Synthesis of functionalized new conjugates of batracylin with tuftsin/retro-tuftsin derivatives and their biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.10.012

New batracylin conjugates with tuftsin/retro-tuftsin derivatives were designed and synthesized using T3P as a coupling agent. The conjugates possess an amide bond formed between the carboxyl group of heterocyclic molecule and the N-termini of the tuftsin/retro-tuftsin chain. The in vitro cytotoxic activity of the new analogues and their precursors was evaluated using a series of human and murine tumor cells. BAT conjugates containing retro-tuftsin with branched side aminoacid chain, in particular with leucine or isoleucine, were about 10-fold more cytotoxic toward two human tumor cell lines (lung adenocarcinoma (A549) and myeloblastic leukemia (HL-60)). These compounds showed about 10-fold increased cytotoxicity against the two types of tumor cells compared to parent BAT. We have not observed important differences in the mechanism of action between BAT and its cytotoxic tuftsin/retro-tuftsin conjugates. We propose that high biological activity of the most active BAT conjugates is a result of their greatly increased intracellular accumulation.

Full text of DOI:10.1016/j.ejmech.2015.10.012

European Journal of Medicinal Chemistry 106 (2015) 85e94
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis of functionalized new conjugates of batracylin with tuftsin/
retro-tuftsin derivatives and their biological evaluation
,
*
a
Wioleta Januchta a, Marcin Serocki b, Krystyna Dzierzbicka a , Grzegorz Cholewinski ,
ꢀ
Andrzej Skladanowski b
a Department of Organic Chemistry, Faculty of Chemistry, Gdansk University of Technology, 11/12 G. Narutowicza Street, 80-233 Gdansk, Poland
b Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, 11/12 G. Narutowicza Street, 80-233
Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
New batracylin conjugates with tuftsin/retro-tuftsin derivatives were designed and synthesized using
T3P as a coupling agent. The conjugates possess an amide bond formed between the carboxyl group of
heterocyclic molecule and the N-termini of the tuftsin/retro-tuftsin chain. The in vitro cytotoxic activity
of the new analogues and their precursors was evaluated using a series of human and murine tumor
cells. BAT conjugates containing retro-tuftsin with branched side aminoacid chain, in particular with
leucine or isoleucine, were about 10-fold more cytotoxic toward two human tumor cell lines (lung
adenocarcinoma (A549) and myeloblastic leukemia (HL-60)). These compounds showed about 10-fold
increased cytotoxicity against the two types of tumor cells compared to parent BAT. We have not
observed important differences in the mechanism of action between BAT and its cytotoxic tuftsin/retro-
tuftsin conjugates. We propose that high biological activity of the most active BAT conjugates is a result of
their greatly increased intracellular accumulation.
Received 5 August 2015
Received in revised form
29 September 2015
Accepted 6 October 2015
Available online 21 October 2015
Keywords:
Batracylin
BAT
Tuftsin
Retro-tuftsin
Topoisomerase
Cytotoxic activity
Bioavailability
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
pharmacologies properties, including increased bioavailability and
lowered general toxicity of the conjugate. We recently set out a
program aimed at the synthesis of new BAT-tuftsin/retro-tuftsin
Batracylin
(8-aminoisoindolo[1,2-b]quinasolin-12(10H)-one,
BAT) [1] is a heterocyclic amine identified by the drug screening
system at the National Institutes of Health (Bethesda, USA) [2]. In
preclinical studies, BAT showed high antitumor and cytotoxic ac-
tivity toward several experimental tumor cell models, including
cells which are resistant to standard chemotherapeutics, such as
doxorubicin, methotrexate and cisplatin [3e8]. In the recently
completed phase I clinical studies, safety profiles of BAT were
evaluated in human cancer patients. These studies showed that BAT
is well tolerated by human patients up to 400 mg/kg and provided
some encouraging data concerning its therapeutic potential [9].
One of the new directions in the synthesis of novel chemical
entities with antitumor activity is to combine several different
molecules with different functions and/or activities to produce
functionalized derivatives [10,11]. The aim of this approach is to
obtain compounds with enhanced activity/specificity and improved
conjugates which were expected to have improved pharmacolog-
ical features (such as increased water solubility, lowered general
toxicity in vivo) and potentially have additional mechanisms of ac-
tion, including immunostimulatory effect of tuftsin. Tuftsin is a
tetrapeptide Thr-Lys-Pro-Arg (TKPR) that been shown to possess
immunologic, tumoricidal, and bactericidal activities [12e14].
Accordingly, tuftsin has been successfully used in combination with
different antibiotics to treat opportunistic infections caused by
bacteria, fungi, and viruses. In addition, it also showed antineo-
plastics properties [15e32]. Moreover, tuftsin binds to the receptor
neuropillin-1 (NRP1) on the surface of cells that participates in
several different signalling pathways controlling cell migration and
survival [33].
We report here the synthesis of a new series of BAT analogues
with tuftsin/retro-tuftsin derivatives containing isopeptide bond
between ε-amino group of lysine and carboxyl group of aliphatic
amino acids such as Gly, Ala, Val, Leu, Ile. In our method, synthesis
of new analogues is based on the modification in the C-terminus of
the peptide residue by the formation of an amide bond between the
* Corresponding author.
E-mail address: krydzier@pg.gda.pl (K. Dzierzbicka).
http://dx.doi.org/10.1016/j.ejmech.2015.10.012
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

86
W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
carboxylic group of the respective peptide and the amine group
of BAT. We hypothesized that combination of BAT and tuftsin/retro-
tuftsin derivatives will allow us to obtain analogues with increased
anticancer activity and improved selectivity toward tumor cells. In
this paper, we provide data concerning evaluation of the cytotoxic
activity as well as other biological effects induced by these com-
pounds in in vitro tests and in tumor cells.
NMM or triethylamine (TEA) in anhydrous DMF was added into
solution. The reaction mixture was stirred at ꢀ15 ꢁC for 4 h then at
room temperature for 24 h. After evaporating the solvent in vacuo,
the crude products were purified by chromatography (SiO2).
Structures of synthesized derivatives 2ael were established by
spectroscopic methods (1H NMR, 13C NMR, MS), optical rotation and
melting point.
The Boc protecting groups were removed by treatment with
trifluoroacetic acid (TFA). The benzyloxycarbonyl (Z) protecting
groups were cleaved by hydrogenolysis (H2/PdeC). For recovering
free carboxylic group we performed hydrolysis methyl esters of
peptides 2ael (Scheme 3) under mild conditions using LiOH [35] as
a reagent.
2. Results and discussion
2.1. Chemistry
The synthesis of BAT with tuftsin/retro-tuftsin derivatives was
carried out according to reaction presented in Scheme 1, using
highly reactive n-propanephosphonic acid anhydride (T3P) [34] as
a coupling agent in solvent mixture. The condensation between
BAT and C-termini of tuftsin/retro-tuftsin derivatives 3ael was
achieved during reaction in anhydrous dimethylformamide (DMF)
under N2 for 24 h. T3P (50% solution in DMF) was added to a
mixture of 1 and 3ael in anhydrous pyridine and DMF, and the
resulting homogenous solution was held at ꢀ15 ꢁC for 4 h. After this
time, the reaction was carried out at 45 ꢁC. After 24 h, DMF was
evaporated under vacuum. The products 4ael were purified with
preparative TLC, and their identities were confirmed by high res-
olution 1H NMR (500 MHz) spectroscopy and MALDI-TOF mass
spectrometry analysis. The tert-butoxycarbonyl (Boc) protecting
groups were removed by treatment with HCl in anhydrous Et2O to
give the corresponding hydrochloride as an oil. The presence of
final products 5ael were confirmed by MALDI-TOF mass spec-
trometry analysis and their purity by HPLC.
BAT 1 was synthesized via modified method based on the
CzerniakeEinhorn reaction [20]. In this method, a symmetrically
protected 1,4-phenylenediamine derivative has undergone the
CzerniakeEinhorn reaction, and after hydrolysis of the protecting
groups, BAT 1 was obtained.
The protected tuftsin and retro-tuftsin derivatives 2ael, tetra-
and pentapeptides, were synthesized by the mixed anhydride
method with isobutyl chloroformate and N-methylmorpholine
(NMM) in anhydrous DMF (Scheme 2) [20e26]. A solution of Boc
protected amino acid in anhydrous DMF was cooled to ꢀ15 ꢁC and
NMM followed by isobutyl chloroformate were added. Five minutes
later, amino acid with free carboxyl group or respective peptide in
the later stages of a synthesis, neutralized by equivalent amount of
2.2. Cytotoxic properties
We performed cytotoxic activity testing using different human
tumor cells: lung adenocarcinoma (A549), two colon carcinomas
(HCT116, HT-29), prostate carcinoma (LNCaP), breast carcinoma
(MCF-7), human promyelocytic leukemia (HL-60), as well as two
murine leukemias (P388, L1210) and two other murine fibroblast-
derived cells (WEHI 1640, NIH-3T3). As presented in Table 1, BAT
showed moderate cytotoxic properties, represented by its IC50
values between 46.3
mM for A549 lung adenocarcinoma and
90.2 M for LNCaP prostate cancer. Interestingly, no cytotoxicity
m
was observed for human breast cancer cells (MCF-7) and murine
leukemia cells (L1210, P388) even at the highest concentration
tested.
For further investigations, we selected two types of tumor cells,
which were the most sensitive to BAT from all tumor cells tested i.e.
A549 lung adenocarcinoma cells, as a solid tumor and the most
sensitive from leukemia cell lines, human promyelocytic leukemia
HL-60 cells. Conjugation of BAT with tuftsin alone did not appre-
ciably change the cytotoxicity of the conjugate, compared to the
parent compound. In contrast, retro-tuftsin-BAT conjugate was
about 2-fold (for HL-60 cells) and about 5-fold (for A549 cells) more
cytotoxic that BAT. The majority of BAT conjugates with branched
tuftsin or retro-tuftsin were more cytotoxic toward both investi-
gated cell lines than the parent BAT (see Table 2). This was partic-
ularly striking for retro-tuftsin conjugates branched with leucine or
isoleucine (compounds 5k and 5l) which showed about 10 times or
more increased cytotoxicity against both tumor cells than the
parent compound BAT and its branched BAT-tuftsin analogues.
Importantly, branching tuftsin but also retro-tuftsin with other
non-polar aliphatic aminoacids (Gly, Ala, Val) had a very variable
effect on the cytotoxic activity of BAT conjugates and this was also
tumor cell type dependent.
HN NH
Boc
NO2
NH
X
NH
O
2.3. Effect of studied compounds on the catalytic activity of human
type I and II DNA topoisomerases
OH
NH
NH
O
N
O
O
Previous studies showed that BAT acts as an inhibitor of both
type I and II DNA topoisomerases [29,30]. Therefore, we next
evaluated whether BAT conjugates with tuftsin and retro-tuftsin,
branched with the same chain aminoacids (Leu and Ile) influence
the activity of purified DNA topoisomerases. Our results presented
on Fig. 1 (upper panel) show that studied compounds did not
appreciably inhibit DNA relaxation mediated by type I DNA topo-
isomerase. Effect of studied compounds on DNA relaxation medi-
NH
NH
OH
O
Boc
3a-f
OH
OH
LiOHxH2O
2a-l
MeOH, H2O
24 h, rt.
Boc
NH
N
NH
O
O
NH
HN
O
HN
ated by topoisomerase IIa enzyme was much more pronounced and
drug dose-dependent (Fig. 1, lower panel). In this case, the inhibi-
tion of DNA topoiosmerase II correlated with the cytotoxicity to-
ward tumor cells. BAT and its 5f derivative which showed moderate
cytotoxic properties, were able to a certain degree inhibit DNA
NO2
HN
3g-l
X = H; Gly; Ala; Val; Leu; Ile
X
Boc
Scheme 1. Synthesis and chemical structures of BAT analogues 5a-l.
relaxation in vitro, at the concentration 10 mM. In contrast, 100 mM

W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
87
Scheme 2. Preparation of Boc-Thr-Lys(BocX)-Pro-Arg(NO2)eOMe 2a-f and Boc-Arg(NO2)-Pro-Lys(BocX)-Thr-OMe 2g-l.
Table 2
Cytotoxic properties of new BAT analogs 5a-l toward tumor cells.
Compound
IC50 value [
A549 cells
mM]
HL-60 cells
BAT
46.3 7.9
52.8 11.9
>100
66.6 4.4
77.3 12.6
>100
5a (BAT-tuftsin)
5b
5c
5d
5.9 0.9
>100
19.6 3.8
>100
5e
5f
>100
83.7 25.8
15.4 3.1
30.8 6.1
28.7 6.1
11.1 2.4
11.0 1.5
5.3 1.8
4.7 0.6
17.4 2.0
8.7 2.9
>100
5.6 0.9
13.1 3.9
4.0 0.5
4.8 0.4
5g (BAT-retro-tuftsin)
5h
5i
5j
5k
5l
branched conjugates (compound 5a) and Leu-branched conjugate
(compound 5k) and compared it with the effect exerted by the
parent compound. Treatment of tumor cells with BAT and its two
conjugates, at doses corresponding to their IC90 concentrations,
resulted in a rapid (within 3 h) phosphorylation of the H2AX his-
tone (Suppl. Fig. 1) which is a typical marker of double-stranded
DNA breaks. Analysis of cell cycle progression (Fig. 2) and nuclear
morphology (Fig. 3) after treatment of tumor cells with studied
compounds revealed that biological action of BAT and its tuftsin or
Scheme 3. Deprotection of a methyl ester groups in BAT-tuftsin (2a-l) and BAT-retro-
tuftsin (2g-l) conjugates.
of a much less cytotoxic analog 5e was sufficient to achieve the
same inhibition rate. Two most cytotoxic analogs, 5k and 5l, where
also the most potent topoisomerase II
enzyme at a concentration close to 10
a
m
inhibitors, able to inhibit
M, that was similar to the
effect induced by a positive control, etoposide (VP-16).
2.4. Effect of BAT and its analogues on cell cycle progression and
induction of DNA damage
To evaluate the effect induced by studied BAT conjugates in
tumor cells, we selected two representative compounds of un-
Table 1
Cytotoxic properties of BAT toward different types of human and
murine cells.
Cell lines
BAT e IC50 SD [mM]
A549
HCT 116
HT-29
LNCaP
MCF-7
HL-60
46.3 7.91
88.7 23.46
64.3 12.42
90.2 2.58
>100
66.6 4.26
>100
Fig. 1. In vitro inhibition of the catalytic activity of type I (upper panel) and type II
a
P388
(lower panel) DNA topoisomerases human pure enzymes by batracylin and its tuftsin
conjugates. For comparison, effect of two standard inhibitors of the two enzymes
(active metabolite of irinotecan, compound SN-38, and etoposide) is shown, as a
positive. R e relaxed DNA; T e DNA topoisomers; S e supercoiled DNA.
L1210
WEHI 1640
NIH-3T3
>100
58.4 4.17
69.9 18.14

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W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
retro-tuftsin analogs was cell-type specific. In A549 cells, these
compounds arrested cell cycle progression in G2/M phase already
after 24h, and this effect was further increased after the next four
days of treatment. Additionally, for 5a and 5k analogs an increasing
fraction of cell debris was observed in drug-treated cell population,
which can be attributed to the induction of cell death by these
compounds and fragmentation of dying cells (Fig. 2). The 5a BAT-
tuftsin conjugate increased debris fraction by about 3.5-fold,
compared to BAT. Lack of mitotic cell in drug-treated tumor cells, as
revealed by fluorescence microscopy after DNA staining with
Hoechst 33342 (Fig. 3), together with our results obtained by flow
cytometry analysis, indicated that A549 cells arrest their prolifer-
ation in G2 phase. Interestingly, A549 cells which did not undergo
apoptosis after further prolonged drug exposure finally acquired
features of premature senescent cells. As can be seen on Fig. 3, cell
nuclei become progressively much larger after drug exposure than
these observed in non-treated cells, also majority of cells become
flat and greatly enlarged (Suppl. Fig. 2). Furthermore, increased
increased water solubility and lower general toxicity that results
from acetylated amino group of BAT. In our conjugates, this amine
position is used to covalently link tuftsin/retro-tuftsin derivatives
the BAT so its acetylation and consequently acetyl-BAT toxicity
should be decreased. We confirmed experimentally our initial hy-
pothesis that by conjugation of BAT with tuftsin and in particular
retro-tuftsin derivatives we were able to increase the cytotoxicity of
these compounds more that 10-fold.
We showed that BAT but also cytotoxic tuftsin/retro-tuftsin
conjugates inhibit the catalytic activity of type II DNA topoisom-
erase and at cytotoxic concentrations produce rapidly DNA damage
(double stranded DNA breaks) in drug-treated tumor cells. This was
associated with the activation DNA damage response and induction
of cells death by apoptosis or premature senescence, depending of
the type of tumor cells used and p53 functionality. If this is true,
induction of p53 by DNA damage produced by BAT and its conju-
gates in A549 cells (functional p53 pathway) should lead to pre-
mature senescence [32] but in cells with inactivated p53 (HL-60)
where G2 arrest induced in the presence of DNA damage is only
transient, drug-treated tumor cells enter abnormal mitosis and
undergo cells death. We concluded that the increased cytotoxicity
of tuftsin/retro-tuftsin conjugates of BAT is most probably not
associated with important differences in the mechanism of their
action on the cellular level. We propose this greatly enhanced
cytotoxic effect should be rather related to increased bioavailability
of new BAT derivatives that leads to their much higher accumula-
tion in tumor cells thus much effective tumor cell killing.
The molecular mechanism of increased accumulation of BAT-
tuftsin/retro-tuftsin conjugates can only be speculated and re-
quires further studies. Tuftsin was shown to be translocated into
living cells, most probably by endocytosis [33]. Tuftsin receptor has
been isolated and partially characterized many years ago [35] but
its function and particularly amino acid sequence remains still
obscure although it has been found to be expressed in human tu-
mor cells [36]. More recent studies have shown that tuftsin binds
specifically to neuropilin-1 receptor (NRP-1), a tyrosine kinase
VEGF co-receptor, and this binding inhibits the interaction between
NRP-1 and VEGFR [37]. It was even proposed that biological effect
of tuftsin is directly associated with its binding to NRP-1 [38]. There
is an intriguing possibility that interaction of tuftsin with NRP-1
may stimulate tuftsin internalization and lead to the increased
bioavailability of tuftsin conjugates.
activity of senescence-associated b-galactosidase was observed in
drug-treated cells (Suppl. Fig. 3). All these features confirm that
premature senescence was induced by BAT and its tuftsin/retro-
tuftsin conjugates in A549.
In contrast to A549 tumor cell model, HL-60 leukemia cells after
24 h treatment with BAT and 5a and 5k analogs do not stop their
cell cycle in G2 phase similar to A549 cells, but enter mitosis. Lack of
prolonged G2M arrest in HL-60 after cell may be related to the
absence of p53 in this cells, which leads to aberrant mitoses and
polyploidy [31]. As shown on Fig. 1 and Suppl. Fig. 2, a high fraction
of abnormal mitotic figures was observed in HL-60 cells treated
with studied compounds. These included lagging telophase cells,
cells with abnormal segregation of chromosomes during mitosis as
well as multipolar mitoses. Most of these abnormal cell divisions
resulted in mitotic catastrophe and led eventually to cell death by
apoptosis. However, some of drug-treated HL-60 cells underwent
polyploidization or micronucleation and only those cells had intact
cell membrane and were still alive after 120 h of treatment (stained
with fluorescein diacetate) (Suppl. Fig. 2). Similarly to A549, both
investigated conjugates induced cell death and increased cell
debris fraction by about two times compared to BAT. At the same
time, both conjugates induced much lower fraction of polyploid or
micronucleated cells as compared to BAT.
3. Summary
4. Experimental section
We describe here the synthesis of a series of new BAT analogs
combined with tuftsin/retro-tuftsin derivatives. The aim of our
design and synthesis these compounds as they should have
Melting points (uncorrected) were determined on the Kofler-
block apparatus. All chemicals and solvents were of reagent grade
Fig. 2. Effect of studied compounds on the cell cycle distribution of tumor cells. Cells were exposed to specified drug doses for the time indicated and analyzed by flow cytometry. *
Measured as a fraction of living cells; ** Measured as a fraction of sub-G1 of the total counted events.

W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
89
Fig. 3. Nuclear morphology of tumor cells treated with BAT and 5a, 5k analogs for the time indicated. After drug treatment cells were stained with Hoechst 33342 and analyzed by
fluorescence microscopy. Green arrow mitoses; yellow arrow polyploid cells; orange arrow multiple micronuclei; gray arrow lagging telophases; purple arrow
apoptotic bodies; scale bar 20 m). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
mitotic catastrophes; red arrow
this article.)
m
and were used without further purification. The reactions were
monitored by TLC on Merck F254 silica gel pre-coated plates. The
following solvent systems (by vol.) were used for TLC development:
CHCl3eMeOH (7:1, v/v) (A), CHCl3eMeOH (9:1, v/v) (B),
CHCl3eMeOH (10:1, v/v) (C), CHCl3eMeOH (20:1, v/v) (D). The
detection was carried out using UV and ninhydrin. MS spectra were
recorded on matrix-assisted laser desorption/ionization-time on
flight mass spectrometry (MALDI-TOF MS, Biflex III Bruker). Proton
and carbon NMR spectra were recorded in DMSO-d6 (isotopic
enrichment 99.95%) solutions at 293 K using a Varian Unity 500
Plus spectrometer (500.13 MHz for 1H, 125.76 MHz for 13C) with a
sample concentrations collected in Table 1 (Suppl.), using 5 mm
inverse detection broadband probes and deuterium lock. The cen-
tral peak of DMSO-d6 signals (2.49 ppm for 1H and 39.50 ppm for
13C) was used as the internal reference standard.
4.2. Tuftsin/retro-tuftsin derivatives 2 and 3
The procedure for the synthesis of compounds 2a, 2ced, 2g and
2i by the mixed anhydride method has been published previously
[19e26]. The compounds 2b, 2e,f, 2h, 2jel were prepared to the
same procedure. The Boc-protecting groups in Boc-Pro-Arg(NO2)e
OMe or Boc-Lys(Z)-Thr-OMe were removed by treatment with TFA
and then coupling with Boc-Lys(Z)-OH or Boc-Pro-OH gave com-
pounds: Boc-Lys(Z)-Pro-Arg(NO2)eOMe or Boc-Pro-Lys(Z)-Thr-
OMe. Next the Boc-protecting groups were removed and then
coupling with Boc-Thr-OH or Boc-Arg(NO2)eOH gave tetrapep-
tides. The Z-protecting groups were removed from Boc-Thr-Lys(Z)-
Pro-Arg(NO2)eOMe or Boc-Arg(NO2)-Pro-Lys(Z)-Thr-OMe by cata-
lytic hydrogenation over 10% palladium on charcoal and then
coupled with Boc-X-OH (X: H, Gly, Ala, Val, Leu, Ile) to give pen-
tapetides Boc-Thr-Lys(Boc-X)-Pro-Arg(NO2)eOMe (2aef) or Boc-
Arg(NO2)-Pro-Lys(BocX)-Thr-OMe (2gel). Finally, the OMe-
protecting group was removed from peptides 2ael by treatment
with LiOH/MeOH to give the free C-terminal groups derivatives
3ael. The chemical characteristics of new compounds are pre-
sented below.
The analysis HPLC compounds 5 were performed using Agilent
liquid chromatograph series 1290 (Agilent Technology, Waldbronn,
Germany) consisting of binary pump G4220A, autosampler
G4226A, thermostated column compartment G1316C, diode-array
detector G1315C. Chromatographic column: Supelcosil C-18,
(4.6 ꢂ 150 mm), 3
m
m, Supelco. Analytes were dissolved in a mixture
of DMSO/MeOH (1:1; v/v) and 2
mL of the solution were injected
onto the chromatographic column. A mixture of A: 0.1% HCOOH in
water B: 0.1% HCOOH in ACN/MeOH (1:1; v/v) was used as a mobile
phase in gradient mode at flow rate of 2.0 mL minꢀ1. Gradient: at
5 min 20% B; at 15 min 100% B for analyzed compounds. The UVeVis
detector was operated at 254, 210 and 580 nm (DAD in single
wavelength mode). All analyses were thermostated at 40 ꢁC.
4.2.1. Boc-Thr-Lys(BocGly)-Pro-Arg(NO2)eOMe 2b
Yield 46%, mp. 106e111 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-K5),
-R4a),1.56 (m,1H,
-P4),
-P3), 2.29 (m, 1H, ε-K6a), 3.07 (m, 1H, ε-K6b), 3.20 (m,
1.04 (d, J ¼ 8.4 Hz, 3H,
1.37 (s, 9H, Boc),1.43 (m,1H,
-K3b), 1.74 (m, 1H,
2.03 (m, 2H,
2H, -R5), 3.54 (s, 1H, R2-OH), 3.60 (m, 2H,
P5), 3.78 (m, 1H,
-T3), 3.86 (d, J ¼ 7.6 Hz, 1H,
R2), 4.29 (d, J ¼ 6.3 Hz, 1H, -P2), 4.39 (m, 1H,
J ¼ 7.0 Hz, 1H, -TN), 7.40 (d, J ¼ 7.0 Hz, 1H, -GN), 7.47 (m, 1H,
g
-T4), 1.29 (m, 2H,
g
-K4), 1.31 (m, 2H,
d
b
-K3a),1.56 (m,1H, g
b
g-R4b), 1.74 (m, 2H, b-R3), 1.91 (m, 2H, g
b
d
a-G2), 3.63 (m, 2H,
d
-
4.1. 8-Aminoisoindolo[1,2-b]quinazolin-12(10H)-one (BAT) 1
b
a-T2)), 4.24 (m, 1H,
a
-
a
a-K2), 6.96 (d,
This compound was prepared according to the method
described by Dzierzbicka et al. [20] from N-[2-(phthalimido-
methyl)-1,4-acetyl]phenylenediamine and recrystallized from
DMF.
a
a
d-
KeN), 7.80 (d, J ¼ 7.0 Hz, 1H,
a-KaN), 8.37 (bs, 1H, d-RdN), 8.36 (d,
J ¼ 7.5 Hz, 1H,
a
-RaN). MS [MþH]þ m/z calcd for C34H60N10O13
20
816.43, found 816.80. Rf ¼ 0.52 (B). [
a
]
ꢀ28 (c 0.5, CHCl3).
D

90
W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
4.2.2. Boc-Thr-Lys(BocLeu)-Pro-Arg(NO2)eOMe 2e. Yield 39%, mp.
109e114 ꢁC
9H, Boc), 1.46 (m, 2H,
1.52 (m, 1H, -K3), 1.55 (m, 2H,
-R3), 1.72 (m, 1H, -K3), 1.82 (m, 1H,
-P3), 3.05 (m, 2H, ε-K6), 3.13 (m, 2H,
-P5), 3.55 (s, 3H, T-COOCH3), 4.0 (dt, J ¼ 5.2 Hz,
-L2), 4.13 (m, 1H, -T3), 4.18 (m, 1H, -R2), 4.33 (dd,
J ¼ 3.4 Hz, J ¼ 8.5 Hz, -T2), 4.29 (m, 1H, -K2), 4.4 (dd, J ¼ 4.0 Hz,
J ¼ 8.3 Hz, 1H, -P2), 5.00 (s, 1H, T3-OH), 7.37 (m, 1H, -KeN), 7.55
(bs, 1H, -TN), 7.78 (d, J ¼ 8.5 Hz, 1H,
-LN), 7.74 (d, J ¼ 7.6 Hz, 1H,
TN), 7.92 (d, J ¼ 7.3 Hz, 1H, -KaN), 7.98 (d, J ¼ 7.3 Hz, 1H, -RaN),
8.18 (bs,1H,
KN), 8.44 (bs, 1H,
b
-L3), 1.48 (m, 2H,
-R4), 1.6 (m, 2H,
-P3), 1.93 (m, 2H,
-R5), 3.45 (m, 1H, d-
d
-K5), 1.51 (m, 1H,
-L4), 1.70 (m, 1H,
-P4), 2.06
b-R3),
b
g
g
1H NMR (500 MHz, DMSO)
d
ppm: 0.81 (d, J ¼ 3.7 Hz, 3H,
2-L6), 1.00 (d, J ¼ 8.0 Hz, 3H, -T4), 1.28 (m,
-K4), 1.32 (d, J ¼ 3.9 Hz, 2H, -L3), 1.35 (m, 2H, -K5), 1.36 (s,
9H, Boc), 1.46 (m, 1H, -K3a), 1.54 (m, 1H,
-R4a), 1.57 (d, J ¼ 5.2 Hz,
1H, -L4), 1.60 (m, 1H, -K3b), 1.72 (m, 1H, -R4b), 1.72 (m, 2H,
R3), 1.83 (m, 2H, -P4), 2.03 (m, 2H, -P3), 2.69 (m, 1H, ε-K6a), 2.99
(m, 1H, ε-K6b), 3.13 (m, 2H, -R5), 3.59 (s, 1H, R2-OH), 3.63 (m, 2H,
-P5), 3.85 (m, 1H, -T2)), 4.09 (m, 1H,
-T3), 3.85 (d, J ¼ 7.9 Hz, 1H,
-R2), 4.31 (d, J ¼ 6.3 Hz, 1H, -P2), 4.47 (m, 1H,
-K2), 6.38 (d, J ¼ 7.2 Hz, 1H, -TN), 7.58 (d, J ¼ 7.6 Hz, 1H, -LN),
d1-L5),
b
b
b
g
0.84 (d, J ¼ 4.2 Hz, 3H,
d
g
d
(m, 1H,
b
d
2H,
g
b
P5), 3.55 (m, 1H,
J ¼ 9.0 Hz, 1H,
d
b
g
a
b
a
g
b
g
b
-
a
a
g
b
a
d
d
a
a
a-
d
b
a
a
a
a
a
-L2), 4.21 (m, 1H,
a
a
d
-RdN), 8.03 (t, J ¼ 5.5 Hz, ε-K6), 8.1 (d, J ¼ 7.8 Hz,1H,
a-
a
a
d
-R5). MS [MþH]þ m/z calcd for C38H68N10O13
20
7.67 (m, 1H,
d
a
-KeN), 7.84 (d, J ¼ 7.0 Hz, 1H,
a
-KaN), 8.26 (d,
872.50, found 873.60. Rf ¼ 0.52 (B). [
a
]
D
ꢀ24 (c 0.5, CHCl3).
J ¼ 7.9 Hz, 1H,
-RaN), 8.58 (bs, 1H,
d
-RdN). MS [MþH]þ m/z calcd
20
for C38H68N10O13 872.50, found 873.80. Rf ¼ 0.51 (B). [
a
]
ꢀ26 (c
4.2.7. Boc-Arg(NO2)-Pro-Lys(BocIle)-Thr-OMe 2l
D
0.5, CHCl3).
Yield 37%, mp. 101e104 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-T4), 1.21
-K5), 1.36 (s, 9H, Boc), 1.48 (m, 2H,
-K3), 1.55 (d, J ¼ 3.5, 2H, 2-I4), 1.55 (m, 1H, -R3),
-R4),1.59 (m,1H, -R3),1.68 (m,1H, -K3),1.82 (m,1H,
-P3), 2.02 (m, 2H, -P4), 2.06 (m, 1H, -P3), 3.03 (m, 2H, ε-K6), 3.08
-R5), 3.50 (m, 1H, -P5), 3.61 (m, 1H, -P5), 3.85 (s, 3H, T-
-I2), 4.24 (m, 1H, -K2), 4.29 (m, 1H, -T3),
-R2), 4.86 (d, J ¼ 3.0 Hz, J ¼ 8.3 Hz, -T2), 4.87 (dd,
J ¼ 4.5 Hz, J ¼ 8.9 Hz, 1H, -P2), 5.02 (s, 1H, T3-OH), 6.44 (d,
J ¼ 7.8 Hz, 1H, -RaN), 7.39 (m, 1H, -KeN), 7.65 (d, J ¼ 8.4 Hz, 1H,
IN), 7.74 (d, J ¼ 7.6 Hz, 1H, -TN), 7.78 (d, J ¼ 8.8 Hz, 1H, -TN), 7.98
(d, J ¼ 6.9 Hz, 1H, -KaN), 8.10 (bs, 1H,
-KN), 8.44 (bs, 1H,
0.82 (brs, 6H,
(m, 2H, -K4), 1.38 (m, 2H,
K5), 1.52 (m, 1H,
1.59 (m, 2H,
g
1-IMe3,
d
-IMe5), 1.04 (d, J ¼ 7.9 Hz, 3H,
g
4.2.3. Boc-Thr-Lys(BocIle)-Pro-Arg(NO2)eOMe 2f
g
d
d-
Yield 43%, mp. 108e112 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
2-I4), 1.07
-K5), 1.36
-R4a), 1.54 (m, 1H,
-P4), 2.10
-P3), 2.31 (m, 1H, ε-K6a), 3.04 (m, 1H, ε-K6b), 3.18 (m, 2H,
-R5), 3.57 (s, 1H, R2-OH), 3.60 (m, 2H, -P5), 3.80 (m, 1H, -T3),
-T2), 3.90 (m, 1H, -I2),
-P2), 4.40 (m, 1H, -K2),
-IN), 6.97 (d, J ¼ 7.1 Hz, 1H, -TN), 7.52 (m,
-RdN), 8.44
b
g
b
0.88 (brs, 6H,
(d, J ¼ 8.2 Hz, 3H,
(s, 9H, Boc), 1.48 (m, 1H,
K3b), 1.70 (m, 1H,
(m, 2H,
g
1-IMe3,
d
-IMe5), 1.14 (d, J ¼ 4.0 Hz, 2H,
g
g
b
b
g
-T4), 1.27 (m, 2H,
g
-K4), 1.33 (m, 2H,
d
b
g
b
b
-K3a), 1.54 (m, 1H,
g
b
-
(m, 2H,
d
d
d
g-R4b), 1.70 (m, 2H,
b
-R3), 1.95 (m, 2H,
g
COOCH3), 3.99 (m, 1H,
4.34 (m, 1H,
a
a
b
b
a
a
d
d
b
a
a
3.83 (m, 1H,
4.26 (m, 1H,
b
-I3), 3.84 (d, J ¼ 7.0 Hz, 1H,
a
a
a
d
a-
a-R2), 4.29 (d, J ¼ 6.0 Hz, 1H,
a
a
a
6.20 (d, J ¼ 9.0 Hz, 1H,
1H,
(d, J ¼ 7.4 Hz, 1H,
a
a
a
d
-RdN), 8.10 (t, J ¼ 5.0 Hz,
d-KeN), 7.84 (d, J ¼ 7.7 Hz, 1H,
a
-KaN), 8.41 (bs, 1H,
d
ε-K6), 8.11 (d, J ¼ 7.4 Hz, 1H,
a
d
-R5). MS [MþH]þ
20
a
-RaN). MS [MþH]þ m/z calcd for C38H68N10O13
m/z calcd for C38H68N10O13 872.50, found 873.40; Rf ¼ 0.56 (B). [
ꢀ42.5 (c 0.5, CHCl3).
a]
D
872.50, found 873.10. Rf ¼ 0.52 (B). [
a]
20 ꢀ38 (c 0.5, CHCl3).
D
4.2.4. Boc-Arg(NO2)-Pro-Lys(BocGly)-Thr-OMe 2h
4.3. General procedure for synthesis of BAT-tuftsin/retro-tuftsin
analogues 4ael
Yield 42%, mp. 107e110 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
1.05 (d, J ¼ 8.4 Hz, 3H,
1.40 (m, 2H, -K5), 1.49 (m, 2H,
2H, -R4), 1.84 (m, 2H, -P3), 1.91 (m, 1H,
-R5), 3.43 (d, J ¼ 6.4 Hz, 1H,
-G2), 3.63 (s, 3H, T-COOCH3), 4.12 (m, 1H,
-K2), 4.29 (d, J ¼ 6.8 Hz, 1H,
(s, 1H, T3-OH), 6.92 (d, J ¼ 7.0 Hz, 1H, -RaN), 7.38 (d, J ¼ 8.0 Hz, 1H,
-GN), 7.80 (d, J ¼ 6.8 Hz, 1H,
-KaN), 8.49 (bs, 1H,
g
-T4), 1.36 (m, 2H,
-R3), 1.53 (m, 2H,
-P4), 2.97 (m, 1H, ε-K6),
-P2), 3.55 (m, 2H, -P5),
-T3), 4.16
-T2), 5.00
g
-K4), 1.37 (s, 9H, Boc),
d
b
b-K3), 1.59 (m,
To a mixture of BAT 1 (0.125 mmol) and tuftsin or retro-tuftsin
derivatives 3ael (0.125 mmol) in anhydrous DMF (500 L) was
g
b
g
a
m
3.13 (m, 2H,
3.60 (m, 2H,
(m, 1H,
d
a
d
added anhydrous pyridine (3.09 mmol) followed by T3P
(0.31 mmol, 50% solution in DMF) in drops. The mixture was cooled
to ꢀ15 ꢁC under nitrogen atmosphere for 4 h then for 24 h at room
temperature. After evaporating the solvent in vacuo the residue was
taken up in ethyl acetate. The solution was washed successively
with 10% citric acid, water, 5% NaHCO3 solution and water (3 times
in each case). The product was purified by TLC.
b
a
a
-R2), 4.28 (m, 1H,
a
a
a
a-TN), 7.83 (m, 1H, d-KeN), 8.01 (d,
J ¼ 7.0 Hz, 1H,
a
d
-RdN). MS [MþH]þ m/z calcd
for C34H60N10O13 816.43, found 817.10. Rf ¼ 0.50 (B). [
a
]
20 ꢀ18 (c 0.5,
D
CHCl3).
4.3.1. Boc-Thr-Lys(Boc)-Pro-Arg(NO2)-BAT 4a
4.2.5. Boc-Arg(NO2)-Pro-Lys(BocVal)-Thr-OMe 2j
Yield 41%, mp. 192e195 ꢁC; 1H NMR (500 MHz, DMSO)
d
g
ppm:
-K4),
-K3a), 1.89 (m,
-P4), 2.8 (m, 2H,
-R3), 3.17 (m, 1H, ε-K6a), 3.23 (m, 1H, ε-K6b), 3.42
-R5), 3.86 (d, J ¼ 6.3 Hz, 1H, -P2), 4.34 (d, J ¼ 7.6 Hz, 1H,
T2), 4.36 (m, 1H, -T3), 4.51 (m, 1H, -K2), 4.55 (m, 2H, -P5), 4.78
(m, 1H,
-R2), 4.91 (s, 2H, 10), 4.99 (s, 1H, T3-OH), 6.39 (d, J ¼ 7.8 Hz,
1H, -KeN), 7.54 (bd,
-TN), 7.35 (bdb, J ¼ 8.0 Hz, 1H, 6), 7.37 (m, 1H,
J ¼ 8.0 Hz, 1H, 7), 7.58 (bs, 1H, 9), 7.73 (t, J ¼ 7.0 Hz, 1H, 3), 7.78 (t,
J ¼ 7.0 Hz, 1H, 2), 7.80 (d, J ¼ 7.3 Hz, 1H, -RaN), 7.87 (d, J ¼ 7.0 Hz,
1H, 4), 7.92 (d, J ¼ 7.3 Hz, 1H, -KaN), 7.98 (d, J ¼ 7.0 Hz, 1H, 1), 8.18
(bs, 1H,
-RdN), 10.12 (s, 1H, 800-CONH); 13C NMR (DMSO, 125 MHz,
Yield 32%, mp. 136e138 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-V3),
-K4),1.38 (s, 9H, Boc),1.40
-K3), 1.55 (m, 2H, -R4), 1.62 (m, 2H,
-P3), 1.91 (m, 1H, -P4), 2.97 (m, 1H, ε-K6), 3.14
-R5), 3.57 (m, 2H, -P5), 3.63 (s, 3H, T-COOCH3), 4.13 (m,
-T3), 4.16 (m, 1H, -R2), 4.29 (m, 1H,
-K2), 4.26 (d, J ¼ 6.3 Hz,
-T2), 4.32 (d, J ¼ 5.9 Hz, 1H, -P2), 4.99 (s, 1H, T3-OH), 5.04 (m,
-V2), 7.01 (d, J ¼ 7.9 Hz, 1H, -RaN), 7.02 (d, J ¼ 7.0 Hz, 1H,
1.22 (d, J ¼ 8.6 Hz, 3H,
1.49 (m, 2H, -K5), 1.81 (m, 1H,
1H, -K3b), 1.90 (m, 1H, -R4b), 2.06 (m, 2H,
P3), 2.34 (m, 2H,
(m, 2H,
g
-T4), 1.32 (s, 9H, Boc), 1.49 (m, 2H,
0.97 (d, J ¼ 4.9 Hz, 6H,
1.05 (d, J ¼ 7.6 Hz, 3H,
(m, 2H, -K5), 1.51 (m, 2H,
R3), 1.84 (m, 2H,
(m, 2H,
d
1-V4,
d
2-V5), 0.97 (d, J ¼ 4.6 Hz, 1H,
b
d
g-R4a), 1.79 (m, 1H,
b
g
-T4),1.31 (m, 2H,
g
b
g
g
b-
d
b
g
b
-
b
b
g
d
a
a-
d
d
b
a
d
1H,
1H,
2H,
b
a
a
a
a
a
a
a
a
d
a-
VN), 7.81 (d, J ¼ 7.2 Hz, 1H,
a
-TN), 7.84 (m, 1H,
d-KeN), 8.22 (d,
a
J ¼ 7.0 Hz, 1H, -KaN), 8.49 (bs, 1H,
a
d
-RdN). MS [MþH]þ m/z calcd
a
C
37H56N10O13 858.48, found 856.60. Rf ¼ 0.54 (B); [
a
]
D
20 ꢀ21 (c 0.5,
d
CHCl3).
30.0 ꢁC: 177.55 (C-P1), 172.73 (C-T1), 172.03 (C-R1), 171.55 (C-K1),
167.04 (C-12), 160.70 (C-R6), 156.86 (C-B1), 156. 68 (C-BK1), 148.65
(C-4b), 139.37 (C-5a), 135.33 (C-4a), 135.37 (C-8), 133.65 (C-2),
132.73 (C-3), 131.20 (C-12a), 128.88 (C-6), 123.46 (C-4), 123.19 (C-
9a), 122.53 (C-1), 120.05 (C-7), 118.67 (C-9), 79.47 (C-B2), 79.45 (C-
BK2), 68.32 (C-T2), 61.71 (C-P2), 60.52 (C-T3), 60.34 (C-R2), 57.69
4.2.6. Boc-Arg(NO2)-Pro-Lys(BocLeu)-Thr-OMe 2k
Yield 39%, mp. 106e110 ꢁC; 1H NMR (500 MHz, DMSO)
0.82 (d, J ¼ 6.4 Hz, 3H, 1-L5), 0.83 (d, J ¼ 6.4 Hz, 3H, 2-L6), 1.03 (d,
J ¼ 6.1 Hz, 3H, -T4), 1.34 (m, 2H, -K4), 1.37 (m, 2H, -K5), 1.41 (s,
d
ppm:
d
d
g
g
d

W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
91
(C-K2), 48.32 (C-P5), 47.75 (C-R5), 41.25 (C-10), 40.73 (C-K6), 32.56
(C-K3), 31.51 (C-P3), 30.41 (C-R3), 30.02 (C-B5), 29.95 (C-BK5),
29.80 (C-B4), 29.64 (C-R4), 29.49 (C-K5), 29.33 (C-BK4), 28.63 (C-
B3), 28.48 (C-BK3), 25.93 (C-P4), 23.38 (C-K4), 22.98 (C-T4). MS
[MþH]þ m/z calcd for C46H64N12O12 977.07, found 978.50. Rf ¼ 0.45
(C).
J ¼ 7.3 Hz, 1H,
a
-KaN), 7.98 (d, J ¼ 7.0 Hz, 1H, 1), 8.16 (bs, 1H,
d
-RdN),
10.07 (s, 1H, 800-CONH). MS [MþH]þ m/z calcd for C51H73N13O13
1076.20, found 1077.60. Rf ¼ 0.45 (C).
4.3.5. Boc-Thr-Lys(BocLeu)-Pro-Arg(NO2)-BAT 4e
Yield 39%, mp. 185e188 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
2-L6), 1.28 (d,
-L3) 1.37 (s, 9H, Boc),
-K5), 1.55 (d, J ¼ 4.6 Hz, 1H, -L4),
-K3a), 1.86 (m, 1H, -K3b),1.97 (m,
-P3), 2.35 (m, 2H,
R3), 3.14 (m, 1H, ε-K6a), 3.42 (m, 1H, ε-K6b), 3.44 (m, 2H, -R5), 3.90
-L2), 4.35 (d, J ¼ 7.0 Hz, 1H,
-K2), 4.49 (m, 2H, -P5), 4.92
0.82 (d, J ¼ 2.9 Hz, 3H,
d
1-L5), 0.83 (d, J ¼ 3.9 Hz, 3H,
d
4.3.2. Boc-Thr-Lys(BocGly)-Pro-Arg(NO2)-BAT 4b
J ¼ 7.6 Hz, 3H,
g
-T4), 1.33 (d, J ¼ 4.5 Hz, 2H,
b
Yield 38%, mp. 187e190 ꢁC; 1H NMR (500 MHz, DMSO)
d
g
ppm:
-K4),
-K3a), 1.93 (m,
-P4), 2.29 (m, 2H,
-R3), 3.20 (m,1H, ε-K6a), 3.37 (m,1H, ε-K6b), 3.45
-R5), 3.62 (m, 2H, -P2), 4.28
-G2), 3.89 (d, J ¼ 6.5 Hz, 1H,
(d, J ¼ 7.8 Hz, 1H, -T2), 4.29 (m, 1H, -T3), 4.52 (m, 1H, -K2), 4.53
(m, 2H, -P5), 4.90 (m, 1H, -R2), 4.93 (s, 2H, 10), 5.00 (s, 1H, T3-
OH), 7.35 (bdb, J ¼ 8.0 Hz, 1H, 6), 7.37 (m, 1H, -KeN), 7.39 (d,
J ¼ 7.6 Hz, 1H, -GN), 7.67 (bd, J ¼ 8.0 Hz, 1H, 7), 7.68 (bs, 1H, 9), 7.74
(d, J ¼ 7.6 Hz, 1H, -TN), 7.76 (t, J ¼ 7.4 Hz, 1H, 3), 7.79 (t, J ¼ 7.5 Hz,
1H, 2), 7.80 (d, J ¼ 7.4 Hz, 1H, -RaN), 7.86 (d, J ¼ 7.4 Hz, 1H, 4), 7.91
1.47 (m, 2H,
1.79 (m, 1H,
g
-K4), 1.47 (m, 2H,
d
g
1.28 (d, J ¼ 9.0 Hz, 3H,
1.42 (m, 2H, -K5), 1.88 (m, 1H,
1H, -K3b), 1.97 (m, 1H, -R4b), 2.14 (m, 2H,
P3), 2.32 (m, 2H,
(m, 2H,
g
-T4), 1.37 (s, 9H, Boc), 1.42 (m, 2H,
g-R4a),1.81 (m, 1H,
b
b
d
g-R4a), 1.92 (m, 1H,
b
1H,
g-R4b), 2.05 (m, 2H,
g-P4), 2.28 (m, 2H,
b
b-
b
g
g
b
-
d
b
(d, J ¼ 6.5 Hz, 1H,
a
-P2), 4.08 (m, 1H,
a
a
a-
d
a
a
T2), 4.36 (m, 1H,
b
-T3), 4.39 (m, 1H,
d
a
b
a
(m, 1H,
J ¼ 8.3 Hz,1H, 6), 7.37 (m,1H,
(bs,1H, 9), 7.60 (d, J ¼ 7.0 Hz,1H,
7.74 (t, J ¼ 7.5 Hz, 1H, 3), 7.79 (t, J ¼ 7.0 Hz, 1H, 2), 7.80 (d, J ¼ 7.3 Hz,
1H, -KaN),
-RaN), 7.88 (d, J ¼ 7.8 Hz, 1H, 4), 7.89 (d, J ¼ 7.3 Hz, 1H,
-RdN), 10.05 (s, 1H, 800-
a
-R2), 4.92 (s, 2H, 10), 4.99 (s, 1H, T3-OH), 7.36 (bdb,
-KeN), 7.57 (bd, J ¼ 8.5 Hz,1H, 7), 7.58
-LN), 7.68 (d, J ¼ 7.7 Hz,1H, -TN),
d
a
d
d
a
a
a
a
a
a
a
7.99 (d, J ¼ 7.0 Hz, 1H, 1), 8.17 (bs, 1H,
d
(d, J ¼ 7.3 Hz, 1H,
a
-KaN), 7.99 (d, J ¼ 7.5 Hz, 1H, 1), 8.15 (bs, 1H,
d
-
CONH). MS [MþH]þ m/z calcd for C52H75N13O13 1090.23, found
1091.10. Rf ¼ 0.44 (C).
RdN), 9.54 (s,1H, 800-CONH). MS [MþH]þ m/z calcd for C48H67N13O13
1034.12, found 1035.70. Rf ¼ 0.43 (C).
4.3.6. Boc-Thr-Lys(BocIle)-Pro-Arg(NO2)-BAT 4f
4.3.3. Boc-Thr-Lys(BocAla)-Pro-Arg(NO2)-BAT 4c
Yield 32%, mp. 191e195 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
2-I4), 1.20
-K4), 1.50 (m,
-K3a), 1.91 (m, 1H,
-P4), 2.25 (m, 2H, -P3),
-R3), 3.12 (m, 1H, ε-K6a), 3.25 (m, 1H, ε-K6b), 3.38 (m,
-R5), 3.83 (m, 1H, -I3), 3.89 (m, 1H,
-I2), 4.29 (d, J ¼ 6.0 Hz,
-P2), 4.35 (d, J ¼ 7.5 Hz, 1H, -T2), 4.53 (m, 1H, -T3), 4.55 (m,
-K2), 4.64 (m, 2H, -P5), 4.66 (m, 1H, -R2), 4.94 (s, 2H, 10),
-TN), 6.18 (d, J ¼ 9.0 Hz,
-IN), 7.35 (bdb, J ¼ 8.0 Hz, 1H, 6), 7.45 (m, 1H, -KeN), 7.57 (bd,
J ¼ 8.6 Hz, 1H, 7), 7.59 (bs, 1H, 9), 7.74 (t, J ¼ 7.0 Hz, 1H, 3), 7.79 (t,
J ¼ 7.0 Hz, 1H, 2), 7.80 (d, J ¼ 7.0 Hz, 1H, -RaN), 7.86 (d, J ¼ 7.0 Hz,
Yield 29%, mp. 195e198 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-A3),1.39 (s, 9H, Boc),1.48
-K5), 1.83 (m, 1H, -R4a), 1.82 (m, 1H,
-K3b), 1.89 (m, 1H, -R4b), 2.10 (m, 2H, -P4),
-R3), 2.46 (m, 2H, -P3), 3.18 (m, 1H, ε-K6a), 3.44 (m,
1H, ε-K6b), 3.49 (m, 2H, -P2), 4.25 (m,
-R5), 3.86 (d, J ¼ 6.3, 1H,
-A2), 4.34 (d, J ¼ 7.5 Hz, 1H, -T2), 4.36 (m, 1H, -T3), 4.52 (m,
-K2), 4.55 (m, 2H, -P5), 4.71 (m, 1H, -R2), 4.92 (s, 2H, 10),
-AN), 7.36 (bdb,
-KeN), 7.57 (bd, J ¼ 8.0 Hz,1H, 7), 7.59
(bs,1H, 9), 7.69 (d, J ¼ 7.3 Hz,1H, -TN), 7.74 (t, J ¼ 7.0 Hz,1H, 3), 7.79
(t, J ¼ 7.0 Hz, 1H, 2), 7.80 (d, J ¼ 7.3 Hz, 1H, -RaN), 7.87 (d, J ¼ 7.0 Hz,
1H, 4), 7.92 (d, J ¼ 7.3 Hz, 1H, -KaN), 7.98 (d, J ¼ 7.0 Hz, 1H, 1), 8.17
(bs, 1H,
-RdN), 10.15 (s, 1H, 800-CONH). 13C NMR (DMSO, 125 MHz,
0.90 (brs, 6H,
(d, J ¼ 7.8 Hz, 3H,
2H, -K5), 1.77 (m, 1H,
K3b), 1.91 (m, 1H, -R4b), 2.03 (m, 2H,
2.25 (m, 2H,
g
1-IMe3,
-T4), 1.37 (s, 9H, Boc), 1.48 (m, 2H,
-R4a), 1.79 (m, 1H,
d
-IMe5), 1.16 (d, J ¼ 3.9 Hz, 2H,
g
1.22 (d, J ¼ 8.4 Hz, 3H,
(m, 2H, -K4), 1.48 (m, 2H,
K3a), 1.89 (m, 1H,
2.34 (m, 2H,
g
-T4),1.31 (m, 3H,
b
g
g
g
d
g
b-
d
g
b
b-
b
g
g
g
g
b
b
b
b
d
a
2H,
1H,
1H,
d
a
a
b
a
1H,
1H,
a
a
a
b
a
b
d
a
d
a
4.99 (s, 1H, T3-OH), 6.49 (d, J ¼ 7.0 Hz, 1H,
a
5.01 (s, 1H, T3-OH), 6.06 (d, J ¼ 7.5 Hz, 1H,
a
J ¼ 8.0 Hz,1H, 6), 7.37 (m,1H,
d
1H,
a
d
a
a
a
a
1H, 4), 7.92 (d, J ¼ 7.5 Hz, 1H, -KaN), 7.97 (d, J ¼ 7.0 Hz, 1H, 1), 8.02
a
d
(bs, 1H,
d
-RdN), 9.50 (s, 1H, 800-CONH). MS [MþH]þ m/z calcd for
30.0 ꢁC: 178.48 (C-A1), 177.33 (C-P1), 177.29 (C-T1), 175.67 (C-R1),
172.29 (C-K1), 166.87 (C-12), 160.56 (C-R6), 156.89 (C-B1), 155.99
(C-BA1), 148.10 (C-4b), 139.37 (C-5a), 134.18 (C-4a), 135.96 (C-8),
133.14 (C-2),132.20 (C-3),130.82 (C-12a),128.43 (C-6),123.78 (C-4),
122,94 (C-9a), 122.25 (C-1), 119.41 (C-7), 118.17 (C-9), 80.86 (C-B2),
78.68 (C-BA2), 70.37 (C-T2), 67.78 (C-P2), 61.34 (C-T3), 59.64 (C-R2),
59.64 (C-K2), 50.40 (C-A2), 47.72 (C-P5), 40.73 (C-R5), 40.74 (C-10),
40.72 (C-K6), 32.64 (C-K3), 38.30 (C-P3), 29.74 (C-R3), 29.64 (C-B5),
29.49 (C-BA5), 29.33 (C-B4), 28.87 (C-B3), 28.72 (C-BA3), 27.93 (C-
P4), 25.35 (C-K4), 22.22 (C-T4), 18.39 (C-A3). MS [MþH]þ m/z calcd
for C49H69N13O13 1048.15, found 1048.90. Rf ¼ 0.43 (C).
C52H75N13O13 1090.23, found 1091.80. Rf ¼ 0.47 (C).
4.3.7. Boc-Arg(NO2)-Pro-Lys(Boc)-Thr-BAT 4g
Yield 41%, mp. 198e204 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-K4),1.89
-P4b), 2.07 (m, 2H,
-K3b), 3.01 (m, 2H,
K5), 3.14 (m, 1H, ε-K6a), 3.15 (m, 1H, ε-K6b), 3.41 (m, 2H, -R5), 3.56
-T2), 4.15 (m, 1H,
-P5), 4.67 (m, 1H, -R2), 4.92
-RaN), 7.37
-KeN), 7.52 (bd, J ¼ 8.0 Hz, 1H,
7), 7.54 (bs, 1H, 9), 7.74 (t, J ¼ 7.3 Hz, 1H, 3), 7.76 (d, J ¼ 7.5 Hz, 1H,
TN), 7.80 (t, J ¼ 7.0 Hz, 1H, 2), 7.88 (d, J ¼ 7.3 Hz, 1H, 4), 7.89 (d,
1.22 (d, J ¼ 7.8 Hz, 3H,
(m, 2H, -R4), 1.97 (m, 1H,
-R3), 2.35 (m, 2H, -P3), 2.98 (m, 2H,
g
-T4),1.33 (s, 9H, Boc),1.45 (m, 2H,
-P4a), 2.06 (m, 1H,
-K3a,
g
g
g
g
b
b
b
b
d-
d
(d, J ¼ 7.7 Hz, 1H,
a
-P2), 4.12 (d, J ¼ 7.7 Hz, 1H,
a
b-
T3), 4.45 (m, 1H,
a-K2), 4.64 (m, 2H,
d
a
(s, 2H, 10), 5.00 (s, 1H, T3-OH), 7.11 (d, J ¼ 7.0 Hz, 1H,
a
(bdb, J ¼ 7.9 Hz, 1H, 6), 7.38 (m, 1H,
d
4.3.4. Boc-Thr-Lys(BocVal)-Pro-Arg(NO2)-BAT 4d
a-
Yield 25%, mp. 183e187 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-V3),
-K4),1.47
-K3a), 1.89 (m, 1H,
-P4), 2.32 (m, 2H, -P3),
-R3), 3.17 (m, 1H, ε-K6a), 3.45 (m, 1H, ε-K6b), 3.41 (m,
-R5), 3.86 (d, J ¼ 6.3 Hz, 1H, -P2), 4.35 (d, J ¼ 7.3 Hz, 1H, -T2),
4.36 (m, 1H, -T3), 4.37 (m, 1H, -K2), 4.61 (m, 2H, -P5), 4.90 (m,
1H, -R2), 4.92 (s, 2H, 10), 4.94 (m, 1H, -V2), 4.99 (s, 1H, T3-OH),
7.04 (d, J ¼ 7.0 Hz, 1H, -VN), 7.36 (bdb, J ¼ 8.0 Hz, 1H, 6), 7.37 (m,
1H,
-KeN), 7.57 (bd, J ¼ 9.0 Hz, 1H, 7), 7.59 (bs, 1H, 9), 7.74 (d,
J ¼ 7.8 Hz, 1H, -TN), 7.75 (t, J ¼ 7.0 Hz, 1H, 3), 7.78 (t, J ¼ 7.0 Hz, 1H,
2), 7.80 (d, J ¼ 7.3 Hz, 1H, -RaN), 7.88 (d, J ¼ 7.0 Hz, 1H, 4), 7.91 (d,
0.97 (d, J ¼ 4.8 Hz, 6H,
1.17 (d, J ¼ 8.0 Hz, 3H,
(m, 2H, -K5), 1.80 (m, 1H,
-K3b), 1.96 (m, 1H, -R4b), 2.16 (m, 2H,
2.34 (m, 2H,
2H,
d
1-V4,
-T4), 1.37 (s, 9H, Boc),1.47 (m, 2H,
-R4a), 1.82 (m, 1H,
d
2-V5), 1.02 (d, J ¼ 4.5 Hz, 1H,
b
J ¼ 7.3 Hz,1H,
a
-KaN), 7.99 (d, J ¼ 6.8 Hz, 1H,1), 8.28 (bs, 1H,
d-RdN),
g
g
9.92 (s, 1H, 800-CONH). MS [MþH]þ m/z calcd for C46H64N12O12
977.07, found 978.40. Rf ¼ 0.45 (C).
d
g
b
b
g
g
b
b
4.3.8. Boc-Arg(NO2)-Pro-Lys(BocGly)-Thr-BAT 4h
d
a
a
Yield 30%, mp. 202e206 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-K4),1.85
-P4b), 2.05 (m, 2H,
-K3b), 2.68 (m, 2H,
K5), 3.17 (m, 1H, ε-K6a), 3.23 (m, 1H, ε-K6b), 3.39 (m, 2H, -R5), 3.64
(m, 2H, -P2), 4.34 (d, J ¼ 6.5 Hz,1H,
-G2), 3.97 (d, J ¼ 6.0 Hz,1H,
T2), 4.35 (m, 2H, -P5), 4.35 (m, 1H, -R2), 4.39 (m, 1H, -T3), 4.45
b
a
d
1.17 (d, J ¼ 8.0 Hz, 3H,
(m, 2H, -R4), 2.02 (m, 1H,
-R3), 2.34 (m, 2H, -P3), 2.62 (m, 2H,
g
-T4),1.37 (s, 9H, Boc),1.44 (m, 2H,
-P4a), 2.04 (m, 1H,
-K3a,
g
a
a
g
g
g
a
b
b
b
b
d-
d
d
a
a
a
a-
a
d
a
b

92
W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
(m, 1H,
a
-K2), 4.91 (s, 2H, 10), 5.05 (s, 1H, T3-OH), 6.48 (d, J ¼ 7.3 Hz,
-KeN), 7.43 (d,
-GN), 7.57 (bd, J ¼ 8.0 Hz, 1H, 7), 7.59 (bs, 1H, 9), 7.69
(d, J ¼ 7.0 Hz, 1H, -TN), 7.74 (t, J ¼ 7.2 Hz, 1H, 3), 7.81 (t, J ¼ 6.9 Hz,
1H, 2), 7.87 (d, J ¼ 7.3 Hz, 1H, 4), 7.89 (d, J ¼ 7.0 Hz, 1H, -KaN), 7.98
6), 7.44 (m, 1H,
7.59 (d, J ¼ 8.4 Hz, 1H,
J ¼ 7.0 Hz, 1H, 3), 7.80 (t, J ¼ 7.1 Hz, 1H, 2), 7.87 (d, J ¼ 7.2 Hz, 1H, 4),
d
-KeN),7.56 (bd, J ¼ 7.9 Hz, 1H, 7), 7.58 (bs, 1H, 9),
1H,
J ¼ 7.3 Hz, 1H,
a
-RaN), 7.35 (bdb, J ¼ 8.2 Hz, 1H, 6), 7.37 (m, 1H,
d
a
-LN), 7.68 (d, J ¼ 7.8 Hz, 1H, -TN), 7.74 (t,
a
a
a
7.92 (d, J ¼ 7.0, 1H,
a-KaN), 7.98 (d, J ¼ 7.3 Hz, 1H, 1), 8.17 (bs, 1H,
d-
a
RdN), 10.03 (s, 1H, 800-CONH). MS [MþH]þ m/z calcd for 1090.23,
found 1090.70. Rf ¼ 0.41 (C).
(d, J ¼ 7.2 Hz, 1H, 1), 8.18 (bs, 1H,
d
-RdN), 10.10 (s, 1H, 800-CONH). MS
[MþH]þ m/z calcd for C48H67N13O13 1034.12, found 1035.50.
Rf ¼ 0.45 (C).
4.3.12. Boc-Arg(NO2)-Pro-Lys(BocIle)-Thr-BAT 4l
Yield 24%, mp. 207e209 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
2-I4), 1.22
g-K4), 1.84 (m,
4.3.9. Boc-Arg(NO2)-Pro-Lys(BocAla)-Thr-BAT 4i
0.85 (brs, 6H,
(d, J ¼ 8.4 Hz, 3H,
2H, -R4), 1.95 (m, 1H,
R3), 2.35 (m, 2H, -P3), 2.92 (m, 2H,
K5), 3.13 (m, 1H, ε-K6a), 3.15 (m, 1H, ε-K6b), 3.37 (m, 2H,
-I3), 3.86 (m, 1H,
-K2), 4.34 (d, J ¼ 6.5 Hz, 1H,
-R2), 4.41 (m, 1H,
g
1-IMe3,
-T4), 1.35 (s, 9H, Boc), 1.49 (m, 2H,
-P4a), 1.97 (m, 1H, -P4b), 2.07 (m, 2H,
-K3a, -K3b), 2.94 (m, 2H,
d
-IMe5), 1.11 (d, J ¼ 3.9 Hz, 2H,
g
Yield 28%, mp. 213e217 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
g
1.20 (d, J ¼ 8.4 Hz, 3H,
(m, 2H, -K4),1.80 (m, 2H,
P4b), 2.11 (m, 2H, -R3), 2.30 (m, 2H,
K3b), 2.87 (m, 2H,
g
-T4),1.34 (m, 3H,
-R4), 2.01 (m,1H,
-P3), 2.85 (m, 2H, b
b-A3),1.37 (s, 9H, Boc),1.48
g
g
g
b
d
-
-
g
g
g-P4a), 2.04 (m,1H,
g-
-
b
b
b
b
d
b
-K3a,
b
d-R5), 3.77
-K5), 3.18 (m, 1H, ε-K6a), 3.25 (m, 1H, ε-K6b),
-R5), 3.92 (d, J ¼ 6.0, 1H, -P2), 4.28 (m, 2H, -A2),
-K2), 4.38 (d, J ¼ 6.5 Hz, 1H, -T2), 4.55 (m, 2H, -P5),
-R2), 4.42 (m, 1H, -T3), 4.92 (s, 2H, 10), 5.00 (s, 1H,
-RaN), 6.79 (d, J ¼ 7.9 Hz, 1H, -AN),
7.36 (bdb, J ¼ 8.0 Hz, 1H, 6), 7.50 (m, 1H, -KeN),7.56 (bd, J ¼ 7.9 Hz,
1H, 7), 7.58 (bs,1H, 9), 7.68 (d, J ¼ 7.5 Hz,1H, -TN), 7.74 (t, J ¼ 7.2 Hz,
1H, 3), 7.79 (t, J ¼ 7.4 Hz, 1H, 2), 7.88 (d, J ¼ 7.6 Hz, 1H, 4), 7.93 (d,
-RdN),
(m, J ¼ 6.4 Hz, 3H,
b
a
-I2), 3.86 (d, J ¼ 6.0 Hz, 1H,
a
d
-
-
3.38 (m, 2H,
4.30 (m, 1H,
4.55 (m, 1H,
d
a
a
P2), 4.28 (m, 1H,
P5), 4.49 (m, 1H,
a
a
-T2), 4.49 (m, 2H,
a
a
d
a
b
-T3), 4.92 (s, 2H, 10), 4.99 (s,
-IN), 6.39 (d, J ¼ 7.6 Hz, 1H,
RaN), 7.36 (bdb, J ¼ 7.9 Hz, 1H, 6), 7.38 (m, 1H, -KeN), 7.57 (bd,
J ¼ 7.9 Hz,1H, 7), 7.59 (bs,1H, 9), 7.76 (d, J ¼ 7.8 Hz,1H, -TN), 7.74 (t,
J ¼ 7.0 Hz, 1H, 3), 7.80 (t, J ¼ 7.1 Hz, 1H, 2), 7.88 (d, J ¼ 7.2 Hz, 1H, 4),
a
b
1H, T3-OH), 6.38 (d, J ¼ 8.4 Hz, 1H,
a
a-
T3-OH), 6.33 (d, J ¼ 7.6 Hz, 1H,
a
a
d
d
a
a
7.94 (d, J ¼ 7.0 Hz, 1H,
a
-KaN), 7.98 (d, J ¼ 7.3 Hz, 1H, 1), 8.17 (bs, 1H,
d
C
-RdN), 10.08 (s, 1H, 800-CONH). MS [MþH]þ m/z calcd for
J ¼ 7.0 Hz, 1H,
a
-KaN), 7.99 (d, J ¼ 7.5 Hz, 1H, 1), 8.18 (bs, 1H,
d
9.92 (s, 1H, 800-CONH). 13C NMR (125 MHz, DMSO, 30.0 ꢁC: 173.52
(C-A1), 172.16 (C-P1), 169.50 (C-T1), 163.52 (C-R1), 169,30 (C-K1),
166.57 (C-12), 160.51 (C-R6), 155.99 (C-B1), 155.97 (C-BA1), 148.06
(C-4b), 138.70 (C-5a), 136.58 (C-4a), 135.04 (C-8), 133.16 (C-2),
132.21 (C-3), 130.84 (C-12a), 128.35 (C-6), 122.97 (C-4), 122,63 (C-
9a), 122.64 (C-1), 119.48 (C-7), 118.09 (C-9), 78.72 (C-B2), 78.55 (C-
BA2), 67.33 (C-T2), 67.32 (C-P2), 61.34 (C-T3), 59.97 (C-R2), 59.20
(C-K2), 50.55 (C-P5), 47.79 (C-R5), 40.78 (C-10), 38.51 (C-K6), 30.69
(C-K3), 30.61 (C-P3), 29.74 (C-R3), 29.43 (C-B5), 29.34 (C-BA5),
29.18 (C-B4), 28.88 (C-B3), 28.72 (C-BA3), 27.91 (C-P4), 23.03 (C-
K4), 20.06 (C-T4). MS [MþH]þ m/z calcd for C51H73N13O13 1076.20,
found 1077.50. Rf ¼ 0.46 (C).
52H75N13O13 1090.23, found 1091.50. Rf ¼ 0.44 (C).
4.4. General procedure of removal of the Boc-protecting groups
The mixture of analogues 4a-l in diethyl ether (Et2O) saturated
with hydrochloric acid was stirred in 0 ꢁC for 30 min. The progress
of the reaction was monitored with TLC in solvent system C. After
evaporating the solvent in vacuo the residue was taken up in
anhydrous diethyl ether. The solution was decanted, and the re-
sidual yellow solid was dried.
4.4.1. 2HClxThr-Lys-Pro-Arg(NO2)-BAT 5a as a yellow solid
Yield 87%, mp. 207e210 ꢁC. MS [MþH]þ m/z calcd for
4.3.10. Boc-Arg(NO2)-Pro-Lys(BocVal)-Thr-BAT 4j
Yield 25%, mp. 203e206 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
-V3), 1.22 (d,
g-K4), 1.85 (m,
C
36H50Cl2N12O8 848.33, found 849.70. Rf ¼ 0.06 (C); 92.45% pure by
HPLC.
0.85 (d, J ¼ 3.9 Hz, 6H,
J ¼ 8.4 Hz, 3H, -T4), 1.35 (s, 9H, Boc), 1.49 (m, 2H,
2H, -R4), 2.02 (m, 1H, -P4a), 2.04 (m, 1H, -P4b), 2.07 (m, 2H,
R3), 2.34 (m, 2H, -P3), 2.85 (m, 2H, -K3a, -K3b), 2.85 (m, 2H,
K5), 3.06 (m, 1H, ε-K6a), 3.10 (m,1H, ε-K6b), 3.39 (m, 2H,
d1-V4, d2-V5), 1.01 (m, 3H, b
g
g
g
g
b
b
d
-
-
4.4.2. 2HClxThr-Lys(Gly)-Pro-Arg(NO2)-BAT 5b as a yellow solid
Yield 82%, mp. 199e204 ꢁC. MS [MþH]þ m/z calcd for
b
b
d
-R5), 4.06
-K2), 4.35 (d, J ¼ 6.5 Hz, 1H,
-R2), 4.40 (m, 1H, -T3), 4.92
-V2), 6.55 (d, J ¼ 7.6 Hz,
-VN), 7.35 (bdb, J ¼ 7.9 Hz, 1H,
-KeN), 7.56 (bd, J ¼ 7.9 Hz, 1H, 7), 7.58 (bs, 1H, 9),
7.68 (d, J ¼ 7.8 Hz, 1H, -TN), 7.74 (t, J ¼ 7.0 Hz, 1H, 3), 7.80 (t,
J ¼ 7.1 Hz, 1H, 2), 7.88 (d, J ¼ 7.2 Hz, 1H, 4), 7.89 (d, J ¼ 7.0 Hz, 1H,
C
38H53Cl2N13O9 906.35, found 907.30. Rf ¼ 0.08 (C); 98.87% pure by
(d, J ¼ 6.0 Hz, 1H,
a
-P2), 4.30 (m, 1H,
a
a
-
HPLC.
T2), 4.37 (m, 2H,
d-P5), 4.38 (m, 1H,
a
b
(s, 2H, 10), 4.98 (s, 1H, T3-OH), 5.02 (m, 1H,
1H,
-RaN), 6.96 (d, J ¼ 8.4 Hz, 1H,
6), 7.37 (m, 1H,
a
4.4.3. 2HClxThr-Lys(Ala)-Pro-Arg(NO2)-BAT 5c as a yellow solid
Yield 85%, mp. 201e205 ꢁC. MS [MþH]þ m/z calcd for
a
a
d
C
39H55Cl2N13O9 920.40, found 920.60. Rf ¼ 0.04 (C); 95.36% pure by
a
HPLC.
a
-
KaN), 7.98 (d, J ¼ 7.3 Hz, 1H, 1), 8.16 (bs, 1H,
d
-RdN), 9.90 (s, 1H, 800-
4.4.4. 2HClxThr-Lys(Val)-Pro-Arg(NO2)-BAT 5d as a yellow solid
Yield 70%, mp. 195e199 ꢁC. MS [MþH]þ m/z calcd for
CONH). MS [MþH]þ m/z calcd for 1076.20, found 1077.50. Rf ¼ 0.46
(C).
C
41H57Cl2N13O9 948.36, found 948.80. Rf ¼ 0.04 (C); 93.72% pure by
HPLC.
4.3.11. Boc-Arg(NO2)-Pro-Lys(BocLeu)-Thr-BAT 4k
Yield 30%, mp. 210e214 ꢁC; 1H NMR (500 MHz, DMSO)
d
ppm:
2-L6), 1.15 (d,
-L3), 1.35 (s, 9H, Boc),
-R4), 2.01 (m, 1H, -P4a), 2.06 (m,
-R3), 2.34 (m, 2H, -P3), 2.87 (m, 2H,
-K5), 3.00 (m, 1H, ε-K6a), 3.11 (m, 1H,
-R5), 4.06 (m, 1H,
-L2), 4.08 (d, J ¼ 6.0 Hz, 1H,
-K2), 4.38 (d, J ¼ 6.5 Hz, 1H, -T2), 4.55 (m, 2H,
-R2), 4.44 (m, 1H, -T3), 4.91 (s, 2H, 10), 4.98 (s,
1H, T3-OH), 6.46 (d, J ¼ 7.6 Hz, 1H, -RaN), 7.35 (bdb, J ¼ 7.9 Hz, 1H,
4.4.5. 2HClxThr-Lys(Leu)-Pro-Arg(NO2)-BAT 5e as a yellow solid
Yield 73%, mp. 194e198 ꢁC. MS [MþH]þ m/z calcd for
0.83 (d, J ¼ 2.9 Hz, 3H,
J ¼ 8.4 Hz, 3H, -T4), 1.34 (m, J ¼ 6.4 Hz, 3H,
1.48 (m, 2H, -K4), 1.85 (m, 2H,
1H, -P4b), 2.10 (m, 2H,
K3a, -K3b), 2.85 (m, 2H,
ε-K6b), 3.38 (m, 2H,
-P2), 4.30 (m, 1H,
-P5), 4.55 (m, 1H,
d
1-L5), 0.84 (d, J ¼ 3.8 Hz, 3H,
d
g
b
C
42H61Cl2N13O9 962.38, found 962.70. Rf ¼ 0.02 (C); 95.67% pure by
g
g
g
HPLC.
g
b
b
b
b-
d
d
a
4.4.6. 2HClxThr-Lys(Ile)-Pro-Arg(NO2)-BAT 5f as a yellow solid
Yield 69%, mp. 203e205 ꢁC. MS [MþH]þ m/z calcd for
a
d
a
a
a
b
C
42H61Cl2N13O9 962.38, found 962.90. Rf ¼ 0.05 (C); 90.58% pure by
a
HPLC.

W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
93
4.4.7. 2HClxArg(NO2)-Pro-Lys-Thr-BAT 5g as a yellow solid
ethanol at ꢀ20 ꢁC. Cells were stained in PBS containing 20
m
g/mL
Yield 84%, mp. 207e210 ꢁC. MS [MþH]þ m/z calcd for
36H50Cl2N12O8 848.33, found 848.70. Rf ¼ 0.03 (C); 97.03% pure by
propidium iodide and 100 mg/mL ribonuclease A for 30 min at room
C
temperature. Samples were analyzed by a Guava EasyCyte™ 8 flow
cytometer (Merck-Millipore) equipped with a 488 nm laser, and the
distribution of cells in the cell cycle was calculated using MultiCycle
software (Phoenix Flow Systems).
HPLC.
4.4.8. 2HClxArg(NO2)-Pro-Lys(Gly)-Thr-BAT 5h as a yellow solid
Yield 78%, mp. 210e215 ꢁC. MS [MþH]þ m/z calcd for
C
38H53Cl2N13O9 906.35, found 907.40. Rf ¼ 0.08 (C); 98.04% pure by
4.8. Inhibition of DNA relaxation mediated by topoisomerases
HPLC.
Supercoiled plasmid pBR322 DNA (>95% form I) was purchased
from Thermo Scientific, human type I topoisomerase was pur-
4.4.9. 2HClxArg(NO2)-Pro-Lys(Ala)-Thr-BAT 5i as a yellow solid
Yield 76%, mp. 220e223 ꢁC. MS [MþH]þ m/z calcd for
chased from MoBiTec and human type II
a topoisomerase was
C
39H55Cl2N13O9 920.40, found 921.20. Rf ¼ 0.05 (C); 96.48% pure by
purchased from TopoGen. Positive controls for topoisomerases
type I and II activity assays, SN-38 and etoposide, respectively
compounds were purchased for SigmaeAldrich. The reaction
mixture contained 200 ng of pBR322 DNA in reaction buffer
(20 mM TriseHCl, pH 7.5, 7.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl,
1 mM ATP) as well as studied compounds. The reaction was
HPLC.
4.4.10. 2HClxArg(NO2)-Pro-Lys(Val)-Thr-BAT 5j as a yellow solid
Yield 65%, mp. 208e212 ꢁC. MS [MþH]þ m/z calcd for
C
41H57Cl2N13O9 948.36, found 949.00. Rf ¼ 0.04 (C). 94.54% pure by
HPLC.
initiated by the addition of topoisomerase IIa enzyme and allowed
to proceed at 37 ꢁC for 30 min. Reactions were terminated by
addition of a loading buffer (0.1% SDS, 0.05% bromophenol blue,
2.5 mM EDTA, 10% sucrose, final concentrations). The samples
were separated in 1% agarose gels at 0.5 V/cm for 18 h in TBE
buffer (90 mM Tris-base, 70 mM boric acid, 1 mM EDTA, pH 8).
4.4.11. 2HClxArg(NO2)-Pro-Lys(Leu)-Thr-BAT 5k as a yellow solid
Yield 61%, mp. 222e227 ꢁC. MS [MþH]þ m/z calcd for
C
42H61Cl2N13O9 962.38, found 962.50. Rf ¼ 0.03 (C); 96.89% pure by
HPLC.
Gels were stained with 0.5 mg/mL ethidium bromide to visualize
4.4.12. 2HClxArg(NO2)-Pro-Lys(Ile)-Thr-BAT 5l as a yellow solid
Yield 68%, mp. 213e219 ꢁC. MS [MþH]þ m/z calcd for
DNA and photographed under UV illumination. The conditions for
DNA relaxation assay for type I topoisomerase were the same
except different reaction buffer was used (10 mM TriseHCl, pH 7.5,
7.5 mM MgCl2, 60 mM KCl).
C
42H61Cl2N13O9 962.38, found 962.70. Rf ¼ 0.07 (C); 95.77% pure by
HPLC.
4.5. Cell culture
4.9. Nuclear and cellular morphology of tumor cells
All cell lines were purchased from American Type Culture
Collection (Rockville, MD). The cells A549, HL-60 LNCaP, P388,
L1210, WEHI 1640 were maintained in RPMI 1640 medium and
HCT-116, HT-29, MCF-7, NIH-3T3 were maintained in DMEM high
glucose. Media were supplemented with 10% fetal bovine serum,
Cells were attached to cover slides in 35 mm Petri dishes and
treated with studied compounds for different time periods.
Following drug treatment, cells were stained with 1
mg/mL Hoechst
33342 alone or with 1 g/mL Hoechst 33342 and 7.5
m
mg/mL fluo-
rescein diacetate dyes for 15 min and analyzed by Olympus BX-60
fluorescent microscope equipped with respective optical filters.
Images were recorded using XC50 digital camera and image
acquisition software CellSens.
2 mM
L-glutamine, and antibiotics (100 units/mL penicillin and
100
m
g/mL streptomycin) and cells were cultivated at 37 ꢁC in 5%
(for RPMI 1640) and 10% (for DMEM H/G) CO2/air atmosphere. All
cell lines were screened routinely for Mycoplasma by the PCR
method with Mycoplasma Plus PCR primer set (Stratagene, La Jolla,
CA).
4.10.
b-Galactosidase staining
4.6. Cytotoxicity assay
Cells were treated with studied compounds with dose corre-
sponding to IC90 for 120 h and washed in PBS, fixed for 3e5 min
(room temperature) in 3.7% formaldehyde, washed, and incubated
at 37 ꢁC (no CO2) with fresh stain solution: 1 mg/ml of 5-bromo-4-
The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, expo-
nentially growing cells were continuously exposed to different drug
concentrations and the cellular viability was determined after four
to five generation times (120 h). Cells were exposed to the MTT
tetrazolium salt for 4 h at 37 ꢁC, and the formation of formazan was
chloro-3-indolyl b-D-galactoside (X-Gal) (stock 20 mg/ml X-Gal in
dimethylformamide)/40 mM citric acid/sodium phosphate, pH 6.0;
5 mM potassium ferrocyanide; 5 mM potassium ferricyanide;
150 mM NaCl; 2 mM MgCl2) for 12e16 h. Images were analyzed by
Olympus BX-60 microscope and recorded using XC50 digital cam-
era as well as image acquisition software CellSens.
measured by
a microplate reader UVM340 (Biochrom Asys
Ltd.). The concentrations required to inhibit cell growth by 50%
compared to untreated controls were determined from the curves
plotting survival as a function of dose by use of the GraphPad Prism
5 program. All values are averages of at least two independent
experiments, each done in duplicate.
Acknowledgment
This work was financially supported by the National Science
Center (Poland), grant no 2012/05/B/NZ7/02461.
4.7. Flow cytometry analysis of cell cycle distribution
Distribution in different phases of the cell cycle was analyzed
after treatment of tumor cells with studied drugs. Briefly, following
drug treatment with a dose corresponding to IC90, cells were
washed in ice-cold phosphate buffered saline (PBS) and fixed in 70%
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.012.

94
W. Januchta et al. / European Journal of Medicinal Chemistry 106 (2015) 85e94
batracylin derivatives, J. Med. Chem. 46 (2003) 978e986.
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