A new model of binding of rifampicin and its amino analogues as zwitterions to bacterial RNA polymerase

Article abstract of DOI:10.1039/c2ob26317c

Seven new benzyl (3-9) and four new phenethyl (10-13) amino analogues of ansa-macrolide rifampicin (1) were synthesized using the optimised method of reductive amination. Structures of 3-13 in solution were determined by 1D and 2D NMR and FT-IR methods whereas the energetically most favoured conformation of amino analogues was calculated with the use of the PM5 method. Spectroscopic and semi-empirical studies revealed the presence of zwitterionic forms of all 3-13 analogues in solutions containing water traces. ¹H-¹⁵N HSQC and ¹H-¹⁵N HMBC in combination with ¹H-¹H COSY and ¹H-¹³C HMBC two dimensional spectroscopic methods unambiguously evidenced that the presence of the zwitterionic form of ansa-macrolides was a consequence of proton transfer from the O(8)-H phenolic group to the secondary amine moiety within 3-13 structures. ¹H-¹H NOESY studies indicated two different orientations of the substituent introduced at the C(3) position for benzyl and phenethyl amino analogues of rifampicin and their similar conformation within the ansa-bridges in solution. FT-IR studies of the deprotonation of molecule 1 and comparison of these data with those for 3-13 indicated C(8)O double bond character after formation of zwitterions in solution. Results of an antibacterial test against Gram-(-) and Gram-(+) strains were compared with detailed structural information on new analogues of 3-13 to indicate some structure-activity relationships. Molecular recognition studies of 1 and 12 inhibitors at the binding site of bacterial RNA polymerase (RNAP) as zwitterions revealed key intermolecular interactions and led to the proposition of a new model of RNAP inhibition, which explains significant differences in antibacterial properties of rifampicin and its analogues.

Full text of DOI:10.1039/c2ob26317c

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Organic & Biomolecular Chemistry
New model of binding of rifampicin and its amino analogues as
zwitterions to bacterial RNA polymerase
Krystian Pyta,a Katarzyna Klich,a Piotr Przybylski*a and Joanna Stefańskab
aFaculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
bMedical University of Warsaw, Department of Pharmaceutical Microbiology, Oczki 3, 02-007
Warsaw, Poland
*E-mail: piotrp@amu.edu.pl, tel. +48 61 8291252, fax. +48 61 829 1505
Seven new benzyl (3-9) and four new phenethyl (10-13) amino analogues of ansa-macrolide
rifampicin (1) were synthesized using the optimised method of reductive amination. Structures of
3-13 in solution were determined by 1D and 2D NMR and FT-IR methods whereas the
energetically most favoured conformation of amino analogues was calculated with the use of
PM5 method. Spectroscopic and semi-empirical studies revealed the presence of zwitterionic
1
1
forms of all 3-13 analogues in solutions containing water traces. H-15N HSQC and H-15N
1
1
HMBC in combination with H-1H COSY and H-13C HMBC two dimensional spectroscopic
methods unambiguously evidenced that the presence of the zwitterionic form of ansa-macrolides
was a consequence of proton transfer from O(8)-H phenolic group to secondary amine moiety
1
within 3-13 structures. H-1H NOESY studies indicated two different orientations of the
substituent introduced at C(3) position for benzyl and phenethyl amino analogues of rifampicin
and their similar conformation within the ansa-bridges in solution. FT-IR studies of the
deprotonation of molecule 1 and comparison of these data with those for 3-13 indicated C(8)=O
double bond character after formation of zwitterions in solution. Results of antibacterial test
against Gram-(-) and Gram-(+) strains were compared with detailed structural information on
new analogues of 3-13 to indicate some structure-activity relationships. Molecular recognition
studies of 1 and 12 inhibitors at the binding site of bacterial RNA polymerase (RNAP) as
zwitterions revealed key intermolecular interactions and led to proposition of a new model of

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RNAP inhibition, which explains significant differences in antibacterial properties of rifampicin
and its analogues.
Keywords: rifampicin analogues; ansa macrolides; antibiotics; zwitterions; reductive amination;
13
15
1
1H, C and N NMR, H-1H NOESY; FT-IR; antibacterial activity; bacterial RNA polymerase
inhibitors.
Introduction
Rifampicin (Fig. 1), called in US rifampin, belongs to ansa macrolide group of antibiotics,
characterised by strong antibacterial properties against Gram-positive and Gram-negative bacteria
strains.1 Because of relatively fast resistance of bacteria to this antibiotic, rifampicin is mainly
used as therapeutic agent in combinatorial therapy against tuberculosis together with e.g.
isoniazid and vancomycin.2 Resistance of bacteria strains to rifampicin is mainly achieved via
hydrolysis of hydrazone moiety at C(38) atom and glycosylation of hydroxyl groups of the ansa
bridge or is gained by RNA polymerase (RNAP) mutations.3 At the beginning the mechanism of
rifampicin activity was supposed to depend on the allosteric inhibition of RNA polymerase.4
Since the X-ray structure of rifampicin-RNA polymerase has been determined, it has been known
that this mechanism is based on direct blocking of the elongated RNA transcript path at 5’ end.5
Although the problem of inhibitor binding site to RNA polymerase has been solved, the low
accurancy of X-ray measurements of rifampicin has not allowed drawing conclusions on the
types of interaction between the inhibitor molecule and the enzyme. Earlier studies also indicated
that the arrangement of O(21)H and O(23)H hydroxyls of ansamycin bridge relative to
naphthalene moiety in the rifamycin group of antibiotics influences their antibacterial activity.6
Up to now a series of oxime, hydrazone, semicarbazone, iminium derivatives undergoing
cyclisation processes and obtained via Wittig reaction at C(38) atom have been synthesised as
potential alternative antibacterial agents to rifampicin.7 Simple amino analogues of rifampicin at
C(3), containing at nitrogen methyl, ethyl or propyl groups have also been obtained but not
studied toward antibacterial activity against standard bacteria strains.8 Recently, we have
demonstrated that the protonation site of rifampicin-based antibiotics has strong impact on their
antibacterial activity.9 Taking into account that hydrolysis of hydrazone moiety at C(38) atom is
one of the processes leading to bacteria resistance to rifampicin, to get more information about
the character of the substituent at C(38) atom desired to achieve high antibacterial activity we

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synthesised and biologically tested a new series of rifampicin amino analogues at C(38) atom,
resistant to hydrolysis process, and containing various substituted phenyl ring with halogen.
Recent reinvestigation of the X-ray structure of rifampicin pentahydrate10 questioned the
correctness of the model proposed by Darst et al.5 of binding of rifampicin to bacterial RNA
polymerase formulated on the basis of rifampicin pentahydrate structure11 presented in the
incorrect non-ionic form. Furthermore, the non-ionic structure of rifampicin was further used for
molecular dynamics calculations of rifampicin and RNAP interactions.12 Earlier we have
demonstrated that formation of important for biological activity strong intermolecular hydrogen
bond between E445 and protonated piperazine nitrogen atom of rifampicin is possible.9 To get
more insight into the molecular mechanism of inhibition of this important enzyme, the total
optimisation of interactions between the inhibitor (compounds 1 and 12) and the enzyme
(bacterial RNA polymerase) at the semi-empirical level of theory has been performed with the
use of PM5 method.
Results and discussion
Synthesis of new rifampicin amino analogues 3-13
Rifampicin was hydrolyzed into rifaldehyde and next converted into its new benzyl and
phenethyl amino analogues (Fig. 2) via four the reductive amination methods (Fig. 1, Table 1).
New amino analogues of rifampicin were characterised by 1D and 2D NMR, FT-IR and MALDI-
TOF MS methods (see Experimental section, Figures 4 and 7). As shown in Table 1, application
of NaBH4 as reductant leads to the lowest yield, whereas the use of NaBH3CN in the presence of
HCl/EtOH as a catalyst is shown to be the most efficient method. It should be emphasised that
the use of cyanoborohydride on polymer support (method 4) as a reductant is not as efficient as
the use of NaBH3CN (method 3), because of the absorption processes of rifaldehyde and
respective rifampicin amino analogues on molecular sieves. Reductive amination via the
application of a relatively “soft” reductant as NaBH(CH3COO)3 in the presence of HCl/EtOH
was also a good choice comparing the yields of methods 2 and 3.
Structure of new rifampicin amino analogues 3-13 in solution
Earlier, on the basis of our X-ray and NMR studies, the intramolecular proton transfer
process for 1 structure was indicated.9 FT-IR spectra of 1 and 8 in aprotic and protic systems,

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with the detailed assignment of the bands on the basis of deuteration, are presented in Figures 3a
and b. Comparison of the spectra of 1 (Fig. 3a) in two different solvents and after addition of
stoichiometric amount of KOH reveals important changes in the ν(C=O) and ν(C=C) band
regions. In the FT-IR spectrum of 1 in CHCl3 in the stretching vibration region characteristic of
carbonyl group, three bands at 1715, 1660 and 1646 cm-1 appear assigned to ν(C35=O) of acetate,
ν(C11=O) of ketone and ν(C15=O) of amide group, respectively. The presence of these bands at
these frequencies demonstrates that in CHCl3 1 exists exclusively in the non-ionic form (Fig. 2),
which is in agreement with the previously obtained NMR data for 1 in CHCl3.9 Strong changes in
FT-IR spectrum are observed upon addition of KOH to 1 in equimolar ratio (Fig. 2). The most
spectacular difference is the appearance of bands at 1655, 1584, 1478 and 1476 cm-1. A slight
shift toward lower frequencies and a significant increase in the band absorption at 1655 cm-1 are
evoked by deprotonation of the most acidic phenolic group at C(8) and formation of phenolate
anion of 1 in CHCl3 solution. The overlapping of ν(C11=O) and ν(C8=O) bands in the spectrum,
indicates that the electron density of oxygen atom of C(8)-O‾ phenolate group is strongly shared
with the naphthalene ring and therefore at C(8) and C(11) positions two carbonyl groups are
13
present. NMR spectra of deprotonated 1, recorded in CDCl3, are given in Figure 4. In the C
NMR spectrum of deprotonated 1 (Fig. 4b) there are two signals at 183. 2 and 188.6 ppm.
Assignment of these signals, on the basis of 2D NMR spectra, to δC8 and δC11 resonances,
confirms a similar character of C11=O and C8=O bonds. The increased electron density in the
naphthalene ring, as a consequence of the proton transfer, influences also the position of ν(C=C)
naphthalene skeleton vibrations i.e. two new bands of ν(C=C) from naphthalene ring appear at
1502 and 1476 cm-1. It should be noted that as a result of the strong resonance effect also the
ν(C=N) band shifts from 1567 to 1584 cm-1 toward higher frequencies revealing more double
C=N bond character of hydrazone moiety. The appearance of ν(C35=O) vibrations as two separate
bands at 1719 and 1713 cm-1 can be explained by the conformational equilibrium of ansa bridge
and bonding of acetate carbonyl group with O(23)H hydroxyl with different strength. The FT-IR
spectrum of 1 in DMSO/H2O seems to be very similar to the respective spectrum of 1 in CHCl3,
recorded after addition of KOH, in 1800-1450 cm-1 analytical range. The presence of a
characteristic band at 1655 cm-1 assigned to ν(C11=O) and ν(C8=O) vibrations along with bands at
1511 and 1478 cm-1 assigned to ν(C=C) naphthalene ring vibrations confirms the formation of
zwitterionic form of 1 in DMSO/H2O, in which our antibacterial tests have been performed.

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Similar double character of C(8)=O and C(11)=O bonds for 1 is manifested by similar chemical
shifts of δC8 (184.2 ppm) and δC11 (184.5 ppm) in DMSO-d6/H2O. In the FT-IR spectra of
rifampicin analogues 3-13 (Fig. 3b) the above discussed bands, characteristic of zwitterionic
form, also are found in the 1800-1450 cm-1 region, irrespectively whether recorded in CHCl3 or
DMSO/H2O. Thus, as a consequence of intramolecular proton transfer process and resonance
phenomenon for rifampicin analogues 3-13 the double character of C(8)-O bond is predominant
in solution. This conclusion is supported by similar δC8 values for 1 and 3-13 amino analogues in
different solvents: in DMSO-d6/H2O (184.1-184.2 ppm) and in CDCl3 (181.7-183.0 ppm9). The
C(8)=O and C(11)=O bond parameters in the calculated structures of 8 and 12 (Fig. 5) support a
similar character of these bonds because C(8)=O and C(11)=O bond length parameters are almost
the same for 8 and for 12, of 1.24 Å and 1.23 Å, respectively.
Localisation of the transferred proton in 1 in DMSO/H2O has been established earlier as
the tertiary nitrogen atom of the piperazine ring.9 Essential information about the localisation of
the transferred proton of O(8)-H group in the new rifampicin analogues 3-13 is provided by the
1
2D NMR spectra (Fig. 6). One bond H-15N couplings of the two protons giving signals at 9.20
1
and 8.56 ppm together with the H-1H spin-spin couplings of the same protons through three
bonds with H(38) and H(39) protons reveal unambiguously that in 3-13 analogues the proton
from O(8)-H group is at the secondary nitrogen atom N(38). Furthermore, chemical shifts of
15
N(38) signals in N NMR spectra of 3-13 are in a narrow range from -329 to -334 ppm (see
1
Experimental section), typical of protonated amines.13 The H NMR chemical shifts of the two
geminal (38)N+-H protons are different (Fig. 7) because of the fact that these protons are
diastereotopic due to the chirality of the molecule combined with the prochiral quaternary
nitrogen center14 and different character of hydrogen bonds in which these protons are involved.
As shown in Figure 5, one of the protons, localised at the positively charged nitrogen atom, is
engaged in a weak intramolecular hydrogen bond with the oxygen atom of amide group (length
~2.9 Å, angle ~150°), whereas the second one is intermolecularly bonded to solvent molecules.
Zwitterionic form of 3-13 compounds is also well reflected in deshielding of the proton signal of
O(1)-H hydroxyl group by about 3 ppm (δO(1)-H = 16.01-16.05 ppm) relative to its position in the
spectrum of non-ionic form of 1 δO(1)-H = 13.19 ppm.9 This spectral difference is a result of
formation of a stronger O(1)-H···O=C(8) hydrogen bond (length ~2.5 Å, ~150°, Fig. 5) in a six
membered system in zwitterions of 3-13 than the respective one O(1)-H ··· O=C(15) in a seven-

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membered system, assumed by the non-ionic form of 1 in solution. The signal assigned to the
phenolic group O(4)-H is found in the range 12.82-12.86 ppm in the spectra of 3-13 and its
position indicates participation of this hydroxyl in intramolecular (11)O···H-O(4) hydrogen bond
(length ~2.6 Å, angle ~178°, Fig. 5).
The orientation of ansa bridge relative to the naphthalene ring, which strongly contributes
to biological activity, in structures of new derivatives 3-13 in DMSO-d6 solution was determined
1
on the basis of H-1H NOESY contacts (Fig. 8). Strong contacts between H(34) and H(14)
protons of methyl groups for all derivatives clearly indicate the close vicinity of these groups and
suggests the orientation of ansa bridge relative to naphthalene moiety as in the calculated
structure shown in Figure 8a [distance H(34) … H(14) is 4 Å]. Furthermore, a strong shielding of
H(34) signal to about -0.3 ppm in all 1H NMR spectra of 3-13 is a result of the location of H(34)
protons over the naphthalene ring π electron cloud. The spatial contacts of H(33) proton with the
1
H(18) and H(38) protons, determined from H-1H NOESY spectra [distances: H(33) … H(18) is
3.8 Å; H(33) … H(38) is 2.5 Å], confirm the conformation of ansa bridge in solution as presented
in Figure 8.
It should be noted that for 3-13 analogues there are several possible combinations of
different C(16)=C(17) and C(18)=C(19) double bond configurations with different conformations
of diene moiety (s-cis and s-trans). Assignment of H(18) and H(19) proton signals with the use of
1
1H-13C HSQC and H-13C HMBC methods and determination of three-bond coupling constants
3
3JH18-H19≈15.5 Hz and JH19-H20≈7.5 Hz for 3-13 compounds evidenced the E- configuration of
C(18)=C(19) bond. The signal at 6.55 ppm assigned to H(18) proton is a double of doublets with
3
one coupling constant as mentioned earlier and the other JH18-H17=10.8 Hz, characteristic of s-
trans conformation of diene moiety.15 The determination of C(16)=C(17) bond configuration on
the basis of one-dimmensional NMR spectra was difficult because of the absence of proton at
C(16) atom. Instead, the 1H-1H NOESY spectra were found to be more informative and revealed
that the resonance at 2.08 ppm of H(30) protons strongly correlates with H(17) and weakly with
H(18) signals. This situation is well reflected in the following distances: H(30)…H(17) 2.31Å and
H(30)…H(18) 4.56 Å, determined for all calculated structures (Fig. 8), in which Z- configuration
of C(16)=C(17) is energetically favoured. Indirect evidence of C(16)=C(17) bond configuration
as Z- are proton-proton contacts of H(18) with H(38), H(21) and N(2)-H protons (Fig. 8b), which

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should not be observed if the double bond assumed the E- configuration in 3-13 structures. It is
3
worth adding that JH28-H29 close to 13 Hz indicates the E- configuration of C(28)=C(29) double
bond.
1
Comparison of H NMR resonances of diene moiety for 1 as well as benzyl (compounds
3-9) and phenethyl (compounds 10-13) amino derivatives (Fig. 7) indicates that different
intramolecular interactions stabilize the structures of 1 and 3-13 in solution. Significant
difference in δH18 values between 1 and 3-13 is a result of different orientation of O=C(15)-N(2)-
H amide group in 1 [intramolecular N(2)-H…N=C(38) bond] and in 3-13 [intramolecular
C(15)=O…H-+N(38) bond] structures and changes within the diene fragment conformation.
Interesting are also the differences in δH17, δH18 and δH19 chemical shifts between 3-9 and 10-13
spectra indicating that for 10-13 the π-π stacking interaction between the electrons of diene with
those of phenyl ring takes place. This difference is probably due to different lengths of the linker
in phenethyl (-CH2-CH2-) and benzyl (-CH2-) derivatives (Fig. 5). The π-π stacking interaction
also weakens the intramolecular hydrogen bond C(15)=O…H-+N(38) in the structure of 10-13 in
solution, which is manifested by lower δN+H values, if compared to those recorded for 3-9 (Fig. 7,
Experimental section).
Antimicrobial activity of 1 and 3-13 macrolides
Rifampicin (1) and a series of its amino analogues (3-13) containing benzyl and phenethyl
moieties substituted with halogen were tested in vitro for their antibacterial properties against
standard bacteria strains. The results collected in Table 2 demonstrate that all compounds studied
are significantly more active against Gram-(+) than Gram-(-) negative bacteria strains. Newly
synthesised benzyl amino analogues comprising fluorine atom at o- and m- positions (compounds
4 and 5) are characterised by moderate antibacterial activity against two Gram-(-) negative strains
E. coli (MIC = 64 μg/ml). The other amino analogues are rather less active or inactive against
Gram-(-) bacteria strains. Generally, lower activity of 3-13 against Gram-(-) strains in
comparison to that of 1 can be explained by structural differences of the substituent at C(3)
position and different orientation of amide moiety resulting in lower lipophilicity and weaker
penetration of Gram-(-) bacteria membranes. Rifampicin shows high antibacterial activity (MIC
= 0.004-0.016 μg/ml) against Gram-(+) strains. The antibacterial activity of 3-13 amino
analogues is lower (MIC = 0.125-4 μg/ml) than that of 1 against Gram-(+) bacteria strains but

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comparable with that determined earlier (MIC = 0.125-0.5 μg/ml)16 for another antibacterial
agent - ciprofloxacin, a member of fluoroquinolones group, widely used for treatment of a
number of infections.17 The lowest activity against Gram-(+) strains is shown by compounds 3-13
against S. aureus ATCC 25923 strain, similarly as by 1. Almost all of 3-13 compounds, reveal a
very interesting high activity against both S. epidermidis ATCC 12228 and ATCC 35984 bacteria
strains (MIC equal 0.5, 0.25 or 0.125 μg/ml), belonging to a group of bacteria evoking the
infections accompanying insertion of plastic elements in the body, e.g. heart valves, medical
prostheses. Combined analysis of the structures of 1 and 3-13 in DMSO/H2O solution, derived
from spectroscopic studies, with the biological data allowed evaluation of the influence of the
substituent structure at C(3) atom on antibacterial properties of this type of derivatives. A
comparison of the biological test results against Gram-(+) bacteria strains indicates that the
analogues comprising chlorine atom at m- and p- positions of phenyl ring (compounds 8 and 9)
are the least active of all other amino analogues synthesised. Furthermore, as follows from
analysis of the biological data of all 3-13 amino analogues, the ones containing substituents at m-
and p- positions at phenyl ring are slightly less active than those comprising unsubstituted or
substituted phenyl ring at o-position. Thus, slightly increased biological properties of compounds
3, 4, 7, 10 and 11 are result of the presence of the less bulky substituents at C(3) position, i.e.
there is no substituted halogen at phenyl ring or the halogen atom is less distanced from ipso
position. Taking into account antibacterial properties of 3-13 amino analogues of rifampicin and
the fact that the substituent at C(3) due to the presence of a secondary amine linker cannot be
easily hydrolized by bacteria (one of the processes evoking bacteria resistance to rifampicin) this
group of derivatives can be interesting for further search for rifampicin alternatives. Our studies
have shown that not only the structure of ansa bridge but also the structure of the substituent at
C(3) atom strongly influences the biological properties of this type of ansa macrolides.
New model of bacterial RNAP inhibition via rifampicin-based ansa macrolides
The fact that 1 shows much higher antibacterial properties against standard bacteria strains than
3-13 analogues and that in DMSO/H2O solution the former is present in the zwitterionic form
with the proton localised at the piperazine ring9 and the latter ones are present in similar
conditions as zwitterions with the proton localised at the nitrogen atom of secondary amine,
suggests that localisation of proton in the structures of the antibiotics studied plays a crucial role

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in inhibition of bacterial RNA polymerase. Taking into account the recently corrected rifampicin
pentahydrate structure10 and the fact that incorrect phenolic form was used for explanation of
RNAP inhibition and that the role of the substituent structure at C(3) was neglected5, it was
necessary to propose a new model of inhibition of RNA polymerase via zwitterionic forms of 1
and 3-13, whose presence is unambiguously evidenced by FT-IR and 1D and 2D NMR in
solution. Results of molecular docking for 1 and 12 at the binding site of RNAP (which is
distanced about 12Å from Mg2+ cation and localised at the β subunit deep within DNA/RNA
channel) together with the results of optimisation of the interactions between inhibitor and RNAP
complex via PM5 method are shown in Figures 9 and 10. Intermolecular hydrogen bond
parameters calculated for the complexes’ structures with RNAP are collected in Table 3. PM5
calculations revealed that the following aminoacids: Q393, S411, F394, R409 and D396 are involved in
stabilisation of both 1 and 12 molecules at RNAP binding site. Difference in bonding of 1 and 12
to RNAP is related to participation of the substituent at C(3) atom in intermolecular hydrogen
bond, so instead of the interaction with E445 for 1, that with N448 is realised for 12. As follows
from the calculations, the side chains of the following amino acids: I452, Q390, L413 and G414
participate mainly in hydrophobic stabilisation of the complex between the inhibitors and RNAP,
which is in agreement with the model of RNAP inhibition proposed by Darst et al.5 It has been
earlier suggested that also H406 is involved in 1 binding to RNAP5 but our calculations do not
support its role in hydrogen-bonding of the inhibitor molecule (parameters collected in Table 3).
A comparison of hydrogen bond parameters in Table 3 demonstrates that for 1 and 12 docked as
zwitterions, the ansa bridge bonding to aminoacids of RNAP is realised in a similar way, i.e. the
type and strength of the interactions are comparable. A very important role in stabilisation of the
inhibitor molecule in the complex with RNAP is played by F394 which acts simultaneously as
donor and acceptor of proton in the two hydrogen bonds formed with the oxygen of C(35)=O of
acetate and with the proton of O(23)H hydroxyl of the ansa-bridge, respectively. Higher electron
density at O(8) atom in the zwitterionic forms of 1 and 3-13 favours the enhancement of
intermolecular hydrogen bonds with the proton of S411 hydroxyl group and with the proton of
Q393 amide group. Thus, if the ansa bridge is bonded in a similar way in 1 and 3-13, the presence
or absence of intermolecularly hydrogen bonded substituent at C(3) is essential. If compared the
nature and parameters of the intermolecular hydrogen bonds formed between RNAP polymerase
and the protons transferred from O(8)H group in 1 or 12: for RNAP-12 complex hydrogen bond

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with N448 is of medium strength (donor-acceptor distance 2.73 Å, angle 140°, Fig. 9a) whereas
the respective one with E445 for RNAP-1 complex is very strong and of acid-amine salt character
(donor-acceptor distance 2.57 Å, angle 176°, Fig. 9b), the significant differences in antibacterial
properties between 1 and 3-13 are understandable. For recently obtained alkyl analogues
containing terminal hydroxyls9 the flexible aliphatic chains with OH group will be rather directed
outside and probably solvated by water molecules than bonded to E445 via a definitely weaker
interaction of different character alkylCH2O-H…‾Ocarboxylate(E445) than that of salt character
postulated for 1 [piperazineN(40)+-H…‾Ocarboxylate(E445)]. Thus, the rigidity of the moiety at C-3
position which acts as proton donor is also an important factor which influences additionally
formation of intermolecular hydrogen bond with E445. For 1 spatial fit and intermolecular
bonding with E445 of RNAP contributes to exposition of hydrophobic part of the piperazine ring
to external environment which additionally protects this structural part of the antibiotic against
solvation process. It should be mentioned here that E445 at the binding site is unsusceptible to the
mutation processes in bacteria which is explained by too far-reached structural changes in RNAP
resulting in its complete inactivation.5,17,18 If a bulky substituent such as a phenyl ring or a
substituted with halogen phenyl ring, cannot take part in analogous interaction with E445 of salt
character (Fig. 10a), it is directed outside and in consequence destabilises rather than stabilizes
the inhibitor-RNAP complex (Fig. 10b). As shown by the calculated structures (Figs. 9 and 10)
after bonding of 1 or 12 to bacterial RNAP the intramolecular O(8)…H-O(1) hydrogen bonds (for
1 and 12), N(2)-H…N=C(38) bond for 1 and N(38)+-H…O=C(15) bond for 12 are conserved in
relation to the respective ones formed for 1 and 12 molecules in solution. The weak π-π stacking
interaction between electrons of diene and phenyl ring realised in the structure of 12 in solution,
is broken with the formation of 12-RNAP complex.
Conclusions
Reductive amination of 3-formylrifamycin SV toward efficient synthesis of analogues 3-13 has
been optimised. On the basis of FT-IR and 1H, 13C and 15N and 2D NMR studies, in solution the
presence of zwitterionic forms of 1 and its 3-13 analogues with the proton transferred from
O(8)H phenol group to tertiary and secondary nitrogen atom of amine moieties, respectively, has
been evidenced. The zwitterionic forms of 1 and 3-13 are stabilised via several intramolecular
hydrogen bonds of which the one O(1)-H…O(8), formed in a six membered system, is the

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strongest. 1H-1H NOESY studies on conformation of the new amino analogues 3-13 revealed two
different orientations in solution of the substituent at C(3) atom. In solution in 3-9 benzyl
analogues the substituent is distanced away from the ansa-bridge whereas in the structure of 10-
13 phenethyl analogues the phenyl ring of the substituent at C(3) is involved in π-π interactions
with the diene moiety of the ansa-bridge. The activity level of analogues 3-13, comparable to
those of other antibiotics as e.g. ciprofloxacin (especially against S. epidermidis strains) and the
presence of C(38)-N bond resistant to hydrolysis by bacteria, make this type of derivatives
promising for further search to enhance the biological properties. Taking into account the
presence of zwitterionic forms of 1, 3-13 and other earlier reported derivatives9 in protic and
aprotic solvents with addition of water and comparison of their biological data with the results of
respective molecular recognition studies, it has been revealed that in effective RNAP inhibition
the strongest intermolecular hydrogen bond of salt character between protonated amine moiety of
the substituent at C(3) and E445 plays a very important role. The calculated interactions between
the ansa-bridge of the antibiotics and the enzyme suggests that this structural fragment is
relatively flexible and is bonded similarly both for 1 and for its analogues, irrespectively of the
structure substituent at C-3 atom (o-, m-, p- substitution). New model of inhibition of bacterial
RNAP proposed here is in agreement with our earlier structural spectroscopic, X-ray and
antibacterial studies9,10 and, in contrast to earlier proposed model, explains the differences in
antibacterial properties between 1 and its aliphatic9 and aromatic amino analogues. Furthermore,
our results suggested that optimisation of biological activity of this group of antibiotics should be
performed via introducing into chain at C-3 position additional secondary or tertiary amines in a
relatively rigid moiety, which will facilitate strong interaction of salt character with E445. This
issue is currently under investigation by us and will be reported later.
Experimental
General
DMSO-d6, CDCl3, DMSO, and CHCl3 for spectroscopic measurements as well as benzylamine,
2-chlorobenzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 2-fluorobenzylamine, 3-
fluorobenzylamine, 4-fluorobenzylamine, phenethylamine, 2-fluorophenethylamine, 3-
fluorophenethylamine, 4-fluorophenethylamine used for syntheses of new rifampicin analogues

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were purchased from Aldrich. The reductants NaBH4, NaBH3CN, NaBH(CH3COO)3 and
BH3CN‾ on polymer support and ZnCl2 were purchased from Aldrich.
The 1H, 13C and 15N measurements of 1-13 were performed in CDCl3 and DMSO-d6 using Varian
Mercury 400 MHz and Bruker Avance 600 MHz spectrometers. The operating frequencies for 1H
measurements were 400.075 and 600.08 MHz; pulse width corresponding to the flip angle of 450;
spectral width, swh = 9842.5 Hz; acquisition time at= 0.2 sec; relaxation delay d1=1.0 s; T =
293.0 K, TMS was used as the internal standard. No window function or zero filling were used.
13
Digital resolution was 0.2 Hz/point. C NMR spectra were recorded at the operating frequency
150.454 MHz; pulse width corresponding to the flip angle of 600; sw = 19000 Hz; at = 1.8 s;
d1=1.0 s; T = 293.0 K and TMS as the internal standard. Line broadening parameters of 0.5 or 1
Hz were applied. 1H, 13C and 15N NMR resonances were unambiguously assigned on the basis of
the HMBC, HSQC, COSY and NOESY correlation spectra.
The FT-IR spectra of 1-13 compounds were recorded in KBr pellet (1.5 mg/200 mg) and DMSO
(0.05 mol dm-3) whereas the spectrum of 1:1 1-KOH mixture was measured in CHCl3. All FT-IR
spectra were recorded with an IFS 113v FT-IR spectrophotometer (Bruker, Karlsruhe) equipped
with a DTGS detector; resolution 2 cm–1, NSS = 125, range 4000-400 cm-1. The Happ-Genzel
apodization function was used.
The HR-MALDI-TOF spectra of 2 and 3-13 analogues were obtained on Water/Micromass
(Manchester, UK) Q-TOF Premier mass spectrometer (software MassLynx V4.1, Manchester,
UK) fitted with a 200 Hz repetition rate Nd/YAG (λ = 355 nm, power density 107 W/cm2). The
compounds analysed were solids and the matrix used was DHB.
The elemental analysis of new rifampicin analogues 3-13 and 2 was carried out on Vario ELIII
(Elementar, Germany).
PM5 calculations of amino analogues of rifampicin structures as well as interactions between 1 or
12 and bacterial RNA polymerase (RNAP) binding site were performed with the use of Cache
WorkSystem Pro version 7.5.0.85 (WinMopac 2007 program).19 The initial model of 1 and 12
structures was built on the basis of the previously determined X-ray structures of zwitterions9 and
X-ray structure of polymerase RNA5 and then optimised via PM5 calculations at the semi-
empirical level of theory. The docking of initially optimised structures of 1 and 12 compounds

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via PM5 method was performed via “dock into active site” function of WinMopac 2007 program.
Docking of inhibitor molecules 1 and 12 was performed on previously reported coordinates of
naphthalene ring of rifampicin, known from X-ray structure of RNAP5. The interactions between
1 molecule, docked as zwitterion, and the aminoacid residues at the binding site to RNAP were
modelled via geometry calculation in MOPAC using PM5 parameters (Cache Work System Pro
Version 7.5.085 – Fujitsu), with the energy gradient not exceeding 2 kcal mol-1 at one step (3678
steps). The interactions between 12 inhibitor molecule, docked as zwitterion, and the aminoacid
residues at the binding site to RNAP were performed via geometry calculation in MOPAC using
PM5 parameters, with the energy gradient not exceeding 2 kcal mol-1 at one step (4122 steps).
Peptide chains of RNAP subunits were locked on α carbon atoms and nitrogen atoms of peptide
bonds and the other part of RNA polymerase at 30 Å distance from the binding site was locked
totally before ligand-enzyme complex calculations. Structures of complexes made by 1 or 12
inhibitors and RNAP were further optimised with the PM5 MOZYME geometry algorithm
suitable for large molecules. In the new postulated model of RNAP inhibition the orientation and
conformation of the key E445 aminoacid residue was conserved as in the X-ray structure of
RNAP5.
Procedure for hydrolysis of rifampicin
To rifampicin (1.06 g, 1.25 mmol) dissolved in diethyl ether (1000 ml), a portion of 250 ml of 0.2
M HCl/H2O was added and the mixture was stirred for four days at room temperature. Then the
organic layer was separated, twice washed with 300 ml of water and evaporated. To crude
rifaldehyde, 150 ml CH2Cl2 was added and the contents were extracted with 100 ml of brine and
separated. Organic layer was evaporated to dryness yielding rifaldehyde (2) as red powder (830.2
mg; Yield: 88.9%).
1
Spectral data for 2: m.p. = 180-185°C; HR-MALDI-TOF [M+H]+ 726.3111; H NMR
(δ ppm in CDCl3): 13.75 (1H, s, NHamide), 12.65 (1H, s, HO-4), 12.30 (1H, s, HO-1), 10.65 (1H,
s, H-38), 6.57 (1H, dd, 3JH18-H19=14.9 Hz, H-18), 6.51 (1H, d, 3JH17-H18=11.4 Hz, H-17), 6.24 (1H,
d, H-29), 6.07 (1H, dd, 3JH19-H20=5.0 Hz, H-19), 4.94 (1H, d, 3JH25-H26=10.0 Hz, H-25), 5.12 (1H,
3
3
dd, JH28-H29=12.7 Hz, H-28), 3.78 (1H, d, JH17-H18=9.5 Hz, H-21), 3.63 (1H, bs, HO-23), 3.55
3
(1H, bs, HO-21), 3.51 (1H, d, JH27-H28=7.0 Hz, H-27), 3.05 (3H, s, H-37), 3.05 (1H, m, H-23),

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2.43 (1H, m, H-20), 2.27 (3H, s, H-14), 2.07 (3H, s, H-30), 2.06 (3H, s, H-36), 1.82 (3H, s, H-
3
3
13), 1.76 (1H, m, H-22), 1.54 (1H, qd, JH24-H33=6.8 Hz, JH23-H24=14.0 Hz, H-24), 1.37 (1H, m,
3
H-26), 1.03 (3H, d, JH22-H32=7.0 Hz, H-32), 0.91 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.67 (3H, d,
3
3JH24-H33=6.9 Hz, H-33), -0.30 (3H, d, JH26-H34=6.9 Hz, H-34); 13C NMR (δ ppm in CDCl3):
196.6 (C-11), 194.1 (C-38), 174.8 (C-8), 172.1 (C-35), 170.4 (C-15), 168.6 (C-6), 156.0 (C-4),
143.7 (C-19), 142.8 (C-29), 137.8 (C-1), 136.9 (C-17), 127.8 (C-16), 122.6 (C-18), 120.6 (C-2),
119.4 (C-28), 119.1 (C-10), 117.4 (C-9), 109.7 (C-3), 109.5 (C-7), 109.2 (C-12), 105.5 (C-5),
77.0 (C-23), 76.5 (C-27), 74.2 (C-25), 70.7 (C-21), 57.1 (C-37), 39.6 (C-26), 38.6 (C-20), 37.6
(C-24), 33.3 (C-22), 21.5 (C-13), 20.7 (C-36), 20.5 (C-30), 16.9 (C-31), 10.9 (C-32), 9.1 (C-34),
1
13
8.6 (C-33), 7.8 (C-14); H and C resonance assignments were confirmed by COSY, HSQC,
HMBC and NOESY 2D correlations; FT-IR (KBr, 1.5 mg): 3415 cm-1 ν(O21-H)+ν(O23-H), 3200
cm-1 ν(O8-H…O1)+ν(O4-H…O11)+ ν(O1-H…O15), 2550 cm-1 ν(N-Hamide), 1726 cm-1 ν(C35=O),
1654 cm-1 ν(C38=O)aldehyde+ ν(C15=O)amide I, 1642 cm-1 ν(C11=O)ketone, 1575 cm-1 ν(C-N)amide II
,
1538 cm-1 ν(C=C)naphthalene, 1464 cm-1 ν(C=C), 1249 cm-1 ν(C-O); Elemental analysis
C38H47NO13: calculated C=62.89%, H=6.53%, N=1.93%; found C=62.81%, H=6.49%, N=1.85%.
6.3. Methods of synthesis of new halogen benzyl and pheneethyl amino analogues of rifampicin
(3-13):
Method 1: Rifaldehyde (2) (290.0 mg, 0.4 mmol) was dissolved in 30 ml CH2Cl2 and p-
chlorobezylamine (0.41 mmol) in 5 ml of C2H5OH with a catalyst (0.2 mmol ZnCl2 or 0.02 mmol
HCl/EtOH) added. The mixtures were stirred at 45°C for half an hour and after that a half of
solvent volume was distilled off. To the cooled reaction mixture (room temperature) the reductant
NaBH4 (6.8 mg, 0.18 mmol) was added portionwise over 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 the respective synthesised analogue of
rifampicin (compound 9) was purified by column chromatography with silica gel (25 cm × 1 cm,
silica gel 60, 0.040-0.063 mm/230-400 mesh ASTM, Fluka) and ethyl acetate/methanol as eluent
(from 100:0 to 15:1). TLC (15:1 ethyl acetate:methanol as eluent) was developed. Compound 9
was obtained as orange solid.
Method 2: Rifaldehyde (2) (290.0 mg, 0.4 mmol) was dissolved in 30 ml CH2Cl2 and the p-
chlorobezylamine (0.41 mmol) in 5 ml of C2H5OH with a catalyst (0.2 mmol ZnCl2 or 0.02 mmol

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HCl/EtOH) added. The 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 mixture (room temperature), the
reductant NaBH(CH3COO)3 (93.3 mg, 0.44 mmol) was added portionwise over 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 the
respective synthesised analogue of rifampicin (compound 9) was purified by column
chromatography with silica gel (25 cm × 1 cm, silica gel 60, 0.040-0.063 mm/230-400 mesh
ASTM, Fluka) and ethyl acetate/methanol as an eluent (from 100:0 to 15:1). TLC (15:1 ethyl
acetate:methanol as eluent) was developed. Compound 9 was obtained as orange solid.
Method 3: Rifaldehyde (2) (290.0 mg, 0.4 mmol) was dissolved in 30 ml CH2Cl2 and the
respective benzyl or phenethyl amine (0.41 mmol) in 5 ml of C2H5OH with a catalyst (0.2 mmol
ZnCl2 or 0.02 mmol HCl/EtOH) added. The 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 mixture (room
temperature), the reductant NaBH3CN (25.1 mg, 0.4 mmol) was added portionwise over 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 the
respective synthesised analogues of rifampicin (compounds 3-13) were purified by column
chromatography with silica gel (25 cm × 1 cm, silica gel 60, 0.040-0.063 mm/230-400 mesh
ASTM, Fluka) and ethyl acetate/methanol as eluent (from 100:0 to 15:1). TLC (15:1 ethyl
acetate:methanol as an eluent) was developed. Compounds 3-13 were obtained as yellow, orange
and red solids.
Method 4: Rifaldehyde (2) (290.0 mg, 0.4 mmol) was dissolved in 30 ml CH2Cl2 and the p-
chlorobezylamine (0.41 mmol) in 5 ml of C2H5OH with a catalyst (0.2 mmol ZnCl2 or 0.02 mmol
HCl/EtOH) added. The 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 mixture (room temperature), the
reductant BH3CN- on polymer support (210.0 mg) was added in one step. The organic layer 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 the respective synthesised analogue of
rifampicin (compound 9) was purified by column chromatography with silica gel (25 cm × 1 cm,
silica gel 60, 0.040-0.063 mm/230-400 mesh ASTM, Fluka) and ethyl acetate/methanol as eluent

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(from 100:0 to 15:1). TLC (15:1 ethyl acetate:methanol as eluent) was developed. Compound 9
was obtained as orange solid.
Spectral data for (3): Yield 57% /method 3 with HCl(EtOH)/; m.p. 134-135 °C; ESI-MS (m/z):
1
817 [M+H]+; HR-MALDI-TOF (m/z) 817.3917 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.02
(1H, bs, OH-1), 12.83 (1H, s, OH-4), 9.27 (1H, s, NHamide), 8.94 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.54 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.49 (2H, m, H-41 + H-45), 7.37 (3H, m, H-
3
3
42 + H-43 + H-44), 6.38 (1H, dd, JH18-H19=15.8 Hz, JH17-H18=10.8 Hz, H-18), 6.26 (1H, d, H-
3
3
17), 6.27 (1H, d, JH28-H29=13.0 Hz, H-29), 6.00 (1H, dd, JH19-H20=7.4 Hz, H-19), 5.06 (1H, d,
3
3JH25-H26=11.0 Hz, H-25), 5.03 (1H, d, JH21-OH=3.1 Hz, OH-21), 4.90 (1H, dd, H-28), 4.24 (2H,
m, H-39), 4.20 (1H, m, H-38a), 3.95 (1H, d, 3JH23-HO=8.2 Hz, OH-23), 3.58 (1H, m, H-38b), 3.56
3
3
(1H, m, H-21), 3.23 (1H, d, JH27-H28=8.9 Hz, H-27), 2.88 (3H, s, H-37), 2.80 (1H, dd, JH22-
H23=8.4 Hz, 3JH23-H24=9.4 Hz, H-23), 2.24 (1H, m, H-20), 1.97 (3H, s, H-36), 1.94 (3H, s, H-30),
1.92 (3H, s, H-14), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.22 (1H, m, H-24), 0.95 (1H, m, H-
26), 0.89 (3H, d, 3JH22-H32=7.0 Hz, H-32), 0.78 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.39 (3H, d, 3JH24-
13
H33=7.0 Hz, H-33), -0.37 (3H, d, 3JH26-H34=7.0 Hz, H-34); C NMR (δ ppm in DMSO-d6): 185.3
(C-11), 184.2 (C-8), 171.8 (C-6), 170.3 (C-15), 169.4 (C-35), 148.7 (C-1), 145.0 (C-4), 143.1 (C-
29), 139.2 (C-19), 131.8 (C-17) , 131.4 (C-40), 131.0 (C-16), 129.8 (C-41 + C-45), 129.0 (C-43),
128.7 (C-44), 128.4 (C-42), 126.0 (C-18), 118.0 (C-2), 117.6 (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), 76.2 (C-27), 75.7 (C-23), 73.1 (C-25), 72.6
(C-21), 55.6 (C-37), 50.2 (C-39), 42.1 (C-38), 40.2 (C-26), 38.1 (C-24), 37.8 (C-20), 32.6 (C-22),
22.1 (C-13), 20.6 (C-36), 19.9 (C-30), 18.2 (C-31), 11.1 (C-32), 9.0 (C-34), 8.6 (C-33), 7.4 (C-
1
14); 15N NMR (δ ppm in DMSO-d6): -255 (N-2), -329 (N-38); H, 13C and 15N resonance
assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr
pellet): 3432 and 3401 cm-1 ν(O21-H) + ν(O23-H), 3344 cm-1 ν(N-H)amide, 3143 cm-1 ν(O4-
+
H···O11), 2740 cm-1 ν(N38 -H···O15), 2468 cm-1 ν(O1-H···O8‾), 1724 cm-1 ν(C35=O), 1646 cm-1
ν(C15=O)amide I, 1597 cm-1 ν (C=C)naphthtalene, 1562 cm-1 δ (N-H)amide II, 1543 cm-1 ν(C=C), 1457
and 1444 cm-1 ν(C=C)diene, 1247 and 1239 cm-1 ν(C-O); Elemental analysis (%): Calcd. for
C45H56N2O12: C, 66.16; H, 6.91, N, 3.43; Found: C, 66.22; H, 6.87, N, 3.49.
Spectral data for (4): Yield 54% /method 3 with HCl(EtOH)/; m.p. 212-213 °C; ESI-MS (m/z):
1
835 [M+H]+; HR-MALDI-TOF (m/z) 835.3818 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.04

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(1H, bs, OH-1), 12.85 (1H, s, OH-4), 9.29 (1H, s, NHamide), 9.01 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.53 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.61 (1H, dt, 3JH44-H45=8.8 Hz, 3JH45-F=5.5 Hz,
H-45), 7.46 (1H, tdd, 3JH42-H43=7.7 Hz, 3JH43-H44=7.7 Hz, 3JH43-F=5.6 Hz, 4JH43-H45=1.8 Hz, H-43),
3
3
7.23 (1H, m, H-44), 7.21 (1H, m, H-42), 6.40 (1H, dd, JH18-H19=15.9 Hz, JH17-H18=10.8 Hz, H-
18), 6.25 (1H, d, 3JH28-H29=12.9 Hz, H-29), 6.22 (1H, d, H-17), 6.03 (1H, dd, 3JH19-H20=7.2 Hz, H-
3
3
19), 5.08 (1H, d, JH25-H26=11.0 Hz, H-25), 5.03 (1H, d, JH21-HO=3.1 Hz, OH-21), 4.91 (1H, dd,
H-28), 4.30 (2H, m, H-39), 4.27 (1H, m, H-38a), 3.98 (1H, d, 3JH23-HO=8.6 Hz, OH-23), 3.65 (1H,
3
m, H-21), 3.61 (1H, m, H-38b), 3.24 (1H, d, JH27-H28=8.7 Hz, H-27), 2.89 (3H, s, H-37), 2.83
(1H, m, H-23), 2.26 (1H, m, H-20), 1.98 (3H, s, H-36), 1.92 (3H, s, H-30), 1.89 (3H, s, H-14),
1.70 (1H, m, H-22), 1.65 (3H, s, H-13), 1.22 (1H, m, H-24), 0.97 (1H, m, H-26), 0.91 (3H, d,
3
3JH22-H32=7.2 Hz, H-32), 0.86 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.45 (3H, d, JH24-H33=7.0 Hz, H-
3
13
33), -0.36 (3H, d, JH26-H34=6.8 Hz, H-34); C NMR (δ ppm in DMSO-d6): 185.3 (C-11), 184.1
2
(C-8), 171.8 (C-6), 170.2 (C-15), 169.3 (C-35), 160.8 (d, JC-F=247.0 Hz, C-41), 148.6 (C-1),
3
145.0 (C-4), 143.0 (C-29), 139.0 (C-19), 132.1 (bs, C-45), 131.9 (C-17), 131.8 (d, JC-F=8.4 Hz,
C-43), 130.7 (C-16), 125.8 (C-18), 124.8 (bs, C-44), 118.4 (d, 2JC-F=14.4 Hz, C-40), 117.9 (C-2),
2
117.6 (C-28), 116.4 (C-10), 115.5 (d, JC-F=20.9 Hz, C-42), 115.1 (C-3 + C-9), 108.8 (C-12),
101.3 (C-7), 98.5 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25), 72.6 (C-21), 55.6 (C-37), 43.4 (C-
39), 42.3 (C-38), 40.2 (C-26), 38.2 (C-24), 37.8 (C-20), 32.6 (C-22), 22.0 (C-13), 20.7 (C-36),
15
19.8 (C-30), 18.1 (C-31), 11.0 (C-32), 8.9 (C-34), 8.5 (C-33), 7.3 (C-14); N NMR (δ ppm in
1
13
15
DMSO-d6): -252 (N-2), -333 (N-38); H, C and N resonance assignments were confirmed by
COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr pellet): 3435 and 3408 cm-1 ν(O21-
+
H) + ν(O23-H), 3352 cm-1 ν(N-H)amide, 3151 cm-1 ν(O4-H···O11), 2746 cm-1 ν(N38 -H···O15), 2465
cm-1 ν(O1-H···O8‾), 1721 cm-1 ν(C35=O), 1641 cm-1 ν(C15=O)amide I, 1601 cm-1 ν(C=C)naphthalene
,
1567 cm-1 δ(N-H)amide II, 1541 cm-1 ν(C=C)diene, 1459 and 1443 cm-1 ν(C=C), 1243 and 1231 cm-1
ν(C-O); Elemental analysis (%): Calcd. for C45H55FN2O12: C, 64.73; H, 6.64, N, 3.36; Found: C,
64.80; H, 6.58, N, 3.32.
Spectral data for (5): Yield 59% /method 3 with HCl(EtOH)/; m.p. 189-190 °C; ESI-MS (m/z):
1
835 [M+H]+; HR-MALDI-TOF (m/z) 835.3805 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.02
(1H, bs, OH-1), 12.85 (1H, s, OH-4), 9.26 (1H, s, NHamide), 9.03 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.52 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.39 (1H, m, H-44), 7.37 (1H, m, H-45), 7.33
3
3
(1H, m, H-41), 7.20 (1H, m, H-43), 6.32 (1H, dd, JH18-H19=15.6 Hz, JH17-H18=11.0 Hz, H-18),

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6.24 (1H, d, H-17), 6.26 (1H, d, 3JH28-H29=13.0 Hz, H-29), 6.00 (1H, dd, 3JH19-H20=7.4 Hz, H-19),
5.06 (1H, d, 3JH25-H26=11.0 Hz, H-25), 5.03 (1H, d, 3JH21-HO=3.1 Hz, OH-21), 4.90 (1H, dd, H-28),
4.26 (2H, m, H-39), 4.20 (1H, m, H-38a), 3.92 (1H, d, 3JH23-HO=8.2 Hz, OH-23), 3.56 (1H, m, H-
21), 3.51 (1H, m, H-38b), 3.23 (1H, d, 3JH27-H28=8.9 Hz, H-27), 2.88 (3H, s, H-37), 2.80 (1H, dd,
3JH22-H23=8.4 Hz, 3JH23-H24=9.4 Hz, H-23), 2.22 (1H, m, H-20), 1.98 (3H, s, H-36), 1.93 (3H, s, H-
30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.61 (1H, m, H-22), 1.21 (1H, m, H-24), 0.96 (1H, m,
3
H-26), 0.89 (3H, d, JH22-H32=7.0 Hz, H-32), 0.75 (3H, d, 3JH20-H31=6.8 Hz, H-31), 0.41 (3H, d,
3
13
3JH24-H33=6.7 Hz, H-33), -0.38 (3H, d, JH26-H34=6.7 Hz, H-34); C NMR (δ ppm in DMSO-d6):
185.3 (C-11), 184.2 (C-8), 171.8 (C-6), 170.2 (C-15), 169.4 (C-35), 162.0 (d, 1JC-F=244.8 Hz, C-
42), 148.7 (C-1), 145.1 (C-4), 143.2 (C-29), 139.0 (C-19), 133.9 (d, 3JC-F=8.0 Hz, C-40), 131.9
(C-17), 130.9 (C-16), 130.8 (d, 3JC-F=8.2 Hz, C-44), 126.1 (d, 4JC-F=2.9 Hz, C-45), 125.9 (C-18),
118.0 (C-2), 117.6 (C-28), 116.8 (d, 2JC-F=21.9 Hz, C-41), 116.5 (C-10), 115.9 (d, 2JC-F=19.8 Hz,
C-43), 115.2 (C-9), 114.8 (C-3), 108.9 (C-12), 101.4 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23),
73.2 (C-25), 72.7 (C-21), 55.7 (C-37), 49.6 (C-39), 42.0 (C-38), 40.3 (C-26), 38.2 (C-24), 37.7
(C-20), 32.6 (C-22), 22.1 (C-13), 20.7 (C-36), 19.9 (C-30), 18.2 (C-31), 11.0 (C-32), 9.1 (C-34),
8.7 (C-33), 7.4 (C-14); 15N NMR (δ ppm in DMSO-d6): -252 (N-2), -334 (N-38); 1H, 13C and 15N
resonance assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-
IR (KBr pellet): 3439 and 3413 cm-1 ν(O21-H) + ν(O23-H), 3339 cm-1 ν(N-H)amide, 3154 cm-1
ν(O4-H···O11), 2751 cm-1 ν(N38+-H···O15), 2461 cm-1 ν(O1-H···O8‾), 1719 cm-1 ν(C35=O), 1639
cm-1 ν(C15=O)amide I, 1598 cm-1 ν(C=C)naphthalene,1561 cm-1 δ(N-H)amide II, 1539 cm-1 ν(C=C), 1451
and 1438 cm-1 ν(C=C), 1249 and 1232 cm-1 ν(C-O); Elemental analysis (%): Calcd. for
C45H55FN2O12: C, 64.73; H, 6.64, N, 3.36; Found: C, 64.66; H, 6.69, N, 3.44.
Spectral data for (6): Yield 56% /method 3 with HCl(EtOH)/; 169-171 °C; ESI-MS (m/z): 835
1
[M+H]+; HR-MALDI-TOF (m/z) 835.3821 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.03 (1H,
bs, OH-1), 12.84 (1H, s, OH-4), 9.26 (1H, s, NHamide), 8.95 (1H, vbs, N+Hintramolecularly hydrogen
3
bonded), 8.45 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.55 (2H, dd, 3JH41-H42/H44-H45=8.8 Hz, JH41-
F/H45-F=5.5 Hz, H-41 + H-45), 7.39 (2H, t, 2JH42-F/H44-F=8.6 Hz, H-42 + H-44), 6.32 (1H, dd, 3JH18-
H19=15.2 Hz, 3JH17-H18=10.7 Hz, H-18), 6.26 (1H, d, 3JH28-H29=12.8 Hz, H-29), 6.25 (1H, d, H-17),
3
3
3
6.02 (1H, dd, JH19-H20=7.5 Hz, H-19), 5.07 (1H, d, JH25-H26=11.0 Hz, H-25), 5.03 (1H, d, JH21-
HO=3.1 Hz, OH-21), 4.90 (1H, dd, H-28), 4.24 (2H, m, H-39), 4.19 (1H, d, =12.0 Hz, H-38a),
3
3.99 (1H, d, JH23-HO=8.7 Hz, OH-23), 3.57 (1H, m, H-21), 3.50 (1H, d, H-38b), 3.24 (1H, d,

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3JH27-H28=8.9 Hz, H-27), 2.88 (3H, s, H-37), 2.81 (1H, m, H-23), 2.24 (1H, m, H-20), 1.98 (3H, s,
H-36), 1.92 (6H, s, H-14 + H-30), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.22 (1H, m, H-24),
3
0.96 (1H, m, H-26), 0.90 (3H, d, JH22-H32=7.0 Hz, H-32), 0.78 (3H, d, 3JH20-H31=7.0 Hz, H-31),
3
3
13
0.43 (3H, d, JH24-H33=6.7 Hz, H-33), -0.37 (3H, d, JH26-H34=6.7 Hz, H-34); C NMR (δ ppm in
1
DMSO-d6): 185.5 (C-11), 184.2 (C-8), 171.9 (C-6), 170.4 (C-15), 169.4 (C-35), 162.4 (d, JC-
F=245.4 Hz, C-43), 148.7 (C-1), 145.1 (C-4), 143.1 (C-29), 139.0 (C-19), 132.3 (d, 3JC-F=8.2 Hz,
C-41 + C-45), 131.9 (C-17), 130.9 (C-16), 127.6 (d, 4JC-F=2.2 Hz, C-40), 125.9 (C-18), 118.0 (C-
2), 117.6 (C-28), 116.5 (C-10), 115.6 (d, 2JC-F=21.7 Hz, C-42 + C-44), 115.2 (C-9), 114.8 (C-3),
108.8 (C-12), 101.5 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25), 72.7 (C-21), 55.6
(C-37), 49.4 (C-39), 41.8 (C-38), 40.2 (C-26), 38.2 (C-24), 37.7 (C-20), 32.6 (C-22), 22.1 (C-13),
20.7 (C-36), 19.9 (C-30), 18.1 (C-31), 11.0 (C-32), 9.0 (C-34), 8.6 (C-33), 7.4 (C-14); 15N NMR
1
(δ ppm in DMSO-d6): -253 (N-2), -332 (N-38); H, 13C and 15N resonance assignments were
confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr pellet): 3429 and
3410 cm-1 ν(O21-H) + ν(O23-H), 3337 cm-1 ν(N-H)amide, 3148 cm-1 ν(O4-H···O11), 2744 cm-1
+
ν(N38 -H···O15), 2457 cm-1 ν(O1-H···O8‾), 1720 cm-1 ν(C35=O), 1644 cm-1 ν(C15=O)amide I, 1596
cm-1 ν(C=C)naphthalene, 1559 cm-1 δ(N-H)amide II, 1542 cm-1 ν(C=C), 1449 and 1435 cm-1 ν(C=C),
1251 and 1236 cm-1 ν(C-O); Elemental analysis (%): Calcd. for C45H55FN2O15: C, 64.73; H, 6.64,
N, 3.36; Found: C, 64.80; H, 6.61, N, 3.41.
Spectral data for (7): Yield 51% /method 3 with HCl(EtOH)/; 165-167 °C; ESI-MS (m/z): 835
1
[M+H]+; HR-MALDI-TOF (m/z) 851.3522 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.02 (1H,
bs, OH-1), 12.86 (1H, s, OH-4), 9.30 (1H, s, NHamide), 9.00 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.68 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.68 (1H, dd, 3JH42-H43=7.1 Hz, H-42), 7.49
3
4
3
(1H, dd, JH44-H45=7.5 Hz, JH43-H45=1.9 Hz,H-45), 7.42 (1H, td, JH43-H44=7.5 Hz H-43), 7.37
3
3
3
(1H, td, H-44), 6.38 (1H, dd, JH18-H19=16.1 Hz, JH17-H18=10.8 Hz, H-18), 6.25 (1H, d, JH28-
H29=12.9 Hz, H-29), 6.21 (1H, d, H-17), 6.02 (1H, dd, 3JH19-H20=7.4 Hz, H-19), 5.08 (1H, d, 3JH25-
H26=10.8 Hz, H-25), 5.03 (1H, d, 3JH21-HO=3.1 Hz, OH-21), 4.91 (1H, dd, H-28), 4.38 (2H, m, H-
3
39), 4.30 (1H, d, =11.9 Hz, H-38a), 4.00 (1H, d, JH23-HO=8.7 Hz, OH-23), 3.65 (1H, m, H-21),
3
3.61 (1H, m, H-38b), 3.24 (1H, d, JH27-H28=8.7 Hz, H-27), 2.89 (3H, s, H-37), 2.82 (1H, dd,
3JH22-H23=8.4 Hz, 3JH23-H24=9.4 Hz, H-23), 2.25 (1H, m, H-20), 1.98 (3H, s, H-36), 1.92 (3H, s, H-
30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.22 (1H, m, H-24), 0.98 (1H, m,
3
H-26), 0.91 (3H, d, JH22-H32=7.0 Hz, H-32), 0.85 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.46 (3H, d,

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3
13
3JH24-H33=7.0 Hz, H-33), -0.37 (3H, d, JH26-H34=6.8 Hz, H-34); C NMR (δ ppm in DMSO-d6):
185.5 (C-11), 184.2 (C-8), 171.9 (C-6), 170.3 (C-15), 169.5 (C-35), 148.7 (C-1), 145.1 (C-4),
143.1 (C-29), 139.1 (C-19), 134.1 (C-40), 132.3 (C-41), 132.1 (C-17), 131.2 (C-16), 130.6 (C-
45), 129.7 (C-42), 129.3 (C-43), 127.8 (C-44), 125.8 (C-18), 118.0 (C-2), 117.6 (C-28), 116.5 (C-
10), 115.2 (C-9), 114.6 (C-3), 108.9 (C-12), 101.5 (C-7), 98.6 (C-5), 76.3 (C-27), 75.8 (C-23),
73.2 (C-25), 72.6 (C-21), 55.7 (C-37), 47.4 (C-39), 42.4 (C-38), 40.2 (C-26), 38.2 (C-24), 37.9
(C-20), 32.6 (C-22), 22.1 (C-13), 20.7 (C-36), 19.9 (C-30), 18.3 (C-31), 11.0 (C-32), 9.0 (C-34),
8.7 (C-33), 7.4 (C-14); 15N NMR (δ ppm in DMSO-d6): -254 (N-2), -334 (N-38); 1H, 13C and 15N
resonance assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-
IR (KBr pellet): 3432 and 3414 cm-1 ν(O21-H) + ν(O23-H), 3346 cm-1 ν(N-H)amide, 3153 cm-1
+
ν(O4-H···O11), 2741 cm-1 ν(N38 -H···O15), 2452 cm-1 ν(O1-H···O8‾), 1724 cm-1 ν(C35=O), 1649
cm-1 ν(C15=O)amide I, 1599 cm-1 ν(C=C)naphthalene, 1563 cm-1 δ(N-H)amide II, 1544 cm-1 ν(C=C), 1446
and 1432 cm-1 ν(C=C), 1241 and 1232 cm-1 ν(C-O); Elemental analysis (%): Calcd. for
C45H55ClN2O15: C, 63.48; H, 6.51, N, 3.29; Found: C, 63.53; H, 6.46, N, 3.22.
Spectral data for (8): Yield 58% /method 3 with HCl(EtOH)/; m.p. 152-154 °C; ESI-MS (m/z):
1
851 [M+H]+; HR-MALDI-TOF (m/z) 851.3510 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.03
(1H, bs, OH-1), 12.85 (1H, s, OH-4), 9.25 (1H, s, NHamide), 9.02 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.56 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.62 (1H, bs, H-41), 7.47 (1H, m, H-43), 7.39
3
3
(1H, m, H-44), 7.43 (1H, m, H-45), 6.33 (1H, dd, JH18-H19=15.7 Hz, H-18), 6.26 (1H, d, JH17-
H18=10.6 Hz, H-17), 6.23 (1H, d, 3JH28-H29=12.9 Hz, H-29), 6.02 (1H, dd, 3JH19-H20=7.6 Hz, H-19),
5.06 (1H, d, 3JH25-H26=10.5 Hz, H-25), 5.03 (1H, d, 3JH21-HO=3.1 Hz, OH-21), 4.91 (1H, dd, H-28),
4.26 (2H, m, H-39), 4.22 (1H, m, H-38a), 3.97 (1H, d, 3JH23-HO=6.2 Hz, OH-23), 3.56 (1H, m, H-
21), 3.54 (1H, m, H-38b), 3.24 (1H, d, 3JH27-H28=8.5 Hz, H-27), 2.88 (3H, s, H-37), 2.81 (1H, dd,
3JH22-H23=8.2 Hz, 3JH23-H24=8.6 Hz, H-23), 2.27 (1H, m, H-20), 1.98 (3H, s, H-36), 1.95 (3H, s, H-
30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.19 (1H, m, H-24), 0.97 (1H, m,
3
H-26), 0.90 (3H, d, JH22-H32=7.0 Hz, H-32), 0.77 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.43 (3H, d,
3
13
3JH24-H33=7.0 Hz, H-33), -0.36 (3H, d, JH26-H34=6.8 Hz, H-34); C NMR (δ ppm in DMSO-d6):
185.3 (C-11), 184.1 (C-8), 171.8 (C-6), 170.2 (C-15), 169.3 (C-35), 148.7 (C-1), 145.0 (C-4),
143.1 (C-29), 138.9 (C-19), 133.7 (C-40), 133.3 (C-42), 131.8 (C-17), 130.9 (C-16), 130.5 (C-
44), 129.9 (C-41), 129.0 (C-45), 128.7 (C-43), 125.9 (C-18), 118.0 (C-2), 117.7 (C-28), 116.4 (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),

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73.2 (C-25), 72.8 (C-21), 55.6 (C-37), 49.5 (C-39), 42.1 (C-38), 40.2 (C-26), 38.1 (C-24), 37.7
(C-20), 32.6 (C-22), 22.0 (C-13), 20.6 (C-36), 19.8 (C-30), 18.2 (C-31), 11.0 (C-32), 9.0 (C-34),
8.6 (C-33), 7.3 (C-14); 15N NMR (δ ppm in DMSO-d6): -251 (N-2), -330 (N-38); 1H, 13C and 15N
resonance assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-
IR (KBr pellet): 3437 and 3396 cm-1 ν(O21-H) + ν(O23-H), 3338 cm-1 ν(N-H)amide, 3150 cm-1
ν(O4-H···O11), 2733 cm-1 ν(N38+-H···O15), 2460 cm-1 ν(O1-H···O8‾), 1721 cm-1 ν(C35=O), 1641
cm-1 ν(C15=O)amide I, 1600 cm-1 ν(C=C)naphthalene, 1558 cm-1 δ(N-H)amide II, 1543 cm-1 ν(C=C), 1452
and 1440 cm-1 ν(C=C), 1241 and 1229 cm-1 ν(C-O); Elemental analysis (%): Calcd. for
C45H55ClN2O12: C, 63.48; H, 6.51, N, 3.29; Found: C, 63.41; H, 6.47, N, 3.26.
Spectral data for (9): Yield 50% /method 3 with HCl(EtOH)/; m.p. 148-150 °C; ESI-MS (m/z):
1
851 [M+H]+; HR-MALDI-TOF (m/z) 851.3514 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.01
(1H, bs, OH-1), 12.84 (1H, s, OH-4), 9.24 (1H, s, NHamide), 9.00 (1H, vbs, N+Hintramolecularly hydrogen
3
bonded), 8.43 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.40 (2H, d, JH41-H42/H44-H45=8.3 Hz, H-41 +
H-45), 7.40 (2H, d, H-44), 6.30 (1H, dd, 3JH18-H19=14.9 Hz, 3JH17-H18=10.8 Hz, H-18), 6.26 (1H, d,
3JH28-H29=12.9 Hz, H-29), 6.24 (1H, d, H-17), 6.01 (1H, dd, 3JH19-H20=7.1 Hz, H-19), 5.06 (1H, d,
3
3JH25-H26=10.2 Hz, H-25), 5.03 (1H, d, JH21-HO=3.1 Hz, OH-21), 4.90 (1H, dd, H-28), 4.24 (2H,
2
3
m, H-39), 4.19 (1H, d, J=12.1 Hz, H-38a), 3.95 (1H, d, JH23-HO=8.2 Hz, OH-23), 3.57 (1H, m,
3
H-38b), 3.56 (1H, m, H-21), 3.24 (1H, d, JH27-H28=8.8 Hz, H-27), 2.88 (3H, s, H-37), 2.81 (1H,
dd, 3JH22-H23=8.9 Hz, 3JH23-H24=9.5 Hz, H-23), 2.24 (1H, m, H-20), 1.98 (3H, s, H-36), 1.92 (3H, s,
H-30), 1.91 (3H, s, H-14), 1.65 (3H, s, H-13), 1.62 (1H, m, H-22), 1.18 (1H, m, H-24), 0.97 (1H,
m, H-26), 0.90 (3H, d, 3JH22-H32=7.1 Hz, H-32), 0.78 (3H, d, 3JH20-H31=7.1 Hz, H-31), 0.43 (3H, d,
3
13
3JH24-H33=6.8 Hz, H-33), -0.38 (3H, d, JH26-H34=6.8 Hz, H-34); C NMR (δ ppm in DMSO-d6):
185.4 (C-11), 184.2 (C-8), 171.8 (C-6), 170.1 (C-15), 169.4 (C-35), 148.7 (C-1), 145.1 (C-4),
143.2 (C-29), 139.0 (C-19), 132.0 (C-40), 131.9 (C-17), 130.8 (C-16), 130.3 (C-41 + C-45),
128.7 (C-42 + C-44), 125.9 (C-18), 123.9 (C-43), 118.0 (C-2), 117.6 (C-28), 116.5 (C-10), 115.1
(C-9), 114.8 (C-3), 108.9 (C-12), 101.4 (C-7), 98.6 (C-5), 76.3 (C-27), 75.7 (C-23), 73.2 (C-25),
72.6 (C-21), 55.7 (C-37), 49.4 (C-39), 41.8 (C-38), 40.3 (C-26), 38.2 (C-24), 37.7 (C-20), 32.6
(C-22), 22.1 (C-13), 20.7 (C-36), 19.9 (C-30), 18.1 (C-31), 11.0 (C-32), 9.1 (C-34), 8.7 (C-33),
1
7.4 (C-14); 15N NMR (δ ppm in DMSO-d6): -253 (N-2), -331 (N-38); H, 13C and 15N resonance
assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr
pellet): 3437 and 3396 cm-1 ν(O21-H) + ν(O23-H), 3338 cm-1 ν(N-H)amide, 3150 cm-1 ν(O4-

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H···O11), 2733 cm-1 ν(N38+-H···O15), 2460 cm-1 ν(O1-H···O8‾), 1721 cm-1 ν(C35=O), 1641 cm-1
ν(C15=O)amide I, 1600 cm-1 ν(C=C)naphahlene, 1558 cm-1 δ(N-H)amide II, 1543 cm-1 ν(C=C), 1452 and
1440 cm-1 ν(C=C), 1241 and 1229 cm-1 ν(C-O); Elemental analysis (%): Calcd. for
C45H55ClN2O15: C, 63.48; H, 6.51, N, 3.29; Found: C, 63.57; H, 6.44, N, 3.21.
Spectral data for (10): Yield 59% /method 3 with HCl(EtOH)/; m.p. 134-136 °C; ESI-MS (m/z):
1
831 [M+H]+; HR-MALDI-TOF (m/z) 851.4067 [M+H]+; H NMR (δ ppm in DMSO-d6): 16.05
(1H, bs, OH-1), 12.83 (1H, s, OH-4), 9.31 (1H, s, NHamide), 8.59 (1H, vbs, N+Hintramolecularly hydrogen
bonded), 8.36 (1H, vbs, N+Hhydrogen bonded with the solvent), 7.32 (2H, m, H-43 + H-45), 7.24 (3H, m, H-
3
3
42 + H-44 + H-46), 6.61 (1H, dd, JH18-H19=15.9 Hz, JH17-H18=10.8 Hz, H-18), 6.34 (1H, d, H-
3
3
17), 6.25 (1H, d, JH28-H29=12.9 Hz, H-29), 6.09 (1H, dd, JH19-H20=7.9 Hz, H-19), 5.08 (1H, d,
3JH25-H26=11.1 Hz, H-25), 5.03 (1H, d, 3JH21-HO=3.1 Hz, OH-21), 4.91 (1H, dd, H-28), 4.35 (1H, d,
3
2J=12.4 Hz, H-38a), 3.94 (1H, d, JH23-HO=7.6 Hz, OH-23), 3.69 (1H, m, H-21), 3.73 (1H, d, H-
38b), 3.22 (1H, d, 3JH27-H28=9.1 Hz, H-27), 3.18 (1H, m, H-39a), 2.94 (2H, m, H-40), 2.89 (3H, s,
3
3
H-37), 2.83 (1H, dd, JH22-H23=8.8 Hz, JH23-H24=9.0 Hz, H-23), 2.28 (1H, m, H-20), 1.98 (3H, s,
H-36), 1.97 (3H, s, H-30), 1.91 (3H, s, H-14), 1.68 (1H, m, H-22), 1.65 (3H, s, H-13), 1.26 (1H,
m, H-24), 0.96 (1H, m, H-26), 0.91 (3H, d, 3JH22-H32=7.1 Hz, H-32), 0.86 (3H, d, 3JH20-H31=7.1 Hz,
3
3
13
H-31), 0.51 (3H, d, JH24-H33=6.8 Hz, H-33), -0.33 (3H, d, JH26-H34=6.8 Hz, H-34); C NMR (δ
ppm in DMSO-d6): 185.3 (C-11), 184.2 (C-8), 171.8 (C-6), 170.2 (C-15), 169.4 (C-35), 148.9 (C-
1), 145.0 (C-4), 143.1 (C-29), 139.1 (C-19), 137.0 (C-41), 131.6 (C-17), 131.4 (C-16), 128.7 (C-
43 + C-45), 128.6 (C-42 + C-46), 126.8 (C-44), 126.2 (C-18), 118.0 (C-2), 117.7 (C-28), 116.7
(C-10), 115.1 (C-9), 114.8 (C-3), 108.8 (C-12), 101.3 (C-7), 98.6 (C-5), 76.3 (C-27), 75.8 (C-23),
73.2 (C-25), 72.8 (C-21), 55.6 (C-37), 48.1 (C-39), 43.0 (C-38), 40.3 (C-26), 38.1 (C-24), 37.9
(C-20), 32.7 (C-22), 31.7 (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); 15N NMR (δ ppm in DMSO-d6): -254 (N-2), -332 (N-38); 1H,
15
13C and N resonance assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D
spectra; FT-IR (KBr pellet): 3448 and 3401 cm-1 ν(O21-H) + ν(O23-H), 3345 cm-1 ν(N-H)amide
,
+
3147 cm-1 ν(O4-H···O11), 2733 cm-1 ν(N38 -H···O15), 2458 cm-1 ν(O1-H···O8‾), 1721 cm-1
ν(C35=O), 1642 cm-1 ν(C15=O)amide I, 1599 cm-1 ν(C=C)naphthalene, 1553 cm-1 δ(N-H)amide II, 1536
cm-1 ν(C=C), 1454 and 1438 cm-1 ν(C=C), 1241 and 1228 cm-1 ν(C-O); Elemental analysis (%):
Calcd. for C45H58N2O15: C, 66.49; H, 7.04, N, 3.37; Found: C, 66.55; H, 6.93, N, 3.40.

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Spectral data for (11): Yield 57% /method 3 with HCl(EtOH)/; m.p. 150-151°C; ESI-MS (m/z):
849 [M+H]+; HR-MALDI-TOF 849.3963 [M+H]+ ;1H NMR (δ ppm in DMSO-d6): 16.03 (1H, bs,
OH-1), 12.82 (1H, s, OH-4), 9.29 (1H, s, NHamide), 8.65 (1H, bs, N+Hintramolecularly hydrogen bonded),
8.37 (1H, bs, N+Hhydrogen bonded with the solvent), 7.30 (1H, m, H-44), 7.19 (1H, m, H-45), 7.15 (1H, m,
3
3
H-43), 7.34 (1H, m, H-46), 6.61 (1H, dd, JH18-H19=16.0 Hz, H-18), 6.33 (1H, d, JH17-H18=10.6
3
3
Hz, H-17), 6.25 (1H, d, , JH28-H29=12.9 Hz, H-29), 6.09 (1H, dd, JH19-H20=7.5 Hz, H-19), 5.08
3
3
(1H, d, JH25-H26=11.0Hz, H-25), 5.03 (1H, d, JH21-HO=3.1 Hz, OH-21), 4.91 (1H, ddH-28), 4.38
(1H, d, 2J=12.3 Hz, H-38a), 3.98 (1H, d, 3JH23-HO=8.1 Hz, OH-23), 3.73 (1H, d, H-38b), 3.69 (1H,
3
m, H-21), 3.28 (1H, m, H-39a), 3.23 (1H, d, JH27-H28=8.6 Hz, H-27), 3.17 (1H, m, H-39b), 2.98
3
3
(2H, m, H-40), 2.89 (3H, s, H-37), 2.83 (1H, dd, JH22-H23=8.8 Hz, JH23-H24=9.1 Hz, H-23), 2.27
(1H, m, H-20), 1.98 (3H, s, H-36), 1.96 (3H, s, H-30), 1.92 (3H, s, H-14), 1.68 (1H, m, H-22),
1.65 (3H, s, H-13), 1.25 (1H, m, H-24), 0.97 (1H, m, H-26), 0.91 (3H, d, 3JH22-H32=7.1 Hz, H-32),
3
3
0.85 (3H, d, 3JH20-H31=6.9 Hz, H-31), 0.50 (3H, d, JH24-H33=6.9 Hz, H-33), -0.33 (3H, d, JH26-
H34=6.8 Hz, H-34); 13C NMR (δ ppm in DMSO-d6): 185.3 (C-11), 184.2 (C-8), 171.8 (C-6), 170.2
1
(C-15), 169.4 (C-35), 160.6 (d, JC-F=244.3 Hz, C-42), 148.9 (C-1), 145.0 (C-4), 143.1 (C-29),
3
3
139.1 (C-19), 131.6 (C-17), 131.4 (C-16), 131.1 (d, JC-F=4.4 Hz, C-46), 129.2 (d, JC-F=8.2 Hz,
4
2
C-44), 126.2 (C-18), 124.8 (d, JC-F=1.2 Hz, C-45), 123.7 (d, JC-F=15.7 Hz, C-41), 118.0 (C-2),
2
117.7 (C-28), 116.7 (C-10), 115.4 (d, JC-F=21.7 Hz, C-43), 115.1 (C-9), 114.8 (C-3), 108.8 (C-
12), 101.4 (C-7), 98.6 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.8 (C-21), 55.7 (C-37),
46.8 (C-39), 43.1 (C-38), 40.3 (C-26), 38.2 (C-24), 37.9 (C-20), 32.7 (C-22), 25.2 (C-40), 22.1
(C-13), 20.7 (C-36), 19.8 (C-30), 18.2 (C-31), 11.2 (C-32), 9.0 (C-34), 8.8 (C-33), 7.4 (C-14);
1
15N NMR (δ ppm in DMSO-d6): -254 (N-2), -333 (N-38); H, 13C and 15N resonance assignments
were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr pellet): 3447
and 3415 cm-1 ν(O21-H) + ν(O23-H), 3337 cm-1 ν(N-H)amide, 3151 cm-1 ν(O4-H···O11), 2743 cm-1
+
ν(N38 -H···O15), 2468 cm-1 ν(O1-H···O8‾), 1724 cm-1 ν(C35=O), 1646 cm-1 ν(C15=O)amide I, 1597
cm-1 ν(C=C)naphthalene, 1561 cm-1 δ(N-H)amide II, 1544 cm-1 ν(C=C), 1457 and 1442 cm-1 ν(C=C),
1244 and 1235 cm-1 ν(C-O); Elemental analysis (%): Calcd. for C46H57FN2O12: C, 65.08; H, 6.77;
N, 3.30; Found: C, 64.94; H, 6.71; N, 3.34.
Spectral data for (12): Yield 53% /method 3 with HCl(EtOH)/; m.p. 154-156°C; ESI-MS (m/z):
849 [M+H]+; HR-MALDI-TOF 849.3968 [M+H]+ ;1H NMR (δ ppm in DMSO-d6): 16.05 (1H, bs,
OH-1), 12.84 (1H, s, OH-4), 9.30 (1H, s, NHamide), 8.59 (1H, bs, N+Hintramolecularly hydrogen bonded),

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8.34 (1H, bs, N+Hhydrogen bonded with the solvent), 7.36 (1H, m, H-45), 7.12 (1H, m, H-42), 7.09 (1H, m,
3
3
H-46), 7.08 (1H, m, H-44), 6.60 (1H, dd, JH18-H19=16.0 Hz, H-18), 6.33 (1H, d, JH17-H18=11.0
3
3
Hz, H-17), 6.26 (1H, d, H-29), 6.09 (1H, dd, JH19-H20=7.5 Hz, H-19), 5.09 (1H, d, JH25-
3
3
H26=11.0Hz, H-25), 5.03 (1H, d, JH21-HO=3.1 Hz, OH-21), 4.94 (1H, dd, JH28-H29=12.8 Hz, H-
28), 4.25 (1H, d, 2J=12.4 Hz, H-38a), 4.01 (1H, d, 3JH23-HO=6.7 Hz, OH-23), 3.72 (1H, d, H-38b),
3
3.69 (1H, m, H-21), 3.34 (1H, m, H-39a), 3.24 (1H, d, JH27-H28=8.4 Hz, H-27), 3.23 (1H, m, H-
39b), 2.95 (2H, m, H-40), 2.90 (3H, s, H-37), 2.83 (1H, dd, 3JH22-H23=8.8 Hz, 3JH23-H24=9.1 Hz, H-
23), 2.29 (1H, m, H-20), 1.98 (3H, s, H-36), 1.96 (3H, s, H-30), 1.93 (3H, s, H-14), 1.68 (1H, m,
H-22), 1.65 (3H, s, H-13), 1.26 (1H, m, H-24), 0.99 (1H, m, H-26), 0.92 (3H, d, 3JH22-H32=7.0 Hz,
3
H-32), 0.87 (3H, d, 3JH20-H31=7.0 Hz, H-31), 0.50 (3H, d, JH24-H33=7.0 Hz, H-33), -0.32 (3H, d,
3JH26-H34=6.8 Hz, H-34); 13C NMR (δ ppm in DMSO-d6): 185.3 (C-11), 184.1 (C-8), 171.8 (C-6),
170.3 (C-15), 169.4 (C-35), 162.3 (d, 1JC-F=240.1 Hz, C-43), 148.9 (C-1), 145.0 (C-4), 143.0 (C-
3
29), 139.8 (d, JC-F=7.2 Hz, C-41), 139.1 (C-19), 131.6 (C-17), 131.4 (C-16), 130.5 (d, 3JC-F=8.4
Hz, C-45), 126.2 (C-18), 124.8 (d, 4JC-F=2.2 Hz, C-46), 118.0 (C-2), 117.7 (C-28), 116.6 (C-10),
2
2
115.5 (d, JC-F=22.2 Hz, C-42), 115.1 (C-9), 114.6 (C-3), 113.6 (d, JC-F=21.1 Hz, C-44), 108.8
(C-12), 101.3 (C-7), 98.5 (C-5), 76.3 (C-27), 75.8 (C-23), 73.2 (C-25), 72.8 (C-21), 55.6 (C-37),
47.6 (C-39), 43.0 (C-38), 40.3 (C-26), 38.1 (C-24), 37.9 (C-20), 32.7 (C-22), 31.3 (C-40), 22.0
(C-13), 20.6 (C-36), 19.8 (C-30), 18.2 (C-31), 11.2 (C-32), 8.9 (C-34), 8.7 (C-33), 7.3 (C-14);
1
15N NMR (δ ppm in DMSO-d6): -255 (N-2), -332 (N-38); H, 13C and 15N resonance assignments
were confirmed by COSY, HSQC, HMBC and NOESY 2D spectra; FT-IR (KBr pellet): 3442
and 3406 cm-1 ν(O21-H) + ν(O23-H), 3331 cm-1 ν(N-H)amide, 3144 cm-1 ν(O4-H···O11), 2737 cm-1
ν(N38+-H···O15), 2472 cm-1 ν(O1-H···O8‾), 1720 cm-1 ν(C35=O), 1642 cm-1 ν(C15=O)amide I, 1599
cm-1 ν (C=C)naphthalene, 1555 cm-1 δ (N-H)amide II, 1541 cm-1 ν(C=C), 1453 and 1440 cm-1 ν(C=C),
1247 and 1239 cm-1 ν(C-O); Elemental analysis (%): Calcd. for C46H57FN2O12: C, 65.08; H, 6.77;
N, 3.30; Found: C, 64.99; H, 6.71; N, 3.26.
Spectral data for (13): Yield 58% /method 3 with HCl(EtOH)/; m.p. 157-158°C; ESI-MS (m/z):
849 [M+H]+; HR-MALDI-TOF 849.3979 [M+H]+ ;1H NMR (δ ppm in DMSO-d6): 16.04 (1H, bs,
OH-1), 12.84 (1H, s, OH-4), 9.31 (1H, s, NHamide), 8.33 (1H, bs, N+Hintramolecularly hydrogen bonded),
3
8.26 (1H, bs, N+Hhydrogen bonded with the solvent), 7.28 (1H, dd, JH42/46-F=5.7 Hz, 3JH42-H43/H45-H46=8.3
3
Hz, H-42+H-46), 7.14 (1H, dd, 3JH43/H45-F=8.8 Hz, H-43+H-45), 6.60 (1H, dd, JH18-H19=15.9 Hz,
H-18), 6.34 (1H, d, 3JH17-H18=11.0 Hz, H-17), 6.26 (1H, d, 3JH28-H29=12.9 Hz, H-29), 6.09 (1H, dd,

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3JH19-H20=7.5 Hz, H-19), 5.09 (1H, d, 3JH25-H26=11.0 Hz, H-25), 5.03 (1H, d, 3JH21-HO=3.1 Hz, OH-
3
21), 4.93 (1H, dd, H-28), 4.35 (1H, m, H-38a), 3.95 (1H, d, JH23-HO=6.8 Hz, OH-23), 3.71 (1H,
3
m, H-38b), 3.70 (1H, m, H-21), 3.29 (1H, m, H-39a), 3.25 (1H, d, JH27-H28=8.7 Hz, H-27), 3.19
(1H, m, H-49b), 2.92 (2H, m, H-40), 2.90 (3H, s, H-37), 2.84 (1H, m, H-23), 2.29 (1H, m, H-20),
1.99 (3H, s, H-36), 1.97 (3H, s, H-30), 1.93 (3H, s, H-14), 1.69 (1H, m, H-22), 1.66 (3H, s, H-
3
13), 1.27 (1H, m, H-24), 1.00 (1H, m, H-26), 0.92 (3H, d, JH22-H32=7.2 Hz, H-32), 0.86 (3H, d,
3
3
3JH20-H31=6.8 Hz, H-31), 0.51 (3H, d, JH24-H33=7.0 Hz, H-33), -0.32 (3H, d, JH26-H34=6.8 Hz, H-
34); 13C NMR (δ ppm in DMSO-d6): 185.4 (C-11), 184.2 (C-8), 171.9 (C-6), 170.4 (C-15), 169.5
2
(C-35), 161.3 (d, JC-F=242.8 Hz, C-44), 149.0 (C-1), 145.1 (C-4), 143.1 (C-29), 139.3 (C-19),
133.2 (d, 4JC-F=1.2 Hz, C-41), 131.7 (C-17), 131.5 (C-16), 130.6 (d, 3JC-F=8.4 Hz, C-42 + C-46),
2
126.3 (C-18), 118.1 (C-2), 117.8 (C-28), 116.7 (C-10), 115.5 (d, JC-F=19.0 Hz, C-43 + C-45),
115.2 (C-9), 114.7 (C-3), 108.9 (C-12), 101.4 (C-7), 98.6 (C-5), 76.4 (C-27), 75.9 (C-23), 73.3
(C-25), 73.0 (C-21), 55.7 (C-37), 45.1 (C-39), 43.1 (C-38), 40.4 (C-26), 38.2 (C-24), 37.9 (C-20),
32.7 (C-22), 30.9 (C-40), 22.5 (C-13), 20.7 (C-36), 19.9 (C-30), 18.2 (C-31), 11.2 (C-32), 9.0 (C-
15
1
13
34), 8.8 (C-33), 7.4 (C-14); N NMR (δ ppm in DMSO-d6): -254 (N-2), -333 (N-38); H, C
and 15N resonance assignments were confirmed by COSY, HSQC, HMBC and NOESY 2D
spectra; FT-IR (KBr pellet): 3436 and 3999 cm-1 ν(O21-H) + ν(O23-H), 3331 cm-1 ν(N-H)amide
,
+
3138 cm-1 ν(O4-H···O11), 2732 cm-1 ν(N38 -H···O15), 2464 cm-1 ν(O1-H···O8‾), 1719 cm-1
ν(C35=O), 1637 cm-1 ν(C15=O)amide I, 1596 cm-1 ν(C=C)naphthalene, 1551 cm-1 δ(N-H)amide II, 1538
cm-1 ν(C=C), 1448 i 1436 cm-1 ν(C=C), 1243 i 1239 cm-1 ν(C-O); Elemental analysis (%): Calcd.
for C46H57FN2O12: C, 65.08; H, 6.77; N, 3.30; Found: C, 65.16; H, 6.69; N, 3.13.
Yield of 2-13 is given after column chromatography purification.
Antibacterial assays of 1 and 3-13
The antibacterial activity of the compounds studied was performed for a series of Gram-positive
and Gram-negative bacteria, including the reference and hospital strains. The following
microorganisms were used in this study: standard strains of Gram-positive cocci: Staphylococcus
aureus NCTC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538,
Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228 and
Staphylococcus epidermidis ATCC 35984; standard strains of Gram-negative rods: Escherichia
coli ATCC 10538, Escherichia coli ATCC 25922, Escherichia coli NCTC 8196, P. aeruginosa

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ATCC 15442, P. aeruginosa NCTC 6749, P. aeruginosa ATCC 27853. The
microorganisms
used were obtained from the collection of the Department of Pharmaceutical Microbiology,
Medical University of Warsaw, Poland. Minimal Inhibitory Concentration (MIC) was determined
by the twofold serial dilution method using Mueller-Hinton II agar medium (Beckton Dickinson)
according to CLSI guidelines.a Concentrated solutions of the compounds tested in DMSO were
made, then they were diluted in water to obtain the required concentration. The concentrations of
the agents tested in a solid medium ranged from 8 to 0.002 µg mL-1 (for Gram-positive bacteria)
and from 256 to 8 µg mL-1 (for Gram-negative bacteria). The final inoculum of all studied
organisms were 104 CFU mL-1 (colony forming units per ml). Minimal inhibitory concentrations
were read after 18h of incubation at 35 °C.
aClinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria That Grow Aerobically; Approved Standard M7-A-7, Clinical and Laboratory
Standards Institute, Wayne, Pa. USA, 2006. The complete data of antibacterial tests were
collected in Table 2.
Acknowledgement
The project was financially supported by Polish National Science Center (NCN), decision
number DEC-2011/03/B/ST5/01014.
References
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2
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method 1
HO
HO
H2NR, NaBH4
HO
method 2
OH
OH OH
OH
OH OH
OH
OH OH
O
O
O
O
O
O
H2NR, NaBH(CH3COO)
Et2O/HCl/H2O
O
O
O
NH
NH
NH
H
O
O
O
O
O
O
method 3
O
N
N
H2NR, NaBH3CN
O
N
O
O
R
OH
N
OH
O
OH
O
method 4
O1
H2NR, BH3CN/polymer
supported
2
3-13
rifampicin
rifaldehyde
rifampicin analogues
Figure 1 Methods for the synthesis of rifampicin halogen benzyl and phenethyl analogues.