Synthesis, Antibacterial, and Anticancer Evaluation of Novel Spiramycin-Like Conjugates Containing C(5) Triazole Arm

Article abstract of DOI:10.1021/acs.jmedchem.6b00764

Huisgen cycloaddition allowed obtaining of novel triazole-bridged antibiotics (6-16) with the reconstructed C(5) arm of spiramycin. ¹H-¹H NOESY couplings indicated the structure of novel derivatives in solution and demonstrated that

Full text of DOI:10.1021/acs.jmedchem.6b00764

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Article
Synthesis, antibacterial and anticancer evaluation of novel
spiramycin-like conjugates containing C(5) triazole arm
Katarzyna Klich, Krystian Pyta, Marcelina M. Kubicka, Piotr
Ruszkowski, Lech Celewicz, Marzena Gajecka, and Piotr Przybylski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00764 • Publication Date (Web): 08 Aug 2016
Downloaded from http://pubs.acs.org on August 9, 2016
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Synthesis, antibacterial and anticancer evaluation of novel spiramycin-like
conjugates containing C(5) triazole arm
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Katarzyna Klich,1 Krystian Pyta,1 Marcelina M. Kubicka,2 Piotr Ruszkowski,3
Lech Celewicz,1 Marzena Gajecka,2,4 Piotr Przybylski1*
1Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61ꢀ614 Poznan, Poland
2Department of Genetics and Pharmaceutical Microbiology, University of Medical Sciences,
Swiecickiego 4, 60ꢀ781 Poznan, Poland
3Department of Pharmacology, University of Medical Sciences, Rokietnicka 5a, 60ꢀ806 Poznan, Poland
4Institute of Human Genetics, Polish Academy of Science, Strzeszynska 32, 60ꢀ479 Poznan, Poland
ABSTRACT: Huisgen cycloaddition allowed obtaining novel triazoleꢀbridged antibiotics (6ꢀ16)
1
with the reconstructed C(5) arm of spiramycin. Hꢀ1H NOESY couplings indicated the structure
of novel derivatives in solution and demonstrated that the rebuilt C(5) arm is slightly differently
oriented relative to the aglycone part, if compared to that of spiramycin (1). Combined analysis of
biological data together with experimentally determined lipophilicity (clogP) and solubility
shown the importance of chemical nature of the newly introduced triazole C(5) arm in the
presence of attractive antibacterial and anticancer potency. The most cytotoxic active triazole
conjugates having hydrophobic and bulky C(5) arm showed higher selectivity towards cancer cell
lines (HeLa, KB, MCFꢀ7, HepꢀG2 and U87) relative to HDF normal cells than that of the parent
spiramycin. Our studies have demonstrated that the aldehyde group is not crucial for the presence
of interesting antibacterial [MIC(S. pneumoniae) ~ 1.2 µM] and anticancer [IC50 (HepG2) ~ 6 µM]
properties of 16ꢀmembered lactone macrolides, based on spiramycin’s aglycone.
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Keywords: 16ꢀmembered lactone macrolides; click chemistry; spectroscopy, clogP and solubility,
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antibacterial and anticancer.
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INTRODUCTION
Spiramycin (Scheme 1) is a natural antibiotic produced by Streptomyces ambofaciens in the form
of a mixture of three compounds, the soꢀcalled spiramycins IꢀIII.1,2 Spiramycin I, having
hydroxyl substituent at C(3) atom, is dominant (~80 %) in the mixture produced by the bacteria.3
Spiramycins are structurally similar to leucomycins in the presence of a common 16ꢀmembered
lactone aglycone and mycaminosylꢀmycarose moiety attached at C(5). However, spiramycins, in
contrast to leucomycins, additionally possess forosamine at C(9) position. The spectrum of
spiramycins’ biological properties comprises mainly bacteriostatic activity against most of Gramꢀ
(+) cocci and rods, mycoplasmas and Toxoplasma gondii .4,5 Comparison of spiramycins’ activity
against different Gramꢀ(+) bacteria strains in vitro with that determined for 14ꢀmembered lactone
macrolides as erythromycins indicates that it is at least twice lower.6 However, the advantage of
spiramycins over erythromycins is their good gastrointestinal tolerance, higher affinity to tissues
and a fewer adverse effects.7,8 To the best of our knowledge, the activity of spiramycins in cancer
cells has never been studied up to now, although literature provides some examples of otherꢀ
group macrolides showing properties of this type e.g. maytansine /ansamacrolide/ or marine 22ꢀ
membered lactone macrolide (ꢀ)ꢀdictyostatin.9–12 The target site of spiramycins’ action is well
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6'''
5'''
7'''
O forosamine
N
4'''
8'''
1'''
7''
3'''
OH
O
2'''
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OH
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8'
8
O
3''
2''
1''
4''
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N
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3'
17
4'
5''
HO
O
O
6''
6
2'
mycarose
5
5'
O
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O
1'
4
O
6'
O
mycaminose
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3
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O
OR
16
2
16-membered lactone ring
Spiramycin I R=H
Spiramycin II R=COCH3
Spiramycin III R=COC2H5
Scheme 1 Structures of Spiramycins IꢀIII together with the carbon atom numbering.
recognized from Xꢀray studies as the exit of the ribosomal tunnel near the peptidyl transferase
loop and the loop of domain II of 23S rRNA, belonging to the large subunit 50S.13–15 Spiramycin
and related 16ꢀmembered lactone macrolides have a common mechanism of activity which
involves binding to 50S subunit and steric blocking of the peptide exit tunnel, which contributes
to inhibiting peptide synthesis at different stages. The stage of peptide synthesis inhibition is
related to the length and chemical nature of the arm attached at C(5) position of the macrolide's
aglycone.16 It is also known that bacteria resistance to 16ꢀmembered lactone macrolides is
achieved when e.g. nucleotides A2058 /E. coli/ or G2099 /H. marismortui/ undergoes Nꢀ
methylation because of the steric clashing between the N,Nꢀdimethyl group of the nucleotide and
the mycaminose part.17,18 Among whole group of mutations, the replacement of A2103 with G in
H. marismortui ribosomes (A2062 in E. coli) seems to be the most important and confers
resistance against spiramycin or tylosin.19 Therefore as reported earlier the modifications of the
aldehyde group, which is usually reversibly bonded to the amine group of A2103 of ribosome
with formation of hemiaminal, for thisꢀtype macrolides gave derivatives at least 100ꢀ1000 fold
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less active over unmodified parent antibiotics.20 Up to know, many modifications, both within
spiramycin and leucomycin groups, have been proposed and tested to obtain comparably or more
active alternative agents able to fight the growing bacterial resistance. Most of the modifications
were related to transformation of: the aldehyde via reduction, nucleophilic addition,21–23 hydroxyl
groups within the aglycone and saccharides parts via etherification, acylation or
sulphonylation,24,25 diene moiety via epoxidation, reduction, DielsꢀAlder reaction or metathesis
with contraction of the lactone ring,26–29 forosamine and/or mycaminose Nꢀoxidation or Nꢀ
substitution30,31 and incorporation of the nitrogen into the lactone macrocyclic ring.32–34
Interesting platform to synthesis and drug discovery among group of macrolide lactone
antibiotics, has been recently proposed by Seiple et al.35 Convenient synthetic strategy leading to
obtain of new chemical entities characterized by attractive biological properties is the use of click
chemistry reactions.36–38 Interesting approach to modification of these structurally complexed
macrolides, in view of their known mechanism of action, has been described by Omura and
Sharpless et al.39 They proposed the functionalization of terminal hydroxyls of mycarose
saccharide with alkyne followed by conversion into respective triazole moieties via Fokinꢀ
Huisgen cycloadditions. However, as a result of this transformation, the obtained derivatives
were significantly less active than the parent leucomycin due to the presence of a too long arm at
C(5) of the aglycone. Recently, we have proposed a novel cascade approach to modification of
the aglycone of spiramycins, which opened a possibility of another pathway to construct the
saccharide part at the aglycone.40 As a continuation of these studies, in the present work we used
a Huisgen cycloaddition to rebuilt spiramycin’s arm at C(5) of the aglycone and to evaluate the
influence of the modification on antibacterial and anticancer potency of novel triazole conjugates
6ꢀ16.
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N
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OH
OH
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δ
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N
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γ
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β
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α
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O
3
1
O
O
1
OH
O
1
2
R2
N
N
N
O
19R
N
R1
O
19R
N
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O
O
*O
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O
5R
O
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O
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*
5R
O
O
for compound 5
3Z
3Z
R2-N3, ascorbic acid, CuOAc
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1
O
THF/DMF (3:1)
rt. 60 min
O
O
O
Novel triazole conjugates (6-16) of
16-membered lactone aglycone
of Spiramycin
R1=
3
R2=
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5
HO
OH
F
11
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7
O
OH
HO
HO
OH
OH
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O
HO
O
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HN
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OH
OH
O
HO
OH
OH
O
N
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HO
HO
O
HN
HO
O
O
HO
OH
OH
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HO
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O
O
Scheme 2 Synthetic route leading to novel triazole conjugates of spiramycin aglycone 6ꢀ16 via
previously obtained compounds 2 and 5 [Ref. 39].
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RESULTS AND DISCUSSION
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In order to rebuilt spiramycin arm at C(5), the earlier obtained compound 2,40 via sequence of
cascade transformations from spiramycin was further subjected to Huisgen dipolar cycloaddition
with the use of different azides containing hydrophobic and hydrophilic substituents in the
presence of CuOAc in THF/DMF mixture of solvents. Novel triazole conjugates of spiramycin’s
aglycone 6ꢀ16 were obtained with the total yields 18ꢀ60 % after purification. The structures of all
triazole conjugates 6ꢀ16 in solution were determined via 1D and 2D NMR (Fig. 1, Supporting
Information) and FTꢀIR (Fig. 49S, Supporting Information) spectroscopic analysis. The use of
HSQC method enabled the assignment of H23 and C23 of triazole ring bonded to the aglycone
structure (Fig. 1a). In turn, application of longꢀrange heteronuclear couplings (HMBC) to
structural analysis of conjugates 6ꢀ16 (Fig. 1b and 1c) revealed simultaneous correlations of
carbon and proton signals between the newly formed triazole ring and those of tetrahydrofuran
bicyclic moiety and R2 terminal substituent (Scheme 2). Furthermore, the FTꢀIR spectrum of
intermediate 2 shows two separated bands at 1722 and 1702 cmꢀ1, assigned to carbonyl stretching
vibrations of aldehyde and double unsaturated lactone, respectively (Fig. 49S). In the spectra of
novel triazole products 6-16 one of these bands /v(C=O)aldehyde/ vanishes, whereas the other one is
shifted towards higher wavenumbers /to 1728 cmꢀ1/. This result clearly indicated the lack of
aldehyde group within structures of 6-16 and the fact that the lactone of the macrocyclic ring is
no longer strongly πꢀπ conjugated with the two double bonds, in contrast to 2. Additionally, for
6ꢀ10 and 16 conjugates the presence of complex bands assigned to ν(OꢀH) stretching vibrations
confirmed successful introduction of the saccharide part into the structure of newꢀtype antibiotics
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(Fig. 49b). Application of Hꢀ1H NOESY method revealed mutual arrangement of newly
introduced triazole ring relative to the modified aglycone part in solution (Fig. 2).
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(c)
Fig. 1 Exemplary 2D NMR correlation spectra of derivative 7, indicating the presence of triazole
bridge between terminal substituent and the newly formed tetrahydofuran bicyclic moiety: (a) 1Hꢀ
13C HSQC revealing H(23)protonꢀC(23)carbon assignment of triazole; (b) Hꢀ13C HMBC
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showing a connection between the newly formed triazole ring and bicyclic moiety; (c) Hꢀ13C
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HMBC revealing a connection between triazole and the terminal substituent, introduced via a
respective azide.
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Fig. 2 The lowestꢀenergy structures of compounds: 1 (Hf°= − 608.2 kcal/mol), 8 (Hf°= − 539.27
kcal/mol) and 11 (Hf°= − 494.2 kcal/mol) showing the orientation of the reconstructed C(5) arm
relative to the aglycone part, calculated by B88ꢀLYP (GGA) DFT /Scigress Explorer package FJ
2.4. EU 3.1.8/.
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Generally, Hꢀ1H NOESY contacts recorded for 6ꢀ16, assigned to 16ꢀmembered aglycone part,
can be divided into two groups i.e. those assigned to the protons oriented above the aglycone
(marked by green ꢀ Fig. 50S) and the others assigned to the protons oriented to the bottom of the
aglycone ring (blue color ꢀ Fig. 50S). Taking into consideration of the following group of
contacts: H(3)ꢀH(6), H(6)ꢀH(11), H(6)ꢀH(19), H(9)ꢀH(11), H(11)ꢀH(13) and H(13)ꢀH(15) all
these protons are suggested to be at same face of the aglycone /above the aglycone/ whereas the
second group of contacts: H(2)ꢀH(14), H(2)ꢀH(17), H(5)ꢀH(17), H(10)ꢀH(12), and H(12)ꢀH(14) is
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assigned to protons which are directed to the bottom of the aglycone (Fig. 50S). Mutual Hꢀ1H
contacts between H(5), H(17) and H(21) show their close vicinity and orientation of the ꢀ
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(21)CH2ꢀ methylene protons to the bottom of the aglycone. As concluded from the presence of
1Hꢀ1H contacts found between H(23) and H(19) and H(1’) in NOESY spectra, the arrangement of
the triazole ring relative to bicyclic tetrahydrofuran moiety is similar for all obtained derivatives
irrespectively of the type of terminal substituent (Fig 2). Comparison of C(5) arm structures
between spiramycin and those of synthesized triazole conjugates 6ꢀ16 shows that they are of
comparable length, whereas the mutual arrangement of C(5) arms relative to the macrocyclic
lactone ring is slightly different in each case (Fig. 2).
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Antibacterial studies. Antibacterial test results as well as experimentally determined
lipophilicity and solubility of 1ꢀ16 compounds are collected in Tables 1 and 1S (Supporting
Information). Analysis of these data shows that 11ꢀ15 derivatives, containing bicyclicꢀtriazole
bridged aglycones, in general have no activity against all tested bacteria. First of all the lack of
aldehyde group (no reversible covalent bonding) and limited possibility of stabilization of the
terminal substituent at C(5) arm via Hꢀbonds are the “weak points” of 11ꢀ15 compounds in view
of the earlier published models.17, 41 Additionally, although compounds 11ꢀ15 show the most
favourable lipophilicity from among all other derivatives (Table 1S), they have the lowest
solubility, which seems to be a very important factor taking into regard the polar walls of the
ribosomal tunnel and filling the tunnel with water molecules (transport to the target site of
action). Compounds 3ꢀ5 also have poor solubility and therefore they show no antibacterial
activity. Similarly, low solubility of triazole conjugate containing AZT (compd. 16) can be an
explanation of its no antibacterial activity. The importance of the role of reversible bonding of the
aldehyde in the presence of antibacterial activity can be concluded from biological data of
derivative 2 (Table 1). The presence of antibacterial activity against Gramꢀpositive
microorganisms on the level 4ꢀ33 µM is mainly a result of forosamine interactions and the
aldehyde bonding with the target site at ribosomes as well as quite wellꢀbalanced solubility and
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lipophilicity of compound 2. However, it should be underlined here, that the activity of 2 is about
8ꢀ28 times lower than that of the parent antibiotic 1, when MICs are expressed in µM. This result
is understandable because compound 2, in contrast to 1, does not have saccharide part involved in
hydrogen bonding with PTC loop nucleotides as G2099 and G2540 /H. marismortui/. Interesting
results were obtained for some of derivatives 6ꢀ10 containing terminal saccharides at the
reconstructed C(5) arm (Table 1 and Table 1S). Taking into regard just their not favourable
lipophilicity and the best of all solubility parameters, no explanation of different antibacterial
properties among them is possible, especially in view of 1. This experimental result suggests
slightly different binding modes among this group of triazoleꢀsaccharide conjugates. Further
comparison of physicoꢀchemical parameters of compounds 6ꢀ10, having relatively hydrophilic
C(5) arm, shows that at good solubility of all these derivatives, just compound 8 has lipophilicity
close to that of 1, which contributes to its generally comparable activity with that of 1 /is 2ꢀ4 less
active than 1 [µM]/. It should be underlined that conjugate 8 is also characterized by much higher
activity than that of 2 having aldehyde (Table 1). All these results clearly show that 16ꢀmembered
macrolide derivatives, even not containing aldehyde group, but having additionally
functionalized saccharide can be active against Gramꢀpositive bacteria at a level comparable to
that of 1.
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Anticancer studies. To the best of our knowledge the leucomycinꢀtype derivatives showed some
anticancer activity.28 However, the anticancer potency of spiramycin and its analogs has not been
studied up to now. Cytotoxic activity determined in six human cell lines: cervical (HeLa),
nasopharyngeal (KB), breast (MCFꢀ7), liver (HepG2), glioblastoma (U87) normal human dermal
fibroblast (HDF) of novel triazole conjugates, compared to those of AZT and cytarabine
standards, are shown in Table 2. Analysis of these data revealed that 1 showed only limited
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cytotoxicity, irrespectively of the type of the cancer cell line. Much lower activities /> 100 µM/ in
comparison to that of the parent compound 1, displayed derivatives 2, 8 and 9. It should be
mentioned here that the most active against Gramꢀpositive bacteria, triazole conjugate 8, has very
weak anticancer activity. The other studied derivatives (compounds 10ꢀ16) show significantly
greater potency than that found for 1 in all cancer cell lines (Table 2). Anticancer properties of
the triazole conjugates containing within the C(5) arm terminal saccharides (8ꢀ10) are varied.
Derivative 10, having 6’ꢀsubstituted saccharide revealed medium anticancer activity in HeLa, KB
and U87 cell lines (IC50 ~ 12 µM), in contrast to the other ones of this group, having saccharide
attached via C(1’) atom. Incorporation of AZT into the C(5)ꢀtriazole arm resulted in increased
activity of 16 in HeLa and KB cancer cell lines (IC50 ~ 15 µM), however, lower than the activity
of AZT itself. Interestingly, all triazole conjugates 11ꢀ15, containing aliphatic or aromatic C(5)ꢀ
terminal substituents, displayed interesting cytotoxic potency in all cancer cell lines (Table 2).
Among relatively hydrophobic triazole conjugates of the favourable logP values, the derivative
bearing cycloheptyl substituent (compound 11) is characterized by the highest potency in the
range IC50 = 6.02 ꢀ 8.63 µM. IC50 values determined for 11 are only 2ꢀ3 times higher than those
determined for cytarabine and are up to 5ꢀtime lower than for 1. Taking into account structures of
10ꢀ15 and their anticancer activity it can be concluded that there is some relationship between the
activity and the bulkiness of the C(5)ꢀarm terminal substituent. The activity order beginning from
the most active is following: 11 with cycloheptyl > 13 with methylene cyclohexyl > 12 with
cyclohexyl > 15 with benzyl group. Another favourable factor of novel hydrophobic triazole
conjugates is their selectivity indexes (SI). Comparison of these indexes for 1 and all synthesized
compounds (Table 3), revealed that those containing hydrophobic triazole arm (compounds 11ꢀ
15) have SI >1, whereas all others have SI ~ 1 or SI < 1. A comparison of SI values determined
for 11ꢀ15 with those of CYT, FUra and FdU standards demonstrates that the most active 11 has
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SI comparable or even higher than those of the standards, whereas also active compound 13 has
even better SI than the standards used (except for HeLa cell line). It should be added that the
most potent against bacteria derivative 8, among all novel triazole conjugates, is 5 fold less toxic
(less active in HDF cell line) than slightly more active parent antibiotic spiramycin (compound
1).
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Results of these studies clearly showed that for anticancer activity of thisꢀgroup of macrolides the
presence of the aldehyde is not so important, whereas the occurrence the hydrophobic and bulky
structure of C(5) arm is crucial. It has been demonstrated also that the use of combined cascade
and click approaches to modification of spiramycin allowed to obtain of the new class of
macrolide derivatives having interesting cytotoxic properties (comparable with FUra and even
better than those of FdU).
CONCLUSION
Novel bicyclicꢀconjugates bearing the reconstructed arm at C(5) of the aglycone have been
synthesized by a combined cascade and click chemistry approach. 1D and 2D NMR and FTꢀIR
spectroscopic studies confirmed the obtaining of these derivatives and revealed their structures in
solution. As indicated by 1Hꢀ1H NOESY studies the reconstructed C(5)ꢀtriazole arm within novel
derivatives has the length comparable with that of spiramycin but is slightly reoriented due to the
presence of bicyclic moiety. Antibacterial activity studies demonstrated that the most active
among novel triazole conjugates is that containing the terminal Nꢀacetylsaccharide moiety
(compound 8). Comparison of the antibacterial activity and physicoꢀchemical parameters of thisꢀ
group of macrolides revealed that for antibacterial potency comparable with that of 1 the
presence of aldehyde is not so important. Furthermore, our studies indicated that for the presence
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of interesting anticancer potency and selectivity toward cancer cells of novel spiramycinꢀlike
conjugates containing C(5) triazole arm, even more attractive than cytotoxic standard FdU or
comparable with FUra, the aldehyde group is not necessary. Comparison of cytotoxic data within
group of new triazole conjugates containing different nature of the rebuilt C(5) arm, revealed that
the most active ones are those bearing hydrophobic and relatively bulky substituents at the end of
the arm.
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EXPERIMENTAL SECTION
General Experimental: Spiramycin was purchased from LKT Laboratories. CH3CN and CD3CN
for spectroscopic measurements as well as NaH, methanol, pirrydynium pꢀtoluenosulfonate,
trimethyl orthoformate, propargyl alcohol, allyl alcohol, 4ꢀfluorobenzyl alcohol, 1ꢀAzidoꢀ1ꢀ
deoxyꢀβꢀ
D
ꢀgalactopyranoside, 1ꢀAzidoꢀ1ꢀdeoxyꢀβꢀ
D
ꢀglucopyranoside, 2ꢀAcetamidoꢀ2ꢀdeoxyꢀβꢀ
ꢀgalactose, sodium
D
ꢀglucopyranosyl azide, 2ꢀAzidoꢀ2ꢀdeoxyꢀ
D
ꢀglucose, 6ꢀAzidoꢀ6ꢀdeoxyꢀD
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azide, benzyl bromide, bromocyclohexane, (bromomethyl)cyclohexane, bromocycloheptane, 4ꢀ
(bromomethyl)tetrahydropyran, AZT, acetonitrile, difluoroacetate acid, acetic anhydride, THF,
DMF used for the syntheses of new Spiramycin derivatives were purchased from Aldrich.
CH3COOC2H5, Et2O, NaCl were purchased from POCH S.A. Gliwice (Poland). H2O HPLC
gradient grade and CH3CN HPLC gradient grade were purchased from J.T. Baker.
HPLC, 1D and 2D NMR and FT-IR measurements: The elemental analysis of new derivatives
of Spiramycin was carried out on Vario ELIII (Elementar, Germany). The purity of the final
compounds, found in all cases as ≥ 95%, was determined by HPLC method using Dionex
Ultimate 3000 equipped with an LPGꢀ3400 SD gradient pump using Thermo GOLD C18
150×4.6 mm (5 ꢁm) and Accucore XL column, TCCꢀ3000SD thermostat to columns (column
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temp. equal 25 ºC) and Dionex VWDꢀ 3400RS variable wavelength UVꢀvis detector (detection at
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λmax=232 nm); the flow rates were 0.5 and 1 mL/min with injection volumes of 10 ꢁL in
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acetonitrile mixtures and the mobile phase: 35:55:10 H2O/CH3CN/0.01M ammonium acetate
buffer prepared in 50:50 mixture CH3CN:H2O.
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The FTꢀIR spectra of new derivatives were recorded in in CH3CN solution. All FTꢀIR spectra
were recorded on an iS50 FTꢀIR spectrophotometer (Thermo, Nicolet, US) equipped with a
DTGS detector and twoꢀcolumnar purge gas generator (Parker, Balston, US); resolution 1 cmꢀ1,
NSS = 150, range 4000ꢀ400 cmꢀ1. The HappꢀGenzel apodization function was used.
The 1H and 13C measurements of new derivatives of Spiramycin were performed in CD3CN using
Varian Mercury 400 MHz and Bruker Avance 600 MHz spectrometers. The operating
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frequencies for H 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. Digital resolution was 0.2 Hz/point. 13C 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 and 13C NMR resonances unambiguously assigned on
the basis of the 1Hꢀ13C HMBC, 1Hꢀ13C HSQC, 1Hꢀ1H COSY couplings.
Antibacterial assays: The antimicrobial activity of novel spiramycin derivatives was studied
against the standard Gramꢀpositive cocci: Micrococcus luteus ATCC 10240, Staphylococcus
epidermidis ATCC 12228, Staphylococcus epidermidis ATCC 49134, Streptococcus pneumoniae
ATCC 49619, Bacillus subtilis ATCC 6635 and two clinical strains of Staphylococcus aureus
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(MRSA strain I and II). The microorganisms used were bought from Argenta company, LGC
Standards or were obtained (MRSA strains) from the collection of the Department of Genetics
and Pharmaceutical Microbiology at the University of Medical Sciences in Poznan, Poland. The
presence of MRSA resistance mechanism among clinical strains was confirmed using a disc
diffusion method with the cefoxitin disc (30 µg), in accordance with the EUCAST
recommendation. In addition, PCR (polymerase chain reaction) reactions were also performed to
verify the presence of the mecA gene, that encodes the protein PBP2A (penicillin binding
protein 2A), responsible for the resistance to all βꢀlactams antibiotics (The PCR method is the
gold standard by the EUCAST). Minimal Inhibitory Concentration (MIC ꢁg/mL) values were
determined independently for 1ꢀ16 compounds according to the recommendations of the
European Society of Clinical Microbiology and Infectious Diseases (EUCAST) using a serial
microdilution method on polystyrene plates with Mueller Hinton II broth MHB (Becton
Dickinson), as a medium. Concentrated solutions of the tested compounds were dissolved in
DMSO (POCH, Gliwice) and diluted in MHB to obtain the required concentration. The
concentrations of the tested compounds in liquid medium ranged from 0.064 to 64 ꢁg mLꢀ1. The
final inoculum of all studied organisms was approximately 5·105 CFU mLꢀ1 (Colony Forming
Units per mL). Minimal inhibitory concentration (MIC ꢀ data shown in Tables 1 and 1S) was
determined after 18 h of incubation at 35 ± 2 °C.
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Cytotoxicity assays: Human cancer cells HeLa (cervical cancer cell line) and KB (carcinoma
nasopharynx) were cultured in RPMI 1640 medium and human cancer cells MCFꢀ7 (breast
cancer cell line) were cultured in DMEM medium. HepG2 (liver cancer cell line) were cultured
in MEM medium. Each medium was supplemented with 10% fetal bovine serum, 1% Lꢀ
glutamine, and 1% penicillin/streptomycin solution. The cell lines were kept in the incubator at
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37◦C. The optimal plating density of cell lines was determined to be 5 × 104. All the cell lines
were obtained from The European Collection of Cell Cultures (ECACC) supplied by Sigmaꢀ
Aldrich. The proteinꢀstaining SRB (SigmaꢀAldrich) microculture colorimetric assay, developed
by the National Cancer Institute (USA) for in vitro antitumor screening was used in this study, to
estimate the cell number by providing a sensitive index of total cellular protein content, being
linear to cell density. The monolayer cell culture was trypsinized and the cell count was adjusted
to 5 × 104 cells. To each well of the 96 well microtiter plate, 0.1 mL of the diluted cell suspension
(approximately 10,000 cells) was added. After 24 hours, when a partial monolayer was formed,
the supernatant was washed out and 100 ꢁL of six different compound concentrations (0.1, 0.2, 1,
2, 10, and 20 ꢁM) were added to the cells in microtitre plates. The tested comꢀpounds were
dissolved in DMSO (containing 10% of water) (100 ꢁL) and the content of DMSO did not
exceed 0.1%; this concentration was found to be nontoxic to the cell lines. The cells were
exposed to compounds for 72 hours at 37 °C in a humidified atmosphere (90% RH) containing
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5% CO . After that, 25 ꢁL of 50% trichloroacetic acid was added to the wells and the plates were
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incubated for 1 hour at 4◦C. The plates were then washed out with the distilled water to remove
traces of medium and next dried by the air. The airꢀdried plates were stained with 100 ꢁL of 0.4%
sulforhodamine B (prepared in 1% acetic acid) and kept for 30 minutes at room temperature. The
unbound dye was removed by rapidly washing with 1% acetic acid and then air dried overnight.
The proteinꢀbound dye was dissolved in 100 ꢁL of 10 mM unbuffered Tris base (pH 10.5) for
optical density determination at 490 nm. All cytotoxicity experiments were performed three
times. Cell survival was measured as the percentage absorbance compared to the control
(nontreated cells). Cytarabine and AZT (SigmaꢀAldrich) were used as the internal standards.
Results of anticancer studies of novel triazole conjugates are shown in Table 2. SI index was
calculated from equation SI = IC50 normal cell line HDF/IC50 respective cancerous cell line (Table 3). A beneficial
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SI > 1.0 indicates a compound with efficacy against tumour cell greater than the toxicity against
normal cells.
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Determination of logP and water solubility: 1 and derivatives 2ꢀ16 were dissolved in HPLC
gradient grade water (pH=7, T=25°C) and the calibration curves were determined by measuring
absorbance as a function of their concentration with Metertech Inc. UVꢀVIS SPꢀ80001
spectrophotometer. Analytical wavelength for the determination of calibration curves A f(c) and
the concentrations cH2O and coctanol on the basis of UVꢀvis measurements was λmax=244 nm. To
determine logPexp the known amounts of 1 and 2ꢀ16 were dissolved in 10 mL of octanol to which
10 mL of H2O was added. The mixture was shaken, vigorously stirred for 1 hour and then
separated. In order to determine the concentration of the compound in the aqueous layer, the
respective measurement of absorbance was performed. Experimental logPexp values were
calculated according to the following equation: logPexp = log(coctanol/cH2O) and shown in Table 1
and Table 2S. To determine the solubility in the gradient grade water, a known amount of each of
1 and 6ꢀ16 derivatives was weighted and respective volumes of H2O were added (by 0.1 mL)
upon vigorous stirring of the solution. The solubility test was performed at least three times for
the derivative studied and its result is given in Table 1 and Table 2S.
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Syntheses and spectral characterization of novel triazole conjugates 6-16: Synthetic
procedures of spiramycin derivatives 2-5 together with the analytical data of them (FTꢀIR,
HRMS, HPLC and Elemental data) are included in ref. 39.
General synthetic procedure of novel triazole conjugates of spiramycin aglycone 6ꢀ16 together
with the analytical data (FTꢀIR, HRMS, HPLC and Elemental data):
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180 mg (0.33 mmol) of 5 was dissolved in 6mL mixture of THF/MeOH (3:1) and respective
mixtures were prepared with each of the following compounds taken separately (0.33 mmol): 1ꢀ
azidoꢀ1ꢀdeoxyꢀβꢀDꢀgalactopyranoside (6), 1ꢀazidoꢀ1deoxyꢀβꢀDꢀglucopyranoside (7), 2ꢀ
Acetamidoꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranosyl azide (8), 2ꢀAzidoꢀ2ꢀdeoxyꢀDꢀglucose (9), 6ꢀAzidoꢀ6ꢀ
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deoxyꢀDꢀgalactose
(10),
azidocycloheptane
(11),
azidocyclohexane
(12),
(azidomethyl)cyclohexane (13), 4ꢀ(azidomethyl)tetrahydropyran (14), benzil azide (15), AZT
(16). Then, to the each mixture 45mg (0.37 mmol) of CH3COOCu(I) and 124.5mg (0.71 mmol)
of ascorbic acid were added.
The mixtures were stirred at room temperature for an hour and after that to the 6-10
diethyl ether was added and extracted twice with 25 ml of water and saturated NaHCO3. In the
next step the water layer was extracted three times with 25ml of ethyl acetate. The organic layer
was evaporated giving the products 6-10.
The mixtures were stirred at room temperature for an hour and after that to the 11-16
diethyl ether was added and extracted three times with 25 ml of water and saturated NaHCO3.
The separated organic layer was evaporated and the synthesized derivatives 11-16 were next
purified by column chromatography with silica gel (25 cm × 1 cm, silica gel 60, 0.040ꢀ0.063
mm/230ꢀ400 mesh ASTM, Fluka) with ethyl acetate as an eluent.
New triazole conjugates of spiramycin’s aglycone 6-11, 15 and 16 were obtained as a
white powders whereas derivatives 12-14 as a colorless oils.
6: (2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ2ꢀ((1ꢀ((2S,3S,4R,5S,6S)ꢀ3,4,5ꢀtrihydroxyꢀ6ꢀ
(hydroxymethyl)tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ
3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (122mg, Yield
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49.4%) mp 93ꢀ95°C. HPLC Rt= 3.480 min. Elemental analysis C37H58N4O12: calculated: C,
59.18; H, 7.79; N, 7.46; measured: C, 59.21; H, 7.77; N, 7.47; HRMS (ESI ꢀ TOF) m/z: [M+H]+
= 751.4104. FTꢀIR (CH3CN): ν(OꢀH) – 3488 cmꢀ1, ν(CꢀH) – 2877 cmꢀ1, ν(C=O)lactone ꢀ1724 cmꢀ1,
1
ν(C=C) ꢀ1673 cmꢀ1, ν(CꢀO)lactone ꢀ1226 cmꢀ1, ν(CꢀO) ꢀ1125 cmꢀ1, ν(CꢀO) ꢀ1092 cmꢀ1, H NMR
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3
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(600 MHz, CD3CN, 25°C), δ= 0.91 (d, JH8,H20=6.7 Hz, 3H, 20ꢀH), 1.11 (d, JH5```,H6```=6.1 Hz,
3
3H, 6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.25 (d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.32 (m, 1H, 2’’’aꢀH),
1.42 (m, 1H, 7bꢀH), 1.43 (m, 1H, 3’’’bꢀH), 1.70 (dt, 3JH18a,H19=5.0 Hz, 3JH6,H18a=5.1 Hz, 2J’=12.4
Hz, 1H, 18aꢀH), 1.78 (m, 1H, 3’’’aꢀH), 1.78 (m, 1H, 2’’’bꢀH), 1.95 (m, 1H, 8ꢀH), 2.11 (m, 1H,
14bꢀH), 2.11 (m, 1H, 18bꢀH), 2.11 (m, 1H, 4’’’ꢀH), 2.15 (s, 6H, 7’’’ꢀH, 8’’’ꢀH), 2.25 (m, 1H, 6ꢀ
H), 2.46 (m, 1H, 14aꢀH), 2.87 (ddd, 4J=1.4 Hz, 3JH2b,H3=6.0 Hz, 2J=15.3 Hz, 1H, 2bꢀH), 3.18 (dd,
3JH2a,H3=7.8 Hz, 2J=15.3 Hz, 1H, 2aꢀH), 3.43 (dq, 3JH4’’’,H5’’’=9.5 Hz, 3JH5’’’,H6’’’=6.1 Hz, 1H, 5’’’ꢀ
3
H), 3.61 (s, 3H, 17ꢀH), 3.68 (m, 1H, 6’ꢀH), 3.68 (m, 1H, 3’ꢀH), 3.79 (t, JH4’,H5’=5.8 Hz,
3JH5’,H6’=5.8 Hz, 1H, 5’ꢀH), 3.95 (m, 1H, 4’ꢀH), 3.99 (d, JH5,H6=9.5 Hz, 1H, 5ꢀH), 4.09 (dd,
3
3JH8,H9’=4.6 Hz, JH9,H10’=9.3 Hz, 1H, 9ꢀH), 4.24 (t, JH1’,H2’=9.6 Hz, JH2’,H3’=9.6 Hz, 1H, 2’ꢀH),
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3
2
4.42 (dd, JH1’’’,H2a’’’=9.4 Hz, JH1’’’,H2b’’’=2.0 Hz, 1H, 1’’’ꢀH), 4.56 (d, J`=11.9 Hz, 1H, 21bꢀH),
2
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3
4.77(d, J`=11.9 Hz, 1H, 21aꢀH), 4.84 (dd, JH2a,H3=7.8 Hz, JH2b,H3=6.0 Hz, 1H, 3ꢀH), 5.20 (m,
3
3
1H, 15ꢀH), 5.20 (d, JH18a,H19=5.0 Hz, 1H, 19ꢀH), 5.54 (d, JH1’,H2’=9.6 Hz, 1H, 1’ꢀH), 5.55 (m,
1H, 13ꢀH), 5.60 (dd, 3JH10,H11=15.2 Hz, 3JH9,H10=9.3 Hz, 1H, 10ꢀH), 6.01 (ddd, JH12,H13=15.2 Hz,
3
3JH11,H12=10.5 Hz, J=1.6 Hz, 1H, 12ꢀH), 6.11 (dd, JH10,H11=15.2 Hz, JH11,H12=10.5 Hz, 1H, 11ꢀ
H), 8.15 (s, 1H, 23ꢀH), 13C NMR (600 MHz, CD3CN, 25°C), δ= 15.7 (20ꢀC), 18.8 (3’’’ꢀC), 19.5
(6’’’ꢀC), 20.5 (16ꢀC), 32.1 (2’’’ꢀC), 32.2 (2ꢀC), 34.5 (8ꢀC), 36.1 (7ꢀC), 39.1 (6ꢀC), 39.3 (18ꢀC),
40.9 (7’’’, 8’’’ꢀC), 41.7 (14ꢀC), 58.5 (17ꢀC), 60.9 (21ꢀC), 62.2 (6’ꢀC), 65.9 (4’’’ꢀC), 69.9 (4’ꢀC),
70.0 (15ꢀC), 70.8 (2’ꢀC), 74.2 (5’’’ꢀC), 74.7 (3’ꢀC), 79.0 (5’ꢀC), 80.5 (9ꢀC), 83.7 (5ꢀC), 89.1 (1’ꢀ
4
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C), 100.8 (1’’’ꢀC), 103.8 (19ꢀC), 104.0 (3ꢀC), 124.0 (23ꢀC), 128.9 (10ꢀC), 132.6 (12ꢀC), 132.7
(13ꢀC), 135.5 (11ꢀC), 145.8 (22ꢀC), 157.7 (4ꢀC), 174.0 (1ꢀC),
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7: (2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ2ꢀ((1ꢀ((2S,3S,4R,5R,6S)ꢀ3,4,5ꢀtrihydroxyꢀ6ꢀ
(hydroxymethyl)tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ
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3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (54 mg, Yield
21.8%) mp 84ꢀ86°C. HPLC Rt= 3.373min. Elemental analysis C37H58N4O12: calculated: C,
59.18; H, 7.79; N, 7.46; measured: C, 59.22; H, 7.80; N, 7.45; HRMS (ESI ꢀ TOF) m/z: [M+H]+
= 751.4130. FTꢀIR (CH3CN): ν(OꢀH) ꢀ3494 cmꢀ1, ν(CꢀH) ꢀ2930 cmꢀ1, ν(C=O)lactone ꢀ1726 cmꢀ1,
ν(C=C) ꢀ1675 cmꢀ1, ν(CꢀO)lactone ꢀ1230 cmꢀ1, ν(CꢀO) ꢀ1125 cmꢀ1, ν(CꢀO) ꢀ1068 cmꢀ1, 1H NMR
(600 MHz, CD3CN, 25°C), δ= 0.91 (d, 3JH8,H20=6.7 Hz, 3H, 20ꢀH), 1.12 (d, 3JH5```,H6```=6.1
Hz, 3H, 6’’’ꢀH), 1.15 (m, 1H, 7aꢀH), 1.25 (d, 3JH15,H16=6.3 Hz, 3H, 16ꢀH), 1.32 (m, 1H, 2’’’aꢀ
H), 1.42 (m, 1H, 3’’’bꢀH), 1.43 (m, 1H, 7bꢀH), 1.70 (m, 1H, 18aꢀH), 1.79 (m, 1H, 3’’’aꢀH), 1.79
(m, 1H, 2’’’bꢀH), 1.94 (m, 1H, 8ꢀH), 2.10 (m, 1H, 4’’’ꢀH), 2.12 (m, 1H, 18bꢀH), 2.12 (m, 1H,
14bꢀH), 2.15 (s, 6H, 7’’’ꢀH, 8’’’ꢀH), 2.26 (m, 1H, 6ꢀH), 2.46 (m, 1H, 14aꢀH), 2.87 (ddd, 4J=1.5
Hz, 3JH2b,H3=6.0 Hz, 2J=15.3 Hz, 1H, 2bꢀH), 3.18 (dd, 3JH2a,H3=7.9 Hz, 2J=15.1 Hz, 1H, 2aꢀ
H), 3.44 (m, 1H, 5’’’ꢀH), 3.48 (m, 1H, 4’ꢀH), 3.54 (m, 1H, 5’ꢀH), 3.55 (m, 1H, 3’ꢀH), 3.61 (s, 3H,
17ꢀH), (α) 3.63 (m, 1H, 6’ꢀH), (β) 3.77 (dd, 3JH5’,H6’=2.4 Hz, 2J =12.0 Hz, 1H, 6’ꢀH), 3.99 (d,
3JH5,H6=9.7 Hz, 1H, 5ꢀH), 4.00 (m, 1H, 2’ꢀH), 4.09 (dd, 3JH8,H9’=4.6 Hz, 3JH9,H10’=9.3 Hz,
1H, 9ꢀH), 4.43 (dd, 3JH1’’’,H2a’’’=9.3 Hz, 3JH1’’’,H2b’’’=1.9 Hz, 1H, 1’’’ꢀH), 4.56 (d,
2J`=11.9 Hz, 1H, 21bꢀH), 4.77 (d, 2J`=11.9 Hz, 1H, 21aꢀH), 4.83 (dd, 3JH2a,H3=7.9 Hz,
3JH2b,H3=5.9 Hz, 1H, 3ꢀH), 5.20 (m, 1H, 15ꢀH), 5.20 (m, 1H, 19ꢀH), 5.55 (ddd,
3JH12,H13=14.9 Hz, 3JH13,H14a=10.8 Hz, 3JH13,H14b=4.2 Hz, 1H, 13ꢀH), 5.59 (d,
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3JH1’,H2’=10.3 Hz, 1H, 1’ꢀH), 5.61 (m, 1H, 10ꢀH), 6.02 (ddd, 3JH12,H13=14.9 Hz,
3JH11,H12=10.5 Hz, 4J=1.6 Hz, 1H, 12ꢀH), 6.11 (dd, 3JH10,H11=15.1 Hz, 3JH11,H12=10.5
Hz, 1H, 11ꢀH), 8.18 (s, 1H, 23ꢀH), 13C NMR (600 MHz, CD3CN, 25°C), δ= 15.7 (20ꢀC), 18.8
(3’’’ꢀC), 19.5 (6’’’ꢀC), 20.5 (16ꢀC), 32.1 (2’’’ꢀC), 32.1 (2ꢀC), 34.4 (8ꢀC), 36.1 (7ꢀC), 39.1 (6ꢀC),
39.2 (18ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.7 (14ꢀC), 58.5 (17ꢀC), 60.9 (21ꢀC), 62.2 (6’ꢀC), 65.9 (4’’’ꢀC),
70.0 (15ꢀC), 70.6 (4’ꢀC), 73.3 (2’ꢀC), 74.0 (5’’’ꢀC), 77.9 (3’ꢀC), 80.2 (5’ꢀC), 80.5 (9ꢀC), 83.7 (5ꢀ
C), 88.6 (1’ꢀC), 100.7 (1’’’ꢀC), 103.8 (19ꢀC), 103.9 (3ꢀC), 123.8 (23ꢀC), 128.8 (10ꢀC), 132.7 (12ꢀ
C), 132.7 (13ꢀC), 135.6 (11ꢀC), 145.9 (22ꢀC), 157.7 (4ꢀC), 173.5 (1ꢀC),
8:
Nꢀ((2S,3S,4S,5R,6S)ꢀ2ꢀ(4ꢀ((((2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ
(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ14ꢀoxoꢀ
3,3a,4,5,6,11,12,14,15,17aꢀdecahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ2ꢀyl)oxy)methyl)ꢀ1Hꢀ
1,2,3ꢀtriazolꢀ1ꢀyl)ꢀ4,5ꢀdihydroxyꢀ6ꢀ(hydroxymethyl)tetrahydroꢀ2Hꢀpyranꢀ3ꢀyl)acetamide (65 mg,
Yield 25%) mp 109ꢀ111°C. HPLC Rt= 3.353 min. Elemental analysis C39H61N5O12: calculated:
C, 59.15; H, 7.76; N, 8.84; measured: C, 59.17; H, 7.78; N, 8.82; HRMS (ESI ꢀ TOF) m/z:
[M+H]+ = 792.4367. FTꢀIR (CH3CN): ν(OꢀH) ꢀ3494 cmꢀ1, ν(NꢀH)amide ꢀ3366 cmꢀ1, ν(CꢀH) ꢀ2931
cmꢀ1, ν(C=O)lactone ꢀ1728 cmꢀ1, ν(C7’=O) ꢀ1687 cmꢀ1, ν(C=C) ꢀ1642 cmꢀ1, δ(NꢀH) ꢀ1537 cmꢀ1,
ν(CꢀO)lactone ꢀ1240 cmꢀ1, ν(CꢀO) ꢀ1122 cmꢀ1, ν(CꢀO) ꢀ1068 cmꢀ1, γ(NꢀH) 570 cmꢀ1, 1H NMR (600
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MHz, CD3CN, 25°C), δ= 0.91 (d, JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.12 (d, JH5```,H6```=6.2 Hz, 3H,
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6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.25 (d, JH15,H16=6.5 Hz, 3H, 16ꢀH), 1.32 (m, 1H, 2’’’aꢀH), 1.42
(m, 1H, 7bꢀH), 1.42 (m, 1H, 3’’’bꢀH), 1.71 (m, 1H, 18aꢀH), 1.72 (s, 3H, 8’), 1.79 (m, 1H, 3’’’aꢀ
H), 1.79 (m, 1H, 2’’’bꢀH), 1.94 (m, 1H, 8ꢀH), 2.10 (m, 1H, 4’’’ꢀH), 2.10 (m, 1H, 18bꢀH), 2.15 (s,
6H, 7’’’ꢀH, 8’’’ꢀH), 2.16 (m, 1H, 14bꢀH), 2.27 (m, 1H, 6ꢀH), 2.44 (m, 1H, 14aꢀH), 2.85 (m, 1H,
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2bꢀH), 3.16 (dd, 3JH2a,H3=8.2 Hz, J=15.4 Hz, 1H, 2aꢀH), 3.43 (m, 1H, 5’’’ꢀH), 3.57 (m, 1H, 5’ꢀ
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H), 3.57 (m, 1H, 4’ꢀH), 3.61 (s, 3H, 17ꢀH), (α) 3.70 (d, 2J =12.0 Hz, 1H, 6’ꢀH), 3.71 (m, 1H, 3’ꢀ
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H), (β) 3.80 (d, J =12.0 Hz, 1H, 6’ꢀH), 3.97 (d, JH5,H6=9.4 Hz, 1H, 5ꢀH), 4.12 (dd, JH8,H9’=4.6
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Hz, JH9,H10’=9.3 Hz, 1H, 9ꢀH), 4.28 (m, 1H, 2’ꢀH), 4.43 (dd, JH1’’’,H2a’’’=9.3 Hz, JH1’’’,H2b’’’=1.7
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Hz, 1H, 1’’’ꢀH), 4.56 (d, J`=11.9 Hz, 1H, 21bꢀH), 4.72 (d, J`=12.2 Hz, 1H, 21aꢀH), 4.83 (dd,
3JH2a,H3=8.0 Hz, JH2b,H3=5.7 Hz, 1H, 3ꢀH), 5.18 (m, 1H, 15ꢀH), 5.15 (d, JH18a,H19=5.2 Hz, 1H,
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19ꢀH), 5.61 (m, 1H, 10ꢀH), 5.61 (m, 1H, 13ꢀH), 5.82 (d, JH1’,H2’=10.0 Hz, 1H, 1’ꢀH), 6.02 (ddd,
3JH12,H13=15.0 Hz, JH11,H12=10.5 Hz, J=1.5 Hz, 1H, 12ꢀH), 6.22 (dd, JH10,H11=15.0 Hz,
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3JH11,H12=10.5 Hz, 1H, 11ꢀH), 8.08 (s, 1H, 23ꢀH), C NMR (600 MHz, CD3CN, 25°C), δ= 15.8
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(20ꢀC), 18.8 (3’’’ꢀC), 19.5 (6’’’ꢀC), 20.5 (16ꢀC), 23.1 (8’ꢀC), 32.1 (2ꢀC), 32.2 (2’’’ꢀC), 34.7 (8ꢀ
C), 36.1 (7ꢀC), 38.7 (6ꢀC), 39.2 (18ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.6 (14ꢀC), 56.0 (2’ꢀC), 58.6 (17ꢀC),
60.8 (21ꢀC), 62.2 (6’ꢀC), 65.9 (4’’’ꢀC), 69.9 (15ꢀC), 71.1 (4’ꢀC), 74.2 (5’’’ꢀC), 75.3 (3’ꢀC), 80.1
(5’ꢀC), 80.6 (9ꢀC), 83.9 (5ꢀC), 87.2 (1’ꢀC), 100.9 (1’’’ꢀC), 103.4 (19ꢀC), 104.2 (3ꢀC), 123.7 (23ꢀ
C), 128.9 (10ꢀC), 132.9 (12ꢀC), 132.9 (13ꢀC), 135.5 (11ꢀC), 145.5 (22ꢀC), 157.3 (4ꢀC), 171.4 (7’ꢀ
C), 173.1 (1ꢀC),
9: (2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ2ꢀ((1ꢀ((2S,3S,4S,5R,6S)ꢀ2,4,5ꢀtrihydroxyꢀ6ꢀ
(hydroxymethyl)tetrahydroꢀ2Hꢀpyranꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ
3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (131 mg,
Yield 53%) mp 114ꢀ116°C. HPLC Rt= 3.347 min. Elemental analysis C37H58N4O12: calculated:
C, 59.18; H, 7.79; N, 7.46; measured: C, 59.17; H, 7.82; N, 7.44; HRMS (ESI ꢀ TOF) m/z:
[M+H]+ = 751.41317. FTꢀIR (CH3CN): ν(OꢀH) ꢀ3482 cmꢀ1, ν(CꢀH) ꢀ2928 cmꢀ1, ν(C=O)lactone
ꢀ
1726 cmꢀ1, ν(C=C) ꢀ1675 cmꢀ1, ν(CꢀO)lactone ꢀ1228 cmꢀ1, ν(CꢀO) ꢀ1121 cmꢀ1, ν(CꢀO) ꢀ1088 cmꢀ1,
1H NMR (600 MHz, CD3CN, 25°C), δ= 0.91 (d, JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.09 (d,
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3JH5```,H6```=5.8 Hz, 3H, 6’’’ꢀH), 1.10 (d, 3JH5```,H6```=5.8 Hz, 3H, 6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.24
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(d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.26 (d, JH15,H16=6.5 Hz, 3H, 16ꢀH), 1.31 (m, 1H, 2’’’aꢀH),
1.39 (m, 1H, 7bꢀH), 1.43 (m, 1H, 3’’’bꢀH), 1.69 (m, 1H, 18aꢀH), 1.74 (m, 1H, 3’’’aꢀH), 1.78 (m,
1H, 2’’’bꢀH), 1.95 (m, 1H, 8ꢀH), 2.09 (m, 1H, 4’’’ꢀH), 2.11 (m, 1H, 18bꢀH), 2.12 (m, 1H, 14bꢀ
H), 2.14 (s, 6H, 7’’’ꢀH, 8’’’ꢀH), 2.27 (m, 1H, 6ꢀH), 2.46 (m, 1H, 14aꢀH), 2.87 (m, 1H, 2bꢀH),
3.16 (m, 1H, 2aꢀH), 3.44 (m, 1H, 5’’’ꢀH), (α) 3.46 (m, 1H, 4’ꢀH), (β) 3.55 (m, 1H, 4’ꢀH), (α) 3.48
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(m, 1H, 5’ꢀH), 3.60 (s, 3H, 17ꢀH), 3.62 (s, 3H, 17ꢀH), (α) 3.72 (d, J =12.0 Hz, 1H, 6’ꢀH), (β)
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3.83 (d, J =12.0 Hz, 1H, 6’ꢀH), (β) 3.88 (m, 1H, 5’ꢀH), 3.98 (d, JH5,H6=9.6 Hz, 1H, 5ꢀH), 4.00
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(d, JH5,H6=9.6 Hz, 1H, 5ꢀH), 4.06ꢀ4.15 (m, 1H, 9ꢀH), (α) 4.09 (m, 1H, 3’ꢀH), (α) 4.20 (dd,
3JH1’,H2’=8.2 Hz, JH2’,H3’=10.5 Hz, 1H, 2’ꢀH), (β) 4.42 (m, 1H, 3’ꢀH), 4.43 (m, 1H, 1’’’ꢀH), 4.52
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(d, J`=11.6 Hz, 1H, 21bꢀH), 4.54 (d, J`=11.8 Hz, 1H, 21bꢀH), (β) 4.64 (dd, JH1’,H2’=3.3 Hz,
3JH2’,H3’=11.0 Hz, 1H, 2’ꢀH), 4.75 (d, J`=11.8 Hz, 1H, 21aꢀH), 4.78 (d, 2J`=11.6 Hz, 1H, 21aꢀH),
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4.84 (m, 1H, 3ꢀH), (α) 5.14 (d, JH1’,H2’=8.1 Hz, 1H, 1’ꢀH), 5.16 (m, 1H, 15ꢀH), 5.19 (d,
3JH18a,H19=4.9 Hz, 1H, 19ꢀH), 5.25 (ddq, JH15,H16=6.3 Hz, JH15,H14a=10.1 Hz, JH15,H14b=2.8 Hz,
1H, 15ꢀH), (β) 5.32 (d, 3JH1’,H2’=3.3 Hz, 1H, 1’ꢀH), 5.61 (m, 1H, 10ꢀH), 5.68 (ddd, 3JH12,H13=15.0
Hz, 3JH13,H14a=10.9 Hz, 3JH13,H14b=5.5 Hz, 1H, 13ꢀH), 6.04 (m, 1H, 12ꢀH), 6.12 (dd, 3JH10,H11=15.0
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Hz, JH11,H12=10.4 Hz, 1H, 11ꢀH), 6.13 (dd, JH10,H11=15.1 Hz, JH11,H12=10.5 Hz, 1H, 11ꢀH), (β)
7.95 (s, 1H, 23ꢀH), (α) 8.03 (s, 1H, 23ꢀH), 13C NMR (600 MHz, CD3CN, 25°C), δ= 15.6 (20ꢀC),
15.7 (20ꢀC), 18.8 (3’’’ꢀC), 19.5 (6’’’ꢀC), 20.5 (16ꢀC), 32.1 (2ꢀC), 32.1 (2’’’ꢀC), 32.2 (2’’’ꢀC),
34.0 (8ꢀC), 34.5 (8ꢀC), 35.9 (7ꢀC), 36.1 (7ꢀC), 38.8 (6ꢀC), 39.2 (6ꢀC), 39.2 (18ꢀC), 40.9 (7’’’,
8’’’ꢀC), 41.6 (14ꢀC), 41.8 (14ꢀC), 58.6 (17ꢀC), 60.8 (21ꢀC), 60.9 (21ꢀC), 62.5 (6’ꢀC), (β) 65.8 (2’ꢀ
C), 65.9 (4’’’ꢀC), (α) 68.7 (2’ꢀC), 70.0 (15ꢀC), (β) 71.1 (3’ꢀC), (α) 71.9 (4’ꢀC), (β) 72.2 (4’ꢀC),
(β) 72.8 (5’ꢀC), 74.2 (5’’’ꢀC), (α) 75.1 (3’ꢀC), (α) 77.3 (5’ꢀC), 80.2 (9ꢀC), 80.5 (9ꢀC), 83.6 (5ꢀC),
83.8 (5ꢀC), (β) 92.3 (1’ꢀC), (α) 95.7 (1’ꢀC), 100.4 (1’’’ꢀC), 100.7 (1’’’ꢀC), 103.4 (19ꢀC), 104.0 (3ꢀ
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C), (β) 124.0 (23ꢀC), (α) 125.6 (23ꢀC), 128.6 (10ꢀC), 129.0 (10ꢀC), 132.5 (13ꢀC), 132.7 (12ꢀC),
132.7 (13ꢀC), 132.9 (12ꢀC), 135.5 (11ꢀC), 135.8 (11ꢀC), 145.2 (22ꢀC), 145.3 (22ꢀC), 157.6 (4ꢀC),
157.8 (4ꢀC), 173.5 (1ꢀC), 173.6 (1ꢀC),
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10: (2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ2ꢀ((1ꢀ(((2S,3R,4R,5S,6S)ꢀ3,4,5,6ꢀ
tetrahydroxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ
3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (127 mg,
Yield 51.1%) mp 96ꢀ98°C. HPLC Rt= 3.420 min. Elemental analysis C37H58N4O12: calculated:
C, 59.18; H, 7.79; N, 7.46; measured: C, 59.20; H, 7.76; N, 7.48; HRMS (ESI ꢀ TOF) m/z:
[M+H]+ = 751.4147. FTꢀIR (CH3CN): ν(OꢀH) ꢀ3482 cmꢀ1, ν(CꢀH) ꢀ2932 cmꢀ1, ν(C=O)lactone ꢀ1726
1
cmꢀ1, ν(C=C) ꢀ1675 cmꢀ1, ν(CꢀO)lactone ꢀ1226 cmꢀ1, ν(CꢀO) ꢀ1125 cmꢀ1, ν(CꢀO) ꢀ1066 cmꢀ1, H
NMR (600 MHz, CD3CN, 25°C), δ= 0.91 (d, 3JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.10 (d, 3JH5```,H6```=6.2
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Hz, 3H, 6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.25 (d, JH15,H16=6.3 Hz, 3H, 16ꢀH), 1.32 (m, 1H, 2’’’aꢀH),
1.37 (m, 1H, 7bꢀH), 1.42 (m, 1H, 3’’’bꢀH), 1.70 (m, 1H, 18aꢀH), 1.79 (m, 1H, 3’’’aꢀH), 1.79 (m,
1H, 2’’’bꢀH), 1.94 (m, 1H, 8ꢀH), 2.09 (m, 1H, 4’’’ꢀH), 2.09 (m, 1H, 18bꢀH), 2.09 (m, 1H, 14bꢀ
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H), 2.14 (s, 6H, 7’’’ꢀH, 8’’’ꢀH), 2.27 (m, 1H, 6ꢀH), 2.42 (m, 1H, 14aꢀH), 2.85 (dd, JH2,H3=5.9
Hz, 2J=15.2 Hz, 1H, 2bꢀH), (β) 3.07 (m, 1H, 2’ꢀH), (α) 3.13 (m, 1H, 4’ꢀH), 3.14 (dd, 3JH2a,H3=8.1
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Hz, J=15.1 Hz, 1H, 2aꢀH), (β) 3.18 (m, 1H, 4’ꢀH), (α) 3.27 (dd, JH1’,H2’=9.3 Hz, JH2’,H3’=3.7
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Hz, 1H, 2’ꢀH), (β) 3.35 (t, JH2’,H3’’=9.0 Hz, JH3’,H4’’=9.0 Hz, 1H, 3’ꢀH), 3.44 (m, 1H, 5’’’ꢀH),
3.60 (s, 3H, 17ꢀH), (α) 3.60 (m, 1H, 3’ꢀH), (β) 3.70 (m, 1H, 5’ꢀH), 3.97 (d, 3JH5,H6=9.5 Hz, 1H, 5ꢀ
H), (α) 4.08 (m, 1H, 5’ꢀH), 4.13 (dd, 3JH8,H9’=4.6 Hz, 3JH9,H10’=9.3 Hz, 1H, 9ꢀH), (β) 4.41 (m, 1H,
1’ꢀH), 4.43 (dd, 3JH1’’’,H2a’’’=9.3 Hz, 3JH1’’’,H2b’’’=1.7 Hz, 1H, 1’’’ꢀH), (α) 4.46 (m, 1H, 6’bꢀH), (β)
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4.48 (d, J =14.4 Hz, JH5’,H6’=3.7 Hz, 1H, 6’ꢀH), 4.53 (d, J`=11.9 Hz, 1H, 21bꢀH), 4.54 (d,
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2J`=11.9 Hz, 1H, 21bꢀH), 4.72 (d, 2J`=12.1 Hz, 1H, 21aꢀH), (α) 4.73 (m, 1H, 6’aꢀH), (β) 4.77 (m,
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1H, 6’ꢀH), 4.81 (m, 1H, 3ꢀH), (α) 5.02 (d, JH1’,H2’=3.7 Hz, 1H, 1’ꢀH), 5.15 (m, 1H, 15ꢀH), 5.19
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(d, JH18a,H19=4.9 Hz, 1H, 19ꢀH), 5.59 (m, 1H, 13ꢀH), 5.61 (m, 1H, 10ꢀH), 6.03 (m, 1H, 12ꢀH),
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6.13 (dd, JH10,H11=15.0 Hz, JH11,H12=10.5 Hz, 1H, 11ꢀH), 7.87 (s, 1H, 23ꢀH), 13C NMR (600
MHz, CD3CN, 25°C), δ= 15.68 (20ꢀC), 15.71 (20ꢀC), 18.8 (3’’’ꢀC), 19.52 (6’’’ꢀC), 19.55 (6’’’ꢀ
C), 20.5 (16ꢀC), 32.09 (2ꢀC), 32.15 (2ꢀC), 32.09 (2’’’ꢀC), 32.15 (2’’’ꢀC), 34.35 (8ꢀC), 34.40 (8ꢀ
C), 35.86 (7ꢀC), 35.95 (7ꢀC), 38.88 (6ꢀC), 38.94 (6ꢀC), 39.2 (18ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.64 (14ꢀ
C), 41.69 (14ꢀC), 52.14 (6’ꢀC), 52.20 (6’ꢀC), 58.6 (17ꢀC), 60.86 (21ꢀC), 60.91 (21ꢀC), 65.9 (4’’’ꢀ
C), 69.78 (15ꢀC), 69.84 (15ꢀC), (α) 71.0 (5’ꢀC), (β) 72.4 (4’ꢀC), (α) 72.7 (4’ꢀC), (α) 73.3 (2’ꢀC),
74.2 (5’’’ꢀC), (α) 74.4 (3’ꢀC), (β) 75.1 (5’ꢀC), (β) 75.7 (2’ꢀC), (β) 77.2 (3’ꢀC), 80.32 (9ꢀC), 80.36
(9ꢀC), 83.7 (5ꢀC), 83.8 (5ꢀC), (α) 93.3 (1’ꢀC), (β) 97.7 (1’ꢀC), 100.7 (1’’’ꢀC), 103.36 (19ꢀC),
103.42 (19ꢀC), 104.18 (3ꢀC), 104.24 (3ꢀC), (β) 125.6 (23ꢀC), (α) 125.74 (23ꢀC), 128.84 (10ꢀC),
128.91 (10ꢀC), 132.61 (13ꢀC), 132.62 (13ꢀC), 132.74 (12ꢀC), 132.79 (12ꢀC), 135.48 (11ꢀC),
135.59 (11ꢀC), 145.34 (22ꢀC), 145.39 (22ꢀC), 157.41 (4ꢀC), 157.47 (4ꢀC), 173.09 (1ꢀC), 173.12
(1ꢀC),
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(2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ2ꢀ((1ꢀcycloheptylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ6ꢀ
(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀ
dimethylꢀ3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (106
mg, Yield 47%) mp 65ꢀ67°C. HPLC Rt= 15.027 min. Elemental analysis C38H60N4O7: calculated:
C, 66.64; H, 8.83; N, 8.18; measured: C, 66.63; H, 8.85; N, 8.19; HRMS (ESI ꢀ TOF) m/z:
[M+H]+ = 685.4512. FTꢀIR (CH3CN): ν(CꢀH) ꢀ2932 cmꢀ1, ν(C=O)lactone ꢀ1726 cmꢀ1, ν(C=C) ꢀ1675
cmꢀ1, ν(CꢀO)lactone ꢀ1228 cmꢀ1, ν(CꢀO) ꢀ1123 cmꢀ1, ν(CꢀO) ꢀ1068 cmꢀ1, H NMR (600 MHz,
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CD3CN, 25°C), δ= 0.91 (d, JH8,H20=6.7 Hz, 3H, 20ꢀH), 1.10 (d, JH5```,H6```=6.2 Hz, 3H, 6’’’ꢀH),
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1.14 (m, 1H, 7aꢀH), 1.25 (d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.33 (m, 1H, 2’’’aꢀH), 1.43 (m, 1H, 7bꢀ
H), 1.43 (m, 1H, 3’’’bꢀH), 1.72ꢀ1.51 (m, 2H, 27ꢀH), 1.72ꢀ1.51 (m, 2H, 28ꢀH), 1.72ꢀ1.51 (m, 2H,
26ꢀH), 1.72ꢀ1.51 (m, 2H, 29ꢀH), 1.67 (m, 1H, 18aꢀH), 1.68 (m, 1H, 25bꢀH), 1.68 (m, 1H, 30bꢀH),
1.80 (m, 1H, 3’’’aꢀH), 1.80 (m, 1H, 2’’’bꢀH), 1.80 (m, 1H, 25aꢀH), 1.80 (m, 1H, 30aꢀH), 1.94 (m,
1H, 8ꢀH), 2.08 (m, 1H, 14bꢀH), 2.08 (m, 1H, 18bꢀH), 2.08 (m, 1H, 4’’’ꢀH), 2.15 (s, 6H, 7’’’ꢀH,
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8’’’ꢀH), 2.25 (m, 1H, 6ꢀH), 2.46 (m, 1H, 14aꢀH), 2.88 (ddd, J=1.4 Hz, 3JH2b,H3=6.1 Hz, J=15.3
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Hz, 1H, 2bꢀH), 3.14 (ddd, J=0.9 Hz, JH2a,H3=7.8 Hz, J=15.3 Hz, 1H, 2aꢀH), 3.43 (dq,
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3JH4’’’,H5’’’=9.4 Hz, JH5’’’,H6’’’=6.2 Hz, 1H, 5’’’ꢀH), 3.60 (s, 3H, 17ꢀH), 3.97 (d, JH5,H6=9.5 Hz,
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1H, 5ꢀH), 4.08 (dd, JH8,H9’=4.6 Hz, JH9,H10’=9.4 Hz, 1H, 9ꢀH), 4.41 (dd, JH1’’’,H2a’’’=9.3 Hz,
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3JH1’’’,H2b’’’=1.9 Hz, 1H, 1’’’ꢀH), 4.49 (d, J`=11.6 Hz, 1H, 21bꢀH), 4.69 (m, 1H, 24ꢀH), 4.73 (d,
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2J`=11.7 Hz, 1H, 21aꢀH), 4.84 (dd, J=0.8 Hz, JH2a,H3=7.8 Hz, JH2b,H3=6.1 Hz, 1H, 3ꢀH), 5.16
(m, 1H, 15ꢀH), 5.17 (d, 3JH18a,H19=4.9 Hz, 1H, 19ꢀH), 5.52 (ddd, 3JH12,H13=14.6 Hz, 3JH13,H14a=10.3
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Hz, JH13,H14b=4.2 Hz, 1H, 13ꢀH), 5.61 (dd, JH10,H11=14.5 Hz, JH9,H10=9.3 Hz, 1H, 10ꢀH), 6.02
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(ddd, JH12,H13=14.5 Hz, JH11,H12=10.3 Hz, J=1.5 Hz, 1H, 12ꢀH), 6.09 (dd, JH10,H11=14.5 Hz,
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3JH11,H12=10.5 Hz, 1H, 11ꢀH), 7.94 (s, 1H, 23ꢀH), C NMR (600 MHz, CD3CN, 25°C), δ= 15.8
(20ꢀC), 18.8 (3’’’ꢀC), 19.5 (6’’’ꢀC), 20.5 (16ꢀC), 24.9 (26ꢀC), 24.9 (29ꢀC), 28.4 (27ꢀC), 28.4
(28ꢀC), 32.1 (2’’’ꢀC), 32.2 (2ꢀC), 34.5 (8ꢀC), 36.0 (7ꢀC), 36.2 (25ꢀC), 36.2 (30ꢀC), 39.2 (6ꢀC),
39.3 (18ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.7 (14ꢀC), 58.6 (17ꢀC), 60.9 (21ꢀC), 63.1 (24ꢀC), 65.9 (4’’’ꢀC),
69.6 (15ꢀC), 74.2 (5’’’ꢀC), 80.6 (9ꢀC), 83.7 (5ꢀC), 100.9 (1’’’ꢀC), 103.4 (19ꢀC), 104.0 (3ꢀC),
122.5 (23ꢀC), 129.1 (10ꢀC), 132.3 (13ꢀC), 132.9 (12ꢀC), 135.5 (11ꢀC), 145.2 (22ꢀC), 157.8 (4ꢀC),
173.0 (1ꢀC),
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(2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ2ꢀ((1ꢀcyclohexylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ6ꢀ
(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀ
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dimethylꢀ3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ14(3H)ꢀone (65
mg, Yield 29.4%) HPLC Rt= 11.667 min. Elemental analysis C37H58N4O7: calculated: C, 66.24;
H, 8.71; N, 8.35; measured: C, 66.21; H, 8.73; N, 8.34; HRMS (ESI ꢀ TOF) m/z: [M+H]+ =
671.4354. FTꢀIR (CH3CN): ν(CꢀH) ꢀ2932 cmꢀ1, ν(C=O)lactone ꢀ1726 cmꢀ1, ν(C=C) ꢀ1675 cmꢀ1,
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ν(CꢀO)lactone ꢀ1228 cmꢀ1, ν(CꢀO) ꢀ1123 cmꢀ1, ν(CꢀO) ꢀ1068 cmꢀ1, H NMR (600 MHz, CD3CN,
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25°C), δ= 0.91 (d, JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.10 (d, JH5```,H6```=6.1 Hz, 3H, 6’’’ꢀH), 1.14 (m,
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1H, 7aꢀH), 1.26 (d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.32 (m, 1H, 27bꢀH), 1.32 (m, 1H, 2’’’aꢀH),
1.45 (m, 1H, 3’’’bꢀH), 1.45 (m, 1H, 7bꢀH), 1.45 (m, 1H, 25bꢀH), 1.45 (m, 1H, 29bꢀH), 1.51 (m,
1H, 27aꢀH), 1.71 (m, 2H, 26ꢀH), 1.71 (m, 2H, 28ꢀH), 1.71 (m, 1H, 18aꢀH), 1.81 (m, 1H, 3’’’aꢀH),
1.81 (m, 1H, 2’’’bꢀH), 1.88 (m, 1H, 8ꢀH), 1.88 (m, 1H, 25aꢀH), 1.88 (m, 1H, 29aꢀH), 2.08 (m,
1H, 14bꢀH), 2.08 (m, 1H, 18bꢀH), 2.08 (m, 1H, 4’’’ꢀH), 2.15 (s, 6H, 7’’’ꢀH, 8’’’ꢀH), 2.26 (m, 1H,
6ꢀH), 2.47 (m, 1H, 14aꢀH), 2.87 (ddd, J=1.4 Hz, 3JH2b,H3=6.1 Hz, J=15.3 Hz, 1H, 2bꢀH), 3.14
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(ddd, J=0.9 Hz, JH2a,H3=7.9 Hz, J=15.3 Hz, 1H, 2aꢀH), 3.43 (dq, JH4’’’,H5’’’=9.4 Hz,
3JH5’’’,H6’’’=6.1 Hz, 1H, 5’’’ꢀH), 3.60 (s, 3H, 17ꢀH), 3.97 (d, JH5,H6=9.5 Hz, 1H, 5ꢀH), 4.08 (dd,
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3JH8,H9’=4.6 Hz, 3JH9,H10’=9.3 Hz, 1H, 9ꢀH), 4.41 (dd, 3JH1’’’,H2a’’’=9.3 Hz, JH1’’’,H2b’’’=1.9 Hz, 1H,
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1’’’ꢀH), 4.45 (m, 1H, 24ꢀH), 4.50 (d, 2J`=11.6 Hz, 1H, 21bꢀH), 4.73 (d, 2J`=11.7 Hz, 1H, 21aꢀH),
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4.84 (dd, J=0.8 Hz, JH2a,H3=7.8 Hz, JH2b,H3=6.0 Hz, 1H, 3ꢀH), 5.15 (m, 1H, 15ꢀH), 5.18 (d,
3JH18a,H19=4.9 Hz, 1H, 19ꢀH), 5.52 (ddd, JH12,H13=14.7 Hz, JH13,H14a=10.3 Hz, JH13,H14b=4.2 Hz,
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1H, 13ꢀH), 5.61 (dd, 3JH10,H11=14.6 Hz, 3JH9,H10=9.3 Hz, 1H, 10ꢀH), 6.02 (ddd, 3JH12,H13=14.6 Hz,
3JH11,H12=10.4 Hz, J=1.5 Hz, 1H, 12ꢀH), 6.10 (dd, JH10,H11=14.5 Hz, JH11,H12=10.5 Hz, 1H, 11ꢀ
H), 7.92 (s, 1H, 23ꢀH), 13C NMR (600 MHz, CD3CN, 25°C), δ= 15.8 (20ꢀC), 18.8 (3’’’ꢀC), 19.5
(6’’’ꢀC), 20.5 (16ꢀC), 25.8 (27ꢀC), 25.9 (26ꢀC), 25.9 (28ꢀC), 32.1 (2’’’ꢀC), 32.2 (2ꢀC), 34.1 (25ꢀ
C), 34.1 (29ꢀC), 34.5 (8ꢀC), 36.0 (7ꢀC), 39.2 (6ꢀC), 39.3 (18ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.7 (14ꢀC),
58.6 (17ꢀC), 60.7 (24ꢀC), 60.9 (21ꢀC), 65.9 (4’’’ꢀC), 69.6 (15ꢀC), 74.2 (5’’’ꢀC), 80.5 (9ꢀC), 83.7
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(5ꢀC), 100.9 (1’’’ꢀC), 103.4 (19ꢀC), 104.0 (3ꢀC), 122.5 (23ꢀC), 129.1 (10ꢀC), 132.3 (13ꢀC), 132.9
(12ꢀC), 135.5 (11ꢀC), 145.2 (22ꢀC), 157.7 (4ꢀC), 173.0 (1ꢀC),
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(2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ2ꢀ((1ꢀ(cyclohexylmethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀ
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yl)methoxy)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀ
methoxyꢀ5,12ꢀdimethylꢀ3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀf][1]oxacyclohexadecinꢀ
14(3H)ꢀone (81 mg, Yield 36%) HPLC Rt= 15.793 min. Elemental analysis C38H60N4O7:
calculated: C, 66.64; H, 8.83; N, 8.18; measured: C, 66.67; H, 8.81; N, 8.19; HRMS (ESI ꢀ TOF)
m/z: [M+H]+ = 685.4553. FTꢀIR (CH3CN): ν(CꢀH) ꢀ2930 cmꢀ1, ν(C=O)lactone ꢀ1724 cmꢀ1, ν(C=C) ꢀ
1673 cmꢀ1, ν(CꢀO)lactone ꢀ1226 cmꢀ1, ν(CꢀO) ꢀ1123 cmꢀ1, ν(CꢀO) ꢀ1066 cmꢀ1, 1H NMR (600 MHz,
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CD3CN, 25°C), δ= 0.91 (d, JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.00 (m, 1H, 26bꢀH), 1.00 (m, 1H, 30bꢀ
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H), 1.10 (d, JH5```,H6```=6.1 Hz, 3H, 6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.24 (m, 1H, 27bꢀH), 1.24 (m,
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1H, 29bꢀH), 1.25 (d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.34 (m, 1H, 2’’’aꢀH), 1.44 (m, 1H, 3’’’bꢀH),
1.44 (m, 1H, 7bꢀH), 1.57 (m, 1H, 26aꢀH), 1.57 (m, 1H, 30aꢀH), 1.60 (m, 2H, 28ꢀH), 1.72 (m, 1H,
18aꢀH), 1.74 (m, 1H, 27aꢀH), 1.74 (m, 1H, 29aꢀH), 1.79 (m, 1H, 3’’’aꢀH), 1.79 (m, 1H, 2’’’bꢀH),
1.94 (m, 1H, 25ꢀH), 1.95 (m, 1H, 8ꢀH), 2.08 (m, 1H, 14bꢀH), 2.08 (m, 1H, 18bꢀH), 2.08 (m, 1H,
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Hz, 3JH2b,H3=6.1 Hz, J=15.3 Hz, 1H, 2bꢀH), 3.15 (ddd, 5J=0.9 Hz, JH2a,H3=8.0 Hz, J=15.3 Hz,
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1H, 2aꢀH), 3.42 (dq, JH4’’’,H5’’’=9.4 Hz, JH5’’’,H6’’’=6.1 Hz, 1H, 5’’’ꢀH), 3.60 (s, 3H, 17ꢀH), 3.97
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(d, JH5,H6=9.5 Hz, 1H, 5ꢀH), 4.09 (dd, JH8,H9’=4.6 Hz, JH9,H10’=9.3 Hz, 1H, 9ꢀH), 4.21 (d,
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3JH24,H25=7.2 Hz, 2H, 24ꢀH), 4.41 (dd, JH1’’’,H2a’’’=9.3 Hz, JH1’’’,H2b’’’=1.9 Hz, 1H, 1’’’ꢀH), 4.52
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(d, J`=11.8 Hz, 1H, 21bꢀH), 4.73 (d, J`=11.8 Hz, 1H, 21aꢀH), 4.83 (dd, J=0.8 Hz, JH2a,H3=8.0
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Hz, JH2b,H3=6.0 Hz, 1H, 3ꢀH), 5.16 (m, 1H, 15ꢀH), 5.17 (d, JH18a,H19=4.9 Hz, 1H, 19ꢀH), 5.53
(ddd, 3JH12,H13=14.6 Hz, 3JH13,H14a=10.3 Hz, 3JH13,H14b=4.2 Hz, 1H, 13ꢀH), 5.62 (dd, 3JH10,H11=14.6
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Journal of Medicinal Chemistry
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Hz, JH9,H10=9.4 Hz, 1H, 10ꢀH), 6.02 (ddd, JH12,H13=14.6 Hz, JH11,H12=10.4 Hz, J=1.5 Hz, 1H,
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12ꢀH), 6.10 (dd, JH10,H11=14.5 Hz, JH11,H12=10.5 Hz, 1H, 11ꢀH), 7.86 (s, 1H, 23ꢀH), C NMR
(600 MHz, CD3CN, 25°C), δ= 15.7 (20ꢀC), 18.8 (3’’’ꢀC), 19.5 (6’’’ꢀC), 20.5 (16ꢀC), 26.3 (27ꢀC),
26.3 (29ꢀC), 26.9 (28ꢀC), 31.1 (26ꢀC), 31.1 (30ꢀC), 32.1 (2ꢀC), 32.2 (2’’’ꢀC), 34.5 (8ꢀC), 36.1 (7ꢀ
C), 39.0 (6ꢀC), 39.3 (18ꢀC), 39.6 (25ꢀC), 40.9 (7’’’, 8’’’ꢀC), 41.7 (14ꢀC), 56.8 (24ꢀC), 58.5 (17ꢀ
C), 61.0 (21ꢀC), 65.9 (4’’’ꢀC), 69.6 (15ꢀC), 74.2 (5’’’ꢀC), 80.6 (9ꢀC), 83.7 (5ꢀC), 100.9 (1’’’ꢀC),
103.4 (19ꢀC), 104.0 (3ꢀC), 124.9 (23ꢀC), 129.1 (10ꢀC), 132.4 (13ꢀC), 132.9 (12ꢀC), 135.4 (11ꢀC),
145.5 (22ꢀC), 157.7 (4ꢀC), 173.0 (1ꢀC),
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14: (2R,3aR,5R,6R,7E,9E,12R,16Z,17aR)ꢀ6ꢀ(((2R,5S,6R)ꢀ5ꢀ(dimethylamino)ꢀ6ꢀmethyltetrahydroꢀ
2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ17ꢀmethoxyꢀ5,12ꢀdimethylꢀ2ꢀ((1ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)ꢀ1Hꢀ
1,2,3ꢀtriazolꢀ4ꢀyl)methoxy)ꢀ3a,4,5,6,11,12,15,17aꢀoctahydroꢀ2Hꢀfuro[2,3ꢀ
f][1]oxacyclohexadecinꢀ14(3H)ꢀone (41 mg, Yield 18.1%) HPLC Rt= 6.673 min. Elemental
analysis C37H58N4O8: calculated: C, 64.70; H, 8.51; N, 8.16; measured: C, 64.67; H, 8.53; N,
8.18; HRMS (ESI ꢀ TOF) m/z: [M+H]+ = 687.4357. FTꢀIR (CH3CN): ν(CꢀH) ꢀ2932 cmꢀ1,
ν(C=O)lactone ꢀ1726 cmꢀ1, ν(C=C) ꢀ1675 cmꢀ1, ν(CꢀO)lactone ꢀ1226 cmꢀ1, ν(CꢀO) ꢀ1121 cmꢀ1, ν(CꢀO)
ꢀ1092 cmꢀ1, 1H NMR (600 MHz, CD3CN, 25°C), δ= 0.91 (d, 3JH8,H20=6.8 Hz, 3H, 20ꢀH), 1.10 (d,
3JH5```,H6```=6.2 Hz, 3H, 6’’’ꢀH), 1.14 (m, 1H, 7aꢀH), 1.25 (d, JH15,H16=6.4 Hz, 3H, 16ꢀH), 1.31
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(m, 1H, 2’’’aꢀH), 1.32 (m, 1H, 26bꢀH), 1.32 (m, 1H, 29bꢀH), 1.39 (m, 1H, 7bꢀH), 1.41 (m, 1H,
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3’’’bꢀH), 1.45 (m, 1H, 25ꢀH), 1.45 (m, 1H, 26aꢀH), 1.45 (m, 1H, 29aꢀH), 1.69 (dt, JH18a,H19=5.1
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Hz, JH6,H18a=5.1 Hz, J’=12.4 Hz, 1H, 18aꢀH), 1.76 (m, 1H, 3’’’aꢀH), 1.80 (m, 1H, 2’’’bꢀH),
1.95 (m, 1H, 8ꢀH), 2.09 (m, 1H, 4’’’ꢀH), 2.10 (m, 1H, 18bꢀH), 2.12 (m, 1H, 14bꢀH), 2.15 (s, 6H,
7’’’ꢀH, 8’’’ꢀH), 2.26 (m, 1H, 6ꢀH), 2.47 (m, 1H, 14aꢀH), 2.86 (ddd, J=1.5 Hz, 3JH2b,H3=5.9 Hz,
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2J=15.3 Hz, 1H, 2bꢀH), 3.15 (dd, 3JH2a,H3=8.2 Hz, J=15.4 Hz, 1H, 2aꢀH), 3.30 (m, 1H, 27bꢀH),
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