Structure and evaluation of antibacterial and antitubercular properties of new basic and heterocyclic 3-formylrifamycin SV derivatives obtained via 'click chemistry' approach

Article abstract of DOI:10.1016/j.ejmech.2014.07.066

Thirty four novel derivatives of 3-formylrifamycin SV were synthesized via reductive alkylation and copper(I)-catalysed azide-alkyne cycloaddition. According to the obtained results, 'click chemistry' can be successfully applied for modification of struct

Full text of DOI:10.1016/j.ejmech.2014.07.066

European Journal of Medicinal Chemistry 84 (2014) 651e676
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure and evaluation of antibacterial and antitubercular properties
of new basic and heterocyclic 3-formylrifamycin SV derivatives
obtained via ‘click chemistry’ approach
*
Krystian Pyta, Katarzyna Klich, Joanna Domagalska, Piotr Przybylski
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 March 2014
Received in revised form
Thirty four novel derivatives of 3-formylrifamycin SV were synthesized via reductive alkylation and
copper(I)-catalysed azideealkyne cycloaddition. According to the obtained results, ‘click chemistry’ can
be successfully applied for modification of structurally complex antibiotics such as rifamycins, with the
formation of desired 1,2,3-triazole products. However, when azideealkyne cycloaddition on 3-
formylrifamycin SV derivatives demanded higher amount of catalyst, lower temperature and longer
reaction time because of the high volatility of substrates, an unexpected intramolecular condensation
with the formation of 3,4-dihydrobenzo[g]quinazoline heterocyclic system took place. Structures of new
derivatives in solution were determined using one- and two-dimensional NMR methods and FT-IR
spectroscopy. Computational DFT and PM6 methods were employed to correlate their conformation
and acidebase properties to biological activity and establish SAR of the novel compounds. Microbio-
logical, physico-chemical (logP, solubility) and structural studies of newly synthesised rifamycins indi-
cated that for the presence of relatively high antibacterial (MIC ~0.01 nmol/mL) and antitubercular (MIC
16 July 2014
Accepted 20 July 2014
Available online 21 July 2014
Keywords:
Rifamycin antibiotics
Click chemistry
Dipolar cycloaddition
DFT and PM6 calculations
FT-IR and NMR spectroscopy
Antibacterial and antitubercular activity
~
0.006 nmol/mL) activities, a rigid and basic substituent at C(3) arm, containing a protonated nitrogen
atom “open” toward intermolecular interactions, is required.
©
2014 Elsevier Masson SAS. All rights reserved.
1
. Introduction
some overlooked important structural details of the rifamycins
bound to bacterial polymerases, having similar amino acids at the
binding site [5]. Recently, the X-ray structures of the complexes
between RNAP from Escherichia coli and other important de-
rivatives of rifalazil, known as benzoxazinorifamycins comprising a
rigid moiety joining C(3)eC(4) positions, have been reported [6].
Docking of benzoxazinorifamycins to low resolution X-ray structure
of RNAP (ranging from 30 to 3.69 Å) suggested that a relatively rigid
tail, located between C(3)eC(4) positions, is directed to the active
site of the enzyme but appears to make no interactions with any of
RNAP amino-acid residues. Unfortunately, the postulated lack of
interaction of the substituent tail at C(3)eC(4) positions for e.g.
benzoxazinorifamycins does not explain the significant differences
in antibacterial activity (against Gram positive strains) found be-
tween other phenolic rifamycins e rifampicin and its analogues
synthesized by us recently [5]. In view of the above, binding of any
of rifamycins to RNAP, irrespectively of the rifamycin type: quinone
or phenolic containing rigid substituent at C(3)eC(4) position,
should result in a comparable and extremely high antibacterial
activity of these compounds, both against Gram-(ꢀ) and Gram-(þ)
strains, which is not true as the antibacterial activity seems to be
The antibiotic 3-formylrifamycin SV (Ral e Fig. 1) belongs to
rifamycins, showing characteristic “basket-like” structures imposed
by the presence of ansa bridge, which is strongly related to the
antibacterial properties of these compounds [1]. Rifamycin group of
antibiotics was discovered and isolated in 1959 by Professor Sensi
research group, from the bark of maritime pine [2]. Well known and
active semisynthetic modifications of 3-formylrifamicin SV are
rifampicin (US. rifampin) and rifapentine (Fig. 2), comprising in
their structures basic moieties linked to C(3) carbon atom. The level
of antibacterial activity of rifampicin and rifapentine allowed their
use as frontline antibiotics against tuberculosis [3]. In 2002, the
mechanism of rifamycin antibiotics activity was proposed by
Campbell et al. on the basis of X-ray structure of the complex be-
tween rifampicin and RNA polymerase dependent on DNA (RNAP)
from Thermus aquaticus [4]. However, our recent works pointed out
*
Corresponding author.
E-mail address: piotrp@amu.edu.pl (P. Przybylski).
http://dx.doi.org/10.1016/j.ejmech.2014.07.066
223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
0

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K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
HO
O
OH
OH
O
O
O
H
N
N
O
O
OH
O
N
N
N
31-34
R
Higher amount of CuOAc
at least 24 hour in r.T.
HO
HO
O
OH
O
OH
R
O
O
2
H N
N
3
O
O
O
O
THF/MeOH (5:1)
%mol CuOAc
5%mol ascorbic acid
CH
2
Cl2, HCl/EtOH
O
OH
O
OH
5
7
NaBH
3
CN
NH
H
NH
H
H H
N
N
N
N
O
O
N
R
O
OH
O
OH
O
O
1
21-24
HO
HO
HO
O
OH
O
OH
O
OH
O
O
O
H
2
N
N
3
O
O
R
O
O
O
O
O
OH
O
OH
2
,3
OH OH
NH
H
N
NH
H
NH
CH
2
Cl2, HCl/EtOH
THF/MeOH (5:1)
0%mol CuOAc
150%mol ascorbic acid
R
H
H
1
NaBH
3
CN
N
O
O
N
n
3
O
N
N
O
n
N
O
OH
O
OH
O
OH
O
O
O
1
1
5-17 (n=2)
8-20 (n=3)
3-formylrifamycin SV (Ral)
2 (n=2)
3
(n=3)
Higher amount of CuOAc
at least 24 hour in r.T.
HO
O
OH
HO
O
O
H
2
N
O
O
OH
OH
R
O
O
O
NH
H
O
OH
H
N
CH
NaBH
2
Cl2, HCl/EtOH
CN
H
3
N
O
R
N
O
OH
O
n
O
O
OH
N
O
N
4
-14
N
2
2
5-27 (n=2)
8-30 (n=3)
R
Fig. 1. Synthetic routes for new basic derivatives of 3-formylrifamycin SV, by reductive alkylation yielding secondary amine derivatives comprising alkyne or azide groups modified
in the next step by 1,3-copper(I)-catalyzed azideealkyne cycloaddition and condensation.
dependent on the nature of substituents at C(3) carbon of naph-
thalene “core”. Recently, we have synthesized a series of benzyl-
halogen and hydroxyleoxaalkyl secondary amine analogues of
rifampicin and we have found that these compounds show defi-
nitely lower activity (100e1000 times) in comparison with that of
the parent compound e rifampicin. We have postulated that,
except for ansa-docking interactions, a very strong additional salt-
type interaction for rifampicin takes place between a protonated
piperazine ring and highly conserved glutamate E445, which is not
realised for the other derivatives of rifamycins studied by us earlier
[5].
It is well known that the ‘click chemistry’ up to now has pro-
vided a number of useful and fast synthetic methods for modifi-
cation of natural products or compounds of biological interest [7].
One of the well known and widely used methods, discovered in the
1960s, is 1,3-dipolar Huisgen cycloaddition between azide and

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
653
having alkyne or azide terminal groups by our earlier optimized
method with NaBH CN as a reductant, with the addition of HCl in
3
2
31
3
methylene chloride [5b] (Figs. 1 and 3). Basic derivatives 1e12 were
obtained by equimolar addition of amine to 3-formylrifamycin SV,
while dimeric derivatives 13 and 14 were obtained by addition of
two fold excess of 3-formylrifamycin SV relative to the respective
diamine. All synthesised compounds 1e14 were characterized by
HO 23
21
3
6
20 19
2
2
3
5
24
25
O
OH
18
O
33
17
16
26
O
34
OH OH
8
O
1
13
37
27
30
2
8
15
NH
HPLC, MALDI-TOF MS, FT-IR, H and C NMR methods and these
data are given in the Experimental section.
14
1
9
29
7
2
Compounds 4e14 have basic properties and were obtained to
check whether significant basicity of a substituent introduced at
C(3) would improve antibacterial properties of rifamycins. Taking
into account the results of our earlier studies [5], we expected that
the introduction of tertiary nitrogen atom within C(3) arm to the
structure of rifamycins should induce the proton transfer process
from O(8)H phenolic group to a seemingly more basic tertiary ni-
trogen. However, our spectroscopic studies of 4e14 demonstrated
that the structure of these compounds in solution is quite different
than expected. Detailed comparison of chemical shifts of C(11),
C(8), C(6) and C(15) carbon resonances for 4e14 revealed that in all
reductive amination products studied there was a phenolate group
at C(8), irrespectively of the type of substituent at C(3) arm (Fig. 4).
Similar chemical shifts of C(8) and C(11) of about 185 ppm, evi-
3
9
N
4
0
O
6
2
10
3
N
5
4
OH
1
38
O
N
42
1
1
R
O
4
1
13
R
4
3
rifampicin
rifapentine
CH
3
4
3
44
45
46
Fig. 2. Structures of clinically used semisynthetic rifamycins rifampicin (US. rifampin)
and rifapentine.
ꢀ
dipolarophile alkyne substrates in which, theoretically, 1,4- and 1,5
regioisomers can be formed [8]. Application of the Meldal variant of
cycloaddition with the use of Cu -catalyst with a classical antiox-
idant system as e.g. sodium ascorbate, contributes to different
stepwise mechanism not involving concerted cycloaddition.
Therefore, the cycloaddition in Meldal variant cannot be considered
as 1,3-dipolar Huisgen cycloaddition but it should be rather called
the copper(I)-catalyzed azideealkyne cycloaddition (CuAAC). This
denced strong resonance effect between C(8)eO and C(11)]O
1
groups in para position at the naphthalene ring. Analysis of H NMR
þ
spectra (Fig. 4) above 8 ppm reveals that for 10e14, two or one
þ
averaged signal of N(38) H protons are observed above 8 ppm
indicating that the proton from phenolic group is transferred to the
secondary nitrogen with the formation of a zwitterion. Further-
more, results of PM6 and DFT electron density theoretical calcula-
tions revealed that within 9 and 12 non-ionic structures the most
probable sites of protonation are secondary nitrogens attached to
C(38) carbon because they have the most negative partial charge
þ
cycloaddition, similarly as that catalyzed by Ag cations (AgAAC),
runs with the formation of 1,2,3-triazoles substituted at 1 and 4
positions. In contrast to these types of cycloadditions, the
ruthenium-catalysed 1,3-dipolar azideealkyne cycloaddition
RuAAC) yields 1,5-regioisomers [9].
In the present work we synthesised by combination of reductive
þ
(Table 1). Chemical shift of N(38) eH signals for compounds 10e14
in the range 8.49e8.60 ppm suggests that protons localised at
positively charged nitrogen are intra- and intermolecularly
hydrogen-bonded. However, the question is what type of structure
(
þ
alkylation and regioselective copper(I)-catalyzed azideealkyne
cycloaddition, three series of basic derivatives of 3-formylrifamycin
SV with different substituents at C(3) atom, which are potentially
resistant to one of the inactivation processes of rifamycin antibi-
otics realised in bacteria e the loss of C(3) substituent via hydro-
lysis. The first group of these derivatives have secondary and
terminal tertiary nitrogens, the second one are characterized by
lower basicity having triazole rings and the third one have triazole
and 3,4-dihydrobenzo[g]quinazoline rings as basic moieties. Such
structural alterations in the substituent at C(3) were performed to
find out whether its basicity, bulkiness or rigidity imply changes in
antibacterial properties of rifamycin derivatives, which could be a
result of different solubility, lipophilicity and different mode of the
inhibitor docking (especially the substituent interactions) at the
binding site of bacterial RNAP. Using biological activity, logP and
solubility together with molecular docking and spectroscopic
studies of synthesized derivatives enabled us to draw some con-
clusions about the desired structure of substituent at C(3) carbon of
rifamycins needed to generate interesting biological properties.
is realised within compounds 4e9, for which no N eH proton
1
signals in ꢀ5e25 ppm range are detected in H NMR spectra. All
1
13
He C HSQC (Fig. 5a) spectra of these compounds, indicated dia-
stereotopic character of H(39), H(40) and H(41) protons of aliphatic
linker as a result of hindered rotation about respective CeC bonds.
1
1
Moreover, He H NOESY experiments (Figs. 5b and 6) indicated
also close arrangement of the introduced basic linker with O(4)eH
phenolic group and the two C(31) and C(33) methyls of the ansa
bridge. The above spectroscopic data and the presence of fast
þ
chemical exchange of labile N eH protons lead to a conclusion that
for 4e9 the intramolecularly hydrogen-bonded pseudocyclic
structure is formed in solution, as shown in Fig. 6. In the most
energetically favourable structures calculated for 4 and 9, the
intramolecular hydrogen bond parameters are as follows: hydrogen
…
þ
…
bond C(15)]O He N(38) the O N distance is of about 2.5 Å and
ꢁ
þ
H-bond angle is of about 145 and in the hydrogen bond N (38)e
H N(40 or 41), the N N distance is of about 2.6 Å and the H-bond
angle is of about 140 . As can be concluded from very comparable
parameters of these two hydrogen bonds, the two N (38)eH
…
…
ꢁ
þ
hydrogen bonded protons fast fluctuate between donor and
acceptor atoms, which is reflected in the lack of the respective
resonances in the H NMR spectra.
2
. Results and discussion
1
2
.1. Synthesis and structure of new basic derivatives of 3-
Compounds 1e3 were obtained as a “platform” for the synthesis
of new type rifamycin derivatives (15e34) comprising triazole
moieties, performed along two ways; with alkyne or azide groups
attached to rifamycin naphthalene “core” (Figs. 1 and 3). Exemplary
monitoring by HPLC of the cycloaddition progress, when alkyne 3-
formylrifamycin derivative was a substrate (compound 1) is given
formylrifamycin SV (compounds 1e34)
The compound 3-formylrifamycin SV was at first subjected to
reductive alkylation with primary amines, comprising in most cases
tertiary nitrogen atoms in their structures, as well as with those

3
2
31
3
2
31
20
HO
2
3
21
HO 23
21
3
6
20 19
36
35
22
19
18
2
2
O
24
3
5
24
25
OH
O
OH
18
25
33
O
O
O
33
9
17
16
2
6
17
16
27
O
OH
1
H
N
2
6
O
34
14 34 8
O
37
2
8
9
3
7
27
O
OH
30
2
8
15
NH
H
7
2
15
30
14
8
29
1
N
29
6
10
H
O
3
R
7
2
5
4
38
12
N
O
OH
O
6
2
10
3
R
40
41
11
5
4
O
1
38
O
OH
15
N
N
43
44
13
1
1
39
O
42
42
45
40
4
1
N
13
2
5
N
N
43
44
4
4
0
0
41
39
39
4
2
45
43
40
41
16
N
N
N
39
45
4
40
40
N
N
41
39
44
43
4
6
26
N
N
4
2
45
41
42
42
N
40
41
17
N
N
43
44
39
46
4
2
45
5
6
N
41
39
42
N
4
4
4
7
43
2
7
N
N
4
4
46
39
39
4
3
45
3
9
41
42
N
4
4
4
0
41
47
4
4
3
1
8
9
N
N
40
46
N
42
43
44
4
6
41
42
3
9
N
4
5
43
4
5
28
29
N
N
39
41
42
40
4
4
44
46
3
1
N
N
N
45
4
1
40
40
46
39
42
44
39
41
42
7
8
N
45
45
N
43
44
4
0
N
N
39
41
42
47
46
40
46
4
3
N
43
45
N
N
42
20
41
3
9
43
44
39
41
47
46
42
N
N
48
4
0
N
N
43
44
3
0
4
7
40
44
45
39
41
4
0
N
21
42
45
48
N
O
4
1
42
43
47
39
39
41
N
N
9
0
N
N
40
50
31
42
4
3
44
4
0
O
O
45
N
43
4
5
49
N
4
4
N
N
46
50
4
4
48
39
O
45
46
41
42
47
49
1
43
N
39
41
O
44
N
46
40
40
47
48
48
4
2
45
O
22
47
4
5
N
39
39
41
O
44
46
4
4
N
N
40
4
3
50
42
45
47
48
5
1
49
32
33
40
N
42
45
1
1
O
N
N
39
41
N
N
40
43
50
49
41
44
42
4
51
23
N
44
N
O
4
3
O
41
N
N
45
40
3
42
N
40
47
5
2
46
N
44
52
3
9
4
1 46
O
45
4
8
O
47
48
N
1
2
N
5
1
43
N
4
5
N
4
3
49
46
5
0
47
4
2
44
39
41
O
5
0
4
9
40
51
50
2
4
42
4
8
1
3
N
43 44
3
9
41
O
10
N
49
1
2
N
40
OH
4
O
34
42
38
2
5
45
4
3
41
39
3
1
0
6
O
48
N
43
4
2
44
N
42
N
H H
40
7
46
N
4
0
N
N
29
14 28
2
47
43
HN
1
9
8
O
34
48
4
5
3
9
41
1
3
15
1
OH
37
3
0
7
O
O
46
6
47
2
6
3
3
25
24
O
1
7
18
OH
O
35
20
2
2
36
1
9
2
1
23 OH
31
32
1
3
O
10 12
OH
O
3
9
H
42
H
3
9
N
38
4
5
6
O
N
N
3
10
14
40
41 41 40
2
7
H
H
HN
15
29
1
9
8 14
2
2
8
37
3
0
OH O34
3
7
1
6
O
26
O
3
25
24
O
1
7
18
OH
O
35
36
20
2
2
1
9
2
1
23 OH
3
1
32
Fig. 3. Structures and atom numbering of new basic derivatives of 3-formylrifamycin SV synthesized via reductive amination (compounds 4e14), copper(I)-catalyzed azideealkyne
cycloaddition (compounds 15e24) and unexpected cyclisation yielding 3,4-dihydrobenzo[g]quinazoline system (compounds 25e34).

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
655
Fig. 4. Comparison of 1H NMR (left) and 13C NMR (right) spectra of 4, 9, 10, 12 basic derivatives of 3-formylrifamycin SV in the range 8e16.5 ppm, recorded in DMSO-d
6
.
ꢁ
in Table 2 and shown in Fig. 7. An increase in temperature to 60 C
shortens the 1,3-dipolar-cycloaddition time to about 2 h. The in-
strongly shifted toward lower ppm values (Fig. 9). In turn, the
He C HMBC spectra of 30 show that the signal at 157.1 ppm is
1
13
ꢁ
crease in the catalyst amount from 5% to 10% at 60 C significantly
correlated with those of the substituent part at C(3) and of diene
moiety simultaneously (Fig. 10). Significant shielding of H(18)
proton signal in the spectra of 25e34 (Fig. 9), relative to its position
in the spectra of 15e24, indicates that H(18) proton is situated over
the newly formed heterocyclic ring within 3-formylrifamycin SV
derivatives, as calculated and shown in Fig. 11. All the above dis-
cussed spectroscopic data indicate unambiguously that for longer
reaction times and at higher amount of CuOAc catalyst used during
the cycloaddition, the subsequent reaction between N(38) amine
and C(15)¼O carbonyl with formation of 3,4-dihydrobenzo[g]qui-
nazoline ring takes place (Fig. 1). The other evidence of formation of
new heterocyclic ring for 25e34 compounds is the decreased in-
accelerated the conversion into expected products; cycloaddition
was completed after 1 h, when the alkyne reagent was 1. In the
cycloaddition between azide attached to the rifamycin “core”
compounds 2 and 3) and more volatile aliphatic alkyne substrates
1-pentyne, 1-hexyne) (in synthesis of 15e20, Figs. 1 and 3), we
noted that the most efficient conditions are: temperature
decreased to 22 C and catalyst amount increased up to 10%.
However, in these reaction conditions requiring longer reaction
times (12e24 h), new products were identified (30 and 3-H O) by
2
(
(
ꢁ
HPLC (Fig. 8a). This result shows that at these specific conditions
during cycloaddition a subsequent reactions with the formation of
ꢀ
1
unexpected dehydrated products (1-H
2
O, 2-H
2
O, 3-H
2
O
and
tensity of complex n(C]O) band of carbonyls at about 1640 cm in
1
2
5e34) is favoured for rifamycins (Fig. 8b). Comparison of the H
and C NMR spectra of isolated compounds 20 and 30 after HPLC
separation, reveals that in the H NMR spectrum of the latter there
are no proton signals of N(2)eH amide and N (38)eH (Fig. 9). In the
their spectra when compared to those of 15e24 (Fig. 12). It should
be mentioned that intramolecular cyclisation was earlier observed
for other 3-formylrifamycin SV derivatives type/Schiff bases or
oxidized to p-quinone system rifamycins/in the presence of AcOH
[10].
13
1
þ
13
C NMR spectrum of 30 the chemical shift of C(15) carbon is
Table 1
Partial charges at oxygen and nitrogen atoms within non-ionic structures of 9, 12, 20 and 30 derivatives, calculated by PM6 and B88LYP DFT methods at the same geometry and
assumed intramolecular interactions (Scigress Explorer package).
(
Mulliken)DFT partial charges
N(2)
O(8)
N(38)
N(41)/N(41)-N]N(triazole)
N(41)-N]N(triazole)
N(41)-N]N(triazole)
O(15)
O(11)
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
PM6
DFT
9
1
2
3
þ0.02
þ0.36
ꢀ0.236
ꢀ0.205
ꢀ0.228
ꢀ0.200
ꢀ0.239
ꢀ0.211
ꢀ0.338
ꢀ0.254
ꢀ0.431
ꢀ0.290
ꢀ0.448
ꢀ0.302
ꢀ0.423
ꢀ0.281
ꢀ0.399
ꢀ0.294
ꢀ0.307
e
e
e
e
e
e
e
e
ꢀ0.301
ꢀ0.221
ꢀ0.304
ꢀ0.241
ꢀ0.301
ꢀ0.241
ꢀ0.390
ꢀ0.264
ꢀ0.393
ꢀ0.272
DFT
ꢀ0.253
PM6
PM6
PM6
2
0
0
þ0.007
ꢀ0.319
ꢀ0.28
ꢀ0.239
ꢀ0.305
ꢀ0.249
e
DFT
DFT
DFT
PM6
DFT
þ0.41
þ0.05
þ0.39
ꢀ0.258
PM6
DFT
PM6
PM6
PM6
þ0.03
ꢀ0.118
ꢀ0.128
DFT
DFT
DFT
þ0.07
ꢀ0.149
ꢀ0.179
PM6
DFT
PM6
PM6
PM6
ꢀ0.461
þ0.01
ꢀ0.120
ꢀ0.133
DFT
DFT
DFT
ꢀ
0.401
þ0.06
ꢀ0.150
ꢀ0.184
e

656
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
Fig. 5. Exemplary 1He13C HSQC (a) and 1He H NOESY (b) spectra of 9 derivative, recorded in DMSO-d
1
solution.
6
In the structures of 15e24 in solution, the proton of phenolic
suggested different sites with the most negative partial charges for
these derivatives e N(38) nitrogen for 20 and N(2) nitrogen for 30
(Table 1). Assignment of the signal at about 11.6 ppm in the
group at C(8) is transferred to secondary N(38) atom with the for-
mation of a zwitterion, as demonstrated by our spectroscopic and
theoretical studies (Table 1), but the question is where is the most
acidic proton localised in the structures of compounds 25e34
having 3,4-dihydrobenzo[g]quinazoline ring. Cyclisation to such a
heterocyclic system proceeds with formation of a basic amidine
moiety, where one of nitrogens N(2) is a very probable site of
intramolecular protonation (Fig. 11). PM6 and DFT calculations
1
respective H NMR spectra of 25e34 to the proton at positively
charged N(2) nitrogen, is a strong evidence of the site of intra-
molecular protonation. Furthermore, comparison of the FT-IR
spectra of 20 and 30 derivatives (Fig. 12), shows in the latter the
ꢀ1
activation of
agreement with the theoretically calculated frequency of this band
n(C]C)aromatic band at 1605 cm , which is in good

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
657
Table 2
Exemplary data of conversion rate [%] for copper(I)-catalyzed azideealkyne cyclo-
addition of compound 1 e alkyne substrate (Fig. 1) with AF3 e 2-(3-azidopropyl)
isoindoline-1,3-dione as azide substrate, determined from HPLC collected at
lmax ¼ 260 nm relative to internal standard (1,2-benzenedimethanol).
Reaction
time
Conversion rate (%)
3% CuOAc 5% CuOAc catalyst
10% CuOAc
catalyst
(min)
catalyst
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
60
C
0
C
22
C
40
C
50
C
60
C
60
C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
4
0
0
0
0
0
0
0
0
e
0
0
0
0
0
0
0
0
<0.1
0.1
0.9
1.6
4.1
6.0
e
0.1
1.8
6.1
1.0
2.5
7.5
3.3
13.3
24.9
44.1
67.5
82.1
90.2
93.9
97.5
98.2
99.0
99.3
99.6
99.9
e
61.0
88.9
97.7
99.5
99.8
99.9
e
2.8
e
7.7
e
20.2
36.9
49.6
58.3
64.0
69.6
73.8
77.1
79.7
82.6
83.3
84.4
85.7
87.9
89.5
e
27.0
43.4
56.3
65.3
74.1
78.8
82.7
86.1
89.6
90.6
92.4
93.1
93.5
93.4
93.7
94.2
94.8
95.1
96.5
97.1
97.4
98.2
98.6
99.0
99.2
>99.0
e
27.6
e
51.3
e
9.6
e
e
0
0
e
00
10
20
30
40
50
60
70
80
90
00
10
20
30
40
70
00
30
60
90
20
64.4
e
e
e
0
e
e
0
e
e
0
e
e
0
e
e
e
e
e
e
e
e
0
12.1
e
e
e
73.2
e
14.8
e
e
e
80.6
e
18.0
e
e
e
84.7
e
23.1
e
e
e
e
e
86.5
e
e
e
e
e
e
e
88.1
e
e
e
e
e
e
92.1
e
e
e
90.3
e
e
e
e
e
e
e
e
91.2
e
35.0
e
93.1
94.7
96.1
97.3
98.1
98.2
99.0
e
e
e
e
e
e
e
e
e
e
e
e
e
50.0
e
e
e
e
e
e
99.0
e
e
e
against Mycobacterium tuberculosis and Mycobacterium bovis as
well as against fungi Candida albicans. Selected results of these
studies are presented in Table 3 and compared to those obtained for
rifampicin (as standard) and 3-formylrifamycin SV. Tests against
bacteria, mycobacteria and fungus were performed only up to a
Fig. 6. Selected and important protoneproton contacts identified on the basis of
He H NOESY spectra, marked on the most energetically favourable pseudocyclic
concentration of 1
mg/mL. All synthesized compounds, irre-
1
1
spectively of whether they have triazole moieties or not, do not
show any interesting antifungal (C. albicans) and antibacterial ac-
tivity against Gram-(ꢀ) strains, similarly as rifampicin.
ꢁ
ꢁ
structures of 4 (H
f
¼ ꢀ451.1 kcal/mol) (a) and 9 (H ¼ ꢀ502.7 kcal/mol) (b), calculated
f
1 1
by MOG-PM6 method at semi-empirical level of theory (Scigress Explorer). He H
contacts marked by black arrows are presented together with the protoneproton
distances [Å] obtained from calculated structures.
Analysis of the data from Table 3 shows that 13e34 compounds
have weak or moderate antibacterial properties against Gram-(þ)
strains. Experimentally determined logP values were compared
with the ones calculated and given in Table 4. As can be seen from
these data there is a significant difference, even by one unit of
magnitude, between the experimentally determined and calcu-
lated values of molecular lipophilicity (logPexp and logPcalc). A
comparison of logPexp determined for derivatives 13e34 and for
rifampicin demonstrates that lipophilicity of the former ones is
more or much more favourable than that for the latter (Table 4).
Thus, the lower activity of compounds 13e34 than rifampicin could
be a result of their lower water solubility (Table 4). The values of
logPexp of compounds 4, 5, 9, 11 comparable with that of rifampicin
and the lower water solubility of the above compounds would
explain why they are more antibacterially active against Gram-(þ)
strains than compounds 13e34 and have lower antibacterial po-
tency in comparison to that of rifampicin. The problem is with
compound 12, which has higher logPexp value than those of
rifampicin and derivatives 4, 5, 9 and 11 and is characterized by low
water solubility. It should be noted that despite much lower
in the spectrum of 3,4-dihydrobenzo[g]quinazoline as zwitterion:
ꢀ
1
ꢀ1
1
611 cm for MO-G PM6 method; 1608 cm for calculations with
ADF package.
The CeH value significantly below 130 ppm, found for 1,2,3-
d
1
13
triazole moiety of 15e34 derivatives, together with He C HMBC
data indicated formation of 1,4-regioisomers as sole cycloaddition
products. In summary, irrespectively of whether 1,3-dipolar Huis-
gen cycloaddition runs with or without intramolecular condensa-
tion to the heterocyclic system, always the zwitterionic structures
are formed with the proton transferred to N(2) nitrogen of amidine
(
1
for 25e34) or to N(38) nitrogen of the introduced substituent (for
5e24).
2.2. Antibacterial and antitubercular activities of 4e34 derivatives
and binding to RNA polymerase
Three series of new basic derivatives: 4e14, 15e24 and 25e34
were studied against Gram-(þ) and Gram-(ꢀ) bacteria strains and

658
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
protonated as a relatively strong bases and therefore would be able
to interact with E445. The significantly lower activity of compounds
e9 than that of rifampicin could be a result of their intramolec-
4
ularly hydrogen-bonded pseudocyclic structures within C(3) sub-
stituent in DMSO, in which the spectroscopic and microbiological
studies were conducted. Fixing of conformationally labile basic and
protonated substituent in intramolecular interaction excludes the
possibility of its hydrogen-bonding with the negatively charged
E445 carboxylate of RNAP (Fig. 6). According to our spectroscopic
studies in solution, for compounds 10 and 11 the pseudocyclic
structures are not realised but these compounds have a con-
formationally labile propyl linker at C(3), instead of the rigid moiety
present in the structure of 12. Furthermore, comparison of the
differences in MIC values against Gram-(þ) strains for 12, 4e11 and
triazole containing rifamycins 15e34, suggests that the kind of
substituent at C(3) plays role in the appearance of antibacterial
activity (Table 3). Additional evidence of high importance of the
interaction between the protonated basic substituent at C(3) of 12
and the highly conserved E445 is the activity of compound 12 higher
in most cases after protonation (MIC 0.03e0.25 mg/mL, Table 3). In
fact, derivative 12 can be protonated spontaneously in biological
systems but the protonation during microbiological tests condi-
tions at pH ~7 is not efficient. Hence, the intentional protonation of
12 increases its water solubility, which further improves the
accessibility of this compound by about 15-fold, in the microbio-
1
13
logical assays' conditions (Table 4). According to our H and
C
NMR spectroscopic data (see Experimental section), an additional
protonation of 12 zwitterionic structure occurs exclusively at the
only one accessible basic nitrogen N(41) atom of piperidine ring,
not involved in any intramolecular interactions.
Molecular docking calculations of 12 þ H at RNAP binding site
revealed similar interactions between ansa-bridge and F394, D396
and R409 as well as between phenolate at C(8) and S411 and Q393
amino acids of RNAP, as for rifampicin [5]. Rigidity of 12 þ H sub-
stituent at C(3) enables effective protection of intermolecular
þ
…
ꢀ
N(41) eH O (E445) hydrogen bond from the outer environment
Fig. 13a, Table 5). When the proton of phenolic group at C(8) is
(
transferred to N(38) but not to N(41) nitrogen within 12, a possi-
bility of new and additional interaction with N448 emerges, in
contrast to rifampicin. Unfortunately, this new interaction weakens
þ
ꢁ
the hydrogen bonding of N(41) eH with E445 for 12 (distance
donoreacceptor 2.9 Å, angle 177 ) if compared to that realized for
ꢁ
rifampicin (2.5 Å, 176 ) [5b], which can be an additional reason why
1
2 þ H is approximately 10-fold less active in comparison with the
clinically used rifampicin (Table 3).
Compounds 25e34, formed by intramolecular condensation
with formation of 3,4-dihydrobenzo[g]quinazoline ring, were the
least active against Gram-(þ) strains of all studied, because their
Fig. 7. HPLC chromatograms of copper(I)-catalyzed azideealkyne cycloaddition
allowing to monitor the reaction progress, analysed at the wavelength
for reaction performed at 60 C and with addition of 5% (molar) CuOAc as catalyst;
l
max ¼ 260 nm
ꢁ
MIC values are above >1 mg/mL. Docking of compound 30 (Fig. 13b,
compound 1 e alkyne substrate (Fig. 1), 23 e product (Fig. 2), AF3 e 2-(3-azidopropyl)
Table 3) at RNAP binding site revealed that besides the ansa bridge
H-bonding, it is not stabilized by any other intermolecular H-bonds,
neither with N448 nor with E445. Similarly, two dimeric derivatives
isoindoline-1,3-dione as azide substrate,
standard.
S e 1,2-benzenedimethanol as internal
13 and 14 also exhibited no activity up to nmol/mL level (Table 3). In
solubility of 12 in comparison with compounds 9 and 11, derivative
2 has the same or higher activity than the other two. To explain
this result it is not enough to analyze the value of logPexp and sol-
ubility of the tested compounds but detailed analysis of the in-
teractions with the target enzyme is also necessary.
Earlier we have proposed that, besides the ansa-docking in-
teractions, a very strong additional salt-type interaction between
protonated nitrogen of piperazine ring at C(3) and highly conserved
glutamate E445 contributes to extremely high activity of rifampicin
fact, compounds 13 and 14 occur in similar zwitterionic structures
to that of 12, but the second half part of the dimer, probably due to
the steric hindrance with the amino acids residues of the enzyme,
prevents stable and effective docking of these ligands at the RNAP
binding site. Compounds 15e24 having triazole substituents are
more antibacterially active against Gram-(þ) strains than com-
pounds 13, 14 and 25e34 (Table 3). Slightly stronger antibacterial
properties of 15e24 (0.5e1 nmol/mL) than those of 25e34 might
be due to the interaction of the former with N448 when docking to
the RNAP (Fig. 13b and c, Table 3). Besides, compounds 13e34 are
characterized by poor water solubility which is also reflected in
their weaker antibacterial activities against Gram-(þ) strains.
1
[5]. In view of this fact, surprising results were obtained for all other
basic derivatives (compounds 4e11) containing amine secondary
linker and terminal tertiary nitrogen, of which the latter could be

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
659
Fig. 8. HPLC chromatograms of copper(I)-catalyzed azideealkyne cycloaddition mixture, recorded after: (a) 12 h, (b) 24 h, at the analytical wavelength
reaction performed at 22 C and with addition of 10% (molar) CuOAc as catalyst; compound 20 e expected cycloaddition product, compound 30 e unexpected cycloaddition product
l
max ¼ 260 nm for the
ꢁ
formed after condensation and cyclisation to 3,4-dihydrobenzo[g]quinazoline system, 3-H
2
O e azide reagent (3) after condensation and cyclisation to 3,4-dihydrobenzo[g]qui-
nazoline system.
Thus, the high antibacterial activity of 12 after protonation, on a
level of 0.03e0.06 nmol/mL, especially against Staphylococcus epi-
dermidis strains, is a sum of the following factors: improved solu-
bility at still good lipophilicity and rigidity of the basic C(3)
substituent influencing the mode of binding with bacterial RNAP.
The derivatives synthesised were also tested against mycobac-
teria (M. tuberculosis
e
MIC's determined in the range
ꢀ
1
1e0.005
e0.125
mg mL and M. bovis e MIC's determined in the range
ꢀ1
1
mg mL ). Surprising results were obtained for 12 and
1
2 þ H against M. tuberculosis because they were found as active as
rifampicin in comparable experimental conditions. Cycloaddition
products 21e23, containing terminal phthalimide ring show ac-
ꢀ
1
tivities of 0.25 mg mL , which is quite a good result among all other
derivatives. In the tests against M. bovis, derivatives 21e23 also
revealed good activity, even comparable with that of rifampicin
ꢀ
1
(0.25
mg mL ). Interestingly, the most active against M. bovis strain
ꢀ1
is compound 12 with the MIC value equal 0.125
mg mL . For
mycobacterial test results of all studied compounds there is no
pronounced relationship between solubility, lipophilicity and the
activity level. In view of the obtained microbiological results de-
rivative 12 is the most active of all other synthesized ones both
against Gram-(þ) and mycobacteria strains.
3. Conclusions
Three new series of natural 3-formylrifamycin SV (Ral) macro-
lide derivatives comprising basic substituents at C(3): secondary
and tertiary nitrogens, secondary nitrogen and substituted triazole
moieties or secondary nitrogen, triazole moiety and a new 3,4-
dihydrobenzo[g]quinazoline system were synthesized using the
reductive alkylation and copper(I)-catalyzed azideealkyne cyclo-
addition synthetic methods. It was found that at a slightly increased
temperature and with a low amount of CuOAc catalyst used in the
presence of L-ascorbic acid the cycloaddition runs with the regio-
selective formation of 1,4-isomers of double substituted 1,2,3-
triazoles, regardless of whether an azide or alkyne group was
attached to the linker at C(3) carbon atom of rifamycins. Longer
Fig. 9. Comparison of 1H (range 5.5e9.5 ppm) and 13C NMR (range 140e190 ppm)
spectra of 20 and 30 compounds recorded in DMSO-d .
6

660
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
Fig. 10. Comparison of 1He13C HMBC spectra of 30 compound for C(15) carbon atom, recorded in DMSO-d
6
.
time of the cycloaddition at room temperature in the presence of a
higher amount of Cu-catalyst, resulted in unexpected formation of
of the structure of the substituent introduced at C(3) position. They
also revealed that the intramolecular protonation can occur at
different sites. Results of antibacterial tests against Gram-(ꢀ),
Gram-(þ) and mycobacteria strains were compared with the
structures of new derivatives 4e34. This combined analysis indi-
cated a correlation between solubility, lipophilicity and the struc-
ture of a substituent at C(3) and the antibacterial properties against
Gram-(þ) strains. The greatest activities both against S. epidermidis
(MIC ¼ 0.03 nmol/mL), M. tuberculosis (MIC ¼ 0.006 nmol/mL) and
3
,4-dihydrobenzo[g]quinazoline system as a new rifamycin “core”.
Structures of synthesized derivatives 1e34 in solution were deter-
mined by 1D and 2D NMR and FT-IR, whereas the energetically
most favourable conformation of newly modified rifamycins was
optimised and visualised with the use of MO-G PM6 method at
semi-empirical level of theory. Spectroscopic studies indicated
formation of zwitterionic structures in all derivatives, irrespectively

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
661
aminobenzylpiperidine, 1-(3-aminopropyl)pyrrolidine, 3-(dime-
thylamino)-1-propylamine,
,4-bis(3-aminopropyl)-piperazine,
3-(diethylamino)propyl-amine,
1
N,N-bis(2-aminoethyl)-1,3-
propanediamine, 1-pentyne, 1-hexyne, 1-heptyne, used for the
syntheses of new 3-formylrifamycin SV derivatives were purchased
from Aldrich. The N-(3-bromopropyl)phthalimide, N-(2-
bromoethyl)phthalimide, N-(bromomethyl)phthalimide, benzyl
bromide, 1-octanol, NaBH
sodium azide, Na SO , N
Aldrich. C OH, CH Cl
were purchased from POCH S.A. Gliwice (Poland). H
gradient grade was purchased from J.T. Baker.
3
CN reductant, CuOAc,
, KOH and ZnCl were purchased from
, CH COOC 5, THF, DMF, Et O,CCl , NaCl
O HPLC
L-ascorbic acid,
2
4
2
H
4
2
2
H
5
2
2
3
2
H
2
4
2
4.2. NMR measurements
The 1H and 13C measurements of new derivatives of 3-
Fig. 11. Superposition of the energetically most favourable structures of selected
copper(I) catalyzed azideealkyne cycloaddition products, calculated by MOG-PM6
formylrifamycin SV were performed in CDCl and DMSO-d using
3
6
ꢁ
Varian Mercury 400 MHz and Bruker Avance 600 MHz spectrome-
method: before condensation e compound 20 (H
f
¼ ꢀ566.9 kcal/mol e pale green
1
colour) and after condensation with the formation of 3,4-dihydrobenzo[g]quinazoline
ters. The operating frequencies for H measurements were 400.075
ꢁ
ring e compound 30 (H
f
¼ ꢀ582.4 kcal/mol e dark grey colour), demonstrating
ꢁ
and 600.08 MHz; pulse width corresponding to the flip angle of 45 ;
significant structural differences within ansa-bridge conformation and the arrange-
ment of hydrophobic substituent at C(3) carbon atom comprising triazole moiety. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
spectral width, swh ¼ 9842.5 Hz; acquisition time at ¼ 0.2 s;
relaxation delay d
standard. No window function or zero filling were used. Digital
resolution was 0.2 Hz/point. C NMR spectra were recorded at the
1
¼1.0 s; T ¼ 293.0 K, TMS was used as the internal
13
operating frequency 150.454 MHz; pulse width corresponding to
ꢁ
the flip angle of 60 ; sw ¼ 19000 Hz; at ¼ 1.8 s; d
1
¼1.0 s; T ¼ 293.0 K
M. bovis (MIC ¼ 0.14 nmol/mL) reveal new derivative 12 comprising
N-benzyl-piperidine moiety and its protonated form. Molecular
docking analyses of the obtained derivatives as zwitterions at the
binding site of bacterial RNA polymerase (RNAP) have shown key
intermolecular interactions contributing to the stability of the
enzymeeinhibitor complexes. The presence of resistant to hydro-
lysis CeN linkage within the introduced substituent at C(3) in the
newly synthesized derivatives, make some of them promising for
future medical applications, especially against S. epidermidis,
M. tuberculosis and M. bovis strains.
and TMS as the internal standard. Line broadening parameters of 0.5
or 1 Hz were applied. H and C NMR resonances were unambig-
1
13
1
13
1
13
uously assigned on the basis of the He C HMBC, He C HSQC,
1
1
1
1
He H COSY and He H NOESY correlation spectra.
4
.3. FT-IR measurements
The FT-IR spectra of new derivatives were recorded in KBr pellet
1.2 mg/200 mg) and in DMSO (0.05 mol dm ), whereas the
spectrum of 1:1 mixture of 3-H O with HClO (Fig. 12a) was
ꢀ3
(
2
4
measured as solid in KBr pellet. All FT-IR spectra were recorded on
an iS50 FT-IR spectrophotometer (Thermo, Nicolet, US) equipped
with a DTGS detector and two-columnar purge gas generator
4
. Experimental section
ꢀ
1
4.1. General
(Parker, Balston, US); resolution 1 cm , NSS ¼ 150, range
ꢀ
000e400 cm . The Happ-Genzel apodization function was used.
1
4
3
-Formylrifamycin SV was purchased from LKT Laboratories,
DMSO and CHCl for spectroscopic
Inc. (ꢂ98%). DMSO-d
6
,
3
4.4. HR-MALDI-TOF mass spectrometry
measurements as well as propargylamine, 1-(2-aminoethyl)piper-
idine, 1-(2-aminoethyl)pyrrolidine, N,N-dimethylethylenediamine,
The HR-MALDI-TOF spectra of new derivatives of 3-
1
-(3-aminopropyl)-imidazole, 3-morpholinopropyl-amine, 4-
formylrifamycin SV were obtained on a Water/Micromass
Fig. 12. FT-IR spectra of 20 (black solid line) and 30 (red dashed line) cycloaddition products recorded in KBr pellet. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

Table 3
Antibacterial and antitubercular test results for selected new 3-formylrifamycin SV derivatives compared to those of Ral and Rif (used as a standard for comparison). MIC values are given in different units: in
m
g/mL and in [nmol/
mL].
Strain
Compound
Ral Rif
>1 [0.62] 0.008 [0.01] 1 [1.25]
4
5
9
11
12
12 þ H
13
15
20
21
25
30
1 [1.27]
31
S. aureus ATCC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. epidermidis ATCC 12228
S. epidermidis ATCC 49134
E. coli ATCC 10538
1 [1.21]
0.5 [0.60] 0.5 [0.60] 0.25 [0.28]
>1 >[1.20] 0.25 [0.28]
0.5 [0.60] 0.5 [0.60] 0.25 [0.28]
0.125 [0.14] >1 [0.62] 1 [1.16]
0.25 [0.14]
0.125 [0.14] >1 [0.62] 1 [1.16]
0.06 [0.067] >1 [0.62] 0.5 [0.58] 0.5 [0.50] 0.5 [0.52] >1 >[1.18] >1 >[1.27] >1 >[1.05]
0.03 [0.034] >1 [0.62] 0.5 [0.58] 0.5 [0.50] 0.5 [0.52] >1 >[1.18] >1 >[1.27] >1 >[1.05]
1 [1.10]
1 [1.03]
1 [1.18]
1 [1.05]
>1
>1
>1
>1
>1
0.016 [0.019] >1 >[1.25] >1 >[1.21] 1 [1.19]
0.008 [0.001] 1 [1.25] 1 [1.21]
0.004 [0.005] 0.5 [0.063] 0.5 [0.61] 0.5 [0.60] 0.5 [0.60] 0.25 [0.28]
0.004 [0.005] 0.5 [0.063] 0.5 [0.61] 0.25 [0.30] 0.25 [0.30] 0.25 [0.28]
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
1 [1.10] 1 [1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
>1 >[1.2]
1 [1.2]
>1 >[1.2]
>1 >[1.2]
>1 >[1.2]
>1 >[1.25] >1 >[1.21] >1 >[1.19] >1 >[1.20] >1 >[1.11]
>1 >[1.25] >1 >[1.21] >1 >[1.19] >1 >[1.20] >1 >[1.11]
>1 >[1.25] >1 >[1.21] >1 >[1.19] >1 >[1.20] >1 >[1.11]
>1 >[1.25] >1 >[1.21] >1 >[1.19] >1 >[1.20] >1 >[1.11]
>1 >[1.25] >1 >[1.21] >1 >[1.19] >1 >[1.20] >1 >[1.11]
N
A
N
A
N
A
N
A
N
A
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
>1 [0.62] >1 >[1.16] >1 >[1.10] >1 >[1.03] >1 >[1.18] >1 >[1.27] >1 >[1.05]
Enterococcus faecalis ATCC 19433 >1
Salmonella abony NCTC 6017
T. rubrum ATCC 28188
C. albicans ATCC 10231
M. tuberculosis H37Rv
M. bovis
>1
>1
>1
>1
>1
0.005 [0.006] 1 [1.25]
0.25 [0.31] 1 [1.25]
0.5 [0.61] 0.5 [0.60] 1 [1.20]
1 [1.21] 1 [1.19] 1 [1.20]
0.005 [0.006] 0.005 [0.006] >1 [0.62] 1 [1.16]
0.125 [0.14] 0.125 [0.14] >1 [0.62] 1 [1.16]
0.5 [0.50] 0.25 [0.26] 1 [1.18]
0.5 [0.50] 0.25 [0.26] 0.5 [0.59] 0.5 [0.64] 0.5 [0.53]
1 [1.27]
0.5 [0.53]
Ral e 3-formylrifamycin SV, Rif e rifampicin, N
A
e not assigned, 12 þ H e protonated compound 12.
Table 4
Experimentally determined water solubilityexp (at pH ¼ 7.0 and T ¼ 25 ꢁC) and logPexp values for rifampicin (Rif), 3-formyrifamycin SV (Ral) and its new derivatives. For comparison the calculated logPcalc values by molinspiration
program are also given.
Compound
Ral
Rif
4
5
9
11
12
12 þ H
13
15
20
21
25
30
31
Solubilityexp 0.1 mg/mL 1.4 mg/mL 0.32 mg/mL 0.24 mg/mL 0.24 mg/mL 0.33 mg/mL <0.01 mg/mL 0.15 mg/mL <0.01 mg/mL <0.01 mg/mL <0.01 mg/mL <0.01 mg/mL <0.01 mg/mL <0.01 mg/mL <0.01 mg/mL
logPexp
1.71
2.673
1.66
2.615
1.48
2.472
1.55
2.875
2.04
3.146
1.78
2.895
3.25
4.261
2.05
1.152
3.83
4.779
2.14
3.28
3.42
4.615
2.24
3.391
3.13
4.203
4.47
5.538
3.26
4.315
logPcalca
a
http://www.molinspiration.com.

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
663
Table 5
ꢁ
Intermolecular hydrogen bond parameters [angle ; distance Å] for calculated
complexes of bacterial polymerase (RNAP) with inhibitors: 12 þ H, 20 and 30 (in-
teractions marked by green on Fig. 13).
ꢁ
RNAP-inhibitor complex
Interatomic distance
D$$$A] [Å]
Angle [D-H$$$A] [ ]
[
RNAP e 12 þ H (Fig. 13a)
(
(
(
(
(
(
(
(
(
(
Q
393) NeH$$$O]C(8)
2.65
2.90
2.78
2.74
4.25
2.81
2.95
2.78
2.85
2.70
165
153
160
165
112
128
135
170
177
167
F
F
D
H
R
R
S
E
N
394) NeH$$$O]C(35)
394) C]O$$$HeO(23)
396) C]O$$$HeO(21)
406) NeH$$$O(23)
409) NeH$$$O(1)
409) NeH$$$O(21)
411) OeH$$$O]C(8)
þ
445) C]O$$$HeN (41)
þ
448) C]O $$$HeN (38)
RNAP e 20 (Fig. 13c)
(
(
(
(
(
(
(
(
(
(
Q
393) NeH$$$O]C(8)
2.68
2.93
2.79
2.90
4.19
2.99
3.14
2.83
e
161
156
148
155
125
143
136
153
e
F
F
394) NeH$$$O]C(35)
394) C]O$$$HeO(23)
D
H
396) C]O$$$HeO(21)
406) NeH$$$O(21)
R
R
409) NeH$$$O(1)
409) NeH$$$O(21)
S
E
N
411) OeH$$$O]C(8)
445) C]O
448) C]O $$$HeN (38)
þ
2.92
151
RNAP e 30 (Fig. 13b)
(
(
(
(
(
(
(
(
(
(
Q
393) NeH$$$O]C(8)
2.69
3.00
2.98
2.96
4.31
2.96
3.39
2.89
e
150
155
129
175
132
138
145
156
e
F
F
394) NeH$$$O]C(35)
394) C]O$$$HeO(23)
D
H
396) C]O$$$HeO(21)
406) NeH$$$O(21)
R
R
409) NeH$$$O(1)
409) NeH$$$O(21)
S
E
N
411) OeH$$$O]C(8)
445) C]O
448) C]O
e
e
D e donor atom in hydrogen bond, A e acceptor atom in hydrogen bond.
(Manchester, UK) Q-TOF Premier mass spectrometer (software
MassLynx V4.1, Manchester, UK) fitted with a 200 Hz repetition rate
2
Nd/YAG (
l
¼ 355 nm, power density 107 W/cm ). The compounds
analysed were solids and the matrix used was DHB.
4.5. HPLC separations
HPLC separations and monitoring of copper(I)-catalyzed azide-
ealkyne cycloaddition were performed with a Dionex Ultimate
000 equipped with an LPG-3400 SD gradient pump using Thermo
3
GOLD C18 150 ꢃ 4.6 mm (5
m
m) Accucore XL column, TCC-3000SD
ꢁ
thermostat to columns (column temp. equal 25 C) and Dionex
VWD-3400RS variable wavelength UVevis detector. The flow rates
were 0.5 and 1 mL/min with injection volumes of 10
water and acetonitrile at 50:50 H O/CH CN were used as the mo-
bile phase, irrespectively of the sample. The analytical wavelengths
were
max ¼ 220 and 260 nm.
mL. Mixtures of
2
3
l
4.6. Elemental analysis
The elemental analysis of new derivatives of 3-formylrifamycin
SV was carried out on Vario ELIII (Elementar, Germany).
Fig. 13. The energetically most favourable intermolecular interactions (marked by
green) between key amino acid residues at the binding site of RNAP and: (a) compound
4
.7. MO-G PM6 calculations of inhibitors and their interactions at
12 þ H, (b) compound 30, (c) compound 20; calculated by MOG-PM6 method at semi-
the binding site of RNAP polymerase
empirical level of theory docking with the mode “dock into active site” (Scigress Ex-
plorer) and taking into account the zwitterionic forms of all synthesized inhibitor
molecules. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
MO-G PM6 calculations of energetically the most favourable
structures of new derivatives of 3-formylrifamycin SV as well as

664
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
interactions between 12 þ H, 20 and 30 and bacterial RNA poly-
merase (RNAP) binding site were performed with the use of Scigress
Explorer package, F.J. 2.4 (EU. 3.1.3., Fujitsu) [11]. FT-IR transitions of
AF3 [2-(3-azidopropyl)isoindoline-1,3-dione]
elemental analysis C11
: calculated: C ¼ 57.39%; H ¼ 4.38%;
N ¼ 24.34%; O ¼ 13.90%; measured C ¼ 57.36%; H ¼ 4.36%;
e yield: 92%;
10 4 2
H N O
þ
1
3
,4-dihydrobenzo[g]quinazoline system were theoretically calcu-
N ¼ 24.31%; O ¼ 13.88%; HR-MALDI-TOF [MþH] ¼ 231.09; H NMR
ppm in DMSO-d ): 7.86 (2H, m); 7.82 (2H, m); 3.63 (2H, t,
J ¼ 6.7 Hz); 3.41 (2H, t, J ¼ 6.7 Hz); 1.83 (2H, quintet); C NMR (
ppm in DMSO-d ): 167.9, 134.3, 131.7, 123.2, 48.4, 35.0, 27.3.
AF4 [1-(azidomethyl)benzene] e yield: 89%; elemental analysis
: C ¼ 63.14%; H ¼ 5.30%; N ¼ 31.56%; measured: C ¼ 63.12%;
lated by two methods: MO-G PM6 Force and ADF program with the
Phase band structure mode. The initial model of 4, 9, 12, 20, 30
structures was built on the basis of the previously determined X-ray
structure of amine zwitterionic analog of rifampicin [5]. Partial
charges of the potential proton-accepting atoms within non-ionic
structures of 9, 12, 20 and 30 were calculated by B88LYP-DFT
method with DGaussComputeEngine FJ 2.4 (EU 3.1.3) and PM6
semi-empirical method of Scigress Explorer package. For calculations
of the interactions at the binding site of the enzyme, the earlier
determined X-raystructure of bacterial RNA polymerase (RNAP) was
used [4]. The docking of 12 þ H, 20 and 30 structures, initially
optimised by MM3 and MO-G PM6 methods, to RNAP binding site
was performed with Scigress Explorer package e with the help of
mode “dock into active site”. The inhibitor docking to the binding
site of RNAP was performed on previously reported coordinates of
naphthalene ring of rifampicin, known from the X-ray structure of
the inhibitor complex with RNAP [4]. Peptide chains of RNAP sub-
(
d
6
3
3
13
d
6
7 7 3
C H N
þ
1
H ¼ 5.27%; N ¼ 31.55%; HR-MALDI-TOF [MþH] ¼ 134.07; H NMR
13
(d
ppm in DMSO-d
6
): 7.58 (5H, m); 4.44 (2H, s); C NMR (d ppm in
DMSO-d ): 135.6, 128.7, 128.4, 128.1, 53.6.
6
4.8.2. Syntheses of 2-azidoethylamine and 3-azidopropylamine
Appropriate AF2 and AF3 (1.5 mmol) were dissolved in 15 mL of
ethanol and hydrazine (3.0 mmol) was added. The mixture was
stirred vigorously at room temperature. After 36 h the reaction was
quenched by adding HCl to acidic pH. The reaction mixture was
evaporated to dryness and extracted twice with 30 mL of chloro-
form and water. To the separated inorganic layer KOH was added to
basic pH and the water layer was extracted with chloroform. To the
combined organic layers, HCl was added to acidic pH and evapo-
rated to dryness. The product was obtained as white solid e its
hydrochloric salt.
units were locked totally on the
a carbon atoms and nitrogen atoms
of peptide bonds at 30 Å distance from the binding site of the
enzyme before structural inhibitoreenzyme complex calculations.
Structures of 12 þ H, 20 and 30 complexes with RNAP were further
optimised with the global minimum search by MM3 and then by
2-Azidoethylamine hydrochloride salt e yield: 79%; elemental
analysis C
2
H
7
ClN
4
: C ¼ 19.60%; H ¼ 5.76%; Cl ¼ 28.93%; N ¼ 45.71%;
“MO-G PM6 large molecule” geometry algorithm, suitable for cal-
measured: C ¼ 19.69%; H ¼ 5.72%; Cl ¼ 28.92%; N ¼ 45.67%; HR-
þ
3
1
culations of huge systems comprising above 100 atoms. In the ob-
tained structures of calculated RNAP complexes with inhibitors the
orientation and conformation of the following amino acid residues:
MALDI-TOF [MþH] ¼ 122.04; H NMR (
d
ppm in DMSO-d
6
): 8.13
13
(3H, s), 3.64 (2H, t, J ¼ 6.7 Hz), 2.95 (2H, m); C NMR (
DMSO-d ): 47.7, 37.5.
3-Azidopropylamine hydrochloride salt e yield: 83%; elemental
analysis C ClN
: C ¼ 26.38%; H ¼ 6.64%; Cl ¼ 25.96%; N ¼ 41.02%;
measured: C ¼ 26.46%; H ¼ 6.60%; Cl ¼ 25.92%; N ¼ 41.02%; HR-
d ppm in
6
Q393, F394, D396, R409, S411, E445, H406, N448, I452 is changed simulta-
neously at their locked carbon positions at the same coordinates as
a
3
H
9
4
in X-ray structure of RifeRNAP complex [4]. The interactions be-
þ
3
1
tween selected zwitterionic inhibitor molecule and RNAP, were
MALDI-TOF [MþH] ¼ 136.05; H NMR (
6
d ppm in DMSO-d ): 8.08
ꢀ1
13
performed with the energy gradient not exceeding 25 kcal mol at
one step (total optimization after about 7000 steps).
(3H, s), 3.49 (2H, t, J ¼ 6.7 Hz), 2.83 (2H, m), 1.83 (2H, m); C NMR
(d
ppm in DMSO-d ): 47.8, 36.3, 26.4.
6
4.8.3. Syntheses of compounds 1e12
4
3
.8. Syntheses of substrates and methods for the syntheses of new
-formylrifamycin SV derivatives (1e34)
Rifaldehyde (290.0 mg, 0.4 mmol) was dissolved in 30 mL
Cl and after the addition of catalyst (0.2 mmol ZnCl or
2 2 2
CH
0.02 mmol HCl/EtOH) respective mixtures were made with each of
4
.8.1. Syntheses of azido-substrates for copper(I)-catalyzed
the following compounds taken separately: propargylamine, 3-
azidopropylamine, 2-azidoethylamine, (1-(2-aminoethyl)piperi-
dine, 1-(2-aminoethyl)pyrrolidine, N,N-dimethylethylenediamine,
1-(3-aminopropyl)-imidazole, 3-morpholinopropyl-amine, 4-
aminobenzylpiperidine, 1-(3-aminopropyl)pyrrolidine or 3-(dime-
azideealkyne cycloaddition
Appropriate bromide (1.5 mmol) was dissolved in 15 mL of DMF
(
dried) and sodium azide (1.6 mmol, 104 mg) was added. The
mixture was stirred vigorously at room temperature. After 12 h the
reaction was quenched by adding 15 mL of distilled water (slightly
exothermic reaction). The cooled reaction mixture (room temper-
ature) was extracted twice with 30 mL of diethyl ether and washed
with water (once with 20 mL). The separated organic layer was
dried over anhydrous sodium sulphate and evaporated to dryness.
The product obtained as white solid was washed with 30 mL of
distilled water.
thylamino)-1-propylamine,
(0.41 mmol) in 5 mL of C
3-(diethylamino)propyl-amine)
OH. The mixtures were stirred at 45 C
ꢁ
2
H
5
for half an hour and after that a half of the solvent volume was
distilled off. To the cooled reaction mixture (room temperature) the
reductant NaBH (6.8 mg, 0.18 mmol) was added portionwise over
4
1 min. The reaction mixture was evaporated to dryness, dissolved
in 50 mL of ethyl acetate and extracted twice with 50 mL of water
and brine. The separated organic layer was evaporated and syn-
thesised derivatives of 3-formylrifamycin SV (compounds 1e12)
were then purified by column chromatography with silica gel
(25 cm ꢃ 1 cm, silica gel 60, 0.040e0.063 mm/230e400 mesh
ASTM, Fluka) and ethyl acetate/methanol was applied as eluent
(from 100:0 to 15:1).
AF1 [2-(azidomethyl)isoindoline-1,3-dione]
elemental analysis C
: calculated: C ¼ 53.47%; H ¼ 2.99%;
N ¼ 27.71%; O ¼ 15.83%; measured: C ¼ 53.43%; H ¼ 2.95%;
e yield: 88%;
9 6 4 2
H N O
þ
1
N ¼ 27.69%; O ¼ 15.82%; HR-MALDI-TOF [MþH] ¼ 203.06; H NMR
13
(d
ppm in DMSO-d
NMR ( ppm in DMSO-d
AF2 [2-(2-azidoethyl)isoindoline-1,3-dione]
elemental analysis C10
: calculated: C ¼ 55.55%; H ¼ 3.73%;
N ¼ 25.91%; O ¼ 14.80%; measured: C ¼ 55.53%; H ¼ 3.70%;
6
): 7.95 (m, 2H); 7.90 (2H, m); 5.06 (2H, s);
): 167.1, 135.1, 131.4, 123.7, 52.3.
yield: 91%;
C
d
6
e
H N O
8 4 2
4.8.4. Syntheses of dimeric compounds 13 and 14
Rifaldehyde (300.0 mg, 0.41 mmol) was dissolved in 30 mL
þ
1
N ¼ 25.88%; O ¼ 14.78%;HR-MALDI-TOF [MþH] ¼ 217.07; H NMR(
d
CH
aminoethyl)-1,3-propanediamine) (0.2 mmol) in 5 mL of C
with a catalyst (0.2 mmol ZnCl or 0.02 mmol HCl/EtOH) added. The
2
Cl
2
and (1,4-bis(3-aminopropyl)-piperazine,
N,N-bis(2-
ppm in DMSO-d
6
): 7.90 (2H, m); 7.85 (2H, m); 3.79 (2H, m); 3.58 (2H,
2 5
H
OH
13
m); C NMR (
d
6
ppm in DMSO-d ): 167.7,134.6,131.4,123.2, 48.5, 36.8.
2

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
665
ꢁ
mixtures were stirred at 45 C for half an hour and after that a half
of the solvent volume was distilled off. To the cooled reaction
4.8.9. Syntheses of compounds 28e30
Compound 3 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
mixture (room temperature) the reductant NaBH
4
(6.8 mg,
and 1.5 mL MeOH. To the mixture 1-pentyne, 1-hexyne, 1-heptyne
0
.18 mmol) was added portionwise over 1 min. The reaction
(0.12 mmol), L-ascorbic acid (0.18 mmol) and catalyst copper (I)
mixture was evaporated to dryness, dissolved in 50 mL of ethyl
acetate and extracted twice with 50 mL of water and brine. The
separated organic layer was evaporated and synthesised derivative
acetate (0.012 mmol) were added. The mixtures were stirred at
room temperature for 48 h. The reaction mixture was evaporated to
dryness, dissolved in 50 mL of ethyl acetate and extracted twice
with 50 mL of water and brine. The separated organic layer was
evaporated and crude products of cycloaddition were purified by
column chromatography with silica gel (25 cm ꢃ 1 cm, silica gel 60,
0.040e0.063 mm/230e400 mesh ASTM, Fluka) and ethyl acetate/
methanol as eluent (from 100:0 to 15:1).
(
compounds 13e14) was purified by column chromatography with
silica gel (25 cm ꢃ 1 cm, silica gel 60, 0.040e0.063 mm/
30e400 mesh ASTM, Fluka) and ethyl acetate/methanol as eluent
from 100:0 to 15:1).
2
(
4.8.5. Syntheses of compounds 15e17
Compound 2 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
4.8.10. Syntheses of compounds 31e34
and 1.5 mL MeOH. To the mixture 1-pentyne, 1-hexyne, 1-heptyne
0.12 mmol), -ascorbic acid (0.0915 mmol) and catalyst Copper (I)
Compound 1 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
and 1.5 mL MeOH. To the mixture of 2-(azidomethyl)isoindoline-
1,3-dione or 2-(2-azidoethyl)isoindoline-1,3-dione or 2-(3-
azidopropyl)isoindoline-1,3-dione or 1-(azidomethyl)benzene
(
L
acetate (0.024 mmol) were added. The mixtures were stirred at
room temperature for 24 h. The reaction mixture was evaporated to
dryness, dissolved in 50 mL of ethyl acetate and extracted twice
with 50 mL of water and brine. The separated organic layer was
evaporated and crude products of cycloaddition were purified by
column chromatography with silica gel (25 cm ꢃ 1 cm, silica gel 60,
(0.12 mmol), L-ascorbic acid (0.18 mmol) and catalyst copper (I)
acetate (0.012 mmol) were added. The mixtures were stirred at
room temperature for 48 h. The reaction mixture was evaporated to
dryness, dissolved in 50 mL of ethyl acetate and extracted twice
with 50 mL of water and brine. The separated organic layer was
evaporated and crude products of cycloaddition were purified by
column chromatography with silica gel (25 cm ꢃ 1 cm, silica gel 60,
0.040e0.063 mm/230e400 mesh ASTM, Fluka) and ethyl acetate/
methanol as eluent (from 100:0 to 15:1).
0
.040e0.063 mm/230e400 mesh ASTM, Fluka) and ethyl acetate/
methanol as eluent (from 100:0 to 15:1).
4.8.6. Syntheses of compounds 18e20
Compound 3 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
and 1.5 mL MeOH. To the mixture 1-pentyne, 1-hexyne, 1-heptyne
0.12 mmol), -ascorbic acid (0.0915 mmol) and catalyst Copper (I)
(
L
4.8.11. Preparation of 12 þ H protonated derivative
acetate (0.024 mmol) were added. The mixtures were stirred at
room temperature with for 24 h. The reaction mixture was evap-
orated to dryness, dissolved in 50 mL of ethyl acetate and extracted
twice with 50 mL of water and brine. The separated organic layer
was evaporated and crude products of cycloaddition were purified
next by column chromatography with silica gel (25 cm ꢃ 1 cm,
silica gel 60, 0.040e0.063 mm/230e400 mesh ASTM, Fluka) and
ethyl acetate/methanol as eluent (from 100:0 to 15:1).
Compound 12 (100 mg, 0.11 mmol) was dissolved in 2 mL of
methanol. To the solution 2.2 mL (0.11 mmol) 0.05 M HCl in
methanol. The mixture was vigorously stirred by 0.5 h and evap-
orated. To dehydrate the sample it was additionally treated with
anhydrous acetonitrile and evaporated (5 times).
4.9. Variants of copper(I)-catalyzed azideealkyne cycloaddition
Method 1. 3% Copper (I) acetate: Compound 1 (30.0 mg,
0.04 mmol) was dissolved in the mixture of 1.5 mL THF with
0.45 mL MeOH. To this mixture 2-(2-azidoethyl)isoindoline-1,3-
4
.8.7. Syntheses of compounds 21e24
Compound 1 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
and 1.5 mL MeOH. To the mixture 2-(azidomethyl)isoindoline-1,3-
dione, 2-(2-azidoethyl)isoindoline-1,3-dione, 2-(3-azidopropyl)
dione (0.04 mmol), L-ascorbic acid (0.0549 mmol) and catalyst
copper (I) acetate (0.00366 mmol) were added. The mixture was
ꢁ
isoindoline-1,3-dione, 1-(azidomethyl)benzene (0.12 mmol),
L-
stirred at 60 C with reflux for five hours.
ascorbic acid (0.0915 mmol) and catalyst Copper (I) acetate
Method 2. 5% Copper (I) acetate: Compound 1 (30.0 mg,
0.04 mmol) was dissolved in the mixture of 1.5 mL THF with
0.45 mL MeOH. To this mixture 2-(2-azidoethyl)isoindoline-1,3-
ꢁ
(
0.0061 mmol) were added. The mixtures were stirred at 60
C
under reflux for two hours. The reaction mixture was evaporated to
dryness, dissolved in 50 mL of ethyl acetate and extracted twice
with 50 mL of water and brine. The separated organic layer was
evaporated and crude products of cycloaddition were purified by
column chromatography with silica gel (25 cm ꢃ 1 cm, silica gel 60,
dione (0.04 mmol), L-ascorbic acid (0,0915 mmol) and catalyst
copper (I) acetate (0.0061 mmol) were added. The mixture was
ꢁ
stirred at 60 C with reflux for two hours.
Method 3. 10% Copper (I) acetate: Compound 1 (30.0 mg,
0.04 mmol) was dissolved in the mixture of 1.5 mL THF with
0.45 mL MeOH. To this mixture 2-(2-azidoethyl)isoindoline-1,3-
0
.040e0.063 mm/230e400 mesh ASTM, Fluka) and ethyl acetate/
methanol as eluent (from 100:0 to 15:1).
dione (0.04 mmol), L-ascorbic acid (0.18 mmol) and catalyst cop-
4
.8.8. Syntheses of compounds 25e27
Compound 2 (100.0 mg, 0.12 mmol) was dissolved in 5 mL THF
per (I) acetate (0.012 mmol) were added. The mixture was stirred at
60 C with reflux for one hour.
ꢁ
and 1.5 mL MeOH. To the mixture 1-pentyne, 1-hexyne, 1-heptyne
0.12 mmol), -ascorbic acid (0.18 mmol) and catalyst Copper (I)
Method 4. Compound 1 (30.0 mg, 0.04 mmol) was dissolved in
the mixture of 1.5 mL THF with 0.45 mL MeOH. To this mixture 2-
(
L
acetate (0.012 mmol) were added. The mixtures were stirred at
room temperature for 48 h. The reaction mixture was evaporated to
dryness, dissolved in 50 mL of ethyl acetate and extracted twice
with 50 mL of water and brine. The separated organic layer was
evaporated and crude products of cycloaddition were purified by
column chromatography with silica gel (25 cm ꢃ 1 cm, silica gel 60,
(2-azidoethyl)isoindoline-1,3-dione (0.04 mmol), L-ascorbic acid
(0.0915 mmol) and catalyst copper (I) acetate (0.0061 mmol) were
ꢁ
added. The mixture was stirred at 0 C for 24 h.
Method 5. Compound 1 (30.0 mg, 0.04 mmol) was dissolved in
the mixture of 1.5 mL THF with 0.45 mL MeOH. To the mixture 2-(2-
azidoethyl)isoindoline-1,3-dione (0.04 mmol),
L-ascorbic acid
0
.040e0.063 mm/230e400 mesh ASTM, Fluka) and ethyl acetate/
(0.0915 mmol) and catalyst copper (I) acetate (0.0061 mmol) were
methanol as eluent (from 100:0 to 15:1).
added. The mixture was stirred at room temperature for five hours.

666
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
Method 6. Compound 1 (30.0 mg, 0.04 mmol) was dissolved in
the mixture of 1.5 mL THF with 0.45 mL MeOH. To the mixture 2-(2-
azidoethyl)isoindoline-1,3-dione (0.04 mmol), -ascorbic acid
0.0915 mmol) and catalyst copper (I) acetate (0.0061 mmol) were
added. The mixture was stirred at 40 C for five hours.
Method 7. Compound 1 (30.0 mg, 0.04 mmol) was dissolved in
the mixture of 1.5 mL THF with 0.45 mL MeOH. To the mixture 2-(2-
(1H, m, H-20), 1.99 (6H, s, H-30 þ H-36), 1.92 (3H, s, H-14), 1.69 (1H,
m, H-22), 1.65 (3H, s, H-13), 1.26 (1H, m, H-24), 0.98 (1H, m, H-26),
3
3
L
0.91 (3H, d, JH22-H32 ¼ 7.0 Hz, H-32), 0.87 (3H, d, JH20-H31 ¼ 7.0 Hz,
3 3
(
H-31), 0.51 (3H, d,
H26 ¼ 6.7 Hz, H-34); C NMR (
J
H24-H33 ¼ 6.7 Hz, H-33), ꢀ0.33 (3H, d,
J
H34-
ꢁ
13
6
d ppm in DMSO-d ): 185.3 (C-11),
184.2 (C-8), 171.8 (C-6), 170.3 (C-15), 169.4 (C-35), 148.8 (C-1), 145.0
(C-4), 143.1 (C-29), 139.1 (C-19), 131.5 (C-16), 131.4 (C-17), 126.2 (C-
18), 118.1 (C-2), 117.7 (C-28), 116.6 (C-10), 115.1 (C-9), 114.7 (C-3),
108.8 (C-12), 101.2 (C-7), 98.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2
(C-25), 72.9 (C-21), 55.6 (C-37), 46.6 (C-40), 44.2 (C-39), 43.8 (C-38),
40.3 (C-26), 38.1 (C-24), 37.9 (C-20), 32.7 (C-22), 22.1 (C-13), 20.7
(C-36), 19.9 (C-30), 18.2 (C-31), 11.2 (C-32), 9.0 (C-34), 8.8 (C-33), 7.4
azidoethyl)isoindoline-1,3-dione (0.04 mmol),
L-ascorbic acid
(
0.0915 mmol) and catalyst Copper (I) acetate (0.0061 mmol) were
ꢁ
added. The mixture was stirred at 50 C for five hours.
Method 8. Compound 1 (30.0 mg, 0.04 mmol) was dissolved in
the mixture of 1.5 mL THF with 0.45 mL MeOH. To the mixture 2-(2-
ꢀ
1
azidoethyl)isoindoline-1,3-dione (0.04 mmol),
L-ascorbic acid
(C-14); FT-IR (KBr pellet): 3440 cm
n
(O21eH) þ
n
(O23eH),
ꢀ1
ꢀ1
ꢀ1
(
0.0915 mmol) and catalyst Copper (I) acetate (0.0061 mmol) were
3240 cm
n
(NeH)amide, 3075 cm
n
(O
4
eH$$$O11), 2720 cm
ꢁ
þ
ꢀ1
ꢀ
ꢀ1
þ
added. The mixtures were stirred at 0 C for two hours.
n
(N38eH$$$O15), 2480 cm
n
(O
1
eH$$$O
8
), 2101 cm
n(N]N ]
ꢀ
ꢀ1
ꢀ1
All reactions were controlled by HPLC (column e Hypersil GOLD,
N ), 1720 cm
n
(C35]O), 1646 cm
n
(C15]O)amide
I
þ
n(C11]
(NeH),
ꢀ
1
ꢀ1
d
Dimension 150 mm ꢃ 4.6 mm) at the 10 min intervals at
l
max ¼ 220
O) þ
n
(C
8
]O), 1625 cm
d
n
(C]C)naphthalene, 1590 cm
(NeH)amide II, 1474 and 1460 cm
(CeO); elemental analysis C40
ꢀ
1
ꢀ1
[nm] and
l
max ¼ 260 [nm]. The mobile phase was H
2
O:CH CN
3
1536 cm
1239 cm
n
(C]C), 1248 and
12: calculated:
ꢁ
ꢀ1
(
50:50) at the flow rate 0.5 mL/min in 25 C.
n
H
53
N
5
O
ꢁ
1
e yield: 65%; HPLC: RT ¼ 6.46 [min]; 25 C; 50:50
C ¼ 60.36%; H ¼ 6.71%; N ¼ 8.80%; O ¼ 24.12%; measured:
þ
(
H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH] 765.3590;
C ¼ 60.31%; H ¼ 6.76%; N ¼ 8.78%; O ¼ 24.15%.
1
ꢁ
þ
H NMR (
d
ppm in DMSO-d
6
): 16.03 (1H, bs, HO-1), 12.80 (1H, s,
3 e yield: 63%; HPLC: RT ¼ 4.63 [min]; 25 C; 50:50
þ
HO-4), 9.30 (1H, s, NHamide), 9.10 (1H, vbs, N H-38hydrogen bonded),
.67 (1H, vbs, N H-38non-hydrogen bonded), 6.61 (1H, dd,
(H
H NMR (
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 810.3921;
þ
3
4
1
8
J
J
H18-
d
ppm in DMSO-d
6
): 16.05 (1H, s, HO-1), 12.83 (1H, s, HO-
3
þ
H19 ¼ 15.9 Hz, H-18), 6.32 (1H, dd,
JH17-H18 ¼ 10.9 Hz,
H17-
4), 9.32 (1H, s, NHamide), 8.53 (1H, bs, N H-38hydrogen bonded), 8.25
H30 ¼ 1.3 Hz, H-17), 6.25 (1H, d, H-29), 6.06 (1H, dd, 3JH19-
(1H, bs, N H-38non-hydrogen bonded), 6.60 (1H, dd, JH18-H19 ¼ 15.9 Hz,
þ
3
3
3
H20 ¼ 7.5 Hz, H-19), 5.08 (1H, d,
J
H25-H26 ¼ 11.1 Hz, H-25), 5.04
H-18), 6.34 (1H, d, JH17-H18 ¼ 11.0 Hz, H-17), 6.26 (1H, d, H-29), 6.09
3
3
3
3
(
1H, d, JHO-H21 ¼ 3.9 Hz, HO-21), 4.92 (1H, dd, JH28-H29 ¼ 12.9 Hz,
(1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.09 (1H, d, JH25-H26 ¼ 11.0 Hz, H-
3 3
H-28), 4.35 (2H, m, H-39), 3.99 (1H, m, H-38), 3.95 (1H, m, HO-23),
25), 5.06 (1H, d,
J
HO-H21 ¼ 4.1 Hz, HO-21), 4.91 (1H, dd,
J
H28-
3
2
3
.69 (1H, m, H-38), 3.59 (1H, t, H-21), 3.24 (1H, d, JH27-H28 ¼ 8.8 Hz,
H-27), 2.89 (3H, s, H-37), 2.83 (2H, m, H-20 þ H-23), 1.99 (4H, s, H-
6 þ H-41), 1.92 (3H, s, H-30), 1.91 (3H, s, H-14), 1.69 (1H, m, H-22),
H29 ¼ 12.9 Hz, H-28), 4.30 (1H, d, J ¼ 12.0 Hz, H-38), 3.98 (1H, d,
3
J
J
HO-H21 ¼ 8.9 Hz, HO-23), 3.69 (2H, m, H-21 þ H-38), 3.45 (2H, t,
3
3
3
H40-H41 ¼ 7.5 Hz, H-41), 3.23 (1H, d, JH27-H28 ¼ 9.0 Hz, H-27), 3.09
1
.64 (3H, s, H-13),1.26 (1H, m, H-24), 0.98 (1H, m, H-26), 0.91 (3H, d,
(1H, m, H-39), 2.98 (1H, m, H-39), 2.89 (3H, s, H-37), 2.83 (1H, dt,
3
3
3
3
3
J
H22-H32 ¼ 7.1 Hz, H-32), 0.87 (3H, d, JH20-H31 ¼ 7.1 Hz, H-31), 0.50
J
H22-H23 ¼ 8.9 Hz, JH23-OH ¼ 8.9 Hz, JH23-H22 ¼ 2.7 Hz, H-23), 2.29
3
3
(
3
(
2
1
(
7
(
3H, d, JH24-H33 ¼ 7.1 Hz, H-33), ꢀ0.33 (3H, d, JH34-H26 ¼ 6.8 Hz, H-
(1H, m, H-20), 1.99 (6H, s, H-36 þ H-30), 1.92 (3H, s, H-14), 1.84 (1H,
4); 13C NMR (
C-6), 170.3 (C-15), 169.4 (C-35), 148.7 (C-1), 145.0 (C-4), 143.1 (C-
9), 138.7 (C-19), 131.5 (C-17), 131.4 (C-16), 126.4 (C-18), 117.9 (C-2),
17.8 (C-28), 116.7 (C-10), 115.1 (C-9), 114.7 (C-3), 108.8 (C-12), 101.3
C-7), 98.5 (C-5), 79.6 (C-40), 76.3 (C-27), 75.8 (C-23), 74.6 (C-41),
3.2 (C-25), 72.9 (C-21), 55.6 (C-37), 42.2 (C-38), 40.3 (C-26), 38.1
C-24), 37.9 (C-20), 36.7 (C-39), 32.7 (C-22), 22.1 (C-13), 20.7 (C-36),
9.9 (C-30),18.2 (C-31),11.3 (C-32), 9.0 (C-34), 8.8 (C-33), 7.4 (C-14);
elemental analysis C41
12: calculated: C ¼ 64.38%; H ¼ 6.85%;
d
ppm in DMSO-d
): 185.3 (C-11), 184.2 (C-8), 171.8
m, H-40), 1.69 (1H, m, H-22), 1.65 (3H, s, H-13), 1.26 (1H, m, H-24),
6
3
0.98 (1H, m, H-26), 0.91 (3H, d, JH22-H32 ¼ 7.0 Hz, H-32), 0.86 (3H, d,
3
3
J
H20-H31 ¼ 7.0 Hz, H-31), 0.51 (3H, d,
J
H24-H33 ¼ 6.7 Hz, H-
3
13
33), ꢀ0.33 (3H, d,
J
H34-H26 ¼ 6.7 Hz, H-34); C NMR (
d ppm in
6
DMSO-d ): 185.3 (C-11), 184.2 (C-8), 171.8 (C-6), 170.3 (C-15), 169.4
(C-35), 148.9 (C-1), 145.0 (C-4), 143.1 (C-29), 139.1 (C-19), 131.5 (C-
16), 131.4 (C-17), 126.2 (C-18), 118.0 (C-2), 117.7 (C-28), 116.6 (C-10),
115.1 (C-9), 114.7 (C-3), 108.8 (C-12), 101.3 (C-7), 98.6 (C-5), 76.3 (C-
27), 75.7 (C-23), 73.1 (C-25), 72.9 (C-21), 55.6 (C-37), 47.8 (C-41),
44.8 (C-39), 43.8 (C-38), 40.3 (C-26), 38.1 (C-24), 37.9 (C-20), 32.7
(C-22), 25.1 (C-40), 22.1 (C-13), 20.7 (C-36), 19.9 (C-30), 18.2 (C-31),
11.2 (C-32), 9.0 (C-34), 8.8 (C-33), 7.4 (C-14); FT-IR (KBr pellet):
1
52 2
H N O
ꢀ1
N ¼ 3.66%; O ¼ 25.10%; FT-IR (KBr pellet): 3437 and 3396 cm
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
n
(O21eH)
þ
n
(O23eH), 3338 cm
n(NeH)amide, 3150 cm
ꢀ1
þ
n
(O
(O
4
eH$$$O11), 2733 cm
eH$$$O ‾), 2136
(C11]O) þ
(C]C), 1539 cm
n
(N38eH$$$O15), 2460 cm
(C^C) 1720 cm
ꢀ
1
ꢀ1
ꢀ1
n
n
1
8
n
n
(C35]O), 1641 cm
3443 cm
3080 cm
n
(O21eH)
þ
n
(O23eH), 3238 cm
(NeH)amide
,
ꢀ
1
ꢀ1
ꢀ1 þ
n(N38eH$$$O15), 2478 cm
ꢀ1
n
(C15]O)amide I
lene, 1561 cm
þ
n
n(C
8
]O), 1600 cm
n
(C]C)naphtha-
n
(O
4
eH$$$O11), 2725 cm
ꢀ1
ꢀ1
ꢀ
ꢀ1
þ
ꢀ
ꢀ1
n
n
d
(NeH)amide II, 1458 and
n(O
1
eH$$$O
8
), 2101 cm
n
(N]N ]N ), 1720 cm
(C
(C35]O),
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
1
446 cm
n
(C]C), 1241 and 1229 cm
n
(CeO); measured:
1646 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
8
]O), 1624 cm
n(C]
ꢀ
1
ꢀ1
d
C ¼ 64.32%; H ¼ 6.87%; N ¼ 3.70%; O ¼ 25.11%.
C)naphthalene, 1591 cm
d
(NeH), 1536 cm
(NeH)amide II, 1474 and
n(CeO); elemental analysis
ꢁ
þ
ꢀ1
ꢀ1
2
e yield: 61%; HPLC: RT ¼ 3.87 [min]; 25 C; 50:50
1462 cm
n
(C]C),1246 and 1240 cm
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 796.3765;
C
41
H
55
N
5
O
12: calculated: C ¼ 60.80%; H ¼ 6.84%; N ¼ 8.65%;
1
H NMR (
d
ppm in DMSO-d
6
): 16.04 (1H, bs, HO-1), 12.85 (1H, s,
O ¼ 23.71%; measured: C ¼ 60.78%; H ¼ 6.87%; N ¼ 8.68%;
þ
HO-4), 9.34 (1H, s, NHamide), 8.55 (1H, bs, N H-38hydrogen bonded),
O ¼ 23.67%.
þ
3
ꢁ
þ
8
.26 (1H, bs, N H-38non-hydrogen bonded), 6.60 (1H, dd,
J
H18-
4 e yield: 48%; HPLC: RT ¼ 6.86 [min]; 25 C; 60:40
H19 ¼ 15.9 Hz, H-18), 6.33 (1H, d, 3JH17-H18 ¼ 11.1 Hz, H-17), 6.25
(H
O:CH
H NMR (
4), 9.23 (1H, brs, NHamide), 6.63 (1H, m, H-18), 6.34 (1H, d,
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 798.4181;
2
3
3
1
(
1H, d, H-29), 6.09 (1H, dd,
J
H19-H20 ¼ 7.5 Hz, H-19), 5.09 (1H, d,
6
d ppm in DMSO-d ): 16.03 (1H, s, HO-1), 12.78 (1H, s, HO-
3
3
3
J
H25-H26 ¼ 11.0 Hz, H-25), 5.07 (1H, d,
J
HO-H21 ¼ 4.0 Hz, HO-21),
J
H17-
3
3
4
.91 (1H, dd,
J
H28-H29 ¼ 12.9 Hz, H-28), 4.15 (1H, m, H-38), 3.99
H18 ¼ 10.3 Hz, H-17), 6.25 (1H, d,
JH28-H29 ¼ 12.6 Hz, H-29), 6.10
1H, d, 3
J
HO-H21 ¼ 8.8 Hz, HO-23), 3.70 (1H, m, H-21), 3.65 (2H, t,
(1H, m, H-19), 5.09 (1H, d, JH25-H26 ¼ 11.5 Hz, H-25), 5.04 (1H, vbs,
3
(
3
3
2
J
H39-H40 ¼ 7.5 Hz, H-40), 3.23 (1H, d, JH27-H28 ¼ 9.0 Hz, H-27), 3.11
HO-21), 4.91 (1H, m, H-28), 4.31 (1H, d, J ¼ 12.0 Hz, H-38), 3.96
3
(
1H, m, H-39), 3.01 (1H, m, H-39), 2.89 (3H, s, H-37), 2.83 (1H, dt,
(1H, bs, HO-23), 3.78 (1H, d, H-38), 3.69 (1H, d, JH20-H21 ¼9.9 Hz, H-
3
3
3
3
JH22-H23 ¼ 8.9 Hz, JH23-OH ¼ 8.9 Hz, JH23-H22 ¼ 2.9 Hz, H-23), 2.28
21), 3.24 (1H, d, JH27-H28 ¼ 8.8 Hz, H-27), 3.08 (2H, m, H-39), 2.89

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
667
(
4
3
3H, s, H-37), 2.86 (1H, d, 3JH23-H24 ¼ 10.7 Hz, H-23), 2.54 (2H, m, H-
(1H, d, 3JH20-H21 ¼ 9.5 Hz, H-21), 3.52 (1H, m, H-38), 3.24 (1H, d,
3
0), 2.28 (1H, m, H-20), 2.20 (6H, s, H-41 þ H-42), 1.98 (6H, s, H-
0 þ H-36),1.92 (3H, s, H-14),1.84 (1H, m, H-40),1.69 (1H, m, H-22),
JH27-H28 ¼ 8.4 Hz, H-27), 3.08 (2H, m, H-39), 2.89 (3H, s, H-37), 2.83
(1H, m, H-23), 2.54 (2H, m, H-40), 2.36 (4H, m, H-41 þ H-45), 2.29
(1H, m, H-20), 1.99 (3H, s, H-36), 1.98 (3H, s, H-30), 1.92 (3H, s, H-
14),1.69 (1H, m, H-22),1.64 (3H, s, H-13),1.50 (4H, m, H-42 þ H-44),
1.35 (2H, m, H-35), 1.26 (1H, m, H-24), 0.99 (1H, m, H-26), 0.92 (3H,
1
.64 (3H, s, H-13),1.27 (1H, m, H-24), 1.00 (1H, m, H-26), 0.92 (3H, d,
3
3
J
H22-H32 ¼ 5.0 Hz, H-32), 0.87 (3H, d, JH20-H31 ¼ 5.7 Hz, H-31), 0.53
3
3
(
3
(
2
1
(
3H, d, JH24-H33 ¼ 6.9 Hz, H-33), ꢀ0.34 (3H, d, JH34-H26 ¼ 7.3 Hz, H-
4); 13C NMR (
C-6), 170.3 (C-15), 169.3 (C-35), 148.9 (C-1), 144.9 (C-4), 143.0 (C-
9), 139.1 (C-19), 131.6 (C-17), 131.4 (C-16), 126.4 (C-18), 118.0 (C-2),
17.5 (C-28), 116.7 (C-10), 115.6 (C-9), 115.0 (C-3), 108.8 (C-12), 101.2
C-7), 98.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 73.1 (C-21),
d
ppm in DMSO-d
): 185.2 (C-11), 184.2 (C-8), 171.8
d,
3
J
H22-H32 ¼ 7.1 Hz, H-32), 0.86 (3H, d,
3
J
H20-H31 ¼ 6.9 Hz, H-31),
6
3
3
0.52 (3H, d,
H26 ¼ 6.7 Hz, H-34); C NMR (
J
H24-H33 ¼ 6.9 Hz, H-33), ꢀ0.34 (3H, d,
J
H34-
13
6
d ppm in DMSO-d ): 185.2 (C-11),
184.2 (C-8), 171.8 (C-6), 170.2 (C-15), 169.3 (C-35), 148.8 (C-1), 144.9
(C-4), 143.1 (C-29), 139.0 (C-19), 131.5 (C-17), 131.4 (C-16), 126.3 (C-
18), 118.0 (C-2), 117.6 (C-28), 116.7 (C-10), 115.3 (C-9), 115.0 (C-3),
108.9 (C-12), 101.3 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2
(C-25), 72.9 (C-21), 55.6 (C-37), 53.7 (C-40), 53.6 (C-41), 53.6 (C-45),
43.4 (C-39),43.0 (C-38), 40.3 (C-26), 38.1 (C-24), 37.8 (C-20), 32.6
(C-22), 25.0 (C-42 þ C-44), 23.7 (C-43), 22.1 (C-13), 20.6 (C-36), 19.8
(C-30), 18.2 (C-31), 11.1 (C-32), 9.0 (C-34), 8.8 (C-33), 7.3 (C-14); FT-
5
4
5.6 (C-37), 54.7 (C-40), 44.5 (C-41 þ C-42), 43.7 (C-39), 43.0 (C-38),
0.2 (C-26), 38.1 (C-24), 37.7 (C-20), 32.6 (C-22), 22.0 (C-13), 20.6
(
C-36), 19.9 (C-30), 18.1 (C-31), 11.0 (C-32), 8.9 (C-34), 8.7 (C-33), 7.3
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
(
C-14); FT-IR (KBr pellet): 3470 cm
þ
3409 cm
ꢀ
1
n
n
n
(O21eH)
þ
n
(O23eH), 3234 cm
n(NeH)amide, 3090 cm
(N38eH$$$O15), 2468 cm
(C35]O), 1649 cm
ꢀ
1
þ
(O
(O
þ
4
eH$$$O11), 2705 cm
n
ꢀ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
n
1
eH$$$O
8
), 1712 cm
n
n
(C15]O)amide
IR (KBr pellet): 3472 cm þ 3408 cm
(O21eH) þ
n
(O23eH),
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
I
d
n
(C11]O) þ
n
(C ]O), 1626 cm
8
n
(C]C)naphthalene, 1588 cm
(NeH)amide II, 1480 and 1460 cm
3239 cm
n
(NeH)amide, 3093 cm
n
(O
4
eH$$$O11), 2711 cm
), 1711 cm
ꢀ
1
ꢀ1
þ
ꢀ1
ꢀ
ꢀ1
(NeH), 1536 cm
d
n
(C]C),
n
(N38eH$$$O15), 2465 cm
n
(O
1
eH$$$O
8
n
(C35]O),
ꢀ
1
ꢀ1
ꢀ1
1
245 and 1237 cm
n
(CeO); elemental analysis C42
59
H N
3
O
12
:
1647 cm
C)naphthalene, 1587 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1626 cm
n(C]
ꢀ1
ꢀ1
d
calculated: C ¼ 63.22%; H ¼ 7.45%; N ¼ 5.27%; O ¼ 24.06%;
d
(NeH), 1536 cm
(NeH)amide II, 1478 and
ꢀ1
ꢀ1
measured: C ¼ 63.20%; H ¼ 7.47%; N ¼ 5.26%; O ¼ 24.07%.
1461 cm
n
(C]C), 1244 and 1239 cm
n(CeO); elemental analysis
ꢁ
5
e yield: 60%; HPLC: RT ¼ 7.01 [min]; 25 C; 60:40
C
45
H
63
N
3
O
12: calculated: C ¼ 64.50%; H ¼ 7.58%; N ¼ 5.01%;
þ
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 824.4330;
O ¼ 22.91%; measured: C ¼ 64.48%; H ¼ 7.60%; N ¼ 5.02%;
1
H NMR (
d
6
ppm in DMSO-d ): 16.01 (1H, s, HO-1), 12.76 (1H, s, HO-
O ¼ 22.90%.
3
ꢁ
þ
4
6
), 9.22 (1H, s, NHamide), 6.63 (1H, dd,
.35 (1H, d,
J
H18-H19 ¼ 15.9 Hz, H-18),
7 e yield: 69%; HPLC: RT ¼ 8.39 [min]; 25 C; 60:40
3
3
J
H17-H18 ¼ 10.7 Hz, H-17), 6.26 (1H, d,
J
H28-
(H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 812.4326;
3
1
H29 ¼ 12.7 Hz, H-29), 6.10 (1H, dd,
J
H19-H20 ¼ 7.5 Hz, H-19), 5.09
H NMR (
d
ppm in DMSO-d
6
): 15.99 (1H, s, HO-1), 12.77 (1H, s, HO-
3
3
(
1H, d, JH25-H26 ¼ 11.0 Hz, H-25), 5.03 (1H, vbs, HO-21), 4.89 (1H, m,
4), 9.21 (1H, s, NHamide), 6.62 (1H, dd, JH18-H19 ¼ 16.0 Hz, H-18), 6.32
2
3
3
H-28), 4.31 (1H, d, J ¼ 12.0 Hz, H-38), 3.96 (1H, d, JHO-H23 ¼ 8.6 Hz,
(1H, d, JH17-H18 ¼ 10.7 Hz, H-17), 6.25 (1H, d, H-29), 6.07 (1H, dd,
3
3
3
HO-23), 3.70 (1H, d, JH20-H21 ¼ 9.5 Hz, H-21), 3.56 (1H, m, H-38),
J
H19-H20 ¼ 7.5 Hz, H-19), 5.08 (1H, d, JH25-H26 ¼ 10.9 Hz, H-25), 5.05
3
3
3
.23 (1H, d, JH27-H28 ¼ 8.5 Hz, H-27), 3.08 (2H, m, H-39), 2.89 (3H, s,
(1H, vbs, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.8 Hz, H-28), 4.20 (1H,
3
2
3
H-37), 2.82 (1H, d, JH23-H24 ¼ 9.2 Hz, H-23), 2.75 (2H, m, H-40), 2.57
d, J ¼ 12.1 Hz, H-38), 3.99 (1H, d, JHO-H23 ¼ 8.7 Hz, HO-23), 3.70
3
(
(
4H, m, H-41 þ H-44), 2.29 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98
3H, s, H-30), 1.92 (3H, s, H-14), 1.72 (4H, m, H-42 þ H-43), 1.70 (1H,
(1H, d, H-21), 3.63 (1H, d, H-38), 3.24 (1H, d, JH27-H28 ¼ 8.5 Hz, H-
27), 3.09 (1H, m, H-39), 2.99 (1H, m, H-39), 2.89 (3H, s, H-37), 2.83
3
m, H-22), 1.63 (3H, s, H-13), 1.27 (1H, m, H-24), 0.99 (1H, m, H-26),
(1H, m, H-23), 2.36 (2H, t,
J
H40-H41 ¼ 6.4 Hz, H-41), 2.28 (1H, h,
3
3
3
3
0
.92 (3H, d, JH22-H32 ¼ 6.9 Hz, H-32), 0.87 (3H, d, JH20-H31 ¼ 6.9 Hz,
J
H20-H31 ¼ 7.1 Hz, JH20-H21 ¼ 7.1 Hz, H-20), 2.18 (6H, s, H-42), 1.99
3
3
H-31), 0.53 (3H, d,
J
H24-H33 ¼ 6.9 Hz, H-33), ꢀ0.35 (3H, d,
J
H34-
(6H, s, H-30 þ H-36), 1.92 (3H, s, H-14), 1.72 (1H, m, H-40), 1.69 (2H,
1
3
3
H26 ¼ 6.9 Hz, H-34); C NMR (
84.2 (C-8), 171.8 (C-6), 170.2 (C-15), 169.3 (C-35), 148.8 (C-1), 144.9
C-4), 143.1 (C-29), 139.1 (C-19), 131.5 (C-17), 131.4 (C-16), 126.4 (C-
8), 118.0 (C-2), 117.5 (C-28), 116.7 (C-10), 114.9 (C-3 þ C-9), 108.8
C-12), 101.2 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25),
d
ppm in DMSO-d
6
): 185.1 (C-11),
m, H-22 þ H-40), 1.64 (3H, s, H-13), 1.26 (1H, dq, JH24-H33 ¼ 7.0 Hz,
3
3
3
1
(
1
(
J
H23-H24 ¼ 13.6 Hz, H-24), 0.98 (1H, m, H-26), 0.91 (3H, d,
J
J
H22-
H32 ¼ 7.0 Hz, H-32), 0.86 (3H, d, H-31), 0.52 (3H, d,
3
H24-
13
H33 ¼ 6.7 Hz, H-33), ꢀ0.33 (3H, d,
NMR ( ppm in DMSO-d
J
H34-H26 ¼ 6.7 Hz, H-34);
C
d
6
): 185.1 (C-11), 184.2 (C-8), 171.8 (C-6),
7
4
3.1 (C-21), 55.6 (C-37), 53.1 (C-41 þ C-44), 51.2 (C-40), 44.6 (C-39),
169.8 (C-15), 169.4 (C-35), 148.7 (C-1), 144.8 (C-4), 143.0 (C-29),
139.0 (C-19),131.6 (C-17),131.4 (C-16),126.1 (C-18),117.8 (C-2),117.6
(C-28),116.8 (C-10),115.9 (C-9),114.8 (C-3),108.8 (C-12),101.2 (C-7),
95.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.7 (C-21), 56.9 (C-
41), 55.6 (C-37), 47.3 (C-39), 44.6 (C-42), 43.6 (C-38), 40.3 (C-26),
38.1 (C-24), 37.9 (C-20), 32.7 (C-22), 22.7 (C-40), 22.1 (C-13), 20.7
(C-36), 19.9 (C-30), 18.3 (C-31), 11.2 (C-32), 9.0 (C-34), 8.9 (C-33), 7.4
3.0 (C-38), 40.3 (C-26), 38.1 (C-24), 37.7 (C-20), 32.6 (C-22), 23.0
(
C-42), 22.1 (C-13), 20.6 (C-36), 19.9 (C-30), 18.2 (C-31), 11.0 (C-32),
8
3
.9 (C-33
474 cm
þ
C-34), 7.3 (C-14); FT-IR (KBr pellet):
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
þ 3406 cm
n
(O21eH) þ
n(O23eH), 3237 cm
ꢀ
ꢀ1
1
n
(NeH)amide
,
3086
cm
n
(O
4
eH$$$O11),
2703
cm
þ
ꢀ
ꢀ1
n
(N38eH$$$O15), 2465 cm
n
(O
1
eH$$$O
8
), 1713 cm
n
(C35]O),
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
1647 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1624 cm
n(C]
(C-14); FT-IR (KBr pellet): 3464 cm
þ
3398 cm
ꢀ1
ꢀ1
ꢀ1
C)naphthalene, 1586 cm
d
(NeH), 1535 cm
d(NeH)amide II, 1482 and
n
n
n
(O21eH)
þ
n
(O23eH), 3230 cm
n(NeH)amide, 3083 cm
ꢀ1
ꢀ1
ꢀ1
þ
1459 cm
n
(C]C),1243 and 1238 cm
n(CeO); elemental analysis
(O
(O
þ
4
eH$$$O11), 2707 cm
eH$$$O
n
(N38eH$$$O15), 2465 cm
ꢀ
ꢀ1
ꢀ1
C
44
H
61
N
3
O12: calculated: C ¼ 64.14%; H ¼ 7.46%; N ¼ 5.10%;
1
8
), 1722 cm
(C
n
(C35]O), 1646 cm
n
(C15]O)amide
ꢀ
1
ꢀ1
O ¼ 23.30%; measured: C ¼ 64.12%; H ¼ 7.48%; N ¼ 5.09%;
I
n
(C11]O) þ
n
ꢀ
8
]O), 1625 cm
n
(C]C)naphthalene, 1592 cm
1
ꢀ1
O ¼ 23.31%.
d
(NeH), 1535 cm
d
(NeH)amide II, 1481 and 1464 cm
n(C]C),
ꢁ
ꢀ1
n
6
e yield: 50%; HPLC: RT ¼ 13.77 [min]; 25 C; 60:40
1248 and 1235 cm
61 3 12
(CeO); elemental analysis C43H N O :
þ
(
H
2
O:CH
H NMR (
), 9.27 (1H, s, NHamide), 6.60 (1H, dd, JH18-H19 ¼ 16.0 Hz, H-18), 6.34
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 838.4490;
calculated: C ¼ 63.61%; H ¼ 7.57%; N ¼ 5.18%; O ¼ 23.65%;
1
d
6
ppm in DMSO-d ): 16.04 (1H, s, HO-1), 12.83 (1H, s, HO-
measured: C ¼ 63.56%; H ¼ 7.61%; N ¼ 5.13%; O ¼ 23.70%.
3
ꢁ
4
8 e yield: 66%; HPLC: RT ¼ 10.49 [min]; 25 C; 60:40
3
þ
(
1H, d, JH17-H18 ¼ 10.5 Hz, H-17), 6.26 (1H, d, H-29), 6.09 (1H, dd,
(H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 840.4643;
3
3
1
J
H19-H20 ¼ 7.4 Hz, H-19), 5.09 (1H, d, JH25-H26 ¼ 11.0 Hz, H-25), 5.04
H NMR (
d
ppm in DMSO-d
6
): 15.98 (1H, s, HO-1), 12.73 (1H, s, HO-
3
3
(
1H, vbs, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.9 Hz, H-28), 4.34 (1H,
4), 9.18 (1H, s, NHamide), 6.63 (1H, dd, JH18-H19 ¼ 16.0 Hz, H-18), 6.31
2
3
3
d, J ¼ 12.0 Hz, H-38), 3.96 (1H, d, JHO-H23 ¼ 8.6 Hz, HO-23), 3.70
(1H, d, JH17-H18 ¼ 10.8 Hz, H-17), 6.24 (1H, d, H-29), 6.06 (1H, dd,

668
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
3JH19-H20 ¼ 7.3 Hz, H-19), 5.08 (1H, d, JH25-H26 ¼ 11.0 Hz, H-25), 5.05
3
C
H
N
O
12: calculated: C ¼ 64.50%; H ¼ 7.58%; N ¼ 5.01%;
45
63
3
3
(
1H, vbs, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.9 Hz, H-28), 4.14 (1H,
O ¼ 22.91%; measured: C ¼ 64.47%; H ¼ 7.62%; N ¼ 4.98%;
2
3
d, J ¼ 11.9 Hz, H-38), 3.99 (1H, d, JHO-H23 ¼ 8.8 Hz, HO-23), 3.70
O ¼ 22.93%.
3
ꢁ
þ
(
1H, d, H-21), 3.61 (1H, d, H-38), 3.24 (1H, d, JH27-H28 ¼ 8.4 Hz, H-
7), 3.05 (1H, m, H-39), 3.00 (1H, m, H-39), 2.90 (3H, s, H-37), 2.83
1H, m, H-23), 2.62 (1H, m, H-41), 2.59 (1H, m, H-41), 2.58 (1H, m.
10 e yield: 61%; HPLC: RT ¼ 7.37 [min]; 25 C; 60:40
2
(H
2
O:CH
3
d
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 854.4441;
1
(
H NMR (
ppm in DMSO-d
6
): 16.04 (1H, s, HO-1), 12.85 (1H, s, HO-
3
3
þ
þ
H-42), 2.53 (1H, m, H-42), 2.29 (1H, h,
J
H20-H31 ¼ 7.1 Hz,
J
H20-
4), 9.30 (1H, s, NHamide), 8.49 (2H, m, N H-38hydrogen bonded þ N H-
3
H21 ¼ 7.1 Hz, H-20), 1.99 (3H, s, H-36), 1.98 (3H, s, H-30), 1.91 (3H,
38non-hydrogen bonded), 6.60 (1H, dd, JH18-H19 ¼ 16.0 Hz, H-18), 6.33
3
3
s, H-14), 1.74 (1H, m, H-40), 1.70 (1H, m, H-40), 1.69 (1H, m, H-22),
(1H, d, JH17-H18 ¼ 10.3 Hz, H-17), 6.25 (1H, d, JH28-H29 ¼ 12.8 Hz, H-
3
3
3
1
.64 (3H, s, H-13), 1.26 (1H, dq,
J
H24-H33 ¼ 7.2 Hz,
J
H23-
29), 6.09 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.10 (1H, m, H-25), 5.07
2
3
H24 ¼ 15.0 Hz, H-24), 1.00 (1H, m, H-26), 0.96 (6H, t, J ¼ 7.1 Hz,
(1H, m, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.8 Hz, H-28), 4.27 (1H, d,
3
2
3
H-43), 0.92 (3H, d, JH22-H32 ¼ 6.9 Hz, H-32), 0.86 (3H, d, H-31), 0.51
J ¼ 12.0 Hz, H-38), 3.98 (1H, d, JHO-H23 ¼ 8.8 Hz, HO-23), 3.70 (1H,
3
3
3
(
3H, d, JH24-H33 ¼ 6.9 Hz, H-33), ꢀ0.33 (3H, d, JH34-H26 ¼ 6.9 Hz, H-
d, JH20-H21 ¼ 9.5 Hz, H-21), 3.49 (5H, m, H-38 þ H-43 þ H-44), 3.24
4); 13C NMR (
C-6), 169.7 (C-15), 169.3 (C-35), 148.7 (C-1), 144.7 (C-4), 143.0 (C-
9), 138.8 (C-19), 131.7 (C-17), 131.3 (C-16), 126.1 (C-18), 117.7 (C-2),
17.6 (C-28), 116.4 (C-10), 116.0 (C-9), 114.7 (C-3), 108.7 (C-12), 101.0
C-7), 98.4 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.7 (C-21),
5.6 (C-37), 50.9 (C-41), 47.8 (C-39), 45.9 (C-42), 43.6 (C-38), 40.3
C-26), 38.1 (C-24), 37.9 (C-20), 32.6 (C-22), 22.4 (C-40), 22.0 (C-13),
d
ppm in DMSO-d
): 185.0 (C-11), 184.2 (C-8), 171.8
(1H, d, JH27-H28 ¼ 8.6 Hz, H-27), 3.18 (1H, m, H-39), 3.05 (1H, m, H-
3
3
6
(
2
1
(
5
(
39), 2.89 (3H, s, H-37), 2.83 (1H, m, H-23), 2.41 (2H, m, H-41), 2.35
(4H, m, H-42 þ H-45), 2.30 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98
(3H, s, H-30), 1.92 (3H, s, H-14), 1.76 (2H, m, H-40), 1.69 (1H, m, H-
3
22), 1.65 (3H, s, H-13), 1.26 (1H, m, H-24), 0.99 (1H, dd,
J
H26-
3
H34 ¼ 7.2 Hz, H-26), 0.92 (3H, d,
J
H22-H32 ¼ 7.1 Hz, H-32), 0.86
3
(3H, m, H-31), 0.52 (3H, d, JH24-H33 ¼ 6.8 Hz, H-33), ꢀ0.33 (3H, d,
3
13
2
3
3
0.6 (C-36), 19.9 (C-30), 18.2 (C-31), 11.2 (C-32), 10.6 (C-43), 9.0 (C-
J
H34-H26 ¼ 6.7 Hz, H-34); C NMR (
6
d ppm in DMSO-d ): 185.3 (C-
4),
473 cm
8.8
(C-33),
7.3
(C-14);
FT-IR
(KBr
pellet):
11), 184.2 (C-8), 171.8 (C-6), 170.0 (C-15), 169.3 (C-35), 148.9 (C-1),
144.9 (C-4), 143.0 (C-29), 139.1 (C-19), 131.4 (C-17 þ C-16), 126.0 (C-
18), 117.9 (C-2), 117.6 (C-28), 116.6 (C-10), 115.1 (C-9), 115.0 (C-3),
108.8 (C-12), 101.3 (C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2
(C-25), 72.8 (C-21), 65.9 (C-43 þ C-44), 55.6 (C-37 þ C-41), 52.9 (C-
42 þ C-45), 47.3 (C-39), 43.5 (C-38), 40.3 (C-26), 38.2 (C-24), 37.8 (C-
20), 32.6 (C-22), 22.1 (C-13), 21.6 (C-40), 20.7 (C-36), 19.9 (C-30),
18.3 (C-31), 11.1 (C-32), 9.0 (C-34), 8.8 (C-33), 7.3 (C-14); FT-IR (KBr
ꢀ
1
ꢀ1
ꢀ
ꢀ1
þ 3405 cm
n
(O21eH) þ
n(O23eH), 3237 cm
1
ꢀ1
n
(NeH)amide
,
3077
cm
n
(O
4
eH$$$O11),
2700
cm
þ
ꢀ1
ꢀ
ꢀ1
n
(N38eH$$$O15), 2474 cm
n
(O
1
eH$$$O
8
), 1723 cm
n
(C35]O),
ꢀ1
ꢀ1
1646 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
d
8
]O), 1624 cm
(NeH)amide II, 1480 and
n(C]
ꢀ
1
ꢀ1
C)naphthalene, 1587 cm
d
(NeH), 1536 cm
ꢀ
1
ꢀ1
1462 cm
n
(C]C), 1245 and 1237 cm
n(CeO); elemental analysis
C
H
45 65
N
3
O
12: calculated: C ¼ 64.34%; H ¼ 7.80%; N ¼ 5.0%;
ꢀ
1
ꢀ1
1
ꢀ1
ꢀ1
O ¼ 22.86%; measured: C ¼ 64.38%; H ¼ 7.77%; N ¼ 5.05%;
pellet): 3473 cm þ 3404 cm
n
(O21eH) þ
n(O23eH), 3234 cm
ꢀ
O ¼ 22.80%.
n(NeH)amide
,
3083
cm
n
(O
4
eH$$$O11),
2724
cm
ꢁ
þ
þ
ꢀ1
ꢀ
ꢀ1
9
e yield: 68%; HPLC: RT ¼ 9.73 [min]; 25 C; 60:40
n(N38eH$$$O15), 2477 cm
n
(O
1
eH$$$O
8
), 1723 cm
n
(C35]O),
ꢀ1
ꢀ1
(
H
2
O:CH
H NMR (
), 9.18 (1H, s, NHamide), 6.62 (1H, dd, JH18-H19 ¼ 16.0 Hz, H-18), 6.31
3
d
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 838.4482;
1647 cm
C)naphthalene, 1588 cm
1463 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1625 cm
n(C]
1
ꢀ1
ꢀ1
d
ppm in DMSO-d
6
): 15.99 (1H, s, HO-1), 12.73 (1H, s, HO-
d
(NeH), 1536 cm
(NeH)amide II, 1481 and
n(CeO); elemental analysis
3
ꢀ1
ꢀ1
4
n
(C]C), 1245 and 1237 cm
3
(
1H, d, JH17-H18 ¼ 10.8 Hz, H-17), 6.24 (1H, d, H-29), 6.07 (1H, dd,
C
45
H
63
N
3
O13: calculated: C ¼ 63.28%; H ¼ 7.44%; N ¼ 4.92%;
3
3
J
H19-H20 ¼ 7.4 Hz, H-19), 5.08 (1H, d, JH25-H26 ¼ 11.0 Hz, H-25), 5.05
O ¼ 24.36%; measured: C ¼ 63.25%; H ¼ 7.47%; N ¼ 4.93%;
3
(
1H, vbs, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.8 Hz, H-28), 4.18 (1H,
O ¼ 24.35%.
2
3
ꢁ
þ
d, J ¼ 12.1 Hz, H-38), 3.99 (1H, d, JHO-H23 ¼ 8.8 Hz, HO-23), 3.70
11 e yield: 42%; HPLC: RT ¼ 4.43 [min]; 25 C; 60:40
3
(
2
1H, d, H-21), 3.63 (1H, d, H-38), 3.24 (1H, d, JH27-H28 ¼ 8.4 Hz, H-
(H
2
O:CH
3
d
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 835.4131;
3
1
7), 3.10 (1H, dt, JH39-H40 ¼ 6.8 Hz, H-39), 3.02 (1H, dt, H-39), 2.89
H NMR (
ppm in DMSO-d
6
): 16.05 (1H, s, HO-1), 12.82 (1H, s, HO-
2
3
þ
þ
(
3H, s, H-37), 2.83 (1H, m, H-23), 2.69 (1H, dt, J ¼ 12.9 Hz
J
H40-
4), 9.32 (1H, s, NHamide), 8.40 (2H, m, N H-38hydrogen bonded þ N H-
H41 ¼ 6.5 Hz, H-41), 2.64 (1H, m, H-41), 2.57 (4H, m, H-42 þ H-
38non-hydrogen bonded), 7.42 (1H, s, H-42), 7.15 (1H, s, H-43), 6.89 (1H,
3
3
3
3
4
5), 2.29 (1H, h, JH20-H31 ¼7.2 Hz, JH20-H21 ¼7.2 Hz, H-20),1.98 (6H,
s, H-44), 6.58 (1H, dd, JH18-H19 ¼ 15.9 Hz, H-18), 6.33 (1H, d, JH17-
3
s, H-30 þ H-36), 1.91 (3H, s, H-14), 1.74 (1H, m, H-40), 1.69 (1H, m,
H18 ¼ 10.5 Hz, H-17), 6.26 (1H, d,
J
H28-H29 ¼ 12.8 Hz, H-29), 6.08
3
3
H-22), 1.68 (4H, m, H-43 þ H-44), 1.64 (3H, s, H-13), 1.27 (1H, dq,
(1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.08 (1H, d, JH25-H26 ¼ 11.0 Hz, H-
3
3
3
3
3
3
J
H24-H33 ¼ 7.7 Hz,
J
H23-H24 ¼ 14.7 Hz, H-24), 0.99 (1H, dq,
J
J
J
H25-
H22-
H24-
25), 5.04 (1H, m, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.8 Hz, H-28),
3
2
3
H26 ¼ 14.1 Hz,
J
H26-H34 ¼ 7.2 Hz, H-26), 0.91 (3H, d,
4.28 (1H, d, J ¼ 12.0 Hz, H-38), 3.99 (1H, d, JHO-H23 ¼ 8.8 Hz, HO-
3
H32 ¼ 7.0 Hz, H-32), 0.86 (3H, d, H-31), 0.51 (3H, d,
23), 3.66 (1H, d, JH20-H21 ¼ 9.5 Hz, H-21), 3.41 (1H, m, H-38), 3.24
3
13
3
H33 ¼ 6.8 Hz, H-33), ꢀ0.32 (3H, d,
NMR ( ppm in DMSO-d
69.8 (C-15), 169.3 (C-35), 148.7 (C-1), 144.7 (C-4), 143.0 (C-29),
38.9 (C-19), 131.6 (C-17), 131.3 (C-16), 126.2 (C-18), 117.7 (C-28),
17.6 (C-2), 116.7 (C-10), 116.4 (C-9), 114.7 (C-3), 108.7 (C-12), 101.1
C-7), 98.4 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.8 (C-21),
J
H34-H26 ¼ 6.9 Hz, H-34);
C
(1H, d, JH27-H28 ¼ 8.6 Hz, H-27), 3.17 (2H, m, H-39), 3.01 (2H, m, H-
d
6
): 185.1 (C-11), 184.2 (C-8), 171.8 (C-6),
40), 2.89 (3H, s, H-37), 2.83 (1H, m, H-23), 2.28 (1H, m, H-20), 2.04
(2H, m, H-41), 1.99 (3H, s, H-36), 1.92 (3H, s, H-14), 1.69 (1H, m, H-
1
1
1
(
5
4
(
3
22), 1.65 (3H, s, H-13), 1.26 (1H, dd, JH24-H33 ¼ 7.1 Hz, H-24), 0.99
3
3
(1H, dd, JH26-H34 ¼ 7.1 Hz, H-26), 0.91 (3H, d, JH22-H32 ¼ 6.9 Hz, H-
3 3
32), 0.85 (3H, d,
J
H20-H31 ¼ 6.8 Hz, H-31), 0.50 (3H, d,
J
H24-
C
3
13
5.6 (C-37), 53.7 (C-41), 53.3 (C-42 þ C-45), 47.7 (C-39), 43.8 (C-38),
H33 ¼ 6.8 Hz, H-33), ꢀ0.33 (3H, d,
J
H34-H26 ¼ 6.8 Hz, H-34);
0.3 (C-26), 38.1 (C-24), 37.8 (C-20), 32.6 (C-22 þ C-43 þ C-44), 23.9
6
NMR (d ppm in DMSO-d ): 185.3 (C-11), 184.2 (C-8), 171.8 (C-6),
C-40), 22.0 (C-13), 20.6 (C-36), 19.9 (C-30), 18.2 (C-31), 11.2 (C-32),
170.3 (C-15), 169.4 (C-35), 148.7 (C-1), 144.9 (C-4), 143.1 (C-29),
139.1 (C-19), 137.1 (C-42), 131.6 (C-17), 131.4 (C-16), 128.3 (C-43),
126.1 (C-18), 119.2 (C-44), 118.0 (C-2), 117.7 (C-28), 116.6 (C-10),
115.0 (C-9), 114.7 (C-3), 108.8 (C-12), 101.3 (C-7), 98.5 (C-5), 76.3 (C-
27), 75.7 (C-23), 73.2 (C-25), 72.8 (C-21), 64.9 (C-41), 55.6 (C-37),
44.5 (C-39), 43.0 (C-26 þ C-38), 38.1 (C-24), 37.9 (C-20), 32.7 (C-22),
27.4 (C-40), 22.1 (C-13), 20.6 (C-36), 19.9 (C-30), 18.2 (C-31), 11.2 (C-
32), 9.0 (C-34), 8.8 (C-33), 7.3 (C-14); FT-IR (KBr pellet):
8
3
.9 (C-34), 8.8 (C-33), 7.3 (C-14); FT-IR (KBr pellet):
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
469 cm
þ 3402 cm
n
(O21eH) þ
n(O23eH), 3230 cm
ꢀ
ꢀ1
1
n
(NeH)amide
,
3086
cm
n
(O
4
eH$$$O11),
2708
cm
þ
ꢀ
ꢀ1
n
(N38eH$$$O15), 2466 cm
n(O
1
eH$$$O
8
), 1722 cm
n
(C35]O),
ꢀ1
ꢀ1
1648 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
ꢀ1
(C
8
]O), 1627 cm
n(C]
ꢀ1
C)naphthalene, 1585 cm
d
(NeH), 1536 cm
d
(NeH)amide II, 1479 and
n(CeO); elemental analysis
ꢀ
1
ꢀ1
1457 cm
n
(C]C), 1249 and 1236 cm

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
ꢀ1
669
ꢀ
ꢀ1
3
n
473 cm 1 þ 3400 cm
n
(O21eH) þ
n(O23eH), 3231 cm
46 þ C-50), 129.2 (C-47 þ C-49), 128.8 (C-48), 126.0 (C-18), 118.1 (C-
28), 117.8 (C-2), 116.2 (C-10), 115.0 (C-9), 114.4 (C-3), 108.7 (C-12),
101.3 (C-7), 98.4 (C-5), 76.2 (C-27), 75.6 (C-23), 73.1 (C-25), 72.9 (C-
21), 59.0 (C-44), 55.6 (C-37), 51.4 (C-39), 49.7 (C-41 þ C-42), 49.5
(C-38) 40.5 (C-26), 38.0 (C-24), 37.8 (C-20), 32.7 (C-22), 26.1 (C-
40 þ C-43), 21.9 (C-13), 20.6 (C-36), 19.7 (C-30), 18.1 (C-31), 11.2 (C-
ꢀ
ꢀ1
1
ꢀ1
(NeH)amide
,
3082
cm
n
(O
4
eH$$$O11),
2730
cm
þ
ꢀ
ꢀ1
n
(N38eH$$$O15), 2476 cm
n
(O
1
eH$$$O
8
), 1720 cm
n
(C35]O),
ꢀ1
ꢀ1
1648 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1624 cm
n(C]
ꢀ
1
ꢀ1
d
C)naphthalene, 1585 cm
d
(NeH), 1536 cm
(NeH)amide II, 1480 and
ꢀ
1
ꢀ1
1461 cm
n
(C]C), 1247 and 1235 cm
n(CeO); elemental analysis
ꢀ
1
C
44
58
H N
4
O
12: calculated: C ¼ 63.29%; H ¼ 7.01%; N ¼ 6.71%;
32), 8.8 (C-34), 8.7 (C-33), 7.2 (C-14); FT-IR (CH
3
CN, 0.05 mol L ):
ꢀ1
ꢀ1
ꢀ1
O ¼ 22.99%; measured: C ¼ 63.27%; H ¼ 7.03%; N ¼ 6.73%;
3472 cm
þ 3400 cm
n
(O21eH) þ
n
(O23eH), 3241 cm
ꢀ1
þ
ꢀ1
O ¼ 22.97%.
n
(NeH)amide, 3153 cm
n
(N41eH), 3092 cm
n
(O
4
eH$$$O11),
ꢁ
þ
ꢀ1
þ
ꢀ1
ꢀ
ꢀ1
1
2 e yield: 66%; HPLC: RT ¼ 21.90 [min]; 25 C; 60:40
2730 cm
n
(N38eH$$$O15), 2471 cm
n
(O
1
n
eH$$$O
8
), 1722 cm
ꢀ1
(
H
2
O:CH
3
d
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 900.4642;
n
(C35]O), 1645 cm
n
(C15]O)amide
I
þ
(C11]O) þ
n
(C
8
]O),
1
ꢀ1
ꢀ1 þ
ꢀ1
ꢀ1
H NMR (
ppm in DMSO-d
6
): 16.00 (1H, s, HO-1), 12.81 (1H, s, HO-
1627 cm
n
(C]C)naphthalene, 1586 cm
d(N eH), 1534 cm
n(C]C), 1250 and 1239 cm
þ
þ
ꢀ1
4
), 9.22 (1H, s, NHamide), 8.60 (2H, bs, N H-38hydrogen bonded þ N H-
d(NeH)amide II, 1485 and 1461 cm
3
3
8
non-hydrogen bonded), 7.29 (5H, m, H-46 e H-50), 6.57 (1H, dd, JH18-
n
(CeO); Elemental analysis C50
H66ClN
3
O
12: calculated: C ¼ 64.12%;
3
H19 ¼ 15.9 Hz, H-18), 6.31 (1H, d, JH17-H18 ¼ 10.7 Hz, H-17), 6.25 (1H,
H ¼ 7.10%; N ¼ 4.49%; measured: C ¼ 64.16%; H ¼ 7.17%; N ¼ 4.46%.
3
3
ꢁ
d, H-29), 6.07 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.08 (1H, d, JH25-
13 e yield: 41%; HPLC: RT ¼ 38.12 [min]; 25 C; 60:40
3
H26 ¼ 11.1 Hz, H-25), 5.06 (1H, m, HO-21), 4.89 (1H, dd,
J
H28-
(H
2
O:CH
3
CN);
flow:
1.0
mL/min;
HR-MALDI-TOF
2
[MþH] 1620.8361; 1H NMR (
þ
H29 ¼ 12.6 Hz, H-28), 4.26 (1H, d, J ¼ 12.0 Hz, H-38), 3.96 (1H, d,
d
ppm in DMSO-d
6
): 16.00 (1H, s,
3
3
þ
J
HO-H23 ¼ 8.9 Hz, HO-23), 3.68 (3H, m, JH20-H21 ¼ 6.7 Hz, H-21 þ H-
HO-1), 12.81 (1H, s, HO-4), 9.22 (1H, s, NHamide), 8.60 (4H, bs, N H-
þ
3
4
3
4), 3.56 (1H, d, H-38), 3.30 (8H, m, H-40 þ H-41 þ H-42 þ H-43),
38hydrogen bonded þ N H-38non-hydrogen bonded), 6.57 (2H, dd,
J
H18-
3
3
.24 (1H, d, JH27-H28 ¼ 8.5 Hz, H-27), 3.16 (1H, m, H-39), 2.98 (1H,
H19 ¼ 15.9 Hz, H-18), 6.31 (2H, d,
J
H17-H18 ¼ 10.7 Hz, H-17), 6.25
3
m, H-39), 2.88 (3H, s, H-37), 2.83 (1H, m, H-23), 2.33 (1H, m, H-41),
(2H, d, H-29), 6.07 (2H, dd,
J
H19-H20 ¼ 7.4 Hz, H-19), 5.08 (2H, d,
3
3
2
.27 (1H, m, H-20), 2.26 (1H, m, H-41), 1.98 (3H, s, H-36), 1.96 (3H, s,
J
H25-H26 ¼ 11.1 Hz, H-25), 5.06 (2H, m, HO-21), 4.89 (2H, dd, JH28-
2
H-30), 1.91 (3H, s, H-14), 1.67 (3H, m, H-22 þ H-40), 1.63 (3H, s, H-
H29 ¼ 12.6 Hz, H-28), 4.26 (2H, d, J ¼ 12.0 Hz, H-38), 3.96 (2H, d,
3
3
3
1
3), 1.25 (1H, m, H-24), 0.99 (1H, m, H-26), 0.91 (3H, d,
J
H22-
JHO-H23 ¼ 8.9 Hz, HO-23), 3.68 (2H, d, JH21-H22 ¼ 6.7 Hz, H-21), 3.56
3
H32 ¼ 7.0 Hz, H-32), 0.84 (3H, d,
J
H20-H31 ¼ 6.7 Hz, H-31), 0.53
(2H, d, H-38), 3.30 (8H, m, H-42 þ H-43 þ H-44 þ H-45), 3.24 (2H,
3
3
3
(
3
(
2
4
2
1
2
3H, d, JH24-H33 ¼ 6.7 Hz, H-33), ꢀ0.35 (3H, d, JH34-H26 ¼ 7.0 Hz, H-
d, JH27-H28 ¼ 8.5 Hz, H-27), 3.16 (2H, m, H-39), 2.98 (2H, m, H-39),
4); 13C NMR (
C-6), 169.8 (C-15), 169.3 (C-35), 148.7 (C-1), 144.7 (C-4), 143.1 (C-
d
ppm in DMSO-d
): 185.1 (C-11), 184.2 (C-8), 171.7
2.88 (6H, s, H-37), 2.83 (2H, m, H-23), 2.33 (2H, m, H-41), 2.27 (2H,
m, H-20), 2.26 (2H, m, H-41), 1.98 (6H, s, H-36), 1.96 (6H, s, H-30),
1.91 (6H, s, H-14), 1.67 (6H, m, H-22 þ H-40), 1.63 (6H, s, H-13), 1.25
6
9), 139.1 (C-19), 138.0 (C-45), 131.5 (C-17), 131.1 (C-16), 128.6 (C-
6 þ C-50), 128.1 (C-47 þ C-49), 126.9 (C-48), 125.9 (C-18), 117.5 (C-
8), 117.3 (C-2), 116.5 (C-10), 115.4 (C-9), 115.0 (C-3), 108.8 (C-12),
3
(2H, m, H-24), 0.99 (2H, m, H-26), 0.91 (6H, d, JH22-H32 ¼ 7.0 Hz, H-
3
3
32), 0.84 (6H, d,
J
H20-H31 ¼ 6.7 Hz, H-31), 0.53 (6H, d,
J
H24-
3
13
01.2 (C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25), 72.9 (C-
H33 ¼ 6.7 Hz, H-33), ꢀ0.35 (6H, d,
J
H34-H26 ¼ 7.0 Hz, H-34);
C
1), 60.3 (C-44), 55.6 (C-37), 54.5 (C-39), 50.7 (C-38 þ C-41 þ C-42),
6
NMR (d ppm in DMSO-d ): 185.1 (C-11), 184.2 (C-8), 171.7 (C-6),
4
2
3
3
0.3 (C-26), 38.2 (C-24), 37.7 (C-20), 32.6 (C-22), 28.3 (C-40 þ C-43),
169.8 (C-15), 169.3 (C-35), 148.7 (C-1), 144.7 (C-4), 143.1 (C-29),
139.1 (C-19), 131.4 (C-16 þ C-17), 126.0 (C-18), 117.5 (C-28), 117.3 (C-
2), 116.5 (C-10), 115.4 (C-9), 115.0 (C-3), 108.8 (C-12), 101.2 (C-7),
98.5 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25), 72.9 (C-21), 55.6 (C-
37), 55.4 (C-41), 51.8 (C-42 þ C-43 þ C-44 þ C-45), 48.1 (C-39), 43.6
(C-38), 40.3 (C-26), 38.2 (C-24), 37.7 (C-20), 32.6 (C-22), 22.1 (C-13),
21.6 (C-40), 20.6 (C-36), 19.8 (C-30), 18.2 (C-31), 11.0 (C-32), 9.0 (C-
2.0 (C-13), 20.6 (C-36), 19.8 (C-30), 18.2 (C-31), 11.0 (C-32), 9.0 (C-
4),
474 cm
8.8
(C-33),
7.3
(C-14);
FT-IR
(KBr
pellet):
ꢀ
1
ꢀ1
ꢀ1
þ 3401 cm
n
1
(O21eH) þ
n(O23eH), 3239 cm
ꢀ
ꢀ1
ꢀ1
n
(NeH)amide
,
3091
cm
n
(O
4
eH$$$O11),
2731
cm
þ
ꢀ
ꢀ1
n
(N38eH$$$O15), 2473 cm
n
(O
1
eH$$$O
8
), 1723 cm
n
(C35]O),
ꢀ1
ꢀ1
1647 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1626 cm
n(C]
ꢀ
1
ꢀ1
d
C)naphthalene, 1586 cm
d
(NeH), 1536 cm
(NeH)amide II, 1483 and
(CeO); elemental analysis
34),
3471 cm
8.8
(C-33),
þ 3411 cm
7.3
(C-14);
FT-IR
(KBr
pellet):
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
1460 cm
n
(C]C), 1248 and 1236 cm
n
n
(O21eH) þ
n
(O23eH), 3230 cm
ꢀ
1
ꢀ1
þ
C
H
50 65
N
3
O
12: calculated: C ¼ 66.72%; H ¼ 7.28%; N ¼ 4.67%;
measured: C ¼ 66.78%; H ¼ 7.24%; N ¼ 4.70%.
2 þ H e yield: 99%; HPLC: RT ¼ 19.5 [min]; 25 C; 60:40
n
(NeH)amide, 3094 cm
n(O
4
eH/O11), 2735 cm
n
(N38eH/O15),
ꢀ1
ꢀ
ꢀ1
ꢀ1
2478 cm
O)amide
1589 cm
n
(O
(C11]O) þ
(NeH), 1535 cm
1
eH/O
8
), 1712 cm
(C
n
(C35]O), 1646 cm
n(C15]
ꢁ
ꢀ1
1
I
þ
n
n
8
]O), 1626 cm
n
(C]C)naphthalene
(NeH)amide II, 1483 and 1458 cm
,
þ
1
ꢀ1
ꢀ1
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [M] 900.4647; H
d
d
ꢀ
1
NMR ( ppm in DMSO-d
d
6
): 16.10 (1H, s, HO-1), 12.87 (1H, s, HO-4),
n
(C]C), 1246 and 1234 cm
n(CeO); elemental analysis
þ
9
.42 (1H, s, NHamide), 8.81 (1H, bs, N H-38hydrogen bonded), 8.52 (1H,
C
86
H
118
N
6
O
24: calculated: C ¼ 63.77%; H ¼ 7.34%; N ¼ 5.19%;
þ
þ
bs, N HeN H-38non-hydrogen bonded), 7.48 (6H, m, H-46 e H-
O ¼ 23.70%; measured: C ¼ 63.75%; H ¼ 7.36%; N ¼ 5.18%;
3
3
5
0 þ N(41)þH), 6.56 (1H, dd, JH18-H19 ¼ 15.9 Hz, JH17-H18 ¼ 10.7 Hz,
O ¼ 23.71%.
3
ꢁ
H-18), 6.34 (1H, d, H-17), 6.23 (1H, d, JH28-H29 ¼ 12.8 Hz, H-29), 6.10
14 e yield: 41%; HPLC: RT ¼ 35.37 [min]; 25 C; 60:40
3
3
(
2
4
(
3
1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.08 (1H, d, JH25-H26 ¼ 10.9 Hz, H-
(H2O:CH3CN);
flow:
1.0
mL/min;
HR-MALDI-TOF
3
[MþH] 1580.8039; 1H NMR (
þ
5), 5.00 (1H, bs, HO-21), 4.92 (1H, dd,
J
H27-H28 ¼ 8.4 Hz, H-28),
d
ppm in DMSO-d
6
): 16.10 (1H, s,
þ
.36 (1H, m, H-38), 4.28 (2H, bs, H-44), 3.96 (1H, bs, HO-23), 3.68
HO-1), 12.85 (1H, s, HO-4), 9.20 (1H, s, NHamide), 8.62 (4H, bs, N H-
3
3
1H, m, H-21 þ H-38), 3.30 (8H, m, H-40 þ H-41 þ H-42 þ H-43),
.23 (1H, d, H-27), 3.02 (2H, m, H-39), 2.89 (3H, s, H-37), 2.82 (1H,
m, H-23), 2.28 (3H, m, H-20 þ H-41), 1.98 (6H, s, H-30 þ H-36), 1.92
3H, s, H-14), 1.76 (1H, m, H-22), 1.68 (2H, m, H-40), 1.65 (3H, s, H-
38), 6.56 (2H, dd,
JH18-H19 ¼ 15.8 Hz, H-18), 6.32 (2H, d,
J
H17-
H18 ¼ 10.8 Hz, H-17), 6.24 (2H, d, H-29), 6.08 (2H, dd, 3JH19-
3
H20 ¼ 7.5 Hz, H-19), 5.09 (2H, d,
J
H25-H26 ¼ 11.2 Hz, H-25), 5.06
3
(
1
(2H, m, HO-21), 4.87 (2H, dd, JH28-H29 ¼ 12.7 Hz, H-28), 4.25 (2H, d,
3
2
3
3), 1.26 (1H, m, H-24), 0.96 (1H, m, H-26), 0.91 (3H, d,
J
H22-
J ¼ 12.0 Hz, H-38), 3.97 (2H, d, JHO-H23 ¼ 8.8 Hz, HO-23), 3.66 (2H,
3
3
3
H32 ¼ 6.9 Hz, H-32), 0.86 (3H, d,
J
H20-H31 ¼ 6.9 Hz, H-31), 0.48
d,
J
H21-H22 ¼ 6.7 Hz, H-21), 3.55 (2H, d, H-38), 3.25 (2H, d,
J
H27-
3
3
(
3H, d, JH24-H33 ¼ 6.8 Hz, H-33), ꢀ0.32 (3H, d, JH34-H26 ¼ 6.8 Hz, H-
H28 ¼ 8.5 Hz, H-27), 3.21 (4H, m, H-41), 3.08 (2H, m, H-39), 2.97
(2H, m, H-39), 2.88 (6H, s, H-37), 2.84 (2H, m, H-23), 2.67 (4H, m, H-
40), 2.28 (2H, m, H-20), 1.99 (6H, s, H-36), 1.96 (6H, s, H-30), 1.91
(6H, s, H-14), 1.78 (2H, m, H-42), 1.66 (2H, m, H-22), 1.64 (6H, s, H-
3
(
2
4); 13C NMR (
C-6), 170.8 (C-15), 169.3 (C-35), 148.9 (C-1), 144.8 (C-4), 142.8 (C-
9), 149.4 (C-19), 131.5 (C-45), 131.3 (C-17), 131.1 (C-16), 129.7 (C-
6
d ppm in DMSO-d ): 185.3 (C-11), 184.0 (C-8), 171.8

670
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
1
3), 1.25 (2H, m, H-24), 1.00 (2H, m, H-26), 0.92 (6H, d, 3JH22-
t, 3
J
H40-H41 ¼ 6.7 Hz, H-40), 4.26 (1H, m, H-38), 3.99 (1H, bs, HO-
3
H32 ¼ 7.1 Hz, H-32), 0.84 (6H, d,
J
H20-H31 ¼ 6.7 Hz, H-31), 0.54
23), 3.72 (2H, m, H-39), 3.66 (1H, m, H-38), 3.63 (1H, m, H-21),
3
3
3
(
6H, d, JH24-H33 ¼ 6.7 Hz, H-33), ꢀ0.36 (6H, d, JH34-H26 ¼ 7.0 Hz, H-
3.23 (1H, d, JH27-H28 ¼ 8.6 Hz, H-27), 2.89 (3H, s, H-37), 2.82 (1H,
4); 13C NMR (
C-6), 169.9 (C-15), 169.4 (C-35), 148.8 (C-1), 144.5 (C-4), 143.1 (C-
9),139.1 (C-19),131.4 (C-16 þ C-17), 126.1 (C-18),117.6 (C-28),117.2
C-2), 116.6 (C-10), 115.4 (C-9), 115.0 (C-3), 108.8 (C-12), 101.3 (C-7),
8.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.8 (C-21), 55.6 (C-
7), 54.3 (C-41), 51.8 (C-40), 44.1 (C-39), 43.7 (C-38), 40.4 (C-26),
8.3 (C-24), 37.7 (C-20), 32.6 (C-22), 28.6 (C-42), 22.1 (C-13), 20.6
d
ppm in DMSO-d
): 185.2 (C-11), 184.2 (C-8), 171.5
d, JH23-H22 ¼ 10.4 Hz, H-23), 2.54 (2H, t, JH39-H40 ¼ 7.6 Hz, H-43),
2.12 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98 (3H, s, H-30), 1.91 (3H, s,
H-14), 1.77 (2H, m, H-44), 1.67 (1H, m, H-22), 1.65 (3H, s, H-13),
1.43 (2H, m, H-45), 1.25 (1H, m, H-24), 0.99 (1H, m, H-26), 0.91
3
3
3
6
(
2
(
9
3
3
3
3
(3H, d, JH22-H32 ¼ 7.0 Hz, H-32), 0.89 (2H, t, JH45-H46 ¼ 7.0 Hz, H-
3
3
46), 0.85 (3H, d,
J
H20-H31 ¼ 7.0 Hz, H-31), 0.51 (3H, d,
J
H24-
C
3
13
H33 ¼ 6.8 Hz, H-33), ꢀ0.34 (3H, d,
J
H34-H26 ¼ 6.7 Hz, H-34);
(
(
n
C-36), 19.8 (C-30), 18.3 (C-31), 11.0 (C-32), 9.0 (C-34), 8.7 (C-33), 7.2
C-14); FT-IR (KBr pellet): 3475 cm
(O21eH)
6
NMR (d ppm in DMSO-d ): 185.5 (C-11), 184.2 (C-8), 171.7 (C-6),
ꢀ
1
ꢀ1
þ
3410 cm
170.4 (C-15), 169.4 (C-35), 148.8 (C-1), 146.8 (C-42), 145.0 (C-4),
143.1 (C-29), 139.1 (C-19), 131.5 (C-16), 131.4 (C-17), 126.2 (C-18),
121.8 (C-41), 118.1 (C-2), 117.7 (C-28), 116.5 (C-10), 115.0 (C-9),
114.6 (C-3), 108.8 (C-12), 101.4 (C-7), 98.5 (C-5), 76.3 (C-27), 75.6
(C-23), 73.2 (C-25), 72.8 (C-21), 55.6 (C-37), 47.5 (C-40), 45.4 (C-
39), 43.2 (C-38), 40.3 (C-26), 38.1 (C-24), 37.9 (C-20), 32.6 (C-22),
29.3 (C-44), 27.6 (C-43), 22.6 (C-45), 22.1 (C-13), 20.7 (C-36), 19.9
(C-30), 18.1 (C-31), 13.8 (C-46), 11.2 (C-32), 8.9 (C-34), 8.8 (C-33),
ꢀ
1
þ
n
(O23eH), 3240 cm
n
(NeH)amide
(N38eH$$$O15), 2476 cm
(C35]O), 1648 cm
þ
n
(NeH),
ꢀ1
ꢀ1
þ
ꢀ1
3
087 cm
n
(O
4
eH$$$O11), 2738 cm
n
ꢀ
ꢀ1
ꢀ1
n
(O
1
eH$$$O
(C11]O) þ
(NeH), 1535 cm
249 and 1235 cm
8
), 1713 cm
n
n
(C15]O)amide
ꢀ1
ꢀ1
I
d
þ
n
n
(C
8
]O), 1625 cm
n
(C]C)naphthalene, 1586 cm
(NeH)amide II, 1484 and 1462 cm
n(CeO); elemental analysis C83H N O
114 6 24:
ꢀ
1
ꢀ1
d
n(C]C),
ꢀ1
1
calculated: C ¼ 63.10%; H ¼ 7.27%; N ¼ 5.32%; O ¼ 24.31%;
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
measured: C ¼ 63.08%; H ¼ 7.29%; N ¼ 5.31%; O ¼ 24.32%.
7.3 (C-14); FT-IR (KBr pellet): 3451 cm
þ
3404 cm
ꢁ
ꢀ1
þ
1
5 e yield: 57%; HPLC: RT ¼ 2.76 [min]; 25 C; 50:50
n
n
n
(O21eH)
þ
n
(O23eH), 3244 cm
n(NeH)amide, 3147 cm
(N38eH$$$O15), 2473 cm
(C35]O), 1647 cm
þ
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 864.4388;
(O
(O
þ
4
1
eH$$$O11), 2741 cm
n
1
ꢀ
ꢀ1
ꢀ1
H NMR (
d
ppm in DMSO-d
6
): 16.01 (1H, bs, HO-1),12.81 (1H, s, HO-
eH$$$O
8
), 1722 cm
(C
n
n
(C15]O)amide
þ
ꢀ1
ꢀ1
4
), 9.32 (1H, s, NHamide), 8.55 (1H, bs, N H-38hydrogen bonded), 8.33
I
d
n
(C11]O) þ
n
8
]O), 1626 cm
n
(C]C)naphthalene, 1585 cm
ꢀ1
d(NeH)amide II,
þ
ꢀ1
n
(
1H, bs, N H-38non-hydrogen bonded), 7.78 (1H, s, H-41), 6.46 (1H, dd,
J
(NeH), 1556 cm
(C]C)triazole, 1536 cm
3
3
ꢀ1
ꢀ1
ꢀ1
n
H18-H19 ¼ 15.9 Hz, H-18), 6.31 (1H, d,
J
H17-H18 ¼ 10.4 Hz, H-17),
1478 cm
1237 cm
þ 1461 cm
and 1442 cm
ꢀ1
n(CeO); elemental analysis C46
(C]C), 1245 and
63 5
H N O12: calculated:
3
6
.24 (1H, d, H-29), 6.08 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.07 (1H,
3
d,
J
H25-H26 ¼ 10.9 Hz, H-25), 5.01 (1H, vbs, HO-21), 4.92 (1H, dd,
C ¼ 62.92%; H ¼ 7.23%; N ¼ 7.98%; O ¼ 21.87%; measured:
3
3
J
H28-H29 ¼ 12.9 Hz, H-28), 4.51 (2H, t, JH40-H41 ¼ 6.9 Hz, H-40), 4.27
C ¼ 62.82%; H ¼ 7.35%; N ¼ 8.02%; O ¼ 21.81%.
ꢁ
þ
(
1H, m, H-38), 3.99 (1H, bs, HO-23), 3.73 (2H, m, H-39), 3.69 (1H, m,
17 e yield: 61%; HPLC: RT ¼ 4.28 [min]; 25 C; 50:50
3
H-38), 3.63 (1H, m, H-21), 3.23 (1H, d, JH27-H28 ¼ 8.4 Hz, H-27), 2.89
(H
H NMR (
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 892.4701;
3
1
(
3H, s, H-37), 2.82 (1H, d,
J
H23-H22 ¼ 10.3 Hz, H-23), 2.56 (2H, t,
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.81 (1H, s,
3
þ
J
H39-H40 ¼ 7.6 Hz, H-43), 2.13 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98
3H, s, H-30), 1.91 (3H, s, H-14), 1.67 (1H, m, H-22), 1.65 (2H, m, H-
4), 1.65 (3H, s, H-13), 1.25 (1H, m, H-24), 0.99 (1H, m, H-26), 0.92
HO-4), 9.32 (1H, s, NHamide), 8.56 (1H, bs, N H-38hydrogen bonded),
8.34 (1H, bs, N H-38non-hydrogen bonded), 7.77 (1H, s, H-41), 6.47 (1H,
þ
(
4
3
3
dd, JH18-H19 ¼ 15.8 Hz, H-18), 6.31 (1H, d, JH17-H18 ¼ 10.5 Hz, H-17),
3
3
3
(
4
3H, d, JH22-H32 ¼ 7.0 Hz, H-32), 0.88 (2H, t, JH45-H46 ¼ 7.0 Hz, H-
6.25 (1H, d, H-29), 6.08 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.07 (1H,
5), 0.85 (3H, d, 3
J
H20-H31 ¼ 7.0 Hz, H-31), 0.50 (3H, d,
3
J
d,
J
3
J
H25-H26 ¼ 10.8 Hz, H-25), 5.01 (1H, vbs, HO-21), 4.91 (1H, dd,
H24-
C
3
13
3
3
H33 ¼ 6.8 Hz, H-33), ꢀ0.35 (3H, d,
J
H34-H26 ¼ 6.7 Hz, H-34);
H28-H29 ¼ 12.8 Hz, H-28), 4.52 (2H, t, JH40-H41 ¼ 6.8 Hz, H-40), 4.28
NMR (
70.3 (C-15), 169.4 (C-35), 148.8 (C-1), 146.6 (C-42), 145.0 (C-4),
43.1 (C-29), 139.1 (C-19), 131.5 (C-16), 131.4 (C-17), 126.3 (C-18),
21.9 (C-41), 118.0 (C-2), 117.7 (C-28), 116.5 (C-10), 115.1 (C-9), 114.7
C-3), 108.8 (C-12), 101.3 (C-7), 98.4 (C-5), 76.4 (C-27), 75.6 (C-23),
3.2 (C-25), 72.8 (C-21), 55.5 (C-37), 47.4 (C-40), 45.3 (C-39), 43.3
C-38), 40.3 (C-26), 38.0 (C-24), 37.8 (C-20), 32.6 (C-22), 27.2 (C-43),
d
ppm in DMSO-d
6
): 185.4 (C-11), 184.1 (C-8), 171.7 (C-6),
(1H, m, H-38), 3.99 (1H, bs, HO-23), 3.73 (2H, m, H-39), 3.67 (1H, m,
3
1
1
1
(
7
(
H-38), 3.62 (1H, m, H-21), 3.22 (1H, d, JH27-H28 ¼ 8.5 Hz, H-27), 2.89
3
(3H, s, H-37), 2.82 (1H, d,
J
H23-H22 ¼ 10.5 Hz, H-23), 2.57 (2H, t,
3
J
H39-H40 ¼ 7.6 Hz, H-43), 2.13 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98
(3H, s, H-30), 1.92 (3H, s, H-14), 1.67 (1H, m, H-22), 1.65 (3H, s, H-
13), 1.59 (2H, m, H-44), 1.32 (2H, m, H-46), 1.29 (2H, m, H-45), 1.25
3
(1H, m, H-24), 0.98 (1H, m, H-26), 0.91 (3H, d, JH22-H32 ¼ 7.0 Hz, H-
3
3
2
2.1 (C-13 þ C-44), 20.7 (C-36), 19.9 (C-30), 18.2 (C-31), 13.6 (C-45),
32), 0.85 (6H, d, JH20-H31 ¼ 7.0 Hz þ t, JH47-H48 ¼ 6.8 Hz, H-31 þ H-
3 3
11.1 (C-32), 9.0 (C-34), 8.8 (C-33), 7.3 (C-14); FT-IR (KBr pellet):
47), 0.51 (3H, d,
J
H24-H33 ¼ 6.8 Hz, H-33), ꢀ0.35 (3H, d,
J
H34-
ꢀ
1
ꢀ1
ꢀ1
13
3
450 cm
þ 3401 cm
n
(O21eH) þ
n
(O23eH), 3245 cm
H26 ¼ 6.7 Hz, H-34); C NMR (
6
d ppm in DMSO-d ): 185.4 (C-11),
ꢀ1
ꢀ1
þ
n
(NeH)amide, 3147 cm
n
(O
4
eH$$$O11), 2740 cm
n
(N38eH$$$O15),
184.2 (C-8), 171.6 (C-6), 170.3 (C-15), 169.4 (C-35), 148.9 (C-1), 146.9
(C-42), 145.0 (C-4), 143.1 (C-29), 139.0 (C-19), 131.5 (C-16), 131.4 (C-
17), 126.2 (C-18), 121.7 (C-41), 118.1 (C-2), 117.7 (C-28), 116.6 (C-10),
115.1 (C-9), 114.7 (C-3), 108.8 (C-12), 101.3 (C-7), 98.4 (C-5), 76.3 (C-
27), 75.7 (C-23), 73.1 (C-25), 72.9 (C-21), 55.5 (C-37), 47.4 (C-40),
45.4 (C-39), 43.3 (C-38), 40.3 (C-26), 38.1 (C-24), 37.8 (C-20), 32.6
(C-22), 30.6 (C-45), 29.9 (C-44), 27.4 (C-43), 22.5 (C-46), 22.1 (C-13),
20.6 (C-36), 19.9 (C-30), 18.1 (C-31), 14.0 (C-47), 11.2 (C-32), 9.0 (C-
ꢀ1
ꢀ
ꢀ1
ꢀ1
2
475 cm
n
(O
1
eH$$$O
8
), 1723 cm
n
(C35]O), 1646 cm
n(C15]
ꢀ1
O)amide
I
þ
n
(C11]O) þ
n
(C
8
]O), 1624 cm
n
(C]C)naphthalene
,
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
1586 cm
d
(NeH), 1558 cm
n
(C]C)triazole
ꢀ1
n
,
1536 cm
(C]C),1247
12: calcu-
ꢀ
1
d
(NeH)amide II,1479 cm þ1462 cm and 1443 cm
ꢀ1
and 1234 cm
n
(CeO); elemental analysis C45
H
61
N
5
O
lated: C ¼ 62.56%; H ¼ 7.12%; N ¼ 8.11%; O ¼ 22.22%; measured:
C ¼ 62.47%; H ¼ 7.15%; N ¼ 8.02%; O ¼ 22.36%.
ꢁ
1
6 e yield: 59%; HPLC: RT ¼ 3.69 [min]; 25 C; 50:50
34),
3448 cm
8.8
(C-33),
7.3
(C-14);
FT-IR
(KBr
pellet):
þ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH]
þ 3398 cm
n
(O21eH) þ
n(O23eH), 3242 cm
1
ꢀ1
8
78.4544; H NMR (
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1),
d(NeH)amide
,
3149
cm
n
(O
4
eH$$$O11),
2738
cm
þ
þ
ꢀ1
ꢀ
ꢀ1
12.81 (1H, s, HO-4), 9.32 (1H, s, NHamide), 8.57 (1H, bs, N H-
n(N38eH$$$O15), 2470 cm
n
(O
1
eH$$$O
8
), 1723 cm
n
(C35]O),
þ
ꢀ1
ꢀ1
3
8
hydrogen bonded), 8.35 (1H, bs, N H-38non-hydrogen bonded), 7.79
1646 cm
C)naphthalene
1535 cm
n
(C15]O)amide I
1589 cm
(NeH)amide II, 1479 cm þ 1459 cm and 1439 cm
þ
n
(C11]O) þ
n
(C
8
]O), 1625 cm
n(C]
3
ꢀ1
ꢀ1
ꢀ1
(
1H, s, H-41), 6.46 (1H, dd, JH18-H19 ¼ 15.9 Hz, H-18), 6.30 (1H, d,
,
d
d
(NeH), 1554 cm
n(C]C)triazole,
3
3
ꢀ1
ꢀ1
ꢀ1
J
H17-H18 ¼ 10.5 Hz, H-17), 6.25 (1H, d, H-29), 6.08 (1H, dd,
J
H19-
3
ꢀ1
H20 ¼ 7.4 Hz, H-19), 5.08 (1H, d,
J
H25-H26 ¼ 10.9 Hz, H-25), 5.01
n(C]C), 1246 and 1235 cm
n(CeO); elemental analysis
3
(
1H, vbs, HO-21), 4.91 (1H, dd, JH28-H29 ¼ 12.8 Hz, H-28), 4.53 (2H,
C
47
H
65
N
5
O
12: calculated: C ¼ 63.28%; H ¼ 7.34%; N ¼ 7.85%;

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
671
O ¼ 21.52%; measured: C ¼ 63,42%; H ¼ 7.27%; N ¼ 7.97%;
22.6 (C-46), 22.1 (C-13), 20.6 (C-36),19.9 (C-30), 18.2 (C-31), 13.8 (C-
47), 11.1 (C-32), 8.9 (C-34), 8.8 (C-33), 7.3 (C-14); FT-IR (KBr pellet):
O ¼ 21.34%.
ꢁ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
1
8 e yield: 67%; HPLC: RT ¼ 3.83 [min]; 25 C; 50:50
3452 cm
þ 3412 cm
n
(O21eH) þ
n(O23eH), 3238 cm
þ
ꢀ1
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 878.4547;
d
(NeH)amide
,
3144 cm
n
(O
4
eH$$$O11),
2742
cm
1
þ
ꢀ
ꢀ1
H NMR (
d
ppm in DMSO-d
6
): 16.01 (1H, bs, HO-1),12.81 (1H, s, HO-
n
(N38eH$$$O15), 2475 cm
n
(O
1
eH$$$O
8
), 1721 cm
n
(C35]O),
þ
ꢀ1
ꢀ1
4
), 9.32 (1H, s, NHamide), 8.56 (1H, bs, N H-38hydrogen bonded), 8.33
1H, bs, N H-38non-hydrogen bonded), 7.77 (1H, s, H-42), 6.47 (1H, dd,
1646 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
(C
8
]O), 1626 cm
n(C]
þ
ꢀ1
ꢀ1
(
C)naphthalene
,
1587 cm
d
(NeH), 1556 cm
n(C]C)triazole,
3
3
ꢀ1
ꢀ1
ꢀ1
ꢀ1
J
H18-H19 ¼ 15.9 Hz, H-18), 6.32 (1H, d,
J
H17-H18 ¼ 10.3 Hz, H-17),
1535 cm
d
(NeH)amide II, 1477 cm þ 1458 cm and 1439 cm
3
ꢀ1
6
.25 (1H, d, H-29), 6.08 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.08 (1H,
n
(C]C), 1246 and 1238 cm
n(CeO); elemental analysis
3
d,
J
H25-H26 ¼ 10.9 Hz, H-25), 5.01 (1H, vbs, HO-21), 4.91 (1H, dd,
C
47
H
65
N
5
O
12: calculated: C ¼ 63.28%; H ¼ 7.34%; N ¼ 7.85%;
3
3
J
H28-H29 ¼ 12.8 Hz, H-28), 4.40 (2H, t, JH40-H41 ¼ 6.8 Hz, H-41), 4.28
O ¼ 21.52%; measured: C ¼ 63.24%; H ¼ 7.39%; N ¼ 7.76%;
(
1H, m, H-38), 3.99 (1H, bs, HO-23), 3.67 (1H, m, H-38), 3.62 (1H, m,
O ¼ 21.61%.
3
ꢁ
H-21), 3.23 (1H, d, JH27-H28 ¼ 8.5 Hz, H-27), 3.04 (2H, m, H-39), 2.89
20 e yield: 63%; HPLC: RT ¼ 6.21 [min]; 25 C; 50:50
3
þ
(
3H, s, H-37), 2.82 (1H, d,
J
H23-H22 ¼ 10.4 Hz, H-23), 2.54 (2H, t,
(H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH]
3
1
J
H39-H40 ¼ 7.5 Hz, H-44), 2.27 (2H, m, H-40), 2.13 (1H, m, H-20),1.99
3H, s, H-36), 1.98 (3H, s, H-30), 1.92 (3H, s, H-14), 1.67 (1H, m, H-
2), 1.66 (2H, m, H-45), 1.65 (3H, s, H-13), 1.25 (1H, m, H-24), 0.98
906.4857; H NMR (
(1H, s, HO-4), 9.33 (1H, s, NHamide), 8.56 (1H, bs, N H-38hydrogen
bonded), 8.34 (1H, bs, N H-38non-hydrogen bonded), 7.78 (1H, s, H-42),
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.81
þ
(
2
þ
3
3
3
(
1H, m, H-26), 0.91 (3H, d,
J
H22-H32 ¼ 7.0 Hz, H-32), 0.89 (2H, t,
6.46 (1H, dd,
J
H18-H19 ¼ 15.9 Hz, H-18), 6.30 (1H, d,
J
H17-
3
3
H18 ¼ 10.5 Hz, H-17), 6.24 (1H, d, H-29), 6.08 (1H, dd, 3JH19-
J
H45-H46 ¼ 7.0 Hz, H-46), 0.85 (3H, d, JH20-H31 ¼ 7.0 Hz, H-31), 0.50
3
3
3
(
3
(
4
1
(
7
(
3H, d, JH24-H33 ¼ 6.8 Hz, H-33), ꢀ0.34 (3H, d, JH34-H26 ¼ 6.7 Hz, H-
4); 13C NMR (
ppm in DMSO-d ): 185.3 (C-11), 184.1 (C-8), 171.8
C-6), 170.3 (C-15), 169.4 (C-35), 148.9 (C-1), 146.7 (C-43), 145.0 (C-
), 143.1 (C-29), 139.1 (C-19), 131.5 (C-16), 131.4 (C-17), 126.2 (C-18),
21.8 (C-42), 118.0 (C-2), 117.7 (C-28), 116.6 (C-10), 115.1 (C-9), 114.6
C-3), 108.8 (C-12), 101.3 (C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-23),
3.1 (C-25), 72.9 (C-21), 55.6 (C-37), 46.2 (C-41), 44.4 (C-39), 43.1
C-38), 40.3 (C-26), 38.1 (C-24), 37.9 (C-20), 32.7 (C-22), 27.0 (C-44),
6.6 (C-40), 22.3 (C-45), 22.1 (C-13), 20.7 (C-36), 19.9 (C-30), 18.2
C-31), 13.6 (C-46), 11.2 (C-32), 9.0 (C-34), 8.8 (C-33), 7.3 (C-14); FT-
H20 ¼ 7.4 Hz, H-19), 5.07 (1H, d,
JH25-H26 ¼ 10.8 Hz, H-25), 5.01
3
d
6
(1H, vbs, HO-21), 4.92 (1H, dd, JH28-H29 ¼ 12.9 Hz, H-28), 4.41 (2H,
3
t, JH40-H41 ¼ 6.8 Hz, H-41), 4.28 (1H, m, H-38), 3.99 (1H, bs, HO-23),
3
3.68 (1H, m, H-38), 3.62 (1H, m, H-21), 3.22 (1H, d,
J
H27-
H28 ¼ 8.5 Hz, H-27), 3.05 (2H, m, H-39), 2.88 (3H, s, H-37), 2.82
3
3
(1H, d, JH23-H22 ¼ 10.4 Hz, H-23), 2.58 (2H, t, JH39-H40 ¼ 7.5 Hz, H-
44), 2.26 (2H, m, H-40), 2.12 (1H, m, H-20), 1.99 (3H, s, H-36), 1.98
(3H, s, H-30),1.91 (3H, s, H-14),1.67 (1H, m, H-22),1.65 (3H, s, H-13),
1.59 (2H, m, H-45), 1.31 (2H, m, H-47), 1.30 (2H, m, H-46), 1.25 (1H,
2
(
3
m, H-24), 0.98 (1H, m, H-26), 0.91 (3H, d, JH22-H32 ¼ 7.0 Hz, H-32),
ꢀ
1
ꢀ1
3
3
IR (KBr pellet): 3449 cm þ 3409 cm
n
(O21eH) þ
n
(O23eH),
0.88 (2H, t, JH45-H46 ¼ 7.0 Hz, H-47), 0.86 (3H, t, JH47-H48 ¼ 6.8 Hz,
3 3
ꢀ
1
ꢀ1
ꢀ1
3
n
241 cm
d
(NeH)amide, 3147 cm
ꢀ1
n
(O
4
eH$$$O11), 2739 cm
H-48), 0.85 (3H, d,
J
H20-H31 ¼ 7.0 Hz, H-31), 0.51 (3H, d,
J
H24-
þ
ꢀ
ꢀ1
3
13
(N38eH$$$O15), 2471 cm
n
(O
1
eH$$$O
8
), 1722 cm
(C
(NeH), 1558 cm
n
(C35]O),
H33 ¼ 6.8 Hz, H-33), ꢀ0.35 (3H, d,
J
H34-H26 ¼ 6.7 Hz, H-34);
C
ꢀ1
ꢀ1
1
647 cm
C)naphthalene
536 cm
n
(C15]O)amide I
þ
n
(C11]O) þ
n
8
]O), 1624 cm
n(C]
6
NMR (d ppm in DMSO-d ): 185.4 (C-11), 184.1 (C-8), 171.6 (C-6),
ꢀ
1
ꢀ1
ꢀ1
,
d
1591 cm
d
n
(C]C)triazole
,
170.3 (C-15), 169.4 (C-35), 148.9 (C-1), 146.8 (C-43), 145.0 (C-4),
143.1 (C-29), 139.1 (C-19), 131.5 (C-16), 131.4 (C-17), 126.2 (C-18),
121.6 (C-42), 118.1 (C-2), 117.7 (C-28), 116.5 (C-10), 115.0 (C-9), 114.7
(C-3), 108.8 (C-12), 101.4 (C-7), 98.4 (C-5), 76.4 (C-27), 75.6 (C-23),
73.3 (C-25), 72.8 (C-21), 55.5 (C-37), 46.1 (C-41), 44.4 (C-39), 43.1
(C-38), 40.3 (C-26), 38.1 (C-24), 37.8 (C-20), 32.5 (C-22), 30.7 (C-46),
29.8 (C-45), 27.5 (C-44), 26.6 (C-40), 22.6 (C-47), 22.1 (C-13), 20.7
(C-36), 19.9 (C-30), 18.1 (C-31), 14.0 (C-48), 11.2 (C-32), 9.0 (C-34),
ꢀ1
ꢀ1
ꢀ1
1
n
(NeH)amide II, 1479 cm þ 1460 cm and 1437 cm
ꢀ
1
(C]C), 1247 and 1236 cm
n
(CeO); elemental analysis
C
47
H
65
N
5
O
12: calculated: C ¼ 62.92%; H ¼ 7.23%; N ¼ 7.98%;
O ¼ 21.87%; measured: C ¼ 63.12%; H ¼ 7.04%; N ¼ 8.12%;
O ¼ 21.72%.
ꢁ
1
9 e yield: 58%; HPLC: RT ¼ 4.96 [min]; 25 C; 50:50
þ
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 892.4703;
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
H NMR (
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.81 (1H, s,
8.8 (C-33), 7.3 (C-14); FT-IR (KBr pellet): 3448 cm þ 3411 cm
þ
ꢀ1
HO-4), 9.33 (1H, s, NHamide), 8.55 (1H, bs, N H-38hydrogen bonded),
n
n
n
(O21eH)
þ
n
(O23eH), 3236 cm
d(NeH)amide, 3142 cm
þ
ꢀ1
þ
8
.34 (1H, bs, N H-38non-hydrogen bonded), 7.76 (1H, s, H-42), 6.48 (1H,
(O
(O
þ
4
eH$$$O11), 2741 cm
n
(N38eH$$$O15), 2473 cm
3
3
ꢀ
ꢀ1
ꢀ1
dd, JH18-H19 ¼ 15.8 Hz, H-18), 6.31 (1H, d, JH17-H18 ¼ 10.4 Hz, H-17),
1
eH$$$O
8
), 1723 cm
(C
n
(C35]O), 1647 cm
n
(C15]O)amide
3
ꢀ1
ꢀ1
6
.25 (1H, d, H-29), 6.08 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.07 (1H,
I
d
n
(C11]O) þ
n
8
]O), 1625 cm
n
(C]C)naphthalene, 1586 cm
ꢀ1
d(NeH)amide II,
3
ꢀ1
n
d,
J
H25-H26 ¼ 10.8 Hz, H-25), 5.01 (1H, vbs, HO-21), 4.92 (1H, dd,
(NeH), 1557 cm
(C]C)triazole, 1536 cm
3
3
ꢀ1
ꢀ1
ꢀ1
n
J
H28-H29 ¼ 12.9 Hz, H-28), 4.43 (2H, t, JH40-H41 ¼ 6.8 Hz, H-41), 4.27
1479 cm
þ 1460 cm
and 1437 cm (C]C), 1247 and
68 5
(CeO); elemental analysis C48H N O12: calculated:
ꢀ1
(
1H, m, H-38), 3.99 (1H, bs, HO-23), 3.68 (1H, m, H-38), 3.63 (1H, m,
1236 cm
n
3
H-21), 3.23 (1H, d, JH27-H28 ¼ 8.5 Hz, H-27), 3.06 (2H, m, H-39), 2.89
C ¼ 63.63%; H ¼ 7.45%; N ¼ 7.73%; O ¼ 21.19%; measured:
3
(
3H, s, H-37), 2.82 (1H, d,
J
H23-H22 ¼ 10.5 Hz, H-23), 2.56 (2H, t,
C ¼ 63.71%; H ¼ 7.37%; N ¼ 7.65%; O ¼ 21.27%.
3
ꢁ
J
H39-H40 ¼ 7.6 Hz, H-44), 2.25 (2H, m, H-40), 2.12 (1H, m, H-20),1.99
3H, s, H-36), 1.98 (3H, s, H-30), 1.91 (3H, s, H-14), 1.77 (2H, m, H-
5), 1.67 (1H, m, H-22), 1.65 (3H, s, H-13), 1.42 (2H, m, H-46), 1.25
21 e yield: 69%; HPLC: RT ¼ 7.23 [min]; 25 C; 50:50
þ
(
4
(H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
1
967.4081; H NMR (
d
ppm in DMSO-d
6
): 16.01 (1H, bs, HO-1), 12.81
3
þ
(
3
1H, m, H-24), 0.99 (1H, m, H-26), 0.92 (3H, d, JH22-H32 ¼ 7.0 Hz, H-
(1H, s, HO-4), 9.19 (1H, s, NHamide), 9.07 (1H, vbs, N H-38hydrogen
2), 0.88 (2H, t, 3
J
H45-H46 ¼ 7.0 Hz, H-47), 0.85 (3H, d,
3
J
bonded), 8.61 (1H, vbs, N H-38non-hydrogen bonded), 8.30 (1H, s, H-41),
þ
H20-
3
H31 ¼ 7.0 Hz, H-31), 0.50 (3H, d,
J
H24-H33 ¼ 6.8 Hz, H-33), ꢀ0.33
7.99 (2H, m, H-45 þ H-48), 7.93 (2H, m, H-46 þ H-47), 6.32 (1H, dd,
3H, d, 3
85.4 (C-11), 184.2 (C-8), 171.7 (C-6), 170.3 (C-15), 169.4 (C-35),
48.8 (C-1), 146.9 (C-43), 145.0 (C-4), 143.1 (C-29), 139.0 (C-19),
31.5 (C-16), 131.4 (C-17), 126.3 (C-18), 121.6 (C-42), 118.0 (C-2),
17.7 (C-28), 116.5 (C-10), 115.1 (C-9), 114.7 (C-3), 108.8 (C-12), 101.4
C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25), 72.8 (C-21),
5.6 (C-37), 46.1 (C-41), 44.5 (C-39), 43.2 (C-38), 40.3 (C-26), 38.0
C-24), 37.9 (C-20), 32.6 (C-22), 29.1 (C-45), 27.5 (C-44), 26.7 (C-40),
JH34-H26 ¼ 6.7 Hz, H-34); C NMR (
13
d
ppm in DMSO-d
):
3
J
H18-H19 ¼ 16.0 Hz, H-18), 6.26 (1H, d, H-29), 6.15 (2H, s, H-42), 6.04
(
1
1
1
1
(
5
(
6
3
3
(1H, d, JH17-H18 ¼ 10.9 Hz, H-17), 5.91 (1H, dd, JH19-H20 ¼ 7.6 Hz, H-
3 3
19), 5.05 (1H, d,
J
H25-H26 ¼ 10.9 Hz, H-25), 5.00 (1H, d,
J
HO-
3
H21 ¼ 3.4 Hz, HO-21), 4.89 (1H, dd, JH28-H29 ¼ 12.5 Hz, H-28), 4.33
3
(2H, m, H-39), 4.20 (1H, m, H-38), 3.89 (1H, d, JHO-H21 ¼8.1 Hz, HO-
3
23), 3.65 (1H, m, H-38), 3.54 (1H, d, JH21-H22 ¼ 9.1 Hz, H-21), 3.22
3
3
(1H, d, JH27-H28 ¼ 8.2 Hz, H-27), 2.86 (3H, s, H-37), 2.73 (1H, t, JH22-
3
H23 ¼ 9.9 Hz, JH23-OH ¼ 9.9 Hz, H-23), 2.17 (1H, m, H-20), 1.99 (3H, s,

672
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
H-36), 1.96 (3H, s, H-30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.50
7.88 (2H, m, H-47 þ H-50), 7.85 (2H, m, H-48 þ H-49), 6.40 (1H, dd,
3
3
(
1H, m, H-22), 1.16 (1H, m, H-24), 1.04 (1H, m, H-26), 0.85 (3H, d,
J
H18-H19 ¼ 16.1 Hz, H-18), 6.25 (1H, d,
JH17-H18 ¼ 10.8 Hz, H-17),
3
3
3
J
H22-H32 ¼ 7.1 Hz, H-32), 0.79 (3H, d, JH20-H31 ¼ 7.1 Hz, H-31), 0.38
6.24 (1H, d, H-29), 6.00 (1H, dd, JH19-H20 ¼ 7.4 Hz, H-19), 5.06 (1H,
3
3
3
3
(
3H, d, JH24-H33 ¼ 6.8 Hz, H-33), ꢀ0.40 (3H, d, JH34-H26 ¼ 6.8 Hz, H-
d, JH25-H26 ¼ 10.8 Hz, H-25), 5.03 (1H, d, JHO-H21 ¼ 3.1 Hz, HO-21),
3 3
3
(
1
4
4
(
7
(
4); 13C NMR (
C-6), 170.1 (C-15), 169.3 (C-35), 166.5 (C-43 þ C-50), 148.7 (C-1),
45.0 (C-4), 143.1 (C-29),138.9 (C-19), 138.5 (C-40), 135.2 (C-46 þ C-
7), 131.9 (C-17), 131.1 (C-16 þ C-44 þ C-49), 126.1 (C-18), 125.9 (C-
1), 123.8 (C-45 þ C-48), 117.9 (C-2), 117.7 (C-28), 116.5 (C-10), 115.1
C-9), 114.8 (C-3), 108.8 (C-12), 101.3 (C-7), 98.6 (C-5), 76.2 (C-27),
5.7 (C-23), 73.1 (C-25), 72.9 (C-21), 67.0 (C-42), 55.6 (C-37), 42.4
C-38), 41.7 (C-39), 40.2 (C-26), 38.1 (C-24), 37.8 (C-20), 32.5 (C-22),
d
ppm in DMSO-d
6
): 185.3 (C-11), 184.1 (C-8), 171.8
4.89 (1H, dd,
J
H28-H29 ¼ 13.0 Hz, H-28), 4.42 (2H, t,
J
H42-
H43 ¼ 7.2 Hz, H-42), 4.31 (2H, m, H-39), 4.22 (1H, m, H-38), 3.93
3
(1H, d, JHO-H21 ¼8.9 Hz, HO-23), 3.62 (3H, m, H-21 þ H-38 þ H-44),
3
3.22 (1H, d, JH27-H28 ¼ 8.6 Hz, H-27), 2.88 (3H, s, H-37), 2.79 (1H, t,
3
3
J
H22-H23 ¼ 7.9 Hz, JH23-OH ¼ 7.9 Hz, H-23), 2.21 (1H, m, H-20), 2.14
(2H, m, H-43), 1.98 (3H, s, H-36), 1.92 (3H, s, H-30), 1.91 (3H, s, H-
14), 1.64 (1H, m, H-22), 1.62 (3H, s, H-13), 1.20 (1H, m, H-24), 0.96
3
(1H, m, H-26), 0.88 (3H, d,
J
H22-H32 ¼ 7.0 Hz, H-32), 0.85 (3H, d,
3
3
2
3
2.1 (C-13), 20.7 (C-36), 19.8 (C-30), 17.8 (C-31), 11.0 (C-32), 9.0 (C-
J
H20-H31 ¼7.0 Hz, H-31), 0.44 (3H, d, JH24-H33 ¼ 7.0 Hz, H-33), ꢀ0.38
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
3
13
4), 8.6 (C-33), 7.4 (C-14); FT-IR (CHCl
3
, 0.045 mol L ): 3470 cm
(3H, d,
J
H34-H26 ¼ 7.0 Hz, H-34); C NMR (
6
d ppm in DMSO-d ):
ꢀ
1
n
n
n
n
(O21eH)
(O
(O
sym(C45]O) þ
þ
n
(O23eH), 3375 cm
d(NeH)amide
,
3120 cm
185.3 (C-11),184.2 (C-8),171.8 (C-6),171.0 (C-15),169.4 (C-35),168.0
(C-45 þ C-52), 148.7 (C-1), 145.0 (C-4), 143.1 (C-29), 138.9 (C-19),
137.7 (C-40), 134.4 (C-48 þ C-49), 131.8 (C-46 þ C-51), 131.6 (C-17),
131.2 (C-16),126.1 (C-41),125.7 (C-18),123.0 (C-47 þ C-50),117.9 (C-
2), 117.7 (C-28), 116.5 (C-10), 115.1 (C-9), 114.7 (C-3), 108.8 (C-12),
101.3 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23), 73.1 (C-25), 72.7 (C-
21), 55.6 (C-37), 47.3 (C-42), 44.5 (C-44), 42.3 (C-38), 41.7 (C-39),
40.2 (C-26), 38.2 (C-24), 37.8 (C-20), 34.8 (C-43), 32.6 (C-22), 22.0
(C-13), 20.6 (C-36), 19.8 (C-30), 17.9 (C-31), 11.1 (C-32), 9.0 (C-34),
ꢀ
1
þ
4
eH$$$O11), 2815 cm
n
(N38eH$$$O15), 2496 cm
ꢀ
ꢀ1
n
1
eH$$$O
8
), 1771 cm
sym(C52]O) þ
(C11]O) þ (C ]O),1556 cm
assym(C45]O) þ (C52]O), 1713 cm
ꢀ
1
n
n
(C35]O), 1644 cm
n
(C15
]
ꢀ1
ꢀ
1
O)amid
þ
n
n
8
n
(C]C), 1536 cm
ꢀ1
ꢀ1
d(NeH)amid II,1500 cm
n
(C]C),1455 and 1439 cm
n
(C]C),1248
14: calcu-
ꢀ
1
and 1237 cm
n
(CeO); elemental analysis C50
H
58
N
6
O
lated: C ¼ 62.10%; H ¼ 6.05%; N ¼ 8.69%; O ¼ 23.16%; measured:
C ¼ 62.16%; H ¼ 5.99%; N ¼ 8.75%; O ¼ 22.90%.
ꢁ
þ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
2
2 e yield: 61%; HPLC: RT ¼ 7.86 [min]; 25 C; 50:50
3
8.7 (C-33), 7.4 (C-14); FT-IR (CHCl , 0.045 mol L ): 3467 cm
ꢀ1
d
(
H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH] 981.4241;
n
n
n
n
(O21eH)
þ
n
(O23eH), 3372 cm
(NeH)amide
, 3142 cm
1
ꢀ1
þ
H NMR (
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.80 (1H, s,
(O
(O
4
1
eH$$$O11), 2812 cm
eH$$$O
n(N38eH$$$O15), 2488 cm
þ
ꢀ
ꢀ1
HO-4), 9.23 (1H, s, NHamide), 8.95 (1H, vbs, N H-38hydrogen bonded),
8
8
), 1773 cm n
assym(C45]O) þ (C52]O), 1711 cm
þ
ꢀ1
.58 (1H, vbs, N H-38non-hydrogen bonded), 8.21 (1H, s, H-41), 7.85
sym(C45]O) þ
n
sym(C52]O) þ
(C
n
(C35]O), 1646 cm
n
(C15]
ꢀ1
3
ꢀ1
(
4H, m, H-46 þ H-47 þ H-48 þ H-49), 6.41 (1H, dd,
J
H18-
O)amid
d
þ
n
(C11]O) þ
n
8
]O), 1557 cm
n
(C]C), 1536 cm
3
ꢀ1
ꢀ1
H19 ¼ 16.1 Hz, H-18), 6.29 (1H, d,
J
H17-H18 ¼ 10.8 Hz, H-17), 6.24
(NeH)amid II,1503 cm
n
(C]C),1452 and 1442 cm
n
(C]C),1249
14: calcu-
3
ꢀ1
n
(
1H, d, H-29), 6.03 (1H, dd,
J
H19-H20 ¼ 7.4 Hz, H-19), 5.07 (1H, d,
and 1238 cm
(CeO); elemental analysis C52
H
62
N
6
O
3
3
J
H25-H26 ¼ 10.7 Hz, H-25), 5.01 (1H, m, HO-21), 4.91 (1H, dd, JH28-
lated: C ¼ 62.76%; H ¼ 6.28%; N ¼ 8.45%; O ¼ 22.51%; measured:
3
H29 ¼ 12.6 Hz, H-28), 4.64 (2H, t, JH42-H43 ¼ 6.1 Hz, H-42), 4.27 (2H,
C ¼ 62.69%; H ¼ 6.21%; N ¼ 8.56%; O ¼ 22.54%.
3
ꢁ
m, H-39), 4.24 (1H, m, H-38), 3.99 (2H, m, H-43), 3.93 (1H, d, JHO-
24 e yield: 70%; HPLC: RT ¼ 8.86 [min]; 25 C; 50:50
þ
H21 ¼ 9.0 Hz, HO-23), 3.65 (1H, m, H-38), 3.62 (1H, m, H-21), 3.23
(H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
3
1
(
2
1H, d, JH27-H28 ¼ 8.2 Hz, H-27), 2.88 (3H, s, H-37), 2.80 (1H, m, H-
898.4232; H NMR (
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.80
þ
3), 2.22 (1H, m, H-20), 1.98 (3H, s, H-36), 1.94 (3H, s, H-30), 1.91
(1H, s, HO-4), 9.24 (1H, s, NHamide), 8.96 (1H, vbs, N H-38hydrogen
þ
(
2
(
3
3H, s, H-14), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.21 (1H, m, H-
bonded), 8.66 (1H, vbs, N H-38non-hydrogen bonded), 8.19 (1H, s, H-41),
3
4), 0.97 (1H, m, H-26), 0.89 (3H, d, JH22-H32 ¼ 7.0 Hz, H-32), 0.81
7.37 (2H, m, H-45 þ H-47), 7.34 (1H, m, H-46), 7.31 (2H, m, H-
3
3
3
3H, d, JH20-H31 ¼ 7.4 Hz, H-31), 0.44 (3H, d, JH24-H33 ¼ 7.4 Hz, H-
44 þ H-48), 6.48 (1H, dd,
J
H18-H19 ¼ 16.2 Hz, H-18), 6.30 (1H, d,
3
13
3
3
3), ꢀ0.36 (3H, d,
J
H34-H26 ¼ 7.0 Hz, H-34); C NMR (
d
ppm in
J
H17-H18 ¼ 11.3 Hz, H-17), 6.25 (1H, d, H-29), 6.06 (1H, dd,
J
H19-
DMSO-d
6
): 185.3 (C-11), 184.1 (C-8), 171.8 (C-6), 170.0 (C-15), 169.3
H20 ¼ 7.5 Hz, H-19), 5.61 (2H, s, H-42), 5.07 (1H, d, 3JH25-
3
(
1
C-35), 167.4 (C-44 þ C-51), 148.7 (C-1), 145.0 (C-4), 142.9 (C-29),
H26 ¼ 11.1 Hz, H-25), 5.01 (1H, d,
J
HO-H21 ¼ 3.9 Hz, HO-21), 4.91
3
38.9 (C-19), 137.8 (C-40), 134.4 (C-47 þ C-48), 131.6 (C-17), 131.4
(1H, dd, JH28-H29 ¼ 12.9 Hz, H-28), 4.34 (1H, m, H-39), 4.28 (1H, m,
3
(
4
C-45 þ C-50), 131.1 (C-16), 126.1 (C-18), 125.8 (C-41), 123.1 (C-
H-39), 4.25 (1H, m, H-38), 3.94 (1H, d,
J
HO-H21 ¼ 8.4 Hz, HO-23),
3
6 þ C-49), 117.9 (C-2), 117.7 (C-28), 116.5 (C-10), 115.1 (C-9), 114.6
3.72 (1H, m, H-38), 3.63 (1H, d,
J
H21-H22 ¼ 8.8 Hz, H-21), 3.23
3
3
(
7
(
C-3), 108.7 (C-12), 101.2 (C-7), 98.5 (C-5), 76.2 (C-27), 75.7 (C-23),
3.2 (C-25), 72.8 (C-21), 55.6 (C-37), 47.4 (C-42), 42.2 (C-38), 41.5
C-39), 40.1 (C-26), 38.1 (C-24), 37.9 (C-20), 37.7 (C-43), 32.6 (C-22),
(1H, d, JH27-H28 ¼ 8.4 Hz, H-27), 2.89 (3H, s, H-37), 2.80 (1H, t, JH22-
3
H23 ¼ 8.3 Hz, JH23-OH ¼ 8.3 Hz, H-23), 2.25 (1H, m, H-20),1.98 (3H, s,
H-36), 1.94 (3H, s, H-30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.63
(1H, m, H-22), 1.22 (1H, m, H-24), 0.97 (1H, m, H-26), 0.91 (3H, d,
2
3
2.0 (C-13), 20.6 (C-36), 19.8 (C-30), 17.8 (C-31), 11.1 (C-32), 8.9 (C-
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
3
3
4), 8.6 (C-33), 7.3 (C-14); FT-IR (CHCl
3
, 0.045 mol L ): 3474 cm
J
H22-H32 ¼ 7.0 Hz, H-32), 0.87 (3H, d, JH20-H31 ¼ 6.8 Hz, H-31), 0.40
ꢀ
1
3
3
n
n
n
n
(O21eH)
(O
(O
sym(C45]O) þ
þ
n
(O23eH), 3369 cm
d
(NeH)amide
(N38eH$$$O15), 2491 cm
assym(C45]O) þ (C52]O), 1712 cm
,
3131 cm
(3H, d, JH24-H33 ¼ 6.8 Hz, H-33), ꢀ0.36 (3H, d, JH34-H26 ¼ 6.8 Hz, H-
ꢀ1
þ
34); 13C NMR (
4
eH$$$O11), 2807 cm
n
d ppm in DMSO-d ): 185.2 (C-11), 184.1 (C-8), 171.8
6
ꢀ
ꢀ1
n
1
eH$$$O
8
), 1772 cm
(C-6), 170.2 (C-15), 169.3 (C-35), 148.7 (C-1), 145.0 (C-4), 143.0 (C-
29), 138.9 (C-19), 138.3 (C-40), 135.7 (C-43), 131.5 (C-17), 131.2 (C-
16), 128.7 (C-45 þ C-47), 128.1 (C-46), 127.7 (C-44 þ C-48), 126.1 (C-
18), 125.5 (C-41), 117.9 (C-2), 117.7 (C-28), 116.6 (C-10), 115.0 (C-9),
114.6 (C-3), 108.8 (C-12), 101.2 (C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-
23), 73.2 (C-25), 72.9 (C-21), 55.6 (C-37), 52.8 (C-42), 42.5 (C-38),
41.7 (C-39), 40.2 (C-26), 38.1 (C-24), 37.9 (C-20), 32.6 (C-22), 22.0
(C-13), 20.6 (C-36), 19.8 (C-30), 17.9 (C-31), 11.1 (C-32), 8.9 (C-34),
ꢀ1
n
sym(C52]O) þ
n
(C35]O), 1645 cm
n
(C15
]
ꢀ1
ꢀ
1
O)amid
þ
n
(C11]O) þ
n
(C
8
]O),1556 cm
n
(C]C), 1536 cm
ꢀ1
ꢀ1
d(NeH)amid II,1502 cm
n
(C]C), 1452 and 1442 cm
n
(C]C), 1249
14: calcu-
ꢀ
1
and 1238 cm
n
(CeO); elemental analysis C51
60
H N
6
O
lated: C ¼ 62.44%; H ¼ 6.16%; N ¼ 8.57%; O ¼ 22.83%; measured:
C ¼ 62.37%; H ¼ 6.19%; N ¼ 8.64%; O ¼ 22.80%.
ꢁ
2
3 e yield: 67%; HPLC: RT ¼ 8.10 [min]; 25 C; 50:50
þ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(
9
H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
8.6 (C-33), 7.3 (C-14); FT-IR (CHCl , 0.045 mol L ): 3474 cm
3
1
ꢀ1
d
95.4395; H NMR (
d
ppm in DMSO-d
6
): 16.02 (1H, bs, HO-1), 12.81
n
n
n
(O21eH)
þ
n
(O23eH), 3368 cm
(NeH)amide, 3151 cm
(N38eH$$$O15), 2493 cm
(C35]O), 1644 cm
þ
ꢀ1
þ
(
1H, s, HO-4), 9.24 (1H, s, NHamide), 9.00 (1H, vbs, N H-38hydrogen
(O
(O
4
1
eH$$$O11), 2809 cm
eH$$$O
n
þ
ꢀ
ꢀ1
ꢀ1
bonded), 8.67 (1H, vbs, N H-38non-hydrogen bonded), 8.19 (1H, s, H-41),
8
), 1712 cm
n
n
(C15
]

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
673
ꢀ
1
ꢀ1
O)amid
þ
n
(C11]O) þ
n
(C
8
]O),1555 cm
n
(C]C), 1536 cm
27.4 (C-43), 22.5 (C-45), 21.5 (C-13), 20.8 (C-36), 20.0 (C-30),17.9 (C-
31), 13.9 (C-46), 12.7 (C-32), 8.5 (C-33), 8.1 (C-34), 7.0 (C-14); FT-IR
ꢀ
1
ꢀ1
d(NeH)amid II, 1501 cm
n
(C]C), 1453 and 1441 cm
(CeO); elemental analysis C48
n
(C]C), 1245
12: calcu-
ꢀ
1
ꢀ1
ꢀ1
d
and 1237 cm
n
H
59
N
5
O
(KBr pellet): 3433 cm
n
(O21eH) þ
n
(O23eH), 3263 cm
(NeH),
ꢀ1
ꢀ1
ꢀ
lated: C ¼ 64.20%; H ¼ 6.62%; N ¼ 7.80%; O ¼ 21.38%; measured:
3135 cm
n
(O
4
eH$$$O11),
2481
cm
n
(O
1
eH$$$O
8
),
ꢀ1
ꢀ1
ꢀ1
n
C ¼ 64.24%; H ¼ 6.59%; N ¼ 7.86%; O ¼ 21.31%.
1725 cm þ1713 cm
n(C35]O), 1642 cm
(C11]O) þ
n
(C
8
]O),
ꢁ
ꢀ1
ꢀ1
ꢀ1
2
5 e yield: 58%; HPLC: RT ¼ 4.89 [min]; 25 C; 50:50
1605 cm
n
(C]C)quinazoline, 1557 cm
n(C]C)triazole, 1532 cm
þ
ꢀ1
ꢀ1
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH]
n
(C]C), 1480 cm þ 1448 cm
n(C]C), 1246 and 1234 cm
1
8
46.4284; H NMR (
d
ppm in DMSO-d
6
): 16.56 (1H, bs, HO-1), 12.37
n
(CeO); elemental analysis C46
61
H N
5
O
11: calculated: C ¼ 64.24%;
þ
(
1H, s, HO-4), 11.60 (1H, bs, N(2) -H), 7.81 (1H, s, H-42), 6.33 (1H, d,
H ¼ 7.15%; N ¼ 8.14%; O ¼ 20.46%; measured: C ¼ 64.32%;
3
3
J
H17-H18 ¼ 11.1 Hz, H-17), 6.12 (1H, d, H-29), 6.00 (1H, dd,
J
H19-
H ¼ 7.07%; N ¼ 8.20%; O ¼ 20.41%.
3
ꢁ
H20 ¼ 7.1 Hz, H-19), 5.70 (1H, dd,
J
H18-H19 ¼ 15.6 Hz, H-18), 5.13
27 e yield: 61%; HPLC: RT ¼ 8.32 [min]; 25 C; 50:50
3
3
þ
(
1H, d, JH25-H26 ¼ 10.6 Hz, H-25), 4.95 (1H, dd, JH28-H29 ¼ 12.7 Hz,
(H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 874.4597;
3
1
J
H28-H27 ¼ 6.6 Hz, H-28), 4.77 (1H, bs, HO-23), 4.69 (2H, m, H-38),
H NMR (
d
ppm in DMSO-d
6
): 16.50 (1H, bs, HO-1), 12.34 (1H, s,
3
þ
3
4
3
.45 (3H, bs þ t, HO-21 þ H-40), 4.06 (2H, t, JH39-H40 ¼ 6.9 Hz, H-
HO-4), 11.69 (1H, bs, N(2) -H), 7.81 (1H, s, H-41), 6.33 (1H, d, JH17-
3
3
3
9), 3.65 (1H, dd, JH21-H22 ¼ 7.5 Hz, JH21-H20 ¼ 3.8 Hz, H-21), 3.32
H18 ¼ 11.1 Hz, H-17), 6.12 (1H, d, H-29), 5.98 (1H, dd,
J
H19-
3
(
1H, m, H-27), 2.95 (3H, s, H-37), 2.93 (1H, m, H-23), 2.54 (2H, t,
H20 ¼ 7.0 Hz, H-19), 5.72 (1H, dd,
JH18-H19 ¼ 15.7 Hz, H-18), 5.13
3
3
3
J
H43-H44 ¼ 7.6 Hz, H-43), 2.11 (1H, m, H-20), 2.01 (3H, s, H-36), 1.96
(1H, d, JH25-H26 ¼ 10.7 Hz, H-25), 4.95 (1H, dd, JH28-H29 ¼ 12.7 Hz,
3
(
(
2
3H, s, H-30), 1.90 (3H, s, H-14), 1.64 (5H, s þ m, H-13 þ H-44), 1.52
J
H28-H27 ¼ 6.6 Hz, H-28), 4.74 (1H, bs, HO-23), 4.68 (2H, m, H-38),
3
3
3
1H, m, H-22), 1.47 (1H, dt, JH23-H24 ¼ 14.0 Hz, JH24-H33 ¼ 7.1 Hz, H-
4.48 (2H, t, H-40), 4.45 (1H, bs, HO-21), 4.09 (2H, t,
J
H39-
J
H21-
3
3
3
3
4), 1.13 (1H, ddd, JH26-H34 ¼ 6.7 Hz, JH26-H27 ¼ 1.9 Hz, H-26), 0.86
H40 ¼ 6.8 Hz, H-39), 3.66 (1H, dd,
J
H21-H22 ¼ 7.4 Hz,
3
3
(
3
2H, t, JH44-H45 ¼ 7.0 Hz, H-45), 0.85 (3H, d, JH22-H32 ¼ 6.8 Hz, H-
H20 ¼ 3.8 Hz, H-21), 3.32 (1H, m, H-27), 2.95 (3H, s, H-37), 2.93
2), 0.72 (3H, d, 3
J
H20-H31 ¼ 6.8 Hz, H-31), 0.51 (3H, d,
3
J
(1H, m, H-23), 2.55 (2H, t, JH43-H44 ¼ 7.6 Hz, H-43), 2.11 (1H, m, H-
3
H24-
ppm in
13
H33 ¼ 6.9 Hz, H-33), ꢀ0.15 (3H, d, H-34); C NMR (
DMSO-d ): 186.3 (C-11), 184.0 (C-8), 171.9 (C-6), 170.1 (C-35), 157.3
C-15), 147.1 (C-42), 142.9 (C-1), 142.6 (C-19), 142.1 (C-29), 141.0 (C-
), 134.5 (C-17), 123.0 (C-16), 122.4 (C-18), 121.6 (C-41), 118.2 (C-2),
16.4 (C-28), 113.3 (C-10), 111.8 (C-9), 108.2 (C-12), 105.1 (C-2), 100.6
C-7), 98.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.0 (C-25), 70.9 (C-21),
6.2 (C-37), 49.2 (C-40), 47.4 (C-39), 44.6 (C-38), 40.0 (C-26), 39.5
C-20), 37.4 (C-24), 33.9 (C-22), 27.1 (C-43), 22.3 (C-44), 21.5 (C-13),
d
20), 2.01 (3H, s, H-36), 1.96 (3H, s, H-30), 1.90 (3H, s, H-14), 1.63 (3H,
3
6
s, H-13), 1.60 (2H, m, H-44), 1.51 (1H, m, H-22), 1.46 (1H, dt, JH23-
3
(
4
1
(
5
(
H24 ¼ 14.1 Hz,
J
H24-H33 ¼ 6.9 Hz, H-24), 1.33 (2H, m, H-46), 1.27
3 3
(2H, m, H-45),1.14 (1H, ddd, JH26-H34 ¼ 6.9 Hz, JH26-H27 ¼ 1.7 Hz, H-
3
3
26), 0.85 (6H, d, JH22-H32 ¼ 6.8 Hz, þ t, JH46-H47 ¼ 6.8 Hz, H-32 þ H-
3 3
47), 0.71 (3H, d,
H33 ¼ 6.9 Hz, H-33), ꢀ0.15 (3H, d, H-34); C NMR (
DMSO-d ): 186.3 (C-11), 183.9 (C-8), 171.8 (C-6), 170.0 (C-35), 157.3
J
H20-H31 ¼ 6.8 Hz, H-31), 0.52 (3H, d,
J
H24-
13
d ppm in
6
2
3
0.7 (C-36), 20.0 (C-30), 17.9 (C-31), 13.4 (C-45), 12.7 (C-32), 8.5 (C-
(C-15), 147.0 (C-42), 142.8 (C-1), 142.6 (C-19), 142.1 (C-29), 141.1 (C-
4), 134.5 (C-17), 122.9 (C-16), 122.5 (C-18), 121.6 (C-41), 118.2 (C-2),
116.4 (C-28), 113.2 (C-10), 111.8 (C-9), 108.1 (C-12), 105.1 (C-2), 100.7
(C-7), 98.5 (C-5), 76.4 (C-27), 75.8 (C-23), 73.0 (C-25), 70.9 (C-21),
56.2 (C-37), 49.2 (C-40), 47.4 (C-39), 44.7 (C-38), 39.9 (C-26), 39.5
(C-20), 37.4 (C-24), 33.9 (C-22), 30.6 (C-46), 30.0 (C-44), 27.3 (C-43),
22.6 (C-46), 21.5 (C-13), 20.8 (C-36), 20.1 (C-30), 17.9 (C-31), 14.1 (C-
47), 12.7 (C-32), 8.4 (C-33), 8.1 (C-34), 7.0 (C-14); FT-IR (KBr pellet):
ꢀ
1
3), 8.1 (C-34), 7.0 (C-14); FT-IR (KBr pellet): 3434 cm
ꢀ
1
ꢀ1
ꢀ1
n
n
n
(O21eH)
(O eH$$$O11), 2479 cm
(C35]O), 1643 cm
þ
n
(O23eH), 3265 cm
d
(NeH), 3136 cm
ꢀ1
ꢀ
ꢀ1
4
n
(O
1
eH$$$O
(C
8
), 1726 cm þ 1715 cm
ꢀ
1
ꢀ1
ꢀ1
n
(C11]O) þ
n
8
]O), 1606 cm
n(C]C)qui-
,
1556 cm 1
ꢀ
n
(C]C)triazole
,
1533 cm
ꢀ1
(C]C), 1247 and 1236 cm n
n
(C]C),
nazoline
ꢀ
1
ꢀ1
1
481 cm þ 1450 cm
n
(CeO);
elemental analysis C45
H
59
N
5
O11: calculated: C ¼ 63.89%; H ¼ 7.03%;
ꢀ1
ꢀ1
ꢀ1
ꢀ1
N ¼ 8.28%; O ¼ 20.80%; measured: C ¼ 63.77%; H ¼ 7.13%;
3433 cm
n
(O21eH) þ
n
(O23eH), 3263 cm
d
(NeH), 3138 cm
ꢀ
1
ꢀ
ꢀ1
N ¼ 8.16%; O ¼ 20.94%.
n
(O
4
eH$$$O11), 2477 cm
n(O
1
eH$$$O
(C
8
), 1725 cm þ 1713 cm
ꢁ
ꢀ1
ꢀ1
ꢀ1
ꢀ1
2
6 e yield: 57%; HPLC: RT ¼ 5.59 [min]; 25 C; 50:50
n
(C35]O), 1643 cm
n
(C11]O) þ
n
8
]O), 1606 cm
n(C]C)qui-
þ
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH]
nazoline
,
1556 cm
n
(C]C)triazole
n
,
1533 cm
ꢀ1
(C]C), 1247 and 1236 cm n
n
(C]C),
1
ꢀ1
ꢀ1
8
60.4441; H NMR (
d
ppm in DMSO-d
6
): 16.59 (1H, bs, HO-1), 12.31
1481 cm þ 1450 cm
(CeO);
þ
(
1H, s, HO-4), 11.67 (1H, bs, N(2) -H), 7.82 (1H, s, H-41), 6.97 (1H,
elemental analysis C47
H
63
N
5
O
11: calculated: C ¼ 64.59%; H ¼ 7.27%;
3
3
dd, JH19-H20 ¼ 7.1 Hz, H-19), 6.31 (1H, d, JH17-H18 ¼ 11.0 Hz, H-17),
N ¼ 8.01%; O ¼ 20.14%; measured: C ¼ 64.51%; H ¼ 7.32%;
3
6
.14 (1H, d, H-29), 5.71 (1H, dd, JH18-H19 ¼ 15.5 Hz, H-18), 5.14 (1H,
N ¼ 8.13%; O ¼ 20.04%.
3
3
3
ꢁ
d, JH25-H26 ¼ 10.6 Hz, H-25), 4.96 (1H, dd, JH28-H29 ¼ 12.8 Hz, JH28-
28 e yield: 59%; HPLC: RT ¼ 6.17 [min]; 25 C; 50:50
þ
H27 ¼ 6.6 Hz, H-28), 4.79 (1H, bs, HO-23), 4.70 (2H, m, H-38), 4.47
(H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH]
3
1
(
3
1H, bs, HO-21), 4.42 (2H, t, H-40), 4.03 (2H, t, JH39-H40 ¼ 6.7 Hz, H-
860.4442; H NMR (
(1H, s, HO-4), 11.61 (1H, bs, N(2) -H), 7.84 (1H, s, H-42), 6.31 (1H, d,
d
ppm in DMSO-d
6
): 16.60 (1H, bs, HO-1), 12.34
3
3
þ
9), 3.64 (1H, dd, JH21-H22 ¼ 7.4 Hz, JH21-H20 ¼ 3.9 Hz, H-21), 3.33
3
3
(
1H, m, H-27), 2.95 (3H, s, H-37), 2.93 (1H, m, H-23), 2.55 (2H, t,
J
J
H17-H18 ¼ 11.0 Hz, H-17), 6.13 (1H, d, H-29), 5.99 (1H, dd,
J
H19-
3
3
H43-H44 ¼ 7.6 Hz, H-43), 2.10 (1H, m, H-20), 2.01 (3H, s, H-36), 1.95
H20 ¼ 7.0 Hz, H-19), 5.72 (1H, dd,
JH18-H19 ¼ 15.5 Hz, H-18), 5.13
3
3
(
3H, s, H-30), 1.91 (3H, s, H-14), 1.78 (2H, m, H-44), 1.62 (3H, s, H-
(1H, d, JH25-H26 ¼ 10.7 Hz, H-25), 4.97 (1H, dd, JH28-H29 ¼ 12.7 Hz,
3
3
3
13), 1.50 (1H, m, H-22), 1.45 (3H, dt,
J
H23-H24 ¼ 14.0 Hz,
J
H24-
JH28-H27 ¼ 6.6 Hz, H-28), 4.80 (1H, bs, HO-23), 4.71 (2H, m, H-38),
3
3
H33 ¼ 6.9 Hz þ m, H-24 þ H-45), 1.14 (1H, ddd, JH26-H34 ¼ 6.9 Hz,
4.42 (1H, bs, HO-21), 4.36 (2H, t, JH40-H41 ¼ 7.2 Hz, H-41), 3.67 (1H,
3
3
3
3
J
H26-H27 ¼ 2.1 Hz, H-26), 0.88 (2H, t, JH45-H46 ¼ 7.0 Hz, H-46), 0.86
dd, JH21-H22 ¼ 7.4 Hz, JH21-H20 ¼ 3.9 Hz, H-21), 3.45 (2H, m, H-39),
3
3
(
3
3H, d, JH22-H32 ¼ 6.8 Hz, H-32), 0.73 (3H, d, JH20-H31 ¼ 6.8 Hz, H-
3.33 (1H, m, H-27), 2.95 (3H, s, H-37), 2.93 (1H, m, H-23), 2.55 (2H,
3
13
3
1), 0.53 (3H, d, JH24-H33 ¼ 6.9 Hz, H-33), ꢀ0.14 (3H, d, H-34);
C
t, JH44-H45 ¼ 7.5 Hz, H-44), 2.24 (1H, m, H-40), 2.12 (1H, m, H-20),
NMR (
d
ppm in DMSO-d
6
): 186.3 (C-11), 184.0 (C-8), 171.8 (C-6),
2.04 (1H, m, H-40), 2.01 (3H, s, H-36), 1.97 (3H, s, H-30), 1.91 (3H, s,
H-14), 1.64 (2H, m, H-45), 1.63 (3H, s, H-13), 1.51 (1H, m, H-22), 1.46
1
1
1
(
70.1 (C-35), 157.2 (C-15), 147.2 (C-42), 142.8 (C-1), 142.7 (C-19),
42.0 (C-29), 141.1 (C-4), 134.5 (C-17), 123.0 (C-16), 122.5 (C-18),
21.5 (C-41), 118.2 (C-2), 116.5 (C-28), 113.2 (C-10), 111.8 (C-9), 108.1
C-12), 105.1 (C-2), 100.6 (C-7), 98.4 (C-5), 76.3 (C-27), 75.8 (C-23),
3
3
(1H, dt, JH23-H24 ¼ 14.1 Hz, JH24-H33 ¼ 6.9 Hz, H-24), 1.15 (1H, ddd,
3
3
3
JH26-H34 ¼ 6.9 Hz,
JH26-H27 ¼ 2.0 Hz, H-26), 0.90 (2H, t,
J
H45-
3
H46 ¼ 7.0 Hz, H-46), 0.84 (3H, d,
JH22-H32 ¼ 6.8 Hz, H-32), 0.74
3
3
7
3
3.1 (C-25), 71.0 (C-21), 56.1 (C-37), 49.3 (C-40), 47.5 (C-39), 44.7 (C-
8), 40.1 (C-26), 39.6 (C-20), 37.3 (C-24), 33.9 (C-22), 29.4 (C-44),
(3H, d, JH20-H31 ¼ 6.9 Hz, H-31), 0.53 (3H, d, JH24-H33 ¼ 6.9 Hz, H-
13
33), ꢀ0.15 (3H, d, H-34); C NMR (
6
d ppm in DMSO-d ): 186.3 (C-

674
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
1
1
1
1
(
4
(
2
3
1), 184.0 (C-8), 171.8 (C-6), 170.0 (C-35), 157.1 (C-15), 147.2 (C-43),
42.8 (C-1), 142.6 (C-19), 142.1 (C-29), 141.0 (C-4), 134.5 (C-17),
23.0 (C-16), 122.5 (C-18), 121.5 (C-42), 118.3 (C-2), 116.4 (C-28),
13.3 (C-10), 111.8 (C-9), 108.1 (C-12), 105.1 (C-2), 100.8 (C-7), 98.3
C-5), 76.2 (C-27), 75.8 (C-23), 73.1 (C-25), 70.8 (C-21), 56.2 (C-37),
9.3 (C-39), 46.4 (C-41), 44.6 (C-38), 40.0 (C-26), 39.6 (C-20), 37.4
C-24), 33.8 (C-22), 27.2 (C-40), 27.1 (C-44), 22.4 (C-45), 21.4 (C-13),
2.07 (1H, m, H-40), 2.01 (3H, s, H-36), 1.97 (3H, s, H-30), 1.90 (3H, s,
H-14), 1.63 (3H, s, H-13), 1.57 (2H, m, H-45), 1.51 (1H, m, H-22), 1.45
3
3
(1H, dt, JH23-H24 ¼ 14.0 Hz, JH24-H33 ¼ 6.9 Hz, H-24), 1.30 (2H, m, H-
3 3
47), 1.29 (2H, m, H-46), 1.15 (1H, ddd,
J
H26-H34 ¼ 6.8 Hz,
J
H26-
3
H27 ¼ 1.8 Hz, H-26), 0.87 (3H, t,
J
H47-H48 ¼ 6.8 Hz, H-48), 0.85
3
3
(3H, d, JH22-H32 ¼ 6.8 Hz, H-32), 0.73 (3H, d, JH20-H31 ¼ 6.8 Hz, H-
3
13
31), 0.52 (3H, d, JH24-H33 ¼ 6.9 Hz, H-33), ꢀ0.17 (3H, d, H-34);
C
0.7 (C-36), 20.0 (C-30), 17.9 (C-31), 13.5 (C-46), 12.7 (C-32), 8.4 (C-
6
NMR (d ppm in DMSO-d ): 186.3 (C-11), 183.9 (C-8), 171.9 (C-6),
ꢀ
1
3), 8.1 (C-34), 7.0 (C-14); FT-IR (KBr pellet): 3433 cm
170.0 (C-35), 157.1 (C-15), 147.1 (C-43), 142.7 (C-1), 142.6 (C-19),
142.0 (C-29), 141.1 (C-4), 134.4 (C-17), 122.9 (C-16), 122.5 (C-18),
121.5 (C-42), 118.2 (C-2), 116.4 (C-28), 113.2 (C-10), 111.8 (C-9), 108.2
(C-12), 105.1 (C-2), 100.8 (C-7), 98.3 (C-5), 76.3 (C-27), 75.8 (C-23),
73.0 (C-25), 70.9 (C-21), 56.1 (C-37), 49.2 (C-39), 46.4 (C-41), 44.7
(C-38), 40.0 (C-26), 39.5 (C-20), 37.3 (C-24), 33.8 (C-22), 30.8 (C-46),
28.7 (C-45), 27.2 (C-40), 24.9 (C-44), 21.8 (C-47), 21.5 (C-13), 20.7 (C-
36), 20.1 (C-30), 17.9 (C-31), 13.8 (C-48), 12.7 (C-32), 8.4 (C-33), 8.1
ꢀ
1
ꢀ1
ꢀ1
n
n
n
(O21eH)
(O eH$$$O11), 2481 cm
(C35]O), 1642 cm
þ
n
(O23eH), 3263 cm
d
(NeH), 3135 cm
), 1725 cm þ 1713 cm
ꢀ
1
ꢀ
ꢀ1
4
n
(O
1
eH$$$O
(C
8
ꢀ
1
ꢀ1
n
(C11]O) þ
n
8
]O), 1605 cm
n(C]C)qui-
,
1557 cm 1
ꢀ
n
(C]C)triazole
,
1532 cm
ꢀ1
(C]C), 1245 and 1234 cm n
ꢀ1
n
(C]C),
(CeO);
nazoline
ꢀ
1
ꢀ1
1
480 cm þ 1450 cm
n
elemental analysis C46
H
61
N
5
O
11: calculated: C ¼ 64.24%; H ¼ 7.15%;
N ¼ 8.14%; O ¼ 20.46%; measured: C ¼ 64.13%; H ¼ 7.25%;
ꢀ
ꢀ1
ꢀ1
1
N ¼ 8.07%; O ¼ 20.55%.
(C-34), 7.0
(C-14); FT-IR (KBr pellet):
3431 cm
ꢁ
ꢀ1
2
9 e yield: 64%; HPLC: RT ¼ 7.10 [min]; 25 C; 50:50
n
n
n
(O21eH)
þ
n
(O23eH), 3264 cm
d
(NeH), 3132 cm
þ
ꢀ1
ꢀ
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 874.4595;
(O
4
eH$$$O11), 2475 cm
n
(O
1
eH$$$O
(C
8
), 1727 cm þ 1713 cm
1
ꢀ1
ꢀ1
ꢀ1
H NMR (
d
ppm in DMSO-d
6
): 16.55 (1H, bs, HO-1), 12.39 (1H, s,
(C35]O), 1641 cm
n
(C11]O) þ
n
8
]O), 1606 cm
n(C]C)qui-
þ
3
ꢀ1
ꢀ1
HO-4), 11.63 (1H, bs, N(2) eH), 7.83 (1H, s, H-42), 6.33 (1H, d, JH17-
nazoline
,
1556 cm
n
(C]C)triazole
,
1535 cm
n
(C]C),
(CeO);
3
ꢀ1
ꢀ1
H18 ¼ 11.1 Hz, H-17), 6.14 (1H, d, H-29), 6.00 (1H, dd,
J
H19-
1482 cm þ 1449 cm
n
(C]C), 1246 and 1235 cm
n
3
H20 ¼ 7.1 Hz, H-19), 5.70 (1H, dd,
J
H18-H19 ¼ 15.6 Hz, H-18), 5.15
elemental analysis C48
H
65
N
5
O
11: calculated: C ¼ 64.92%; H ¼ 7.38%;
3
3
(
1H, d, JH25-H26 ¼ 10.6 Hz, H-25), 4.97 (1H, dd, JH28-H29 ¼ 12.8 Hz,
N ¼ 7.89%; O ¼ 19.82%; measured: C ¼ 64.83%; H ¼ 7.45%;
3
J
H28-H27 ¼ 6.6 Hz, H-28), 4.76 (1H, bs, HO-23), 4.71 (2H, m, H-38),
N ¼ 7.77%; O ¼ 19.95%.
3
ꢁ
4
.44 (1H, bs, HO-21), 4.34 (2H, t, JH40-H41 ¼ 7.1 Hz, H-41), 3.66 (1H,
31 e yield: 65%; HPLC: RT ¼ 9.05 [min]; 25 C; 50:50
3
3
þ
dd, JH21-H22 ¼ 7.3 Hz, JH21-H20 ¼ 3.8 Hz, H-21), 3.46 (2H, m, H-39),
(H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
1
3
.33 (1H, m, H-27), 2.95 (3H, s, H-37), 2.93 (1H, m, H-23), 2.57 (2H,
949.3979; H NMR (
d
ppm in DMSO-d
6
): 16.61 (1H, bs, HO-1), 12.37
3
þ
t, JH44-H45 ¼ 7.5 Hz, H-44), 2.23 (1H, m, H-40), 2.11 (1H, m, H-20),
.06 (1H, m, H-40), 2.01 (3H, s, H-36), 1.97 (3H, s, H-30), 1.90 (3H, s,
H-14), 1.78 (2H, m, H-45), 1.63 (3H, s, H-13), 1.52 (1H, m, H-22), 1.46
(1H, s, HO-4), 11.64 (1H, bs, N(2) eH), 8.34 (1H, s, H-41), 8.01 (2H,
3
2
m, H-45 þ H-48), 7.96 (2H, m, H-46 þ H-47), 6.33 (1H, d,
J
H17-
H18 ¼ 11.0 Hz, H-17), 6.12 (1H, d, H-29), 6.06 (2H, s, H-42), 5.99
3
3
3
3
(
4
1H, dt, JH23-H24 ¼ 14.1 Hz, JH24-H33 ¼ 7.0 Hz, H-24), 1.41 (2H, m, H-
(1H, dd, JH19-H20 ¼ 7.1 Hz, H-19), 5.80 (1H, dd, JH18-H19 ¼ 15.6 Hz,
3 3
3
3
6), 1.14 (1H, ddd, JH26-H34 ¼ 6.8 Hz, JH26-H27 ¼ 1.8 Hz, H-26), 0.89
H-18), 5.14 (1H, d,
H29 ¼ 12.7 Hz,
J
H25-H26 ¼ 10.7 Hz, H-25), 4.98 (1H, dd,
H28-H27 ¼ 6.7 Hz, H-28), 4.83 (2H, m, H-39), 4.79
(1H, bs, HO-23), 4.72 (2H, m, H-38), 4.42 (1H, bs, HO-21), 3.68 (1H,
J
H28-
3
3
3
(
3
2H, t, JH46-H47 ¼ 7.1 Hz, H-47), 0.85 (3H, d, JH22-H32 ¼ 6.8 Hz, H-
J
2), 0.72 (3H, d, 3
J
H20-H31 ¼ 6.8 Hz, H-31), 0.51 (3H, d,
3
J
H24-
13
3
3
H33 ¼ 6.9 Hz, H-33), ꢀ0.18 (3H, d, H-34); C NMR (
DMSO-d ): 186.4 (C-11), 183.9 (C-8), 171.8 (C-6), 170.0 (C-35), 157.2
C-15), 147.1 (C-43), 142.7 (C-1), 142.6 (C-19), 142.0 (C-29), 141.0 (C-
), 134.5 (C-17), 122.9 (C-16), 122.4 (C-18), 121.6 (C-42), 118.3 (C-2),
16.5 (C-28),113.2 (C-10),111.9 (C-9),108.2 (C-12),105.2 (C-2), 100.7
C-7), 98.4 (C-5), 76.3 (C-27), 75.8 (C-23), 73.0 (C-25), 71.0 (C-21),
6.1 (C-37), 49.3 (C-39), 46.5 (C-41), 44.7 (C-38), 40.0 (C-26), 39.6
C-20), 37.3 (C-24), 33.9 (C-22), 29.2 (C-45), 27.5 (C-44), 27.1 (C-40),
2.7 (C-46), 21.5 (C-13), 20.7 (C-36), 20.1 (C-30), 17.9 (C-31), 13.7 (C-
d
ppm in
dd, JH21-H22 ¼ 7.5 Hz, JH21-H20 ¼ 4.0 Hz, H-21), 3.33 (1H, m, H-27),
2.95 (3H, s, H-37), 2.94 (1H, m, H-23), 2.10 (1H, m, H-20), 2.01 (3H, s,
H-36), 1.98 (3H, s, H-30), 1.91 (3H, s, H-14), 1.63 (3H, s, H-13), 1.51
6
(
4
1
(
5
(
2
3
3
(1H, m, H-22), 1.44 (1H, dt, JH23-H24 ¼ 14.1 Hz, JH24-H33 ¼ 6.9 Hz, H-
3 3
24), 1.15 (1H, ddd, JH26-H34 ¼ 6.8 Hz, JH26-H27 ¼ 1.9 Hz, H-26), 0.84
3 3
(3H, d, JH22-H32 ¼ 6.9 Hz, H-32), 0.72 (3H, d, JH20-H31 ¼ 6.9 Hz, H-
3
13
31), 0.51 (3H, d, JH24-H33 ¼ 7.0 Hz, H-33), ꢀ0.16 (3H, d, H-34);
C
6
NMR (d ppm in DMSO-d ): 186.3 (C-11), 184.0 (C-8), 171.8 (C-6),
170.0 (C-35), 166.7 (C-43 þ C-50), 157.4 (C-15), 142.7 (C-1 þ C-19),
142.1 (C-29), 141.0 (C-4), 139.7 (C-40), 135.1 (C-46 þ C-47), 134.5 (C-
17), 131.5 (C-44 þ C-49), 125.1 (C-41), 123.9 (C-45 þ C-48), 123.1 (C-
16), 122.4 (C-18), 118.3 (C-2), 116.4 (C-28), 113.3 (C-10), 111.8 (C-9),
108.1 (C-12), 105.1 (C-2),100.8 (C-7), 98.2 (C-5), 76.3 (C-27), 75.7 (C-
23), 73.1 (C-25), 70.9 (C-21), 67.2 (C-42), 56.2 (C-37), 43.6 (C-39),
42.7 (C-38), 40.1 (C-26), 39.5 (C-20), 37.3 (C-24), 33.8 (C-22), 21.5
(C-13), 20.7 (C-36), 20.0 (C-30), 17.9 (C-31), 12.8 (C-32), 8.4 (C-33),
4
3
7), 12.7 (C-32), 8.5 (C-33), 8.1 (C-34), 7.0 (C-14); FT-IR (KBr pellet):
432 cm
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
n
(O21eH) þ
n
(O23eH), 3261 cm
d
(NeH), 3133 cm
ꢀ
1
ꢀ
ꢀ1
n
(O
4
eH$$$O11), 2480 cm
n
(O
1
eH$$$O
(C
8
), 1726 cm þ 1713 cm
ꢀ
1
ꢀ1
n
(C35]O), 1642 cm
n
(C11]O) þ
n
8
]O), 1605 cm
n(C]C)qui-
,
1558 cm 1
ꢀ
n(C]C)triazole
,
1534 cm
(C]C), 1246 and 1237 cm
ꢀ1
ꢀ1
n
(C]C),
(CeO);
nazoline
ꢀ
1
ꢀ1
1
482 cm þ 1451 cm
n
n
elemental analysis C47
63
H N
5
O
11: calculated: C ¼ 64.59%; H ¼ 7.27%;
ꢀ
1
ꢀ1
ꢀ1
N ¼ 8.01%; O ¼ 20.14%; measured: C ¼ 64.50%; H ¼ 7.35%;
8.1 (C-34), 7.0 (C-14); FT-IR (CHCl
3
, 0.045 mol L ): 3459 cm
ꢀ1
N ¼ 8.12%; O ¼ 20.03%.
n
(O21eH)
þ
n
(O23eH), 3315 cm
d
(NeH), 3133 cm
), 1768 cm
ꢁ
ꢀ1
ꢀ
ꢀ1
3
0 e yield: 67%; HPLC: RT ¼ 9.71 [min]; 25 C; 50:50
n(O eH$$$O11), 2493 cm
4
n
(O
1
eH$$$O
8
n
assym(C45
sym(C52
]
]
þ
ꢀ1
(
H
2
O:CH
3
CN); flow: 1.0 mL/min; HR-MALDI-TOF [MþH] 888.4751;
O) þ (C52]O), 1729 þ 1713 cm
n
sym(C45]O) þ
n
1
ꢀ1
ꢀ1
H NMR (
d
ppm in DMSO-d
6
): 16.57 (1H, bs, HO-1),12.36 (1H, s, HO-
O) þ
n
(C35]O), 1639 cm
n
(C11]O) þ
n
(C
8
]O), 1609 cm
n
(C]
þ
3
ꢀ1
ꢀ1
n
4
), 11.68 (1H, bs, N(2) -H), 7.82 (1H, s, H-42), 6.32 (1H, d,
J
H17-
C)quinazoline, 1553 cm
n
(C]C)triazole, 1503 cm (C]C), 1459 and
ꢀ1
n(CeO); elemental analysis
H18 ¼ 11.0 Hz, H-17), 6.13 (1H, d, H-29), 5.99 (1H, dd, 3JH19-
1436 cm
ꢀ1
n
(C]C), 1247 and 1231 cm
3
H20 ¼ 7.0 Hz, H-19), 5.71 (1H, dd,
J
H18-H19 ¼ 15.6 Hz, H-18), 5.15
C
50
H
56
N
6
O
13: calculated: C ¼ 63.28%; H ¼ 5.95%; N ¼ 8.86%;
3
3
(
1H, d, JH25-H26 ¼ 10.6 Hz, H-25), 4.96 (1H, dd, JH28-H29 ¼ 12.7 Hz,
O ¼ 21.92%; measured: C ¼ 63.35%; H ¼ 6.03%; N ¼ 8.75%;
3
J
H28-H27 ¼ 6.6 Hz, H-28), 4.78 (1H, bs, HO-23), 4.70 (2H, m, H-38),
O ¼ 21.87%.
3
ꢁ
þ
4
.46 (1H, bs, HO-21), 4.35 (2H, t, JH40-H41 ¼ 7.1 Hz, H-41), 3.67 (1H,
32 e yield: 63%; HPLC: RT ¼ 9.72 [min]; 25 C; 50:50
3
3
dd, JH21-H22 ¼ 7.4 Hz, JH21-H20 ¼ 3.8 Hz, H-21), 3.45 (2H, m, H-39),
(H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH] 963.4135;
1
3
.33 (1H, m, H-27), 2.95 (3H, s, H-37), 2.94 (1H, m, H-23), 2.58 (2H,
H NMR (
d
ppm in DMSO-d
6
): 16.57 (1H, bs, HO-1),12.35 (1H, s, HO-
3
þ
t, JH44-H45 ¼ 7.6 Hz, H-44), 2.22 (1H, m, H-40), 2.11 (1H, m, H-20),
4), 11.69 (1H, bs, N(2) eH), 8.31 (1H, s, H-41), 7.85 (4H, m, H-

K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
675
3
ꢀ1
ꢀ1
n(C]C)triazole, 1502 cm
ꢀ1
4
6
6 þ H-47 þ H-48 þ H-49), 6.32 (1H, d, JH17-H18 ¼ 11.1 Hz, H-17),
1609 cm
n
n
(C]C)quinazoline, 1552 cm
ꢀ1
3
ꢀ1
.13 (1H, d, H-29), 6.00 (1H, dd, JH19-H20 ¼ 7.0 Hz, H-19), 5.79 (1H,
(C]C), 1461 and 1437 cm
elemental analysis C52
n
(C]C), 1248 and 1232 cm
n(CeO);
3
3
dd, JH18-H19 ¼ 15.7 Hz, H-18), 5.14 (1H, d, JH25-H26 ¼ 10.6 Hz, H-25),
H
60
N
6
O
13: calculated: C ¼ 63.92%; H ¼ 6.19%;
3
3
4
.96 (1H, dd, JH28-H29 ¼ 12.8 Hz, JH28-H27 ¼ 6.6 Hz, H-28), 4.86 (2H,
N ¼ 8.60%; O ¼ 21.29%; measured: C ¼ 63.87%; H ¼ 6.25%;
3
m, H-39), 4.74 (1H, bs, HO-23), 4.71 (2H, m, H-38), 4.66 (2H, t, JH42-
H43 ¼ 6.2 Hz, H-42), 4.42 (1H, bs, HO-21), 3.97 (1H, m, H-43), 3.63
N ¼ 8.56%; O ¼ 21.32%.
ꢁ
34 e yield: 68%; HPLC: RT ¼ 10.56 [min]; 25 C; 50:50
3
3
þ
(
2
(
1H, dd, JH21-H22 ¼ 7.4 Hz, JH21-H20 ¼ 3.8 Hz, H-21), 3.34 (1H, m, H-
(H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
1
7), 2.95 (3H, s, H-37), 2.93 (1H, m, H-23), 2.11 (1H, m, H-20), 2.01
880.4126; H NMR (
d
ppm in DMSO-d
6
): 16.58 (1H, bs, HO-1), 12.39
þ
3H, s, H-36), 1.96 (3H, s, H-30), 1.90 (3H, s, H-14), 1.62 (3H, s, H-13),
(1H, s, HO-4),11.61 (1H, bs, N(2) -H), 8.30 (1H, s, H-41), 7.44 (2H, m,
3
3
1.52 (1H, m, H-22), 1.47 (1H, dt,
J
H23-H24 ¼ 14.1 Hz,
J
H24-
H-45 þ H-47), 7.40 (1H, m, H-46), 7.35 (2H, m, H-44 þ H-48), 6.33
3
H26-H34 ¼ 6.8 Hz, 3JH26-
3
H33 ¼ 7.1 Hz, H-24), 1.14 (1H, ddd,
J
(1H, d,
J
J
H17-H18 ¼ 11.1 Hz, H-17), 6.12 (1H, d, H-29), 5.99 (1H, dd,
3
3
3
H27 ¼ 1.7 Hz, H-26), 0.84 (3H, d,
J
H22-H32 ¼ 6.8 Hz, H-32), 0.74
H19-H20 ¼ 7.0 Hz, H-19), 5.75 (1H, dd,
J
H18-H19 ¼ 15.7 Hz, H-18),
3
3
3
(
3H, d, JH20-H31 ¼ 6.9 Hz, H-31), 0.53 (3H, d, JH24-H33 ¼ 6.9 Hz, H-
5.59 (2H, s, H-42), 5.15 (1H, d, JH25-H26 ¼ 10.7 Hz, H-25), 4.97 (1H,
13
3 3
3
1
3), ꢀ0.18 (3H, d, H-34); C NMR (
d
ppm in DMSO-d
6
): 186.4 (C-
dd, JH28-H29 ¼ 12.7 Hz, JH28-H27 ¼ 6.6 Hz, H-28), 4.75 (1H, bs, HO-
1), 183.9 (C-8), 171.8 (C-6), 170.0 (C-35), 167.8 (C-44 þ C-51), 157.1
C-15), 142.7 (C-1), 142.5 (C-19), 142.0 (C-29), 141.0 (C-4), 139.8 (C-
0), 134.6 (C-47 þ C-48), 134.5 (C-17), 131.2 (C-45), 131.2 (C-50),
24.9 (C-41), 123.3 (C-46 þ C-49), 122.9 (C-16), 122.5 (C-16), 118.2
C-2), 116.5 (C-28), 113.3 (C-10), 111.8 (C-9), 108.2 (C-12), 105.1 (C-2),
00.8 (C-7), 98.4 (C-5), 76.3 (C-27), 75.8 (C-23), 73.0 (C-25), 71.0 (C-
1), 56.1 (C-37), 47.5 (C-42), 43.7 (C-39), 42.8 (C-38), 40.0 (C-26),
9.5 (C-20), 37.9 (C-43), 37.3 (C-24), 33.9 (C-22), 21.5 (C-13), 20.7
23), 4.70 (2H, m, H-38), 4.47 (1H, bs, HO-21), 4.34 (1H, m, H-39),
3
3
(
4
1
(
1
2
3
3.65 (1H, dd, JH21-H22 ¼ 7.5 Hz, JH21-H20 ¼ 3.8 Hz, H-21), 3.33 (1H,
m, H-27), 2.95 (3H, s, H-37), 2.94 (1H, m, H-23), 2.11 (1H, m, H-20),
2.01 (3H, s, H-36),1.98 (3H, s, H-30),1.91 (3H, s, H-14),1.62 (3H, s, H-
3
3
13), 1.52 (1H, m, H-22), 1.46 (1H, dt,
J
H23-H24 ¼ 13.9 Hz,
J
H24-
H33 ¼ 7.0 Hz, H-24), 1.15 (1H, ddd, 3JH26-H34 ¼ 6.8 Hz, 3JH26-
3
H27 ¼ 7.0 Hz, H-26), 0.85 (3H, d,
J
H22-H32 ¼ 6.8 Hz, H-32), 0.72
3
3
(3H, d, JH20-H31 ¼ 6.9 Hz, H-31), 0.51 (3H, d, JH24-H33 ¼ 6.8 Hz, H-
13
(
(
C-36), 20.1 (C-30), 17.8 (C-31),12.7 (C-32), 8.5 (C-33), 8.2 (C-34), 7.0
33), ꢀ0.15 (3H, d, H-34); C NMR (
6
d ppm in DMSO-d ): 186.4 (C-
ꢀ
1
ꢀ1
ꢀ1
C-14); FT-IR (CHCl
3
,
0.045 mol
L
): 3463 cm
11), 183.9 (C-8), 171.9 (C-6), 170.0 (C-35), 157.2 (C-15), 142.8 (C-1),
142.7 (C-19), 142.1 (C-29), 141.1 (C-4), 139.8 (C-40), 135.5 (C-43),
134.4 (C-17), 129.0 (C-45 þ C-47), 128.3 (C-46), 127.9 (C-44 þ C-48),
125.1 (C-41), 122.9 (C-16), 122.5 (C-18), 118.2 (C-2), 116.5 (C-28),
113.2 (C-10), 111.8 (C-9), 108.2 (C-12), 105.1 (C-2), 100.8 (C-7), 98.3
(C-5), 76.3 (C-27), 75.9 (C-23), 73.0 (C-25), 70.9 (C-21), 56.2 (C-37),
52.4 (C-42), 43.6 (C-39), 42.7 (C-38), 40.1 (C-26), 39.6 (C-20), 37.3
(C-24), 33.7 (C-22), 21.5 (C-13), 20.7 (C-36), 20.1 (C-30), 17.9 (C-31),
ꢀ
1
n
(O21eH)
þ
n
(O23eH), 3321 cm
d
(NeH), 3128 cm
ꢀ
1
ꢀ
ꢀ1
n
(O eH$$$O11), 2487 cm
4
n
(O
1
eH$$$O
8
), 1762 cm
n
assym(C45]
ꢀ
1
O) þ (C52]O), 1728 þ 1714 cm
n
sym(C45]O) þ
n
sym(C52]
ꢀ
1
ꢀ1
O) þ
n
(C35]O), 1640 cm
n
(C11]O) þ
n
(C
8
]O), 1608 cm
n(C]
n(C]C), 1463 and
ꢀ1
ꢀ1
C)quinazoline, 1554 cm
n
(C]C)triazole, 1501 cm
ꢀ1
ꢀ1
1439 cm
n
(C]C), 1249 and 1229 cm
n(CeO); elemental analysis
C
H
51 58
N
6
O
13: calculated: C ¼ 63.61%; H ¼ 6.07%; N ¼ 8.73%;
O ¼ 21.60%; measured: C ¼ 63.54%; H ¼ 6.12%; N ¼ 8.65%;
3
12.7 (C-32), 8.5 (C-33), 8.2 (C-34), 7.0 (C-14); FT-IR (CHCl ,
0.045 mol L ): 3459 cm
(NeH), 3133 cm
ꢀ
1
ꢀ1
ꢀ1
(O23eH), 3337 cm
O ¼ 21,69%.
n
(O21eH) þ
n
ꢁ
ꢀ1
ꢀ1
ꢀ
3
3 e yield: 61%; HPLC: RT ¼ 10.23 [min]; 25 C; 50:50
d
n
(O
4
eH$$$O11), 2487 cm
n(O eH$$$O ),
1
8
þ
ꢀ1
ꢀ1
(
H
2
O:CH
3
CN); flow: 0.5 mL/min; HR-MALDI-TOF [MþH]
1726 þ 1712 cm
n
(C35]O), 1640 cm
n
(C11]O) þ
n
(C
8
]O),
1
ꢀ1
ꢀ1
ꢀ1
977.4290; H NMR (
d
ppm in DMSO-d
6
): 16.53 (1H, bs, HO-1), 12.36
1608 cm
n
(C]C)quinazoline, 1555 cm
n
(C]C)triazole, 1503 cm
(C]C), 1247 and 1235 cm
þ
ꢀ1
ꢀ1
(
1H, s, HO-4),11.65 (1H, bs, N(2) -H), 8.29 (1H, s, H-41), 7.88 (2H, m,
n
(C]C), 1459 and 1440 cm
elemental analysis C48
n
n(CeO);
3
H-47 þ H-50), 7.85 (2H, m, H-48 þ H-49), 6.31 (1H, d,
J
H17-
H
57
N
5
O
11: calculated: C ¼ 65.51%; H ¼ 6.53%;
H18 ¼ 11.1 Hz, H-17), 6.13 (1H, d, H-29), 5.99 (1H, dd, 3JH19-
N ¼ 7.96%; O ¼ 20.00%; measured: C ¼ 65.48%; H ¼ 6.57%;
3
H20 ¼ 7.0 Hz, H-19), 5.79 (1H, dd,
J
H18-H19 ¼ 15.5 Hz, H-18), 5.16
N ¼ 7.89%; O ¼ 20.06%.
3
3
(
1H, d, JH25-H26 ¼ 10.6 Hz, H-25), 4.96 (1H, dd, JH28-H29 ¼ 12.8 Hz,
3
J
H28-H27 ¼ 6.7 Hz, H-28), 4.84 (2H, m, H-39), 4.76 (1H, bs, HO-23),
4.10. Antimicrobial tests of 4e34
3
4
.72 (2H, m, H-38), 4.45 (2H, t, JH42-H43 ¼ 7.1 Hz, H-42), 4.44 (1H,
3
3
bs, HO-21), 3.69 (1H, dd, JH21-H22 ¼ 7.4 Hz, JH21-H20 ¼ 3.9 Hz, H-21),
The antimicrobial activities of novel of 3-formylrifamycin SV
derivatives were studied against a series of bacteria strains such as
standard strains of Gram-positive cocci: Staphylococcus aureus
ATCC 25923, S. aureus ATCC 4163, S. aureus ATCC 6538, S. epi-
dermidis ATCC 12228 and S. epidermidis ATCC 49134; standard
strains of Gram-negative rods: E. coli ATCC 10538, Enterococcus
faecalis ATCC 19433, Salmonella abony NCTC 6017, Trichophyton
rubrum ATCC 28188 and M. tuberculosis H37Rv ATCC 27294 (strain
susceptible to all first line drugs) and M. bovis ATCC 19210 as well as
against fungus C. albicans ATCC 10231. The microorganisms used
were bought from Argenta company (first passage), LGC Standards
or obtained from the collection of the Medical University of Pozna nꢀ ,
Poland. Minimal Inhibitory Concentration (MIC) values were
determined independently for compounds 4e34 according to the
recommendations of the European Society of Clinical Microbiology
and Infectious Diseases (EUCAST) by the serial microdilution
method using polystyrene plates and MuellereHinton II agar me-
dium (Beckton Dickinson). Concentrated solutions of the com-
pounds were dissolved in DMSO (POCH, Gliwice) and diluted in
water to obtain the required concentration. The concentrations of
3
.67 (1H, m, H-44), 3.32 (1H, m, H-27), 2.95 (3H, s, H-37), 2.93 (1H,
m, H-23), 2.17 (2H, m, H-43), 2.12 (1H, m, H-20), 2.01 (3H, s, H-36),
.97 (3H, s, H-30), 1.91 (3H, s, H-14), 1.63 (3H, s, H-13), 1.52 (1H, m,
1
3
3
H-22),1.45 (1H, dt, JH23-H24 ¼ 14.2 Hz, JH24-H33 ¼ 6.9 Hz, H-24),1.14
3
3
(
1H, ddd, JH26-H34 ¼ 6.9 Hz, JH26-H27 ¼ 1.8 Hz, H-26), 0.85 (3H, d,
3
3
J
H22-H32 ¼ 6.9 Hz, H-32), 0.74 (3H, d, JH20-H31 ¼ 6.7 Hz, H-31), 0.52
3H, d, 3JH24-H33 ¼ 7.0 Hz, H-33), ꢀ0.16 (3H, d, H-34); C NMR (
13
d
(
ppm in DMSO-d
3
(
1
(
9
(
4
6
): 186.3 (C-11), 183.8 (C-8), 171.9 (C-6), 170.0 (C-
5),168.1 (C-45 þ C-52),157.3 (C-15),142.8 (C-1),142.6 (C-19),142.0
C-29), 141.1 (C-4), 139.6 (C-40), 134.6 (C-48 þ C-49), 134.4 (C-17),
31.0 (C-46 þ C-51), 125.0 (C-41), 123.0 (C-47), 123.0 (C-50), 122.8
C-16), 122.6 (C-18), 118.2 (C-2), 116.4 (C-28), 113.2 (C-10), 111.9 (C-
), 108.2 (C-12), 105.2 (C-2), 100.8 (C-7), 98.4 (C-5), 76.2 (C-27), 75.8
C-23), 73.1 (C-25), 70.8 (C-21), 56.1 (C-37), 47.7 (C-42), 44.8 (C-44),
3.5 (C-39), 42.6 (C-38), 40.0 (C-26), 39.6 (C-20), 37.4 (C-24), 34.6
C-43), 33.9 (C-22), 21.4 (C-13), 20.7 (C-36), 20.1 (C-30), 17.8 (C-31),
(
1
3
2.8 (C-32), 8.4 (C-33), 8.1 (C-34), 7.0 (C-14); FT-IR (CHCl ,
ꢀ
1
ꢀ1
ꢀ1
(O23eH), 3324 cm
0
.045 mol L ): 3455 cm
n
(O21eH) þ
eH$$$O11), 2497 cm
assym(C45]O) þ (C52]O), 1726 þ 1712 cm
n
ꢀ1
ꢀ1
ꢀ
d(NeH), 3131 cm
n
(O
4
n
(O
1
eH$$$O
nsym(C45
8
),
]
ꢀ
1
ꢀ1
1759 cm
n
the compounds tested in a solid medium ranged from 1 to
ꢀ
1
ꢀ1
O) þ
n
sym(C52]O) þ
n
(C35]O), 1642 cm
n(C11]O) þ
n
(C
8
]O),
0.002
m
g mL (for S. aureus ATCC 25923, S. aureus ATCC 4163,

676
K. Pyta et al. / European Journal of Medicinal Chemistry 84 (2014) 651e676
S. aureus ATCC 6538, S. epidermidis ATCC 12228 and S. epidermidis
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polymerase, Cell 104 (2001) 901e912.
ꢀ
1
ATCC 49134) and 1e2
ATCC 19433, S. abony NCTC 6017, T. rubrum ATCC 28188). The final
mg mL (for E. coli ATCC 10538, E. faecalis
[
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5
ꢀ1
inoculum of all studied organisms was 5$10 CFU mL (colony
forming units per mL). Minimal inhibitory concentrations (MIC)
2
(
385e2388;
ꢁ
were read after 18 h of incubation at 35e37 C. In in vitro tests
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[
[
ꢁ
growth after 7 days of incubation at 37 C. MIC values in microbi-
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(
2013) 319e331;
d) S.G. Agalave, S.R. Maujan, V.S. Pore, Click chemistry: 1,2,3-triazoles as
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ranged from 1 to 0.125
m
g mL . Solutions of antibiotics were
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(
initially prepared in DMSO (POCH, Gliwice) and diluted in sterile
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(
f) E.N. da Silva Júnior, I.M.M. de Melo, E.B.T. Diogo, V.A. Costa, J.D. de Souza
4
.11. Determination of logP and water solubility for newly
Filho, W.O. Valença, C.A. Camara, R.N. de Oliveira, A.S. de Araujo, F.S. Emery,
M.R. dos Santos, C.A. de Simone, R.F.S. Menna-Barreto, S.L. de Castro, On the
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synthesised derivatives of 3-formylrifamycin SV
The derivatives analysed were dissolved in HPLC gradient grade
ꢁ
water (pH ¼ 7, T ¼ 25 C) and the calibration curves were deter-
(2012) 304e312;
mined by measuring absorbance as a function of their concentra-
tion on Metertech Inc. UVevis SP-80001 spectrophotometer.
Analytical wavelengths for the determination of calibration curves
Af(c) and the concentrations cH2O and coctanol on the basis of UVevis
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Hsp90 molecular chaperone inhibitors, Eur. J. Med. Chem. 65 (2013)
measurements were respectively: for Rif
max ¼ 322.5 nm and for 4e34 derivatives
known amount of 3-formylrifamycin SV derivative was dissolved in
0 mL of octanol to which 10 mL of H O was added. The mixture
l
max ¼ 332.6 nm, for Ral
l
l
max ¼ 314.4 nm. Next, a
4
(
64e476;
i) S.N. Sunassee, C.G.L. Veale, N. Shunmoogam-Gounden, O. Osoniyi,
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Silva Júnior, M.T. Davies-Coleman, Cytotoxicity of lapachol, -lapachone and
1
2
b
was shaked, vigorously stirred for 1 h and then separated. In order
to determine the concentration of the compound in the aqueous
layer, the respective measurement of absorbance was performed on
a UVevis spectrophotometer. LogPexp values were calculated ac-
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2
O
compared to those calculated by the Molinspiration program
(
http://www.molinspiration.com). To determine the solubility in
the gradient grade water, a known amount of given derivative was
weighted and respective volumes of H O were added (by 0.1 mL)
2
(l) P.K. Mandal, Copper-catalyzed one-pot synthesis of glycosylated imino-
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4
(2014)
upon vigorous stirring of the solution. The solubility test was per-
formed at least three times for the derivative studied.
5
[
3
Acknowledgement
[
PP, KP and KK wish to thank for the project financial support of
the Polish National Science Center (NCN), grant decision number
DEC-2011/03/B/ST5/01014.
(
2
(
V.V. Fokin, Ruthenium-catalyzed azide-alkyne cycloaddition: scope and
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