ar), 2.72–2.79 (d/dd, 1.4H, J
= 6.7 Hz, J = 13.7/6.1 Hz, –CH–CHH–Ar), 3.97–4.5 (m, 2H, –O–
CH2–COO–), 4.10–4.24 (m, 2H, –CH–CH2–Ar, –CH–NH–CO–),
4.51–4.60 (m, 2H, –O–CH2–), 5.09 (2s, 2H, –CH2–Ar), 5.28 (2bd,
0.8H, –NH), 5.33–5.43 (m, 1H, –CH–O–CO–), 6.2 (2bd, 1H, –NH),
7.12–7.36 (m, 15H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 15.7, 16.1,
28.2, 28.3, 28.5, 29.1, 30.9, 41.08, 41.1, 50.3, 50.5, 66.4, 66.5,
67.2, 67.3, 70.55, 70.6, 73.29, 73.3, 76.8, 77.0, 77.2, 77.3, 80.5,
126.61, 126.64, 128.06, 128.1, 128.2, 128.27, 128.3, 128.4, 128.48,
128.49, 128.5, 128.5, 129.3, 135.7, 135.8, 136.9, 137.35, 137.4,
155.6, 168.4, 168.8, 173.2; ESI-MS: calcd. 632.74, found 655.32
[M+Na]+; Anal. calcd. for C36H44N2O8: C 68.34, H 7.01, N 4.43;
Found C 68.32; H 7.42; N 4.35.
5.3.8. Benzyl 4-[(2R,3S)-2-{[(tert-butoxy)carbonyl]amino}-3-
hydroxybutanamido]-5-phenylpentanoate (11)
t-Boc
HN
O
H
N
HO
O
O
DIPEA (4.33 ml, 25.49 mmol), Boc-
L-Thr-OH (9) (1.84 g, 8.4
mmol) and PyBOP (4.96 g, 9.54 mmol) were mixed together. Com-
pound 8b (2.28 g, 7.12 mmol) was added and the mixture was dis-
solved in 120 ml DCM. The solution was stirred for 16 h at ambient
temperature. The solvent was removed in vacuo and the residue
was taken up with EtOAc. The organic layer was washed with 1
M KHSO4 (1ꢂ), 5% NaHCO3 and brine (1ꢂ). After drying over
MgSO4, the organic solvent was evaporated under reduced pres-
sure. Purification by flash chromatography (CHCl3) yielded the pro-
duct (11) as a white solid which was a diastereomeric mixture that
could not be separated (87%, 3.02 g).
5.3.10. Benzyl 4-(3-{[2-(benzyloxy)propanoyl]oxy}-2-{[(tert-
butoxy)carbonyl]amino}butanamido)-5-phenylpentanoate (14b)
t-Boc
HN
O
H
N
O
O
O
Rf (CHCl3/MeOH 9:1): 0.83; MP: 115–118 °C; 1H NMR: (CDCl3) d
[ppm]: 1.03 (d, 1.5H, J = 6.4 Hz, –CH3), 1.08 (d, 1.5H, J = 6.5 Hz,
–CH3), 1.43 (2s, 9H, t-Boc), 1.64–1.74 (m, 1H, –CHH–CH2–), 1.89–
1.98 (m, 1H, –CHH–CH2–), 2.36–2.43 (m, 2H, –CH2–CH2–COO–),
2.76–2.8 (dd, 2H, J = 16.1/6.9 Hz, –CH–CH2–Ar), 3.90 (dd, 0.5H, J =
8.1/2.2 Hz, –CH–CO–NH–), 3.92–3.94 (m, 0.5H, –CH–CO–NH–),
4.16–4.3 (m, 2H, –CH–CH2–Ar, –CH–CH3), 5.09 (d, 2H, J = 5.3 Hz,
–CH2–Ar), 5.39 (2 bd, 1H, –OH/NH), 6.48 (bt, 1H, –NH), 7.15–7.37
(m, 10H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 18.3, 28.2, 28.3, 29.1,
29.2, 30.9, 31.0, 41.3, 41.4, 49.9, 50.0, 66.39, 66.4, 66.6, 80.3,
126.62, 128.20, 128.22, 128.23, 128.3, 128.4, 128.5, 128.52,
129.27, 129.3, 135.78, 135.8, 137.5, 156.5, 171.3, 173.0, 173.1;
ESI-MS: calcd. 484.585, found 507.03 [M+Na]+; Anal. calcd. for
O
O
Compound 11 (3.02 g, 6.23 mmol) was dissolved in 50 ml of
DCM, then added to a solution of (R)-(+)-2-benzyloxypropionic
acid (12b) (1.23 g, 6.84 mmol) and DIPEA (3.7 ml, 27.8 mmol) in
50 ml of DCM at ꢁ20 °C under an argon atmosphere. After stirring
for 15 min, PyBOP (3.56 g, 6.842 mmol), dissolved in 50 ml of DCM,
was added dropwise to the solution above. The mixture was stirred
with gradual warming to RT until TLC indicated no further conver-
sion (4–6 d). The reaction mixture was diluted with DCM and the
organic layer washed with 1 M NaHSO4, 5% NaHCO3 and brine.
The organic layer was dried over MgSO4 and removed in vacuo.
Purification by flash chromatography (CHCl3) obtained the product
as a white solid which was an inseparable diastereomeric mixture
(63%, 3.89 g).
C
27H36N2O6: C 66.92, H 7.49, N 5.78; Found: C 66.92, H 7.36, N 5.82.
5.3.9. Benzyl 4-[(2R,3R)-3-{[2-(benzyloxy)acetyl]oxy}-2-{[(tert-
butoxy)carbonyl]amino}butanamido]-5-phenylpentanoate (14a)
t-Boc
Rf (CHCl3/MeOH 9:1): 0.92; MP: 120–124 °C; 1H NMR: (CDCl3) d
[ppm]: diastereomeric mixture (1:1): 1.06 (d, 1.1H, J = 6.4 Hz,
–CH3), 1.23 (d, 1.8H, J = 6.5 Hz, –CH3), 1.35 (d, 1.9H, J = 6.9 Hz,
–CH3), 1.39 (d, 1.1H, J = 6.9 Hz, –CH3), 1.42 (s, 3.9H, t-Boc), 1.45 (s,
5.6H, t-Boc), 1.52–1.62 (m, 1H, –CHH–CH2–), 1.79–1.89 (m, 1H,
–CHH–CH2–), 2.28–2.39 (m, 2H, –CH2–CH2–COO–), 2.54 (dd, 0.6H,
J = 13.6/7.5 Hz, –CH–CH2–Ar), 2.71–2.78 (m, 1.4H, –CH–CH2–Ar),
4.0 (qui, 1H, J = 6.7 Hz, –CH–CH3), 4.06–4.24 (m, 2H, –CH–CH2–Ar,
–CH–NH–CO–), 4.43 (2d, 1H, J = 11.6 Hz, –OCHH–), 4.6 (2d, 1H, J
= 11.5 Hz, J = 11.6 Hz, –OCHH–), 5.08 (2s, 2H, –CH2–Ar), 5.3–5.41
HN
O
H
N
O
O
O
O
O
Compound 11 (652.9 mg, 1.35 mmol) was dissolved in 15 ml of
DCM, then added to a solution of compound 12a (267 mg, 1.485
mmol) and TEA (721 ml, 5.2 mmol) in 20 ml of DCM at ꢁ20 °C
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
15
(m/dd, 2H, J = 6.4/4.3 Hz, –NH, –CH–OCO–), 6.16–6.24 (2d, 1H, J =
5.4.3. 4-(2-{[(tert-Butoxy)carbonyl]amino}-3-[(2-hydroxyacetyl)oxy]
8.7 Hz, J = 8.5 Hz, –NH), 7.09–7.37 (m, 15H, Ar–H); 13C NMR:
(CDCl3) d [ppm]: 15.8, 16.1, 18.6, 28.2, 28.3, 28.3, 29.1, 30.9, 31.0,
41.1, 41.2, 50.3, 50.6, 57.7, 57.9, 66.4, 70.2, 71.9, 71.9, 74.1, 74.2,
80.4, 126.6, 126.6, 127.0, 127.8, 127.9, 127.9, 128.0, 128.1, 128.2,
128.3, 128.3, 128.5, 128.5, 128.5, 128.6, 128.6, 129.3, 129.3,
135.8, 137.4, 155.7, 168.4, 168.5, 171.8, 171.9, 173.1, 173.1; ESI-
MS: calcd. 646.769, found 669.25 [M+Na]+; Anal. calcd. for
butanamido)-5-phenylpentanoic acid (16a)
Following the general procedure A, compound 14a (595 mg,
0.94 mmol) was dissolved in 60 ml 2-propanol and hydrogenated
as described above. The resulting filtrate was purified by flash
chromatography (MTBE/EtOH: 100:0 ? 50:50) to obtain a white
solid (332 mg).
MP: 123–125 °C; 1H NMR: (CDCl3) d [ppm]: 1.15–1.30 (m, 3H,
–CH3), 1.45 (s, 9H, t-Boc), 1.60–1.80 (2m, je 1H, –CH–CH2–CH2–),
2.14–2.26 (m, 2H, –CH2–CH2–COO–), 2.68–2.84 (m, 2H, –CH–
CH2–Ar), 4.04–4.35 (m, 4H, HO–CH2–COO–, –CH–CH2–Ar, –CH–
NH–CO–), 5.37–5.61 (m, 0.6H, –NH), 7.12–7.29 (m, 5H, Ar–H);
13C NMR: (CD3OD) d [ppm]: 15.6, 15.8, 27.3, 31.9, 32.1, 40.5,
50.5, 59.8, 70.98, 71.0, 79.7, 125.9, 126.0, 127.95, 127.98, 128.93,
138.1, 170.14, 170.16, 171.8, 172.1; ESI-MS: calcd. 452.215, found
475.15 [M+Na]+, 451.18 [MꢁH]ꢁ; HR-MS calcd. 475.2056 [M+Na]+,
451.2079 [MꢁH]ꢁ, found 475.2050; 451.2081.
C37H46N2O8: C 68.71, H 7.17, N 4.33; Found: C 68.49, H 7.23, N 4.37.
5.4. General procedure A: hydrogenolysis
t-Boc
R2
HN
O
H
N
O
HO
OH
5.4.4. 4-(2-{[(tert-Butoxy)carbonyl]amino}-3-[(2-hydroxypropanoyl)
oxy]butanamido)-5-phenylpentanoic acid (16b)
O
O
R1
Following the general procedure A, compound 14b (450 mg,
0.70 mmol) was dissolved in 45 ml 2-propanol and hydrogenated
as described above. The product yielded as white solid and it was
used without further purification (256 mg).
Entry
R1
R2
Yield
15a
-H
-H
-H
97 %
93 %
78 %
79 %
15b
-CH3
-H
MP: 111–114 °C; 1H NMR: (CDCl3) d [ppm]: 1.12 (d, 1H, J = 5.9
Hz, –CH3); 1.24 (d, 2H, J = 6.3 Hz, –CH3), 1.37 (dd, 3H, J = 14.6/7.0
Hz, –CH3), 1.45 (s, 9H, -t-Boc), 1.63–1.77 (m, 1H, –CH–CHH–CH2–),
1.77–1.93 (m, 1H, –CH–CHH–CH2–), 2.25–2.40 (m, 2H,
CH2–CH2–COOH), 2.68–2.88 (m, 2H, –CH2–Ar), 4.07–4.34 (m, 3H,
–CONH–CH–; –CH–CH–CONH-; CH3–CH–COO–), 5.30–5.37 (m,
1H, –COO–CH–CH–), 5.55–5.61 (m, 1H, –NH), 5.83–5.88 (m, 1H,
–NH), 6.60–6-65 (m, 1H, –OH), 6.72–6.78 (m, 1H, –OH), 7.12–7.29
(m, 5H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 16.3, 16.6, 20.3, 20.5,
28.3, 28.9, 30.7, 30.8, 41.1, 41.3, 50.5, 50.9,57.9, 58.0, 59.8, 67.1,
67.3, 71.2, 71.3, 80.8, 80.9, 126.6, 126.6, 128.5, 128.5, 129.3,
129.4, 137.4, 137.4, 156.2, 168.8, 169.1, 173.9, 174.4, 176.9,
177.1; ESI-MS calcd: 466.52, found: 489.18 [M+Na]+, 465.16
[MꢁH]ꢁ; Anal. calcd. for C23H34N2O8: C 59.21, H 7.35, N 6.00;
Found C 58.64, H 7.06, N 6.17.
16a*
16b*
-CH2Ar
-CH2Ar
-CH3
*differing procedure
To a solution of the respective depsipeptide in MeOH, Pd/C was
added (10% Pd). After pressurizing with hydrogen (4 bar), the
heterogeneous mixture was stirred at RT for 24 h. The suspension
was filtered through CeliteÒ, washed with MeOH and the filtrate
was concentrated in vacuo. Further purification was not necessary.
5.4.1. 4-(2-{[(tert-Butoxy)carbonyl]amino}-3-[(2-hydroxyacetyl)oxy]
butanamido)butanoic acid (15a)
Following the general procedure A, compound 13a (500 mg,
0.92 mmol) was dissolved in 60 ml MeOH and hydrogenated as
described above. Compound 15a yielded as a clear syrup (267.5
mg).
5.4.5. Methyl 4-(2-{[(tert-butoxy)carbonyl]amino}-3-[(2-
hydroxypropanoyl)oxy]butanamido)-5-phenylpentanoat (17)
1H NMR: (CDCl3) d [ppm]: 1.25–1.26 (m, 3H, –CH3), 1.42 (s, 9H, -
t-Boc), 1.79–1.86 (m, 2H, –CH2–CH2–CH2–), 2.33–2.36 (m, 2H, –
CH2–COO–), 3.27–3.3 (m, 2H, –NH–CH2–), 4.14–4.15 (m, 2H, –O–
CH2–COO–), 4.32 (bd, 1H, –NH–CH–CH–), 5.43–5.5 (m, 1H, CH3–
CH–CH–), 5.86 (bd, 1H, –NH) 7.4 (bd, 1H, –NH/OH); ESI-MS: calcd.
362.3755, found 385.16 [M+Na]+, 361.06 [MꢁH]ꢁ; Anal. calcd. for
t-Boc
HN
O
H
N
O
HO
O
C15H26N2O8: C 49.72, H 7.23, N 7.73; Found: C 49.54, H 7.44, N 8.08.
O
O
5.4.2. 4-(2-{[(tert-Butoxy)carbonyl]amino}-3-[(2-hydroxypropanoyl)
oxy]butanamido)butanoic acid (15b)
Following the general procedure A, compound 13b (1.76 g, 3.2
mmol) was dissolved in 60 ml MeOH and hydrogenated as
described above. Compound 15b yielded as a white solid (1.12 g).
MP: 96–97 °C; 1H NMR: (CDCl3) d [ppm]: 1.16–1.22 (m, 3H,
–CH3), 1.22–1.28 (m, 3H, –CH3), 1.30 (s, 9H, -t-Boc), 1.67–1.76 (m,
2H, –CH2–CH2–CH2–), 2.24–2.3 (m, 2H, –CH2–COO–), 3.07–3.27
(m, 2H, –NH–CH2–), 4.13–4.22 (m, 1H, HO–CH–CH3), 4.3 (bd, 1H,
–NH–CH–CH–), 5.25 (bs, 1H, –CH3–CH–CH–), 6.15 (bd, 1H, –NH/
OH), 7.4 (bd, 1H, –NH/OH); 13C NMR: (CDCl3) d [ppm]: 16.6, 20.2,
24.1, 28.3, 31.1, 38.9, 57.8, 67.3, 71.5, 80.8, 156.3, 169.8, 174.3,
176.9; ESI-MS: calcd. 376.40, found 399.07 [M+Na]+, 375.13
[MꢁH]ꢁ; Anal. calcd. for C16H28N2O8: C 51.05, H 7.5, N 7.44; Found:
C 51.45, H 7.57, N 7.57.
Compound 14b (2.52 g, 3.89 mmol) was dissolved in 200 ml of
MeOH (HPLC grade) and Pd over charcoal was added. The hetero-
geneous mixture was transferred into an autoclave and pressurized
with H2 (4 bar). The mixture was allowed to stir at RT overnight.
After that time, filtration over celiteÒ followed and MeOH was
removed in vacuo. Compound 17 was obtained after flash chro-
matography (CHCl3) as a white solid (86%, 1.61 g).
Rf (MTBE): 0.70; MP: 115–117 °C; 1H NMR: (CDCl3) d [ppm]:
1.12–1.14 (m, 1H, –CH3), 1.25 (d, 2H, J = 6.5 Hz, –CH3), 1.33 (d,
2.4H, J = 6.8 Hz, –CH3), 1.37 (d, 0.6H, J = 6.8 Hz, –CH3), 1.45 (2s,
9H, t-Boc), 1.61–1.72 (m, 1H, –CHH–CH2–), 1.82–1.91 (m, 1H,
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16
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
–CHH–CH2–), 2.28–2.35 (m, 2H, –CH2–CH2–COO–), 2.67–2.88 (m,
2H, –CH–CH2–Ar), 3.64 (s, 3H, –COO–CH3), 4.06–4.25 (m, 3H,
–CH–CH3, –CH–CH2–Ar, –CH–NH–CO–), 5.32–5.44 (m, 2H, –NH,
–CH–OCO–), 6.42 (bd, 1H, –NH), 7.14–7.29 (m, 5H, Ar–H); 13C
NMR: (CDCl3) d [ppm]: 16.2, 16.4, 20.35, 20.42, 27.0, 28.2, 28.4,
28.9, 30.7, 30.8, 41.1, 41.2, 50.4, 50.7, 51.80, 51.83, 57.8, 58.0,
67.0, 71.1, 71.2, 80.5, 126.6, 128.5, 129.3, 137.3, 137.4, 155.7,
168.6, 168.7, 174.1, 174.3; ESI-MS: calcd. 480.5512, found 503.17
[M+Na]+; Anal. calcd. for C24H36N2O8: C 59.98, H 7.55, N 5.83;
Found: C 60.05, H 7.82, N 5.89.
formed, the procedure would need to be repeated. All material was
directly used for the last reaction step (26%, 289 mg).
MP: 235–239 °C; 1H NMR: (CDCl3) d [ppm]: 1.25 (d, 3H, J = 6.5
Hz, 5-CH3), 1.44 (s, 9H, t-Boc), 1.52 (d, 3H, J = 7 Hz, 2-CH3), 1.87 (m,
1H, H-100), 2.17 (m, 1H, H-10), 2.46 (m, 2H, H-11), 3.15 (m, 1H, H-
90), 3.72 (m, 1H, H-9), 4.18 (m, 1H, H-6), 5.24 (m, 2H, H-2 and H-5),
5.33 (bd, 1H, NH), 5.9 (bd, 1H, NH); ESI-MS: calcd. 358.387, found
381.00 [M+Na]+.
5.5.2. tert-Butyl N-(9-benzyl-5-methyl-3,7,12-trioxo-1,4-dioxa-8-
azacyclododecan-6-yl)carbamate (19)
Following the general procedure B, compound 16a (159.7 mg,
0.353 mmol) was dissolved in 250 ml of DCM. DIPEA (600 ml,
3.53 mmol) was injected and the temperature adjusted to
ꢁ20 °C. After stirring for 15 min, PyBOP (827.4 mg, 1.59 mmol, dis-
solved in 110 ml of DCM) was added dropwise and a concentration
of 16a of less than 10ꢁ3 M has to be maintained. The reaction mix-
ture was stirred for 3 d. The solvent was removed in vacuo and the
residue purified via chromatography (MTBE) to yield the isolated
diastereomers as white solids (41%, 63 mg). ESI-MS: calcd.
434.205, found 457.10 [M+Na]+.
5.5. General procedure B: ring closure
t-Boc
NH
O
6
7
5
Diastereomer 1 (19 DS1): 20%, 30.8 mg; Rf (MTBE): 0.80; MP:
198–201 °C; 1H NMR: (CDCl3) d [ppm]: 1.23 (d, 3H, J = 6.4 Hz, 5-
CH3), 1.43 (s, 9H, t-Boc), 1.75–1.82 (m, 1H, H-100), 1.91–2.00 (m,
1H, H-10), 2.24–2.31 (m, 1H, H-11), 2.53 (ddd, 1H, J =
17.2/7.4/1.5 Hz, H-110), 2.63 (dd, 1H, J = 13.3/8.8 Hz, –CHH–Ar),
3.01 (dd, 1H, J = 13.3/4.6 Hz, CHH–Ar), 3.99–4.08 (m, 1H, H-9),
4.10–4.16 (m, 1H, H-6), 4.31 (d, 1H, J = 15.7 Hz, H-2), 4.99 (d, 1H,
J = 15.7 Hz, H-20), 5.27–5.33 (m, 2H, H-5, –NH), 5.84 (bd, 1H,
–NH), 7.17–7.29 (m, 5H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 13.7,
26.5, 28.2, 29.7, 31.3, 41.8, 53.2, 55.4, 61.6, 62.0, 69.5, 80.3,
126.6, 128.5, 129.4, 137.6, 154.6, 165.3, 166.4, 167.4, 175.1; HR-
MS: calcd. 435.2132 [M+H]+, 457.1951 [M+Na]+; found 435.2122,
457.1942.
R1
8 NH
O 4
O
O
9
3
2
10
12
O
R2
11
1
Entry
18a
R1
-H
-H
R2
Yield
20 %
26 %
20 %
21 %
27 %
17 %
-H
18b
-CH3
-H
CD (MeOH): 216.6 nm,
De = -9.729 mdeg.
Diastereomer 2 (19 DS2): 21%, 32.2 mg; Rf (MTBE): 0.62; MP:
165–175 °C; 1H NMR: (CDCl3) d [ppm]: 1.39 (d, 3H, J = 6.3 Hz, 5-
CH3), 1.5 (s, 9H, t-Boc), 1.79–1.88 (m, 1H, H-100), 1.92–1.99 (m,
1H, H-10), 2.33–2.4 (m, 1H, H-11), 2.47 (ddd, 1H, J = 16.7/8.1/2.7
Hz, H-110), 2.72 (dd, 1H, J = 13.6/7.7 Hz, –CHH–Ar), 2.87 (dd, 1H, J
= 13.7/5.7 Hz, CHH–Ar), 4.13 (d, 1H, J = 12.7 Hz, H-2), 4.22–4.31
(m, 2H, H-6, H-9), 4.95 (bd, 1H, –NH), 5.03 (d, 1H, J = 12.3 Hz, H-
20), 5.24 (m, 1H, H-5), 6.13 (bd, 1H, –NH), 7.15 (d, 2H, J = 7.14 Hz,
Ar–H), 7.21–7.31 (m, 3H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 16.1,
27.6, 28.3, 31.1, 41.7, 50.7, 59.2, 62.0, 73.9, 81.0, 126.7, 128.6,
129.3, 137.1, 155.3, 166.1, 168.5, 172.8; HR-MS: calcd. 435.2132
[M+H]+, 457.1951 [M+Na]+; found 435.2123, 457.1943.
19 DS1* -Bn
19 DS2* -Bn
20 DS1* -Bn
20 DS2* -Bn
-H
-CH3
-CH3
*other procedure
CD (MeOH): De = 218.9 nm, +0.64 mdeg; 225.9 nm, -1.60 mdeg.
The reaction proceeded under argon atmosphere. The hydro-
genated product of general procedure A was dissolved in DCM.
DIPEA was injected and the mixture stirred at ꢁ20 °C for 15 min.
Dissolved PyBOP was added dropwise and the reaction mixture
was allowed to warm gradually to RT and to stir overnight or for
3 d. The solvent was removed under reduced pressure and the resi-
due purified with flash chromatography using either CHCl3 or
MTBE as eluent, or due to recrystallization with EtOAc.
5.5.3. tert-Butyl N-(9-benzyl-2,5-dimethyl-3,7,12-trioxo-1,4-dioxa-8-
azacyclododecan-6-yl)carbamate (20)
Following the general procedure B, compound 16b (235.0 mg,
0.504 mmol) was dissolved in 530 ml of DCM. DIPEA (857 ml,
5.04 mmol) was injected and the temperature adjusted to ꢁ20 °C.
After stirring for 30 min, PyBOP (1.19 g, 2.27 mmol, dissolved in
20 ml of DCM) was added dropwise and a concentration of 16b
of less than 10ꢁ3 M has to be maintained. The reaction mixture
was stirred for 3 d. The solvent was removed in vacuo and the resi-
due purified via chromatography (MTBE) to yield the isolated
diastereomers as white solids (44%, 99 mg). ESI-MS: calcd.
448.51, found 471.18 [M+Na]+.
Diastereomer 1 (20 DS1): 27%; 60 mg; Rf (MTBE): 0.82; MP:
178–180 °C; 1H NMR: (CDCl3) d [ppm]: 1.24 (d, 3H, J = 6.5 Hz, 5-
CH3), 1.45 (s, 9H, t-Boc), 1.50 (d, 3H, J = 7.1 Hz, 2-CH3), 1.71–1.80
(m, 1H, 10-H), 1.91–2.03 (m, 1H, 100-H), 2.25 (dd, 1H, J =
17.4/11.8 Hz, 11-H), 2.51 (dd, 1H, J = 17.4/7.2 Hz, 110-H), 2.63 (dd,
5.5.1. tert-Butyl N-(2,5-dimethyl-3,7,12-trioxo-1,4-dioxa-8-
azacyclododecan-6-yl)carbamate (18b)
Following the general procedure B, compound 15b (1.149 g,
3.06 mmol) was dissolved in 10 ml of DCM. DIPEA (779 ml, 4.59
mmol) was injected, and the mixture was stirred at ꢁ20 °C for
15 min. After stirring, PyBOP (1.59 g, 3.06 mmol) (dissolved in 4
ml of DCM) was added and the reaction mixture was stirred over-
night. The solvent was removed in vacuo and the residue processed
dropwise with EtOAc to obtain a pearlescent solid. If no solid was
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K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
17
1H, J = 13.3/8.8 Hz, Ar–CHH–), 3.03 (dd, 1H, J = 13.2/3.8 Hz, –Ar–
CHH–), 3.96–4.07 (m, 1H, 9-H), 4.12–4.19 (m, 1H, 6-H), 5.20–5.35
(m, 3H, –NH, 5-H, 2-H), 5.89 (d, 1H, J = 8.1 Hz, –NH), 7.16–7.34
(m, 5H, –Ar–H); 13C NMR: (CDCl3) d [ppm]: 13.9, 16.3, 26.3, 28.3,
31.4, 42.0, 53.4, 55.4, 66.8, 69.3, 80.3, 126.6, 128.5, 128.8, 129.4,
137.7, 154.6, 167.6, 168.4, 175.5; Anal. calcd. for C23H32N2O7:
C 61.59, H 7.19, N 6.25; found: C 60.80; H 7.07; N 6.19; HR-MS:
calcd. 449.2289 [M+H]+, 471.2107 [M+Na]+; found: 449.206,
471.2127.
tion was monitored by ESI-MS. The residual brown syrup was dis-
solved in DMF and neutralized with DIPEA while stirring and ice
cooling to give a solution of the twelve-membered ring. PyBOP,
the relevant acid and DIPEA were dissolved in DMF. This was often
accompanied by a color change. The solution of the twelve-mem-
bered ring was slowly added via syringe, and the reaction mixture
was stirred at 37 °C for 4 d. The mixture was purified via flash
chromatography.
CD (MeOH):
D
e
= 215.5 nm, ꢁ12.97 mdeg.
5.6.1. N-(5-Methyl-3,7,12-trioxo-1,4-dioxa-8-azacyclododecan-6-yl)
pyridine-2-carboxamide (22a)
Diastereomer 2 (20 DS2): 17%; 39 mg; Rf (MTBE): 0.46; Rf
(CHCl3/MeOH 9:1): 0.71; MP: 182–186 °C; 1H NMR: (CD3OD) d
[ppm]: 1.21–1.29 (m, 3H, 5-CH3), 1.37–1.48 (m, 12H, t-Boc, 2-
CH3), 1.59–1.69 (m, 1H, 10-H), 2.04–2.12 (m, 1H, 100-H), 2.26–
2.34 (m, 1H, 11-H), 2.53–2.61 (m, 1H, 110-H), 2.65–2.75 (m, 2H,
Ar–CH2–), 4.02–4.13 (m, 1H, 6-H), 4.25–4.34 (m, 1H, 9-H), 4.63–
4.71 (m, 1H, 2-H), 4.94–4.99 (m, 1H, 5-H), 7.09–7.24 (m, 5H, Ar–
H); 13C NMR: (CD3OD) d [ppm]: 14.8, 15.1, 26.1, 27.3, 28.4, 40.9,
48.8, 59.4, 70.3, 72.7, 80.0, 126.1, 128.0, 129.0, 137.9, 156.2,
170.2, 170.3, 172.9; Anal. calcd. for C23H32N2O7: C 61.59, H 7.19,
N 6.25; found: C 60.84; H 7.72; N 6.25; HR-MS: calcd. 449.2289
[M+H]+, 471.2107 [M+Na]+; found: 449.2302, 471.2122.
Following the general procedure C, compound 18a (50 mg,
0.145 mmol) was dissolved in TFA/DCM (248 ml/248 ml) and stirred
for 5 min. Deprotection was monitored by ESI-MS with a mass of
245.09 [M+H]+ (calcd. 244.244). The residue was dissolved in 1.0
ml of DMF and neutralized with DIPEA. PyBOP (85.3 mg, 0.164
mmol), picolinic acid (21a) (35.7 mg, 0.29 mmol), and DIPEA (37
ml, 0.22 mmol) were dissolved in 1.0 ml of DMF. The solution of
the twelve-membered ring was added via syringe and stirred at
37 °C for 4 d. The dark brown mixture yielded a white solid after
flash chromatography with CHCl3 (21.0 mg).
Rf (CHCl3/MeOH 9:1): 0.7; MP: 189 °C; 1H NMR: (CDCl3) d
[ppm]: 1.34 (d, 3H, J = 6.3 Hz, 5-CH3), 1.96 (m, 1H, H-10), 2.16 (m,
1H, H-100), 2.51 (m, 2H, H-11), 3.21 and 3.76 (each m, 2H, H-9),
4.52 and 4.87 (each d, 2H, J = 15.3 Hz, H-2), 4.68 (dd, 1H, J =
8.2/5.0 Hz, H-6), 5.42 (m, 1H, H-5), ca. 6.0 (br d, 1H, –NH), 7.47
(ddd, 1H, J = 7.43/4.7/1.17 Hz, H-500), 7.86 (m, 1H, J = 7.6/1.56 Hz,
H-400), 8.17 (d, 1H, H-300), 8.61 (m, 1H, H-600), 8.76 (d, 1H, –NH);
13C NMR: (CDCl3) d [ppm]: 14.3, 24.0, 32.9, 40.4, 55.1, 61.8, 70.1,
122.6, 126.7, 137.7, 148.15, 148.15, 163.86, 165.6, 167.4, 174.7;
ESI-MS: calcd. 349.339, found 372.22 [M+Na]+; Anal. calcd. for
CD (MeOH):
De = 218.9 nm, +4.28 mdeg.
5.6. General procedure C: final substitution
C
17H19N3O6: C 55.01; H 5.48; N 12.03; Found: C 55.53; H 5.34, N
N
11.72.
R3
5.6.2. N-(2,5-Dimethyl-3,7,12-trioxo-1,4-dioxa-8-azacyclododecan-6-
yl)pyridine-2-carboxamide (23a)
O
O
NH
Following the general procedure C, compound 18b (100 mg,
0.279 mmol) was dissolved in TFA/DCM (477 ml/954 ml) and stirred
for 5 min. Deprotection was monitored by ESI-MS with a mass of
281.07 [M+Na]+ (calcd. 258.271). The residue was dissolved in
1.95 ml of DMF and neutralized with DIPEA. PyBOP (162.5 mg,
0.312 mmol), picolinic acid (21a) (68.7 mg, 0.558 mmol), and
DIPEA (71.1 ml, 0.419 mmol) were dissolved in 1.95 ml of DMF.
The solution of the twelve-membered ring was added via syringe
and stirred at 37 °C for 4 d. The brown mixture was purified with
flash chromatography (CHCl3) to result in a white solid (49.0 mg).
Rf (CHCl3/MeOH 9:1): 0.72; MP: 204–207 °C; 1H NMR: (CDCl3) d
[ppm]: 1.35 (d, 3H, J = 6.65 Hz, 2-CH3), 1.54 (d, 3H, J = 7 Hz, 5-CH3),
1.85–1.92 (m, 1H, H-10), 2.17–2.25 (m, 1H, H-100), 2.42–2.55 (m,
2H, H-11), 3.17–3.22 (m, 1H, H-9), 3.71–3.79 (m, 1H, H-90), 4.66
(dd, 1H, J = 8.2 Hz/5.28 Hz, H-6), 5.25 (q, 1H, J = 7.0 Hz, H-2),
5.35–5.41 (m, 1H, H-5), 6.0 (bd, 1H, –NH), 7.45–7.48 (m, 1H, H-
500), 7.87 (dt, 1H, J = 7.7 Hz/1.56 Hz, H-400), 8.17–8.18 (m, 1H, H-
300), 8.58–8.59 (m, 1H, H-600), 8.76 (bd, 1H, –NH); 13C NMR: (CDCl3)
d [ppm]: 14.4, 16.3, 23.4, 32.8, 40.4, 54.8, 69.43, 69.50, 122.7, 126.6,
137.8, 147.9, 148.7, 163.5, 167.4, 168.5, 175.3; ESI-MS: calcd.
363.36, found 386.18 [M+Na]+; Anal. calcd. for C17H21N3O6: C
56.19; H 5.83; N 11.56; Found: C 55.91, H 5.54, N 11.29.
6
7
5
R1
8 NH
O 4
O
O
9
3
2
10
12
O
R2
11
1
Entry
22a
R1
-H
-H
-H
-H
R2
R3
Yield
42 %
5 %
-H
-H
22b
23a
-H
-OH
-H
-CH3
-CH3
-H
48 %
28 %
49 %
23b
-OH
-OH
24 DS1* -Bn
5.6.3. N-(2,5-Dimethyl-3,7,12-trioxo-1,4-dioxa-8-azacyclododecan-6-
yl)-3-hydroxypyridine-2-carboxamide (23b)
*other procedure
Following the general procedure C, compound 18b (189 mg,
0.528 mmol) was dissolved in TFA/DCM (902 ml/902 ml) and stirred
for 5 min. Deprotection was monitored by ESI-MS with a mass of
281.12 [M+Na]+ (calcd. 258.271). The residue was dissolved in
3.7 ml of DMF and neutralized with DIPEA. PyBOP (308 mg,
The respective N-boc twelve-membered ring was dissolved in a
mixture of TFA/DCM (1:1) at 0 °C. The reaction mixture was stirred
at RT for 5 min. The solvent was evaporated below 40 °C. Deprotec-
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18
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
T-Boc-pregabalin (25) (520 mg, 2.01 mmol) was dissolved in 5
ml of DCM and PyBOP (1.15 g, 2.21 mmol), DIPEA (546 ml, 3.22
mmol) and l-isoleucin benzyl ester (26) (444 mg, 2.01 mmol) were
added to the solution. The mixture stirred at RT for 5 h. The solvent
was removed in vacuo and the residue was purified via column
chromatography (EtOAc/Hexan 1:1). Compound 27 was obtained
as a colorless liquid which solidified under refrigeration (81%,
750 mg).
0.591 mmol), 3-hydroxypicolinic acid (21b) (147 mg, 1.056 mmol),
and DIPEA (134.4 ml, 0.792 mmol) were dissolved in 3.7 ml of DMF.
The solution of the twelve-membered ring was added via syringe
and stirred at 37 °C for 4 d. The brown mixture was purified with
flash chromatography (CHCl3/MeOH (100:0 ? 90/10)) to result in
a white solid (55.3 mg).
Rf (CHCl3/MeOH 9:1): 0.73; MP: 230 °C; 1H NMR: (CDCl3) d
[ppm]: 1.4 (d, 3H, J = 6.5 Hz, 2-CH3), 1.58 (d, 3H, J = 7 Hz, 5-CH3),
1.84–1.92 (m, 1H, H-10), 2.2–2.3 (m, 1H, H-100), 2.46–2.53 (m,
2H, H-11), 3.31–3.37 (m, 1H, H-9), 3.52–3.61 (m, 1H, H-90), 4.68
(dd, 1H, J = 8.25/4.7 Hz, H-6), 5.12 (q, 1H, J = 7.0 Hz, H-2), 5.34 (m,
1H, H-5), 6.44 (bd, 1H, –NH), 7.57(dd, 1H, J = 4.7/4.5 Hz, H-500),
7.62 (m (dd), 1H, H-400), 8.19 (dd, 1H, J = 4.1/1.6 Hz, H-600), 9.29
Rf (EtOAc/Hexan 1:1): 0.93; 1H NMR: (CDCl3): d [ppm]: 0.78–
0.84 (m, 12H, CH3–), 1.05 (t, 2H, J = 7.2 Hz, (CH3)2–CH–CH2–CH–),
1.16 (m, 1H, CH3–CHH–CH–), 1.34 (m, 1H, CH3–CHH–CH–), 1.35
(s, 9H, t-Boc), 1.54 (m, 1H, NH–CH2–CH), 1.82–1.93 (m, 2H, CH3–
CH2–CH–CH–, (CH2)3–CH–), 2.00–2.15 (m, 2H, CH–CH2–CO–),
2.73–2.82 (m, 1H, –NH–CHH–), 3.30–3.39 (m, 1H, –NH–CHH–),
4.49 (m, 1H, –NH2–CH–CO–), 4.66–4.74 (bt, 1H, –NH), 5.03 (d,
1H, J = 12.1 Hz, –COO–CHH–Ar), 5.13 (d, 1H, J = 12.1 Hz, –COO–
CHH–Ar), 7.18–7.27 (m, 5H, –COO–CH2–Ar–H); ESI-MS: calcd.
462.622, found 485.23 [M+Na]+.
(bd, 1H, –NH), 11.66 (s, 1H, –OH); 13C NMR: (CDCl3)
d
[ppm]: 14.4, 16.3, 23.4, 32.8, 40.5, 54.4, 69.1, 69.6, 126.9, 129.0,
130.2, 139.3, 158.0, 166.8, 167.8, 168.5, 175.5; ESI-MS: calcd.
379.3645, found 402.21 [M+Na]+; Anal. calcd. for C17H21N3O7: C
53.82, H 5.54, N 11.08; Found: C 52.43, H 5.18, N 10.66; HR-MS:
calcd. 380.1459 [M+H]+, 402,1277 [M+Na]+; found 380.14495,
402,1269.
5.8. Benzyl 2-[(3S)-3-[(2-{[(tert-butoxy)carbonyl]amino}-3-
hydroxybutanamido)methyl]-5-methylhexanamido]-3-
methylpentanoate (28)
5.6.4. N-(9-Benzyl-5-methyl-3,7,12-trioxo-1,4-dioxa-8-
azacyclododecan-6-yl)-3-hydroxypyridine-2-carboxamide (24 DS1)
Following the general procedure C, compound 19 DS1 (60 mg,
0.138 mmol) was dissolved in TFA/DCM (236 ml/236 ml) and stirred
for 30 min. Deprotection was monitored by ESI-MS with a mass of
357.27 [M+Na]+, 335.07 [M+H]+ (calcd. 334.367). The residue was
dissolved in 1.0 ml of DMF and neutralized with DIPEA. PyBOP
(86.3 mg, 0.166 mmol), 3-hydroxypicolinic acid (21b) (38.4 mg,
0.276 mmol), and DIPEA (81.5 ml, 0.48 mmol) were dissolved in
1.0 ml of DMF under an argon atmosphere. The solution of the
twelve-membered ring was added via syringe and stirred at RT
for 4 d. The mixture was purified with flash chromatography
(CHCl3) to deliver a white solid (29.5 mg).
t-Boc
NH
O
H
N
HO
O
N
H
O
O
Rf (CHCl3/MeOH 9:1): 0.8; MP: 224–225 °C; 1H NMR: (CDCl3) d
[ppm]: 1.35 (d, 3H, J = 6.5 Hz, 5-CH3), 1.80–1.87 (m, 1H, H-100),
1.96–2.06 (m, 1H, H-10), 2.35 (ddd, 1H, J = 17.3/11.6/1.37 Hz, H-
11), 2.58 (ddd, 1H, J = 17.4/7.4/1.8 Hz, H-110), 2.66 (dd, 1H, J =
13.3/8.8 Hz, –CHH–Ar), 3.04 (dd, 1H, J = 13.3/4.6 Hz, CHH–Ar),
4.07–4.16 (m, 1H, H-9), 4.36 (d, 1H, J = 15.6 Hz, H-2), 4.57 (dd,
1H, J = 8.0/5.6 Hz, H-6), 5.06 (d, 1H, J = 15.8 Hz, H-20), 5.45 (m, 1H,
H-5), 5.94 (d, 1H, J = 7.9 Hz, –NH), 7.15–7.28 (m, 5H, Ar–H), 7.33
(dd, 1H, J = 8.5/1.4 Hz, H-500), 7.38 (dd, 1H, J = 8.5/4.3 Hz, H-400),
8.12 (dd, 1H, J = 4.3/1.5 Hz, H-600), 8.7 (d, 1H, J = 8.0 Hz, –NH),
11.72 (s, 1H, –OH); 13C NMR: 14.1, 26.5, 31.3, 41.8, 53.4, 54.1,
61.7, 68.8, 126.5, 126.6, 128.5, 129.0, 129.4, 130.6, 137.5, 139.7,
157.9, 165.3, 166.4, 168.1, 175.4; ESI-MS: calcd. 455.4605,
found 456.11 [M+H]+, 478.25 [M+Na]+, 454.17 [MꢁH]ꢁ. HR-MS:
calcd. 478.1591 [M+Na]+, 454.1595 [MꢁH]ꢁ; found 478.1583,
454.1620.
Compound 27 (1.142 g, 2.47 mmol) was dissolved in TFA/DCM
(2.0 ml/2.0 ml) and stirred at RT for 1 h. The solvents were
removed under reduced pressure and the residue was taken up
with toluene and again evaporated. The process was repeated three
times. CHCl3 was added and the evaporation process was repeated.
The free amine was redissolved in 5 ml of DCM, PyBOP (1.414 g,
2.72 mmol), DIPEA (671 ml, 3.95 mmol) and N-Boc- -Thr-OH (9)
L
(541 mg, 2.47 mmol) were added and stirred at RT for 3 h. The sol-
vent was removed in vacuo. The reaction mixture was purified via
column chromatography (EtOAc/Hexan 1:1) to deliver a white pro-
duct (90%, 1.25 g).
Rf (EtOAc/Hexan 1:1): 0.24; 1H NMR: (CDCl3): d [ppm]: 0.86–
0.89 (m, 9H, CH3–), 0.92 (d, 3H, J = 6.8 Hz, CH3–CH–CH2–CH3),
1.08–1.14 (m, 1H, CH3–CH–CH2–CH3), 1.12 (d, 3H, J = 6.8 Hz, CH3–
CH–OH), 1.26 (t, 2H, J = 7.1 Hz, (CH3)2–CH–CH2–CH–), 1.37–1.43
(m, 1H, CH3–CH–CH2–CH3), 1.46 (s, 9H, t-Boc), 1.67 (m, 1H, CH3–
CH2–CH–CH3), 1.93–2.00 (bs, 1H, (CH2)3–CH–), 2.05–2.15 (m, 2H,
–CH–CH2–CO–), 2.20 (m, 1H, –NH–CH2–CH–), 3.26 (bs, 1H, –NH–
CH2–CH–), 3.92 (d, 1H, J = 6.8 Hz, CH3–CH–OH), 4.32 (dq, 1H, J =
6.5/1.9 Hz, t-Boc–NH–CH–CO–), 4.62 (dd, 1H, J = 8.4/4.9 Hz, –NH–
CH–COO–CH2–Ar), 5.13 (d, 1H, J = 12.2 Hz, –COO–CH2–Ar), 5.22
(d, 1H, J = 12.2 Hz, –COO–CH2–Ar), 5.54 (d, 1H, J = 7.8 Hz, –NH),
7.01–7.08 (bs, 1H, –NH), 7.16 (d, 1H, J = 8.8 Hz, –NH), 7.31–7.38
(m, 5H, –COOCH2–Ar–H); 13C NMR: (CDCl3) d [ppm]: 11.6, 15.8,
18.7, 22.6, 22.7, 25.2, 28.3, 34.2, 37.2, 39.1, 41.7, 42.4, 56.9, 58.0,
66.7, 67.1, 80.3, 128.4, 128.6, 135.4, 156.6, 172.6, 172.7, 172.9;
ESI-MS: calcd. 563.7260, found 586.21 [M+Na]+; Anal. calcd. for
5.7. 2-[(S)-3-(tert-Butoxycarbonylaminomethyl)-5-methl-
hexanoylamino]-3-methyl-pentansäurebenzylester (27)
O
H
N
O
t-Boc
N
H
O
C30H49N3O7:C 63.92, H 8.76, N 7.45; Found: C 63.37, H 8.78, N
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
19
7.26; HR-MS: calcd. 564.3649 [M+H]+, 586.3468 [M+Na]+; found
564.3638, 586.3456.
13C NMR: (CDCl3) d [ppm]: 10.8, 15.8, 16.7, 22.5, 22.9, 24.6, 25.2,
33.1, 34.2, 41.8, 43.1, 45.9, 56.1, 57.9, 71.3, 122.7, 126.9, 137.6,
148.8, 165.3, 168.6, 170.4, 172.7; ESI-MS: calcd. 460.566, found
483.38 [M+Na]+, 459.18 [MꢁH]ꢁ; Anal. cald. for C24H36N4O5: C
62.59, H 7.88, N 12.16; Found: C 61.07, H 7,49, N 11.62; HR-MS:
calcd. 461.2783 [M+H]+, 483.2583 [M+Na]+; found 461.2753,
483.2574.
5.9. N-[(7S)-3-(Butan-2-yl)-12-methyl-7-(2-methylpropyl)-2,5,10-
trioxo-1-oxa-4,9-diazacyclododecan-11-yl]pyridine-2-carboxamide
(31)
5.10. Benzyl 3-(2-{[(tert-butoxy)carbonyl]amino}-3-
hydroxybutanamido)propanoate (33)
N
t-Boc
NH
O
O
NH
O
H
HO
N
O
O
O
NH
O
O
T-Boc-Thr-OH (9) (1.31 g, 6.00 mmol) was dissolved in 12 ml
of DCM and compound 32 (538 mg, 2.50 mmol) was added
while stirring. DCC (748 mg, 3.60 mmol) was added and the
mixture was stirred at RT for 5 h. DCU started to precipitate
immediately as white solid. Afterwards the urea derivative
was separated by filtration and the organic phase was washed
with 1 M HCl, 1 M KHCO3 and water. Then the solution was
dried over MgSO4 and the solvent was removed by evaporation.
After purification by flash chromatography with CHCl3, com-
pound 33 obtained as clear syrup which solidified during
storage (46%, 435 mg).
N
H
Compound 28 (944 mg, 2.0 mmol) and Pd/C, dissolved in 50 ml
of MeOH, were transferred into an autoclave and pressurized with
H2 (5 bar). The final heterogeneous solution was stirred at RT for 6
h. After completion, the catalyst was filtered through celiteÒ and
the filtrate was concentrated in vacuo. The resulting white solid,
compound 29 (150 mg, 0.32 mmol) was dissolved in 5 ml of
DCM. The flask was flushed with argon and cooled at –22 °C. DIPEA
(158 ml, 0.93 mmol) and a preliminarily produced solution of
PyBOP (182 mg, 0.35 mmol) in 5 ml of DCM (stored under argon)
were injected. After stirring at –22 °C for 15 min, the solution
was gradually warmed to RT with additional stirring for 5 h. The
muddy mixture was diluted with 30 ml of DCM and washed with
NaHSO4 (2ꢂ), brine (1ꢂ), sat. NaHCO3-solution (1ꢂ) and brine
(1ꢂ). The organic phase was dried over MgSO4 and the solvent
was removed in vacuo. The oily residue, compound 30 (241 mg,
0.53 mmol), was dissolved in TFA/DCM (1.0 ml/1.0 ml). After stir-
ring for 5 min, the solvent was removed by evaporation below
40 °C. The residue was dissolved in 2.0 ml of DMF and neutralized
with DIPEA with an ice/water bath. A solution of PyBOP (308 mg,
0.59 mmol), picolinic acid (21a) (65 mg, 0.53 mmol) and DIPEA
(135 ml, 0.8 mmol) in 2.0 ml of DMF was slowly added to the solu-
tion of the trifluoroacetate salt. The reaction stirred at 37 °C for 4 d.
The product (31) was obtained after flash chromatography with
CHCl3/MeOH/NH3 (97:3:1) as a colorless solid (16%, 40 mg).
Rf (CHCl3/MeOH/NH3 98:2:0.5): 0.46; MP: 102–109 °C; 1H
NMR: (CDCl3): d [ppm]: 0.84–0.90 (m, 9H, CH3–CH–CH3, CH3–
CH–CH3, CH3–CH2–CH–), 0.96 (d, 3H, J = 6.4 Hz, CH3–CH–CH2–
CH3), 1.06 (m, 2H, (CH3)2–CH–CH2–CH–), 1.11–1.20 (m, 1H, CH3–
CH–CH3), 1.33 (d, 3H, J = 6.4 Hz, CH3–CH–OH), 1.53–1.63 (m, 2H,
CH3–CH–CH2–CH3), 1.82–1.89 (m, 1H, CH3–CH2–CH–CH3), 1.88–
1.93 (m, 1H, –CH–CH2–CO–), 2.27–2.35 (bs, 1H, –NH–CH2–CH–),
2.42 (d, 1H, J = 14.7 Hz, –CH–CH2–CO–), 3.02 (d, 1H, J = 13.7 Hz,
–NH–CH2–CH–), 3.34 (m, 1H, –NH–CH2–CH–), 4.51 (t, 1H, J = 9.5
Hz, –NH–CH–CO-(Ile)), 4.71 (m, 1H, –NH–CH–CO–), 5.52–5.56 (m,
1H, CH3–CH–OOC–), 6.50 (d, 1H, J = 5.9 Hz, –NH), 6.55 (d, 1H, J =
9.8 Hz, –NH), 7.48–7.51 (m, 1H, Ar–H), 7.88 (m, 1H, Ar–H), 8.17
(d, 1H, J = 7.8 Hz, Ar–H), 8.69 (m, 1H, Ar–H), 8.70 (m, 1H, –NH);
Rf (CHCl3/MeOH 9:1): 0.61; 1H NMR: (CDCl3) d [ppm]: 1.15 (d,
3H, J = 6.4 Hz, –CH3), 1.44 (s, 9H, t-Boc), 2.58 (t, 2H, J = 6.2 Hz,
–CO–NH–CH2–CH2–), 2.92 (bs, 1H, –NH), 3.52–3.56 (m, 2H, –CH2–
CH2–COO–), 4.00 (d, 1H, J = 6.0 Hz, –CH–CH–CO–NH–), 4.32 (dq,
1H, J = 6.5/2.3 Hz, CH3–CH–), 5.12 (s, 2H, –CH2–Ar), 5.48 (bd, 1H,
–OH), 7.03 (bs, 1H, –NH), 7.32–7.38 (m, 5H, –Ar–H); 13C NMR:
(CDCl3) d [ppm]: 18.3, 28.3, 34.0, 35.0, 58.3, 66.6, 66.8, 80.3,
128.3, 128.4, 128.6, 135.6, 156.4, 171.5, 171.9; ESI-MS: calcd.
380.435, found 403.03 [M+Na]+, 381.25 [M+H]+; Anal. calcd. for
C19H28N2O6: C 59.98, H 7.42, N 7.36; Found: C 59.63, H 7.45, N 7.53.
5.11. Benzyl 3-{3-[2-(benzyloxy)benzoyloxy]-2-{[(tert-
butoxy)carbonyl]amino}butanamido}propanoate (35)
O
O
O
O
O
N
H
O
HN
t-Boc
Benzyloxy benzoic acid (34) (297 mg, 1.30 mmol) and dimethyl
aminopyridine (37 mg, 0.3 mmol) were dissolved in 10 ml of DCM.
Compound 33 (384 mg, 1.01 mmol) was dissolved in 10 ml of DCM
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

20
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
added at RT under stirring conditions. After 5 min of stirring, DCC
(268 mg, 1.30 mmol) was dissolved in 15 ml of DCM and added
to the mixture. DCU started to precipitate immediately as white
solid. Stirring was continued overnight. DCU was separated by fil-
tration and the organic phase was washed with sat. NH4Cl and
water. The org. phase was dried over MgSO4 and the solvent was
removed in vacuo. The resulting oily liquid was purified by flash
chromatography (heptane/EtOAc 8:2). The product (35) yielded
as a white solid (61%, 363 mg).
5.13. tert-Butyl N-(8-methyl-2,6,10-trioxo-2,3,4,5,6,7,8,10-octahydro-
1,9,5-benzodioxazacyclododecin-7-yl)carbamate (37)
t-Boc
NH
O
MP: 77 °C; Rf (CHCl3/MeOH 9:1): 0.86; 1H NMR: (CDCl3) d
[ppm]: 1.29 (d, 3H, J = 6.4 Hz, –CH3), 1.43 (s, 9H, t-Boc), 2.41–
2.54 (m, 2H, –CO–NH–CH2–CH2–), 3.30–3.38 (m, 1H, –CH2–
CHH–COO–), 3.44–3.52 (m, 1H, –CH2–CHH–COO–), 4.23 (d, 1H,
J = 5.0 Hz, –CH–CH–CONH–), 4.92 (s, 2H, –COO–CH2–Ar), 5.23 (s,
2H, –Ar–O–CH2–Ar), 5.54–5.57 (m, 2H, –NH, –COO–CH–CH–),
6.65–6.75 (m, 1H, –NH), 6.97–7.01 (m, 2H, –Ar–H), 7.24–7.46
(m, 11H, –Ar–H), 7.80 (d, 1H, J = 7.4 Hz, –Ar–H); 13C NMR: (CDCl3)
d [ppm]: 16.0, 28.2, 33.8, 35.0, 57.6, 66.3, 70.7, 71.0, 80.2, 114.0,
120.5, 120.8, 127.2, 128.2, 128.2, 128.3, 128.5, 128.7, 132.3,
133.6, 135.6, 136.3, 155.5, 157.9, 165.1, 168.9, 171.9; ESI-MS:
calcd. 590.663, found 613.17 [M+Na]+; Anal. calcd. for
NH
O
O
O
O
Compound 36 (320 mg, 0.78 mmol) was dissolved in 740 ml of
DCM and flushed with argon. After addition of DIPEA (1.327 ml,
7.80 mmol), the mixture was cooled at ꢁ20 °C with an acetone/
dry ice bath and stirred for 30 min. Additionally, PyBOP (1.83 g,
3.51 mmol) was dissolved in 40 ml of DCM and slowly added to
the solution. The mixture was allowed to warm gradually to RT
and stirred for 5 d. The solvent was removed in vacuo and the
resulting residue was purified by flash chromatography (CHCl3).
After recrystallization with MTBE, the product yielded as a white
solid (15%, 45.1 mg).
C33H38N2O8: C 67.10, H 6.48, N 4.74; Found: C 66.37, H 6.47,
N 5.16.
5.12. 3-(2-{[(tert-Butoxy)carbonyl]amino}-3-(2-hydroxybenzoyloxy)
butanamido)propanoic acid (36)
MP: 201–206 °C (sublimates at 179 °C); Rf (MTBE): 0.77; 1H
NMR: (CDCl3) d [ppm]: 1.35 (d, 3H, J = 6.4 Hz, –CH3), 1.44 (s, 9H,
t-Boc), 2.91 (t, 2H, J = 5.5 Hz, –CH2–CH2–NH–CO–), 3.43–3.60 (m,
1H, –OCO–CHH–CH2–), 3.72–3.83 (m, 1H, –OCO–CHH–CH2–),
4.33–4.42 (m, 1H, –NHCO–CH–CH–), 5.29–5.38 (m, 1H, –NH),
5.45 (td, 1H, J = 6.2/2.1 Hz, –CH–CH–OCO–), 6.59 (t, 1H, J = 6.2 Hz,
–NH), 7.12 (dd, 1H, J = 8.1/0.7 Hz, –Ar–H), 7.35 (dt, 1H, J = 7.6/0.9
Hz, –Ar–H), 7.55 (dt, 1H, J = 7.8/1.7 Hz, –Ar–H), 7.90 (d, 1H, J =
7.5 Hz, –Ar–H); 13C NMR: (CDCl3) d [ppm]: 14.5, 28.2, 34.0, 35.8,
56.3, 72.4, 80.5, 123.6, 124.5, 126.5, 132.0, 133.3, 148.4, 154.9,
164.4, 169.1, 170.2; ESI-MS: calcd. 392.403, found 415.09 [M
+Na]+; Anal. calcd. for C19H24N2O7: C 58.16, H 6.16, N 7.14; Found:
C 57.71, H 5.99, N 7.31.
O
O
O
O
N
H
OH
HN
OH
t-Boc
5.14. 3-Hydroxy-N-(8-methyl-2,6,10-trioxo-2,3,4,5,6,7,8,10-
octahydro-1,9,5-benzodioxazacyclododecin-7-yl)pyridine-2-
carboxamide (38)
Compound 35 (1.06 g, 1.80 mmol) was dissolved in 2-propanol
and Pd over charcoal was added. After pressurizing with hydrogen
(5 bar), the heterogeneous mixture stirred at RT for 5 h. The pro-
duct yielded as a white solid and was used without further purifi-
cation (43%, 320 mg).
MP: 100–104 °C; 1H NMR: (CDCl3) d [ppm]:1.37 (s, 9H, t-
Boc), 1.50 (s, 3H, –CH3), 2.36–2.50 (m, 0.4H, –CO–NH–CH2–
CH2–), 2.53.-2.64 (m, 1.6H, –CO–NH–CH2–CH2–), 3.43–3.54 (m,
1H, –CHH–COOH), 3.54–3.67 (m, 1H, –CHH–COOH), 4.37 (d,
0.2H, J = 9.7 Hz, –CH–CH–CO–NH–), 4.79 (dd, 0.8H, J = 9.1/6.2
Hz, –CH–CH–CO–NH–), 5.41–5.47 (m, 0.8H, –COO–CH–CH–),
5.58 (d, 0.2H, J = 9.6 Hz, –COO–CH–CH–), 5.60–5.70 (m, 0.8H,
–NH), 5.70–5.77 (m, 0.2H, –NH), 6.72 (t, 0.2H, J = 6.9 Hz, –NH),
6.86 (t, 0.8H, J = 7,3 Hz, –NH), 6.89–6.99 (m, 1H, –Ar–H),
7.29–7.36 (m, 0.2H, –Ar–H]), 7.37–7.49 (m, 1.8H, –Ar–H]),
7.79 (d, 0.8H, J = 7.7 Hz, –Ar–H), 7.92 (d, 0.2H, J = 7.4 Hz, –Ar–
N
OH
O
O
NH
NH
O
O
O
H), 10.58–10.65 (m, 1H, –OH); 13C NMR: (CDCl3)
d [ppm]:
O
16.7, 12.1, 33.5, 34.5, 57.4, 71.8, 81.3, 112.3, 117.6, 119.2,
130.0, 135.9, 156.4, 161.7, 169.1, 169.3, 175.9; ESI-MS: calcd.
410.418, found 433.05 [M+Na]+, 409.15 [MꢁH]ꢁ; Anal. calcd.
for C19H26N2O8: C 55.60, H 6.39, N 6.83; Found: C 55.62, H
6.34, N 6.97.
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
21
reduced pressure and the residue extracted with EtOAc. The
organic layer was washed with KHSO4, brine and dried over
MgSO4. The solvent was removed in vacuo. Purification was
achieved by flash chromatography using either MTBE or CHCl3 as
eluent.
Compound 37 (40.4 mg, 0.103 mmol) was dissolved TFA/DCM
(160 ml/160 ml) at 0 °C and stirred for 30 min. The solvent was
removed by evaporation. The residue was dissolved in 700 ml of
DMF and neutralized with DIPEA. Deprotection was monitored
by ESI-MS with a mass of 293.09 [M+H]+ (calcd. 292.29). PyBOP
(64.3 mg, 0.124 mmol), 3-hydroxypyridine-2-carboxylic acid
(21b) (28.7 mg, 0.206 mmol) and DIPEA (61 ml, 0.361 mmol) were
dissolved in 700 ml of DMF and flushed with argon. The resulting
deprotected and neutralized twelve-membered ring of 37 was
added via syringe. The mixture stirred additionally at RT for 3 d.
After purification with flash chromatography (MTBE) the product
resulted in a white solid (53%, 22.4 mg).
5.15.1. Benzyl 4-(3-{[2-(benzyloxy)acetyl]oxy}-2-(pyridin-2-
ylformamido)butanamido)butanoate (39)
According to the general procedure D, compound 13a (165 mg,
0.304 mmol) was dissolved in TFA/DCM (523 ml/1.05 ml) and stir-
red for 1 h. The residue was dissolved in 2.5 ml of DMF and neu-
tralized with DIPEA. PyBOP (190.5 mg, 0.37 mmol), 21a (75.2 mg,
0.61 mmol), and DIPEA (154.8 ml, 0.912 mmol) were dissolved in
2.5 ml of DMF and the solution of the trifluoroacetate salt of 13a
was injected. The mixture was stirred at RT overnight. The workup
followed the general procedure, yielding a clear syrup (45%, 75
mg).
MP: 193–197 °C; Rf (MTBE): 0.53; 1H NMR: (CDCl3) d [ppm]:
1.42 (d, 3H, J = 6.3 Hz, –CH3), 2.87 (t, 0.4H, J = 4.3 Hz, –CH2–CHH–
NHCO–), 2.91 (t, 0.6H, J = 4.3 Hz, –CH2–CHH–NHCO–), 2.99–3.08
(m, 1H, –CH2–CHH–NHCO–), 3.47–3.56 (m, 1H, –OCO–CHH–CH2–),
2.86–3.95 (m, 1H, –OCO–CHH–CH2–), 4.80 (dd, 1H, J = 7.9/4.5 Hz,
–NH–CO–CH–CH–), 5.56–5.63 (m, 1H, –CH–CH–OCO–), 6.60 (bdd,
1H, –NH), 7.15 (d, 1H, J = 8.0 Hz, –Ar–H), 7.34–7.42 (m, 3H,
–Ar–H), 7.60 (dt, 1H, J = 7.7/1.6 Hz, –Ar–H), 8.00 (dd, 1H, J =
7.8/1.5 Hz, –Ar–H), 8.12 (dd, 1H, J = 4.1/1.5 Hz, –Ar–H), 8.80 (bd,
1H, –NH), 11.70 (bs, 1H, –OH); 13C NMR: (CDCl3) d [ppm]: 14.6,
34.1, 35.9, 54.5, 71.3, 123.8, 124.0, 126.4, 126.6, 129.1, 130.6,
132.5, 133.7, 139.9, 148.7, 157.8, 164.3, 168.0, 168.6, 170.3; ESI-
MS: calcd. 413.381, found 436.21 [M+Na]+; Anal. calcd. for
Rf (MTBE): 0.34; 1H NMR: (CDCl3) d [ppm]: 1.33 (d, 3H, J = 6.4
Hz, –CH3), 1.79 (qui, 2H, J = 7.1 Hz, –CH2–CH2–CH2–), 2.35 (t, 2H,
J = 7.2 Hz, –CH2–CH2–COO–), 3.17–3.24 (m, 1H, –CHH–CH2–CH2–),
3.25–3.32 (m, 1H, –CHH–CH2–CH2–), 4.12 (s, 2H, –OCH2COO–),
4.56 (d, 1H, J = 11.7 Hz, –O–CHH–Ar), 4.60 (d, 1H,J = 11.6 Hz, –O–
CHH–Ar), 4.77 (dd,1H,J = 8.8/3.8 Hz, –CH–NH–CO–), 5.04 (s, 2H,
–CH2–Ar), 5.65–5.69 (m, 1H, –CH–CH–OOC–), 6.73 (bt, 1H,J = 5.7
Hz, –NH), 7.36–7.26 (m, 10H, –Ar–H), 7.45 (ddd, 1H, J =
7.6/4.8/1.2 Hz, H-50), 7.85 (dt, 1H,J = 7.7/1.7 Hz, H-40), 8.17 (td,
1H,J = 7.9/1.1 Hz, H-30), 8.57 (ddd, 1H,J = 4.8/1.7/0.9 Hz, H-60), 8.77
(bd, 1H, –NH); 13C NMR: (CDCl3) d [ppm]: 16.7, 24.3, 31.6, 39.1,
56.53, 66.4, 67.4, 70.5, 73.3, 122.5, 126.7, 127.9, 128.2, 128.3,
128.5, 128.6, 135.8, 137.1, 137.5, 148.4, 148.9, 164.8, 168.4,
169.2, 173.1; ESI-MS: calcd. 547.599, found 570.24 [M+Na]+,
548.13 [M+H]+, 546.07 [MꢁH]ꢁ; Anal. calcd. for C30H33N3O7: C
65.8, H 6.07, N 7.67; Found: C 65.92, H 6.00, N 7.831.
C
20H19N3O7: C 58.11, H 4.63, N 10.16; Found: C 57.39, H 4.89, N
9.64; HR-MS: calcd. 414.1301 [M+H]+, 436.11197 [M+Na]+,
412.1143 [MꢁH]ꢁ; found 414.1294, 436.1112, 412.1149.
5.15. General procedure D: open-chain compounds
5.15.2. Benzyl 4-(3-{[2-(benzyloxy)acetyl]oxy}-2-[(3-hydroxypyridin-
2-yl)formamido]butanamido)butanoate (40)
X
According to the general procedure D, compound 13a (300 mg,
0.55 mmol) was dissolved in TFA/DCM (943 ml/1.89 ml) and stirred
for 1 h. The residue was dissolved in 3.9 ml of DMF and neutralized
with DIPEA. PyBOP (343 mg, 0.66 mmol), 21b (153 mg, 1.1 mmol),
and DIPEA (280 ml, 1.65 mmol) were dissolved in 3.9 ml of DMF
and the solution of the trifluoroacetate salt of 13a was injected.
The mixture was stirred at RT overnight. The workup followed
the general procedure, yielding a brown syrup (16%, 46.9 mg).
Rf (MTBE): 0.70; 1H NMR: (CDCl3) d [ppm]: 1.33 (d, 3H, J = 6.4
Hz, –CH3), 1.78 (qui, 2H, J = 7.0 Hz, –CH2–CH2–CH2–), 2.36 (t, 2H,
J = 7.1 Hz, –CH2–CH2–COO–), 3.33–3.16 (m, 2H, –CH2–CH2–CH2–),
4.13 (s, 2H, –O–CH2–COO–), 4.57 (d, 1H, J = 11.6 Hz, –O–CHH–Ar),
4.61 (d, 1H, J = 11.6 Hz, –O–CHH–Ar), 4.67 (dd, 1H, J = 8.6/3.7 Hz,
–CH–NH–CO–), 5.05 (s, 2H, –CH2–Ar), 5.61–5.67 (m, 1H, –CH–
CH–OOC–), 6.63 (bt, 1H, –NH), 7.40–7.27 (m, 12H, H-40, H-50, Ar–
H), 8.09 (dd, 1H, J = 4.2/1.5 Hz, H-60), 8.76 (bd, 1H, –NH), 11.68
(bs, 1H, –OH); 13C NMR: (CDCl3) d [ppm]: 16.5, 24.2, 29.7, 31.7,
39.3, 56.1, 66.4, 67.4, 70.2, 73.4, 126.6, 127.98, 128.04, 128.20,
128.29, 128.49, 128.56, 129.1, 130.5, 157.9, 167.7, 168.9, 169.0,
173.2; ESI-MS: calcd. 563.598, found 564.14 [M+H]+, 586.22 [M
+Na]+, 562.19 [MꢁH]ꢁ; Anal. calcd. for C30H33N3O8: C 63.93, H
5.9, N 7.46; Found: C 63.60, H 5.97, N 7.39.
R2
R1
HN
O
O
H
O
N
O
O
O
O
Entry
39
X
N
N
N
C
R1
R2
-H
Yield
45 %
16 %
26 %
6 %
-H
-H
40
-OH
-F
41
-H
42
-H
-OH
-H
43
N
N
N
C
-CH3
-CH3
-CH3
-CH3
52 %
50 %
48 %
20 %
44
-OH
-F
45
46
-OH
The reaction proceeded under an argon atmosphere. The benzyl
ester of the open-chain depsipeptides were dissolved in TFA/DCM
(1:2) at 0 °C and stirred at RT for 1 h. The solvent was removed
in vacuo below 40 °C to yield the trifluoroacetate salt as a brown
viscous liquid. The residue was collected with DMF and neutralized
with DIPEA with ice cooling. In a second flask, PyBOP, the respec-
tive acid and DIPEA were dissolved in DMF. The dissolved trifluo-
roacetate salt was injected via syringe to the reaction mixture
and stirred at RT overnight. The DMF was evaporated under
5.15.3. Benzyl 4-(3-{[2-(benzyloxy)acetyl]oxy}-2-[(3-fluoropyridin-2-
yl)formamido]butanamido)butanoate (41)
The lithium 3-fluoropyridine-2-carboxylate (120 mg, 0.85
mmol) was dissolved in water. The solution was acidified with
conc. H2SO4 to have a pH of 2. The 3-fluoropyridine-2-carboxylic
acid (21c) was extracted with DCM (3ꢂ), the organic phases were
combined, and the solvent was removed in vacuo. The isolated
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

22
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3-fluoropyridine-2-carboxylic acid could be used without further
purification. According to the general procedure D, compound
13a (200 mg, 0.37 mmol) was dissolved in TFA/DCM (634 ml/1.27
ml) and stirred for 1 h. The residue was dissolved in 2.5 ml of
DMF and neutralized with DIPEA. PyBOP (231 mg, 0.44 mmol),
the free 3-fluoropicolinic acid (21c) and DIPEA (188.4 ml, 1.11
mmol) were dissolved in 2.5 ml of DMF and the solution of the tri-
fluoroacetate salt of 13a was injected. The mixture was stirred at
RT overnight. The workup followed the general procedure, yielding
a light yellow syrup (26%, 54 mg).
11.7 Hz, –O–CHH–), 4.66 (d, 1H, J = 11.54 Hz, –O–CHH–), 4.73–
4.76 (dd, 1H, J = 8.88/3.9 Hz, –CH–NH–CO–), 5.03 (s, 2H, –CH2–
Ar), 5.62–5.68 (m, 1H, –CH–CH–OOC–), 6.62–6-64 (m, 1H, –NH),
7.25–7.36 (m, 10H, Ar–H), 7.43–7.46 (ddd, 1H, J = 7.6/4.9/1.2 Hz,
H-50), 7.84 (dt, 1H, J = 7.7/1.76 Hz, H-40), 8.15–8.17 (td, 1H, J =
7.8/0.98 Hz, H-30), 8.57–8.59 (m, 1H, H-60), 8.76 (bd, 1H, –NH);
13C NMR: (CDCl3) d [ppm]: 16.7, 18.6, 24.2, 31.5, 39.0, 56.4, 66.2,
70.4, 71.8, 74.1, 122.3, 126.5, 127.74, 127.78, 128.06, 128.1,
128.15, 128.33, 128.45, 135.7, 137.31, 137.46, 148.40, 148.84,
164.8, 168.4, 171.9, 172.9; ESI-MS: calcd. 561.63, found 584.14
[M+Na]+, 559.97 [MꢁH]ꢁ; Anal. Calcd. for C31H35N3O7: C 66.3, H
6.28, N 7.48; Found: C 66.68, H 6.58, N 6.81.
Rf (MTBE):0.26; 1H NMR: (CDCl3) d [ppm]: 1.34 (s, 3H, –CH3),
1.79 (qui, 2H, J = 7.0 Hz, –CH2-CH2–CH2–), 2.36 (t, 2H, J = 7.1 Hz,
–CH2–CH2–COO–), 3.16–3.33 (m, 2H, –CH2–CH2–CH2–), 4.12 (s,
2H, –O–CH2–COO–), 4.57 (d, 1H, J = 11.6 Hz, –O–CHH–Ar), 4.61 (d, 1H,
J = 11.7 Hz, –O–CHH–Ar), 4.72 (dd, 1H, J = 8.5/3.9 Hz, –CH–NH–
CO–), 5.07 (s, 2H, –CH2–Ar), 5.65 (dq, 1H, –CH–CH–OOC–), 6.53
(bt, 1H, –NH), 7.3–7.37 (m, 10H, Ar–H), 7.48–7.58 (m, 2H, H-40, H-
50), 8.4 (d, 1H, J = 4.2 Hz, H-60), 8.56 (bd, 1H, J = 8.3 Hz, –NH); ESI-
MS: calcd. 565.589, found 566.12 [M+H]+, 588.24 [M+Na]+,
564.08 [MꢁH]ꢁ; Anal. calcd. for C30H32FN3O7: C 63.71, H 5.7, N
7.43; Found: C 63.33, H 5.74, N 7.09.
5.15.6. Benzyl 4-(3-{[2-(benzyloxy)propanoyl]oxy}-2-[(3-
hydroxypyridin-2-yl)formamido]butanamido)butanoate (44)
According to the general procedure D, compound 13b (350 mg,
0.63 mmol) was dissolved in TFA/DCM (1.08 ml/2.16 ml) and stir-
red for 1 h. The residue was dissolved in 4.4 ml of DMF and neu-
tralized with DIPEA. PyBOP (394 mg, 0.756 mmol), 21b (175.3
mg, 1.26 mmol), and DIPEA (320 ml, 1.89 mmol) were dissolved in
4.4 ml of DMF and the solution of the trifluoroacetate salt of 13b
was injected. The mixture was stirred at RT overnight. The workup
followed the general procedure, yielding a yellow syrup (50%, 181
mg).
5.15.4. Benzyl 4-(3-{[2-(benzyloxy)acetyl]oxy}-2-[(2-hydroxyphenyl)
formamido]butanamido)butanoate (42)
According to the general procedure D, compound 13a (300 mg,
0.55 mmol) was dissolved in TFA/DCM (943 ml/1.89 ml) and stirred
for 1 h. The residue was dissolved in 3.9 ml of DMF and neutralized
with DIPEA. PyBOP (343 mg, 0.66 mmol), 21d (153 mg, 1.1 mmol),
and DIPEA (280 ml, 1.65 mmol) were dissolved in 3.9 ml of DMF
and the solution of the trifluoroacetate salt of 13a was injected.
The mixture was stirred at RT overnight. The workup followed
the general procedure, yielding a colorless syrup (6%, 18.8 mg).
Rf (CHCl3/MeOH 9:1): 0.83; 1H NMR: (CDCl3) d [ppm]: 1.31 (d,
3H, J = 6.5 Hz, –CH3), 1.79 (qui, 2H, J = 7.0 Hz, –CH2–CH2–CH2–),
2.37 (t, 2H, J = 7.0 Hz, –CH2–CH2–COO–), 3.16–3.23 (m, 2H, - CH2–
CH2–CH2–), 4.13 (s, 2H, –O–CH2–COO–), 4.57 (d, 1H, J = 11.8 Hz,
–O–CHH–Ar), 4.58 (d, 1H,J = 11.6 Hz, –O–CHH–Ar), 4.65 (dd,1H,J
= 7.1/4.7 Hz, –CH–NH–CO–), 5.08 (s, 2H, –CH2–Ar), 5.53–5.59 (m,
1H, –CH–CH–OOC–), 6.73 (bt, 1H, –NH), 6.85 (t, 1H, J = 7.6 Hz,
–Ar–H), 6.98 (d, 1H, J = 8.4 Hz, –Ar–H), 7.29–7.47 (m, 15H, –Ar–H,
–NH), 11.93 (bs, 1H, –OH); 13C NMR: (CDCl3) d [ppm]: 16.03,
24.16, 31.67, 39.38, 56.08, 66.51, 67.51, 70.08, 73.42, 113.57,
118.56, 118.95, 125.98, 127.96, 128.17, 128.22, 128.33, 128.57,
134.75, 135.67, 136.81, 161.60, 167.91, 169.19, 169.95, 173.14;
ESI-MS: calcd. 562.61; found 585.24 [M+Na]+, 561.2 [MꢁH]ꢁ; Anal.
Calcd for C31H34N2O8: C 66.18, H 6.09, N 4.98; Found: C 65.00, H
6.107, N 4.946.
Rf (CHCl3/MeOH 9.5:0.5): 0.72; 1H NMR: (CDCl3) d [ppm]: 1.33
(d, 3H, J = 6.35 Hz, –CH3), 1.41 (d, 3H, J = 6.84 Hz, –CH3), 1.72–
1.78 (m, 2H, –CH2–CH2–CH2–), 2.34 (t, 2H, J = 7.1 Hz, –CH2–CH2–
COO–), 3.07–3.14 (m, 1H, –CHH–CH2–CH2–), 3.26–3.33 (m, 1H, –
CHH–CH2–CH2–), 4.09 (q, 1H, J = 6.8 Hz, –CH–OC), 4.46 (d, 1H, J =
11.5 Hz, –O–CHH–), 4.65 (d, 1H, J = 11.72 Hz, –O–CHH–), 4.68
(dd, 1H, J = 8.88/4.15 Hz, –CH–NH–CO–), 5.05 (s, 2H, –CH2–Ar),
5.59–5.64 (m, 1H, –CH-CH–OOC–), 6.64 (bt, 1H, –NH), 7.24–7.37
(m, 12H,H-40, H-50, Ar–H), 8.1 (dd, 1H, J = 4.1/1.47 Hz, H-60), 8.7
(bd, 1H, –NH), 11.67 (s, 1H, –OH); 13C NMR: (CDCl3) d [ppm]:
16.5, 18.52, 24.1, 31.6, 39.2, 55.9, 66.3, 70.1, 70.9, 71.9, 74.23,
126.07, 127.76, 127.85, 128.14, 128.22, 128.40, 128.4, 129.0,
135.7, 137.4, 139.9, 157.7, 167.7, 169.1, 171.8, 173.1; ESI-MS:
calcd. 577.6249, found 578.17 [M+H]+, 600.25 [M+Na]+, 576.18
[MꢁH]ꢁ; Anal. calcd. for C31H35N3O8: C 64.46, H 6.11, N 7.27;
Found: C 64.77, H 6.19, N 7.77.
5.15.7. Benzyl 4-(3-{[2-(benzyloxy)propanoyl]oxy}-2-[(3-fluoropyridin-
2-yl)formamido]butanamido)butanoate (45)
The lithium 3-fluoropyridine-2-carboxylate (200 mg, 1.36
mmol) was dissolved in water. The solution was acidified with
conc. H2SO4 to have a pH of 2. The 3-fluoropyridine-2-carboxylic
acid was extracted with DCM (3ꢂ), the organic phases were com-
bined, and the solvent was removed in vacuo. The isolated 3-fluo-
ropyridine-2-carboxylic acid (21c) could be used without further
purification. According to the general procedure D, compound
13b (350 mg, 0.63 mmol) was dissolved in TFA/DCM (1.08
ml/2.16 ml) and stirred for 1 h. The residue was dissolved in 4.4
ml of DMF and neutralized with DIPEA. PyBOP (394 mg, 0.756
mmol), the free 3-fluoropicolinic acid and DIPEA (183.3 ml, 1.08
mmol) were dissolved in 4.4 ml of DMF and the solution of the tri-
fluoroacetate salt of 13b was injected. The mixture was stirred at
RT overnight. The workup followed the general procedure, yielding
a yellow syrup (48%, 167 mg).
5.15.5. Benzyl 4-(3-{[2-(benzyloxy)propanoyl]oxy}-2-(pyridin-2-
ylformamido)butanamido)butanoate (43)
According to the general procedure D, compound 13b (200 mg,
0.36 mmol) was dissolved in TFA/DCM (617 ml/1.23 ml) and stirred
for 1 h. The residue was dissolved in 2.5 ml of DMF and neutralized
with DIPEA. PyBOP (225 mg, 0.432 mmol), 21a (88.6 mg, 0.72
mmol), and DIPEA (183.3 ml, 1.08 mmol) were dissolved in 2.5 ml
of DMF and the solution of the trifluoroacetate salt of 13b was
injected. The mixture was stirred at RT overnight. The workup fol-
lowed the general procedure, yielding a yellow syrup (52%, 181
mg).
Rf (CHCl3/MeOH 9:1): 0.79–0.86; 1H NMR: (CDCl3) d [ppm]: 1.31
(d, 3H, J = 6.4 Hz, –CH3), 1.37 (d, 3H, J = 6.8 Hz, –CH3), 1.71–1.79 (m,
2H, –CH2–CH2–CH2–), 2.32 (t, 2H, J = 7.2 Hz, –CH2–CH2–COO–),
3.05–3.14 (m, 1H, –CHH–CH2–CH2–), 3.24–3.33 (m, 1H, –CHH–
CH2–CH2–), 4.05 (q, 1H, J = 6.8 Hz, –CH–CH3), 4.43 (d, 1H, J = 11.6
Hz, –O–CHH–), 4.64 (d, 1H, J = 11.6 Hz, –O–CHH–), 4.76 (dd, 1H, J
Rf (CHCl3/MeOH 9.5/0.5): 0.66; 1H NMR: (CDCl3) d [ppm]: 1.33
(d, 3H, J = 6.45 Hz, –CH3), 1.39 (d, 3H, J = 6.84 Hz, –CH3), 1.72–
1.78 (m, 2H, –CH2–CH2–CH2–), 2.33 (t, 2H, J = 7.1 Hz, –CH2–CH2–
COO–), 3.06–3.14 (m, 1H, –CHH–CH2–CH2–), 3.25–3.33 (m, 1H, –
CHH–CH2–CH2–), 4.07 (q, 1H, J = 6.8 Hz, –CH–OC), 4.44 (d, 1H, J =
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
23
= 8.6/4.3 Hz, –CH–NH–CO–), 5.04 (s, 2H, –CH2–Ar), 5.57–5.63 (m,
1H, –CH–OCO–), 6.66 (bt, 1H, –NH), 7.21–7.35 (m, 10H, Ar–H),
7.47 (dd, 1H, J = 8.4/4.1 Hz, H-50), 7.49–7.55 (m, 1H, H-60), 8,38
(dt, 1H, J = 4.2/1.4 Hz, H-40), 8,57 (d, 1H, J = 8.6 Hz, –NH); 13C
NMR: (CDCl3) d [ppm]: 16.8, 18.6, 24.3, 31.6, 39.1, 56.0, 66.3,
70.5, 71.9, 74.2, 126.2, 127.8, 128.1, 128.2, 128.4, 128.5, 135.8,
136.7 (d, J = 4.6 Hz), 137.5, 144.2, 157.9, 160.7, 162.6, 168.4,
172.0, 172.9; ESI-MS: calcd. 579.62, found 602.22 [M+Na]+,
578.14 [MꢁH]ꢁ; Anal. calcd. for C31H34FN3O7: C 64.24, H 5.91, N
7.25; Found: C 64.49, H 6.134, N 6.878.
After stirring for 16 h the solvent was removed in vacuo and the
residue was dissolved in EtOAc. The organic layer was washed with
0.5 M NaHSO4, water, 5% NaHCO3 and brine. After drying over Na2-
SO4 and evaporation of the solvent the residue was chro-
matographed with DCM/EtOAc (2:1), yielding 56 as a white solid
(63%, 435 mg, calculated for compound 53 over both steps).
Rf (DCM/EtOAc 2:1): 0.42; MP: 80–85 °C; 1H NMR: (CDCl3) d
[ppm]: 1.11 (d, 3H, J = 6.4 Hz, –CH3), 1.45 (s, 9H, t-Boc), 1.66–1.76
(m, 1H, –CHH–CH2–), 1.94 (dtd, 1H, J = 15.5/7.6/4.1 Hz, –CHH–
CH2–), 2.39–2.43 (t, 2H, J = 7.5 Hz, –CH2–CH2–COO–), 2.72–2.77
(m, 2H, –CH–CH2–Ar), 3.14 (s, 1H, NH), 3.93 (d, 1H, J = 7.0 Hz,
–CH–CO–NH–), 4.21–4.25 (m, 2H, –CH–CH2–Ar, –CH–CH3), 5.10
(s, 2H, O–CH2–Ar), 5.34 (s, 1H, –OH), 6.47 (d, 1H, J = 9.5 Hz, –NH),
7.15–7.37 (m, 10H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 18.3, 28.3,
28.3, 29.1, 31.0, 41.4, 50.1, 58.1, 66.41, 66.6, 80.4, 126.64, 128.22,
128.24, 128.46, 128.53, 129.28, 135.83, 137.5, 156.55, 171.4,
173.01; ESI-MS: calcd. 484.585, found 507.14 [M+Na]+.
5.15.8. Benzyl 4-(3-{[2-(benzyloxy)propanoyl]oxy}-2-[(2-
hydroxyphenyl)formamido]butanamido)butanoate (46)
According to the general procedure D, compound 13b (300 mg,
0.54 mmol) was dissolved in TFA/DCM (922 ml/1.84 ml) and stirred
for 1 h. The residue was dissolved in 4.0 ml of DMF and neutralized
with DIPEA. PyBOP (337 mg, 0.648 mmol), 21d (149 mg, 1.08
mmol), and DIPEA (275 ml, 1.62 mmol) were dissolved in 4.0 ml
of DMF and the solution of the trifluoroacetate salt of 13b was
injected. The mixture was stirred at RT overnight. The workup fol-
lowed the general procedure, yielding a clear syrup (20%, 161 mg).
Rf (MTBE): 0.72; 1H NMR: (CDCl3) d [ppm]: 1.30 (d, 3H, J = 6.5
Hz, –CH3), 1.40 (d, 3H, J = 6.8 Hz, –CH3), 1.77 (qui, 2H, J = 7.0 Hz,
–CH2–CH2–CH2–), 2.36 (t, 2H, J = 7.0 Hz, –CH2–CH2–COO–), 3.07–
3.14 (m, 1H, –CHH–CH2–CH2–), 3.26–3.33 (m, 1H, –CHH–CH2–
CH2–), 4.11 (q, 1H, J = 6.8 Hz, –CHOC–), 4.49 (d, 1H, J = 11.4 Hz,
–O–CHH–), 4.60–4.63 (m, 2H, –O–CHH-, –CH–NH–CO–), 5.08 (s,
2H, –CH2–Ar), 5.51–5.56 (m, 1H, –CH–CH–OOC–), 6.54 (bt, 1H,
–NH), 6.84–6.87 (m (dt), 1H, H-50), 6.97–6.99 (m(d), 1H, H-30),
7.28–7.43 (m, 13H, H-40, H-60, Ar–H, –NH), 11.93 (s, 1H, –OH); 13C
NMR: (CDCl3) d [ppm]: 16.2, 18.6, 24.2, 27.0, 31.7, 39.3, 49.4,
56.1, 66.5, 70.0, 72.0, 72.8, 74.4, 113.7, 118.5, 119.0, 126.1, 127.8,
128.0, 128.2, 128.3, 128.5, 128.6, 134.7, 135.7, 137.3, 161.5,
168.0, 170.0, 172.1, 173.1; ESI-MS: calcd. 576.6368, found 599.21
[M+Na]+, 575.10 [MꢁH]ꢁ; Anal. calcd. for C32H36N2O8: C 66.65, H
6.29, N 4.86; Found: C 66.10, H 6.23, N 4.88.
5.15.10. Benzyl 4-[(2R,3R,5R)-3-{[2-(benzyloxy)acetyl]oxy}-2-{[(tert-
butoxy)carbonyl]amino}butanamido]-5-phenylpentanoate (57)
t-Boc
HN
O
H
O
N
O
O
O
O
Compounds 56 (1.00 g, 2.06 mmol) and 12a (0.41 g, 2.48 mmol)
were dissolved in 26 ml of DCM. After addition of DMAP (0.2 eq.,
0.048 g, 0.41 mmol) the mixture was cooled to 0 °C on an ice bath.
EDC-HCl (0.481 g, 2.48 mmol) was added in small portions over a
period of 15 min and stirred at 0 °C for 2 h. The reaction mixture
was allowed to heat to RT. The completion of reaction was moni-
tored by TLC. The mixture was diluted with EtOAc, washed with
water, 0.5 M NaHSO4, water, saturated NaHCO3 and brine. After
drying of the organic layer over Na2SO4 and removal of the solvent
with reduced pressure, the residue was purified by flash chro-
matography (DCM/EtOAc 2:1) yielding 57 as a white solid (92%,
1.2 g).
5.15.9. Benzyl 4-[(2R,3S,5R)-2-{[(tert-butoxy)carbonyl]amino}-3-
hydroxybutanamido]-5-phenylpentanoate (56)
t-Boc
HN
O
H
N
O
Rf (DCM/EtOAc 2:1): 0.74; MP: 105–110 °C; 1H NMR: (CDCl3) d
[ppm]: 1.24 (d, 3H, J = 6.4 Hz, –CH3), 1.46 (s, 9H, t-Boc), 1.64 (ddd,
1H, J = 14.3/10.1/7.2 Hz, –CHH–CH2–), 1.88 (dtd, 1H, J =
14.9/7.6/3.8 Hz, –CHH–CH2–), 2.39 (t, 2H, J = 7.4 Hz, –CH2–CH2–
COO–), 2.65 (dd, 1H, J = 13.7/7.2 Hz, –CH–CHH–Ar), 2.78 (dd, 1H,
J = 13.7/6.1 Hz, –CH–CHH–Ar), 4.00 (s, 2H, –O–CH2–COO–), 4.13–
4.21 (m, 2H, –CH–CH2–Ar, –CH–NH–CO–), 4.50–4.61 (m, 2H, –O–
CH2–), 5.10 (s, 2H, COO–CH2–Ar), 5.29 (d, 1H, J = 8.2 Hz, –NH),
5.43 (qd, 1H, J = 6.4/3.8 Hz, –CH–O–CO–), 6.27 (d, 1H, J = 6.28 Hz,
–NH), 7.12–7.38 (m, 15H, Ar–H); 13C NMR: (CDCl3) d [ppm]: 16.2,
28.28, 28.57, 30.95, 41.15, 50.49, 57.67, 66.44, 67.25, 70.62,
73.30, 80.45, 126.63, 128.033, 128.071, 128.09, 128.124, 128.238,
128.264, 128.294, 128.31, 128.317, 128.325, 128.332, 128.34,
128.348, 128.416, 128.423, 128.431, 128.446, 128.454, 128.461,
128.469, 128.503, 128.530, 128.537, 128.545, 128.556, 128.568,
128.587, 128.594, 128.609, 129.31, 135.84, 136.99, 137.45,
155.64, 168.45, 168.92, 173.21; ESI-MS: calcd. 632.74, found
655.14 [M+Na]+.
OH
O
In a round bottom flask, compound 53 (0.545 g, 1.42 mmol) was
dissolved in TFA/DCM (1:1) (1.09 ml, 14.2 mmol) and stirred vigor-
ously for 1 h. The solvents were removed under reduced pressure
and the oily residue was taken up with toluene and evaporated.
This process was repeated three times even with the solvents
CHCl3 and heptane. The sticky oily residue was used for the follow-
ing amide coupling step without further purification.
To a solution of N-Boc- -Thr–OH (9) (c = 0.07 M, 0.38 g, 1.74
mmol), DIPEA (910 ml, 5.22 mmol) and PyBOP (1.02 g, 1.96 mmol)
in DCM a solution of compound 55 (dissolved in DCM) was added.
L
Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045

24
K. Laqua et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5.15.11. (R)-4-((2S,3R)-2-((tert-Butoxycarbonyl)amino)-3-(2-
hydroxyacetoxy)butanamido)-5-phenylpentanoic acid (58)
5.01 (d, 1H, J = 15.7 Hz, H-20), 5.30–5.37 (m, 2H, H-5, –NH), 5.89
(d, 1H, J = 8.2 Hz, –NH), 7.21–7.31 (m, 5H, Ar–H); 13C NMR: (CDCl3)
d [ppm]: 13.7, 26.5, 28.3, 31.3, 41.8, 53.2, 55.4, 61.6, 69.5, 80.3,
126.6, 128.45, 128.51, 128.55, 129.4, 137.6, 154.6, 165.3, 167.4,
175.1; ESI-MS: calcd. 434.48 [M+H]+, found 457.10 [M+Na]+.
t-Boc
HN
O
Acknowledgements
H
O
N
We are grateful to Dr. Michael Ramm, Dr. Kerstin Voigt, Dr.
Hans-Martin Dahse and Christiane Weigel (Leibniz Institute for
Natural Product Research and Infection Biology, Hans Knöll Insti-
tute, Jena) for the evaluation of general antimycobacterial and
antimicrobacterial activities as well as any cytotoxic and antiprolif-
erative effects. We thank Dr. Jürgen Schmidt and Gudrun Hahn
(both Leibniz Institute of Plant Biochemistry, Halle (Saale)) for
the high resolution mass spectrometry analyses as well as the cir-
cular dichroism measurements. We would like to thank Lourdes
Encinas, Eva María Lopéz-Román and Sara Palomo at GSK and
the Tres Cantos Open Lab Foundation (TCOLF) for offering the pos-
sibility to screen our compounds against M. tuberculosis and InhA
overexpressor assays.
HO
OH
O
O
To a solution of 57 (c = 7.5 mM, 0.37 g, 0.59 mmol) in 2-propa-
nol was added Pd/C (10% Pd). The suspension was stirred under a
hydrogen atmosphere (4 bar) for 24 h. Subsequently, the catalyst
was filtered and the solvent was removed in vacuo. The resulting
product 62 (85%, 260 mg) was used without further purification.
MP: 143–151 °C; 1H NMR: (CD3OD) d [ppm]: 1.11–1.23 (m, 3H,
–CH3), 1.45 (s, 9H, t-Boc), 1.61–1.68 and 1.84–1.90 (2m, 1H, –CH–
CH2–CH2–), 2.20–2.40 (m, 2H, –CH2–CH2–COO–), 2.77 (d, 2H, J =
6.9 Hz, –CH–CH2–Ar), 4.04–4.20 (m, 4H, HO–CH2–COO–, –CH–
CH2–Ar, –CH–NH–CO–), 5.21–5.32 (m, 1H, –CH–CH3), 7.14–7.27
(m, 5H, Ar–H); 7.87 (m, 1H, COOH); ESI-MS: calcd. 452.215, found
475.07 [M+Na]+, 451.17 [MꢁH]ꢁ.
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Please cite this article in press as: Laqua K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.045