Synthesis and structure-bactericidal activity relationships of non-ketolides: 9-Oxime clarithromycin 11,12-cyclic carbonate featured with three-to eight-atom-length spacers at 3-OH

Article abstract of DOI:10.1016/j.ejmech.2019.03.037

In general, potent non-ketolide versions of erythromycin possessed conformationally constricted two- or three-atom-length sidechains at 3-OH. Novel 14-membered non-ketolides possessing long spacers beyond three-atom length were evaluated for antibacterial

Full text of DOI:10.1016/j.ejmech.2019.03.037

Accepted Manuscript
Synthesis and structure-bactericidal activity relationships of non-ketolides: 9-Oxime
clarithromycin 11,12-cyclic carbonate featured with three-to eight-atom-length spacers
at 3-OH
Xue-Meng Li, Wei Lv, Si-Yang Guo, Ya-Xin Li, Bing-Zhi Fan, Mark Cushman, Fan-
Sheng Kong, Jun Zhang, Jian-Hua Liang
PII:
S0223-5234(19)30255-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.037
EJMECH 11207
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2018
Revised Date: 14 March 2019
Accepted Date: 15 March 2019
Please cite this article as: X.-M. Li, W. Lv, S.-Y. Guo, Y.-X. Li, B.-Z. Fan, M. Cushman, F.-S. Kong,
J. Zhang, J.-H. Liang, Synthesis and structure-bactericidal activity relationships of non-ketolides: 9-
Oxime clarithromycin 11,12-cyclic carbonate featured with three-to eight-atom-length spacers at 3-OH,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.037.
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ACCEPTED MANUSCRIPT
Synthesis and Structure-bactericidal Activity Relationships of
Non-ketolides: 9-Oxime Clarithromycin 11,12-Cyclic Carbonate
Featured with Three- to Eight-atom-length Spacers at 3-OH
Xue-Meng Li, Wei Lv, Si-Yang Guo, Ya-Xin Li, Bing-Zhi Fan,
*
Mark Cushman, Fan-Sheng Kong, Jun Zhang, Jian-Hua Liang

ACCEPTED MANUSCRIPT
Synthesis and Structure-bactericidal Activity Relationships of
Non-ketolides: 9-Oxime Clarithromycin 11,12-Cyclic Carbonate
Featured with Three- to Eight-atom-length Spacers at 3-OH
a
b
a
a
c
Xue-Meng Li, Wei Lv, Si-Yang Guo, Ya-Xin Li, Bing-Zhi Fan,
b
d
a
a, c *
Mark Cushman, Fan-Sheng Kong, Jun Zhang, Jian-Hua Liang
a
School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the
Purdue Center for Cancer Research, Purdue University 47907 USA
School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081,
China
b
c
d
Beijing Increasepharm Safety & Efficacy Co. Ltd, Beijing 102206, China
_
*
_________________
To whom correspondence should be addressed. J.-H. Liang: E-mail: ljhbit@bit.edu.cn
1

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Abstract: In general, potent non-ketolide versions of erythromycin possessed conformationally
constricted two- or three-atom-length sidechains at 3-OH. Novel 14-membered non-ketolides
possessing long spacers beyond three-atom length were evaluated for antibacterial activity. The
most potent one is 34a, featuring a five-atom-length flexible linker from of a pyridine ring to the
aglycone. Conversion of the pyridine of 34a to other aryl groups, changing the linker’s length of
3
4a to longer or shorter ones, and variation of the linker flexibility to a rigid olefin or alkyne led to
decreased antibacterial activity. The hybrids of macrolides and quinolones 28b, 31 and 34b
possessing various sidechains, unlike their 15-membered counterparts, were ineffective compared
to 34a. Similar to the marketed ketolide telithromycin, the non-ketolide 34a proved to be a
time-dependent bactericidal agent, but it exhibited superior in vivo pharmacokinetic properties
such as longer half-life, higher plasma concentration, lower clearance and shorter time to reach the
highest drug concentration relative to telithromycin. Molecular docking suggested 34a might π - π
interact with the bacterial ribosomal RNA base G2505Er. This study suggested that the
bacteriostatic agent erythromycin can be structurally modified to afford a new bactericidal
chemotype that targets the ribosome and is superior to ciprofloxacin with regard to its minimum
bactericidal concentration.
Keywords: Erythromycin; macrolide; quinolone; ribosome; bactericidal activity; resistant bacteria
2

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1
. Introduction
Erythromycin, consisting of a 14-membered ring to which two sugars are
attached (Fig. 1), has been widely used for curing upper and lower respiratory tract
infections since the 1950s. One of the two sugars, 5-O-desosamine, hydrogen bonds
to the ribosomal RNA base A2058, and plays a crucial role in the bacteriostatic
activity by inhibiting the release of nascent peptides from the bacterial ribosome.
Inevitably, clinically isolated pathogens acquire erm (erythromycin ribosomal
methylation)/mef (macrolide efflux)-mediated resistance to erythromycin. [1]
Expression of an erm gene leads to irreversible mono- or dimethylation at a nitrogen
atom of the A2058, which disrupts its ability to act as a hydrogen bond donor toward
a hydroxyl oxygen of the 5-O-desosamine of the drug. As a result, erythromycin
cannot bind as firmly as before and is easily flushed away when nascent peptides pass
through the binding sites of erythromycin. In addition, a mef gene produces efflux
pump proteins that can export drug molecules outside the cell. Thus, the intracellular
concentration of erythromycin is reduced below the therapeutic level when it
encounters the ribosome. The second-generation erythromycins, clarithromycin and
azithromycin (Fig. 1), are also ineffective against these erm/mef-encoded bacteria.
Meanwhile, more and more bacteria are increasingly resistant to multiple drugs
including methicillin, ciprofloxacin, and erythromycin, which possess different
antibacterial mechanisms. Therefore, strenuous efforts are presently being made to
structurally modify the clinically used antibiotics in order to restore their activities
against resistant pathogens. [2, 3]
<Figure 1>
It was found that the other sugar, namely 3-O-cladinose, is responsible for the
inducible expression of erm and mef genes. [4] Removal of 3-O-cladinose, followed
by oxidation of the resulting 3-OH, led to the so-called ketolides. Telithromycin is the
first marketed third-generation erythromycin, and several other ketolides are currently
in late phases of clinical trials, such as solithromycin, modithromycin and
cethromycin, as illustrated in Fig. 2. [5, 6] Recent studies on high-resolution X-ray
3

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crystal structures of erythromycin derivatives in complex with bacterial ribosomes
have clarified the fact that the cladinose does not directly interact with rRNA, while
the aryl groups anchored the end of the newly introduced sidechains of telithromycin
and solithromycin afford an additional π - π stacking interaction with a base pair
A752/U2609 of the ribosomal RNA. [7-9]
<Figure 2>
In contrast, non-ketolides are less explored and thus far none of them have
entered clinical trials. In particular, the length of the spacers were mainly investigated
within a three-atom distance from the 3-oxygen to distal aryl groups. [10-20] Among
them, 3-O-acylide featured with a pyridyl acetyl (α-methoxyimino) group [12, 19],
3
-O-alkylide featured with a 1-propenyl pyrimidine [17], and 3-O-carbamolide
featured with an (R)-pyrrolidin-2-yl pyridine [18] (compounds A-D as shown Fig. 3)
were found with high potency against erythromycin-resistant pathogens. These potent
non-ketolides have been designed to share common structural characteristics, i.e. the
spacers are conformationally restricted so that they can orient and approach a novel
binding site, the base pair C2620/G2505Er (Escherichia coli numbering). [18, 19, 21,
2
2] On the other hand, the linkers’ length, derived from 8a-aza-lactam, a
fifteen-membered macrolide, has been studied with an extension to eight atoms. [23,
4] It was found that a hybrid of macrolide-quinolone named macrolone (Fig. 4),
possessing a seven-atom-length spacer, was extremely active. [24] However,
5-membered macrolides generally have different structure-activity relationships
SAR) than 14-membered scaffolds. [25] For these reasons, an extensive investigation
2
1
(
involving the extension of the three-atom-length spacers attached to the 3-OH of
clarithromycin, accompanied with various configurations, would be highly desirable.
<
Figure 3>
Figure 4>
<
Erythromycin is bacteriostatic and is a complete inhibitor of protein synthesis.
However, the ketolides such as telithromycin, lacking the bulky cladinose, supplied
the room allowing for the passage of the nascent peptides. Their inability to globally
4

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arrest nascent peptide biosynthesis results in a fatal imbalance of protein production
within the cells, which was attributed to the cause of their bactericidal activity against
some bacterial species. [26] Thus far, less information has been reported on the
bactericidal activity of the non-ketolides. [22] Over half of the clinical antibiotics
target the ribosome, and few of them are bactericidal agents. [27] Therefore, a study
on the structure-bactericidal activity relationships of non-ketolides is important to
unveil the unexplored bactericidal mechanisms associated with their effects on
bacterial ribosomes.
2
. Results and Discussion
2
.1 Chemistry
As incorporation of an 11,12-cyclic carbonate and a 9-oxime are beneficial for
enhancement of antibacterial activity, [28] we took 9-oxime clarithromycin
1,12-cyclic carbonate as the framework, and then endowed it with 3- to
-atom-length linkers from the 3-oxygen to distal heteroaryl groups in order to reveal
1
8
the structure-activity relationships.
As mentioned above, the rigidity of the spacers within a three-atom linker length
is critical for the antimicrobial activity of the potent non-ketolides (Fig. 3). We first
prepared 3a - 3i possessing a propyne linker terminated by a variety of mono aryl
groups from 1 by propargylation to afford 2 and subsequent Sonogashira reaction
(Scheme 1). The starting material 1 was prepared in high yield from commercially
available erythromycin 9-oxime in seven steps. [29] Direct hydrolysis of 1 produced 4
as a comparator of 3a - 3i. Further derivatization at the 9-allyl oxime (5, i.e
2
'-O-acetate of 3b) by Heck reaction led to the 3-OH 6b, instead of the desired 6a,
due to unexpected dealkylation of the original sidechain at 3-O (Scheme 2). The same
compound 6b was also obtained when using 2 as a precursor for the Heck reaction. In
other words, the 3-O-propargyl group attached to 3-O is labile under the Heck
reaction conditions.
<Scheme 1>
5

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<Scheme 2>
Next, we attempted to install two separate aryl groups at the end of the
-sidechain of the aglycone in a linear array to probe the potential binding sites. Thus,
3
the related reagents 8a and 8b were prepared from the acid 7, as depicted in Scheme 3.
A methyl group regioselectively replaced the acetyl group of the 9-oxime 9, which
was prepared according to the reference [30], in the presence of a base (Scheme 4).
The resulting product 10 reacted with bis(trichloromethyl)carbonate (BTC) to afford
the 11,12-carbonate 11. Then, 12 was obtained by propargylation at the 3-O position.
Sonogashira coupling of 12 followed by methanolysis of the 2'-acetate produced the
target compounds 13a - 13e.
<
Scheme 3>
Scheme 4>
<
To achieve the goal of elongation of the spacers, treatment of 11 with
3
-chloropropionyl chloride and triethylamine in MeCN followed by methanolysis
afforded the key intermediate 14 by a modified procedure (Scheme 5). [31] Michael
addition of 3-aminopropyne (or allylamine hydrochloride) to 14 in MeOH gave 15 (or
1
7), which was then converted to the unsaturated compound 16 (or 18) by
Sonogashira (or Heck) coupling reaction, as shown in Scheme 5. It should be noted
that hydrogenation of 16 and 18 in methanol resulted in the removal of the sidechains
with formation of the 3-OH groups due to methanolysis of the esters.
<Scheme 5>
To further compare the effects of the flexibility and the length of the sidechains
on antibacterial activity, a series of amino-capped saturated spacers 23a - 23e and 27
were prepared by employing the Cbz (benzyloxycarbonyl)-protected aminopropyne
(or aminobutyne) 21 (or 25) as starting materials, incorporation of aryl groups in 22
(
or 26), and deprotection under 2-3 atm H , as illustrated in Scheme 6. Following the
2
Michael addition procedure, 23a - 23d reacted with 14, resulting in the
seven-atom-length spacer in compounds 28a - 28d (Scheme 7), while 27 in reaction
with 14 yielded the eight-atom-length spacer in compound 29 (Scheme 7). It was
6

ACCEPTED MANUSCRIPT
previously reported that installation of 30 into a 15-membered 8a-lactam conferred
high efficacy, [24] so a 14-membered counterpart 31 was synthesized by Michael
addition of 30 to 14 (Scheme 7).
<
Scheme 6>
Scheme 7>
<
The length of the spacers was then shortened to five or six atoms by replacement
of the acryloyl group with a substituted carbamate (Scheme 8). Compound 11 was
subjected to CDI (carbonyl diimidazole) and DMAP (4-dimethylaminopyridine) in
DCM (dichloromethane), and then addition of aminopropyne led to the
propargylcarbamate 32. Methanolysis followed by Sonogashira reaction with the
corresponding ArBr or ArI produced 33a - 33b with the rigid linkers. Further
hydrogenation yielded 34a - 34b with the flexible linkers. Compound 34e was
obtained from 11, which was successively treated with CDI, 23e, and MeOH (Scheme
8
). Similarly, 35, which is analogous to 34a but with elongation with a methylene unit
in the spacer, was prepared by treatment of 11 with CDI, 27, and MeOH (Scheme 8).
Scheme 8>
.2 Structure-activity relationships of non-ketolides featured with three- to
<
2
eight-atom-length spacers at 3-OH
The compounds 3a - 3i, 4, 13a - 13e, 15 - 18, 28a - 28d, 29, 31, 33a, 34a - 34b,
3
4e, and 35 were tested against a panel of erythromycin-resistant and
erythromycin-susceptible clinical isolates and ATCC stains, including Gram-positive
bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes
and Staphylococcus epidermidis) and Gram-negative bacteria (Haemophilus
influenzae and Moraxella catarrhalis) by the standard method of two-fold broth
dilution. [22] Clarithromycin, azithromycin, telithromycin and ciprofloxacin were
used as reference compounds.
The antibacterial assays indicated that introduction of 3-O-3-substituted
pyridyl-2-propargyl (3a - 3i) resulted in 2 - 4 fold improvement of activity in
comparison with their 3-OH analog 4 (See Table 1). However, varying the mono aryl
7

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groups did not improve activity against susceptible bacteria relative to the controls,
clarithromycin and azithromycin. Among 3a - 3i, 3b was slightly more active than the
others. To further improve the potency, we initially attempted to synthesize a 3b
derivative, i.e. compound 6a, which features of two sidechains. Unfortunately, it was
not possible to synthesize it because the 3-O-propargyl group of 5 proved to be labile
in the subsequent Heck reaction.
<Table 1>
We substituted 9-methyl oxime for 9-allyl oxime and speculated that two aryl
groups bridged by zero- to two-atom-length intervals (13a -13e) might be suitable for
probing potential rRNA binding sites. Unfortunately, 13a -13e generally had higher
minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations
(MBCs) compared to the controls against most of the tested strains, as presented in
Table 2. Unlike 13a -13b and 13d, 13c and 13e sharing the hydrophobic phenyl group
instead of hydrophilic heteroaryl groups were weakly active against the constitutively
resistant S. aureus (MIC: 32 µg/mL), which was not susceptible to telithromycin and
clarithromycin (MICs: >256 µg/mL). Moreover, 13c and 13e (MBCs: 8-16 µg/mL)
had bactericidal activity comparable to clarithromycin against efflux-resistant S.
pneumonia PU09 and inducibly resistant S. pyogenes 01-968.
<Table 2>
Next, we used the 3-OH to attach an acryloyl group and constructed 7-atom to
-atom-length rigid or flexible spacers terminated by a variety of heteroaryl groups.
8
The antibacterial data are listed in Table 3. The propyl linker in 28a was superior to
the propargyl 16 and the propenyl 18, as emphasized by the 16-fold greater potency
against inducibly resistant S. pyogenes 01-968 (MIC: 0.5 µg/mL). The relative
potencies of 16, 18 and 28a indicated that the rigidity of the spacers at 3-O does not
improve antimicrobial activity. Meanwhile, introduction of a pyridyl group to the
unsubstituted rigid linkers (15 and 17) diminished the efficacy of the resulting
compounds 16 and 18, which confirmed that rigid linkers cannot be well
accommodated by the pocket formed by the bacterial ribosomal RNA bases.
8

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Therefore, we kept the flexible linkers constant and then examined the influence of
various aromatic groups that were anchored at the end of the linkers on activities (28a
-
28d). Of the four compounds, 28c and 28d were weakly active against
multidrug-resistant S. aureus PU20 (MICs: 28c and 28d, 64 µg/mL vs telithromycin, >
56 µg/mL vs ciprofloxacin, 256 µg/mL). However, the pyridine 28a showed a
2
well-balanced antibacterial profile. For example, the quinolone 28b, the
phenylimidazole 28c, and the tetrahydroquinoline 28d were less active than 28a
against inducibly resistant S. pyogenes 01-968 (MICs: 8 - 32 µg/mL vs. 0.5 µg/mL of
2
8a). Elongating the linker of 28a by one more methylene led to the homologue 29,
which generally retained the activities of 28a with the exception of the activity against
inducibly resistant S. pyogenes 01-968 (MICs: 29, 64 µg/mL vs 28a, 0.5 µg/mL) and
the activity against inducibly resistant S. aureus PU32 (MICs: 128 µg/mL of 29 vs 32
µg/mL of 28a). It was reported that when 30 was introduced to a 15-membered
8
a-lactam, the resulting macrolone (compound E as shown in Fig. 4) had amazingly
high potency. [24] For the purpose of comparison, the 14-membered counterpart 31
was prepared. However, the desired macrolone 31, with an ethylenediamine spacer,
was inferior to the pyridine 28a and the quinolone 28b, both possessing a
propylamine linker. This decreased activity of 31, contrary to compound E, can be
attributed to the different macrocyclic conformations of 14-membered macrolides
relative to 15-membered macrolides.
Finally, a shrinkage of linker length to five or six atoms was evaluated with
compounds 33, 34, and 35. The same trend was observed when comparing the rigid
and less potent 33a to the flexible but highly potent 34a. Moreover, 34a significantly
decreased the MIC values, in comparison with the parent clarithromycin, against the
clinically resistant isolates except for H. influenzae. Conversion of the terminal
pyridine 34a to the quinolone 34b or the aminopyridine 34e significantly decreased
the potency. Remarkably decreased activity was observed when the sidechain was
extended by a methylene unit (34a vs 35).
The non-ketolide 34a exhibited at least 32-fold higher bactericidal activity
9

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against mef-encoded resistant S. pneumoniae PU09, erm-mediated resistant S.
pyogenes 01-968, and erm-encoded resistant S. aureus PU32, in comparison with the
parent clarithromycin. Actually, 34a acquired equal and even superior bactericidal
activity in comparison to telithromycin against susceptible S. pneumoniae
ATCC49619 (MICs: 0.125 µg/mL of 34a vs 0.125 µg/mL of telithromycin) and
resistant S. pneumoniae PU09 (MICs: 0.5 µg/mL of 34a vs 4 µg/mL of telithromycin).
It was previously speculated that the ketolide telithromycin, unlike erythromycin,
lacks the bulky 3-O-cladinose so that it supplied the nascent peptides with more space
for the bypass. [27] This structural difference is viewed as an explanation for the
bactericidal activity of telithromycin. However, we proved that introduction of a
pyridylpropyl carbamoyl moiety at 3-OH could enable the non-ketolides to acquire
bactericidal activity against the erythromycin-resistant pathogens.
<Table 3>
2
.3 Molecular modelling
The molecular modelling was based on the X-ray crystal structure of a
carbamolide in complex with the Deinococcus radiodurans 50S ribosomal subunit
PDB ID 4IO9). [18] The molecular docking as presented in Fig. 5 suggested the most
potent compound 34a might interact with the bacterial ribosomal base G2484Dr
G2505, Escherichia coli numbering) via π - π stacking. Meanwhile, a weaker lone
(
(
pair - π contact contributed by the pyridine of 34a and perpendicular U2590 was
observed. In contrast, a little less potent 33a having a rigid linker posed similarly to
flexible 34a, but the nitrogen atom of the pyridine of 33a rotated by 120º relative to
that of 34a. The considerably less potent 35 oriented its pyridine ring away from the
position close to G2484 due to the longer sidechain. The molecular modelling may
provide a rational explanation of the observed structure-activity relationships.
It was previously reported that the non-ketolides with shorter and
conformationally restricted spacers at the 3-postion targeted rRNA base G2484Dr
(G2505Ec). [18, 19, 29] This study suggested the possibility of a structurally different
chemotype 34a with a prolonged and flexible linker interacting at the same binding
1
0

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site G2484Dr (G2505Ec).
<Figure 5>
2
.4 Bactericidal curves
According to time- or concentration-killing bacteria relationships, antibiotics can
be divided into a time-dependent group, a concentration-dependent group, and a
mixture of the two groups. For example, macrolides are generally time-dependent
while quinolones are concentration-dependent. Bactericidal curves indicated that both
3
4a and telithromycin are time-dependent agents because different concentrations
exerted little impact on bactericidal effects (Fig. 6).
Telithromycin possessed bactericidal effects (reducing CFU by no less than three
log units) against erythromycin-susceptible S. pneumoniae ATCC49619 at all MICs.
In contrast, 34a reduced two log units at 1×MIC, but it was bactericidal at the higher
MICs. With regard to efflux-resistant S. pneumoniae PU09, telithromycin and 34a
showed nearly the same bactericidal curve at various MICs. For erm-mediated S.
pyogenes 01-968, 34a presented bactericidal effects at the time point of 12 h, but the
reduced number of colonies bounced up to ≤ three log units at 24 h. A similar trend
was observed for telithromycin at 1×MIC.
<Figure 6>
2
.5 In vivo pharmacokinetic (PK) properties and cytochrome P450 (CYP) inhibition
Pharmacokinetic testing in male SD rats via oral administration suggested 34a
exhibited both longer half-life and higher plasma concentration than telithromycin
Figure S1 in Supplementary Material). Encouragingly, each of the PK properties of
4a is better than telithromycin (Table 4), such as 7-fold higher AUC (area under
curve), 7-fold slower systematic plasma clearance (CL), 2.4-fold longer half-life (t ),
(
3
1
/2
1
.4-fold longer mean residence time (MRT), 5-fold higher Cmax (the highest drug
concentration observed in plasma) and 0.4-fold shorter Tmax (time at which Cmax is
observed). Because the antibacterial effects of time-dependent drugs heavily depend
on the time when the blood concentration is beyond the MIC, the PK properties of
3
4a are favorable to increase the exposure time of pathogens to bactericidal
1
1

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concentrations.
Unfortunately, 34a is a medium inhibitor of human liver CYP 3A4 (IC50: 2.5 µM;
telithromycin, 11.8 µM; Table 4 and Figure S2 in Supplementary Material). As CYP
3
A4 is responsible for metabolism of half of the marketed drugs, [32] the chemical
use of 34a might lead to drug-drug interaction. Therefore, further structural
modification of 34a will be required to find safer non-ketolide candidates while
retaining good PK properties.
<Table 4>
3
. Conclusion
A series of novel erythromycin derivatives were designed that have variable
linker chain lengths and rigidities. The SAR indicated that flexile spacers confer
greater activities when they were examined within a range of 3-atom to 8-atom
lengths. Among the designed non-ketolides, 34a featured with a 5-atom-length
saturated spacer anchored with a pyridyl group is the most potent. Variation of the
rigidity and length of the spacers, and conversion of the terminal pyridine to various
quinolones and other aryl groups, led to decreased activity.
Surprisingly, 34a is even more active than the reference ciprofloxacin against
inducibly resistant S. aureus PU32 (a strain that is resistant to methicillin,
ciprofloxacin and erythromycin), susceptible S. pneumoniae ATCC49619, and
efflux-resistant S. pneumoniae PU09 as revealed by an 8- to 16-fold improvement of
MBC values. Bactericidal curves indicated that 34a, similar to telithromycin, is a
time-dependent antibiotic rather than concentration-dependent one. In vivo tests in SD
rats via oral administration suggested 34a has much superior PK properties over
telithromycin. This study suggested erythromycin can be structurally modified to a
non-ketolide version, serving as a probe for study on less-explored underlying
bactericidal mechanisms associated with bacterial ribosomes.
4
. Experimental Section
1
2

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4
.1 MIC and MBC assays
Each strain of bacteria was purified by plate activation before the experiment, and
fresh bacteria were used in the assays. The final concentration of bacterial solution
5
was about 5 ×10 CFU/ml. Standard ATCC strains and positive antibiotics were used
as quality control in each experiment where the MIC values should be within the
suggested MIC ranges, and bacterial solution without antibiotics was used as growth
control. Staphylococcus and Moraxella catarrhalis were incubated in CAMHB
medium at 35 ℃ for 20 hours. Streptococcus was incubated in CAMHB medium with
5
% horse serum at 35 ℃ for 20 - 24 hours. Haemophilus was incubated in HTM broth
medium with 1% the reagent SR158 at 35 ℃ for 20 - 24 hours. MICs were determined
from 0.002/0.032 to 64/256 µg/mL by the standard two-fold broth dilution method
recommended by the Clinical and Laboratory Standards Institute. [33] The samples
from 1 - 8 fold MIC were transported to the culture medium and subcultured at 35 ℃
for 24 h in the absence of antibiotics. Then, the lowest concentrations where the
number of colonies was reduced to > 3Log were read as MBCs.
1
0
4
.2 Docking methods
The crystal structure of the Deinococcus radiodurans 50S ribosomal subunit in
complex with a carbamolide was retrieved from the Protein Data Bank (code: 4IO9).
Only residues within 30 Å of the carbamolide were kept to simplify the calculation.
The 9-carbonyl group was changed to an oxime group. The carbamate nitrogen on the
11-position was changed to an oxygen. The carbamate side chain on the 3-position
was removed. The docking calculation was performed using the GOLD 3.0.1 program
through the genetic algorithm. The carbamate sidechains of compounds 33a, 34a and
3
5 were docked into the receptor binding site through a covalent attachment to the
oxygen on the 3-position. Each sidechain was docked 10 times and the best docking
solution according to the GoldScore fitness function was chosen as the binding
conformation.
4
.3 Bactericidal curves testing
A certain concentration of tested compounds (1×MIC, 2×MIC, 4×MIC, 8×MIC or
1
3

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1
6×MIC) and bacterial solution (Streptococcus pneumoniae or Streptococcus
6
pyogenes, about 1×10 CFU/mL) were added to the CAMHB medium containing 5%
horse serum. The mixture was incubated at 35 ℃ and the samples were taken at the
time points of 1, 2, 4, 6, 8, 12, and 24 h. The samples were diluted according to the
turbidity observed by the naked eyes. Three- to four-fold dilutions were made and
subcultured at 35 °C for 20 - 24 h. The number of colonies was counted and a
bactericidal curve was drawn in which the number of colonies (log) changes with time
(h).
4
.4 In vivo PK testing and CYP inhibition assays
Telithromycin and 34a were formulated in saline solution containing 1% DMF/ 20%
hydroxypropyl-β-cyclodextrin (w/v). [34] In vivo PK data were obtained in 2-3 male
SD rats by oral administration of a single dose of 20 mg/Kg. The blood samples were
collected at the time points of 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h. The blood samples were
centrifuged at 2500 g for 5 minutes at 4 °C. Quantitative analysis of drug
concentrations was performed by LC-MS/MS. The pharmacokinetic parameters for
the tested compounds such as half-life (t ), the highest drug concentration observed
1
/2
in plasma (Cmax), time at which Cmax is observed (Tmax), systematic plasma clearance
CL), mean residence time (MRT) and the area under curve (AUC) were calculated by
(
WinNonlin software (Version 5.2).
A mixture of the human liver microsome CYP 3A4 (final concentration: 0.03
mg/mL), the substrate (midazolam, final concentration: 5.00 µM) and 0.1 M
potassium phosphate buffer (pH 7.4) with 12 mM MgCl (final concentration: 0.72
2
mg/mL) was warmed at 37 ºC for 5 min. The tested compounds (0 - 25.00 µM ; or
ketoconazole as a positive inhibitor, 0 - 0.25 µM) and 25 mM NADPH solution (final
concentration: 1.5 mM) were added and the mixture was incubated at 37 ºC for 10
min. The metabolites were detected by HPLC (ACQUITY UPLC C18, 1.7 µm, 50 x
2
.1 mm I.D., Waters; gradient acetonitrile/water as eluents at a flow rate of 0.5
mL/min) and a triple quadrupole mass spectrometer (API 5500). The peak area ratio
of metabolite/internal standard was determined for each metabolite. The IC50 values
1
4

ACCEPTED MANUSCRIPT
were calculated using commercial software Graphpad Prism (Version 7).
4
.5 Synthetic procedures
All solvents and reagents were obtained from commercial sources (InnoChem;
J&K scientific) and used without further purification unless otherwise noted. Column
1
13
chromatography was performed on silica gel (100-200 or 200-300 mesh). H and C
spectra were taken in CDCl on Bruker Ascend 400, ARX 500 or Ascend 700 MHz
3
spectrometers with tetramethylsilane (TMS) as an internal standard. High resolution
mass spectra (HRMS) were obtained with Agilent Q-TOF 6520 LC/MS or Bruker
Apex IV FT. The purities of the target compounds for antibacterial evaluation were
examined by high-performance TLC under the conditions of different eluents and
visualization, and further confirmed by NMR spectra by comparing integral
calculation of the number of hydrogen atoms of impurities.
4
.5.1. 3-O-Descladinosyl-3-O-acryloyl-6-O-methylerythromycin
A
9-O-methyl
oxime-11, 12-cyclic carbonate (14)
To a solution of 11 (0.7235 g, 1.05 mmol) and triethylamine (1.468 mL, 10.53
mmol) in acetonitrile (15 mL) was added dropwise a solution of 3-chloropropionyl
chloride (0.288 ml, 2.95 mmol) in acetonitrile (15 mL). The reaction mixture was
stirred at 80 ℃ for 3 h and then the solvent was removed under reduced pressure. The
mixture was extracted with ethyl acetate and the organic layer was washed
successively with water and brine. The organic solvent was removed in vacuum and
dried to afford 2'-OAc-14 (0.9762 g, 1.32 mmol, 125.7%).
A solution of 2'-OAc-14 (0.9762 mg, 1.32 mmol) in MeOH (20 mL) was
stirred at 65 ℃ for 3 h. The organic solvent was removed in vacuum and dried to
afford 14 (0.7905 g, 1.13 mmol, 85.6%) as a dark yellow solid.
4
3
9
.5.2.
-O-Descladinosyl-3-O-[3'-propargylamino-propionyl]-6-O-methylerythromycin
A
-O-methyl oxime-11, 12-cyclic carbonate (15)
To a solution of 14 (0.7905 g, 1.13 mmol) and ethyldiisopropylamine (0.985 mL,
1
5

ACCEPTED MANUSCRIPT
5
.9 mmol) in MeOH (15 mL) was added 3-aminopropyne (0.23 mL, 3.54 mmol). The
reaction mixture was stirred at 60 ℃ for 5 h and then the solvent was removed under
reduced pressure. The mixture was extracted with dichloromethane and the organic
phase was washed successively with water and brine. The organic solvent was
removed in vacuum. The residue was purified by column chromatography on silica
gel (CH Cl /C H OH/NH •H O, 10:0.2:0.1) to afford 15 (0.4926 g, 0.65 mmol,
2
2
2
5
3
2
5
7.5%).
HRMS (ESI) (M+H) m/z 754.4484, Calcd for C H N O 754.4485. H
+
1
3
8
64
3
12
NMR (CDCl , 400 MHz), δ: 5.16 (dt, J = 10.8, 1.7 Hz, 1 H, H-13), 5.07 (d, J =
3
1
3
3
7
2
2
2
1
3
1
1.0 Hz, 1 H, H-3), 4.88 (s, 1 H, H-11), 3.95 (d, J = 7.2 Hz, 1 H, H-1'), 3.82 (s,
H, 9-O-CH ), 3.75-3.69 (m, 1 H, H-8), 3.67 (d, J = 3.3 Hz, 1 H, H-5),
3
.45-3.42 (m, 2 H, 3-O-CH C≡C), 3.41-3.37 (m, 1 H, H-5'), 3.16 (dd, J = 10.1,
2
.2 Hz, 1 H, H-2'), 3.02 (s, 3 H, 6-OCH ), 2.98 (t, J = 6.4 Hz, 2 H, -N-CH ),
3
2
.95-2.85 (m, 1 H, H-2), 2.63 (t, J = 6.4 Hz, 2 H, 3-O-CO-CH -), 2.52-2.4 (m,
2
H, H-10, H-3'), 2.28 (s, 6 H, -N(CH ) ), 2.22 (q, J = 2.1 Hz, 1 H, H-propargyl),
3
2
.14-2.06 (m, 1 H, H-4), 1.96-1.85 (m, 1 H, H-14eq), 1.68-1.62 (m, 1 H, H-4'eq),
.60-1.53 (m, 1 H, H-14ax), 1.49 (s, 3 H, 12-CH ), 1.45-1.37 (m, 1 H, H-7a), 1.34 (s,
3
H, 6-CH ), 1.32-1.27 (m, 1 H, H-7b), 1.22 (t, J = 6.3 Hz, 6 H, 5'-CH , 10-CH ),
3
3
3
.14-1.08 (m, 6 H, 2-CH , 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.86 (t, J = 7.3
3
3
3
1
3
Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ: 173.9, 172.3, 164.5, 154.7, 103.8,
3
3
8
3
8
4
3
4.9, 83.2, 81.8, 78.5, 78.0, 75.7, 71.6, 70.4, 69.6, 65.9, 61.4, 49.8, 43.8, 43.2, 40.3,
8.2, 37.3, 36.5, 34.8, 32.7, 28.4, 25.6, 22.2, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2,
.9.
.5.3.
-O-Descladinosyl-3-O-[3'-(3"-(3"'-pyridyl)propargylamino)-propionyl]-6-O-methyle
rythromycin A 9-O-methyl oxime-11, 12-cyclic carbonate (16)
To a solution of 15 (0.4926 g, 0.65 mmol), CuI (12 mg, 0.063 mmol),
(
PPh ) PdCl (23 mg, 0.033 mmol) and triethylamine (0.136 mL, 0.975 mmol) in
3 2 2
acetonitrile (15 mL) was added 3-bromopyridine (0.189 mL, 1.95 mmol). The
1
6

ACCEPTED MANUSCRIPT
reaction mixture was recharged with argon and stirred at 80 ℃ for 3 h in a sealed tube.
The mixture was extracted with ethyl acetate and the organic layer was washed
successively with water and brine. The organic solvent was removed in vacuum. The
residue
was
purified
by
column
chromatography on
silica
gel
(
CH Cl /C H OH/NH •H O, 10:0.2:0.1) to afford 16 (0.135 g, 0.162 mmol,
2 2 2 5 3 2
2
4.9%).
+
1
HRMS (ESI) (M+H) m/z 831.4760, Calcd for C H N O 831.4750. H
4
3
67
4
12
NMR (CDCl , 400 MHz), δ: 8.64 (s, 1 H, pyridine), 8.53 (d, J = 4.7 Hz, 1 H,
3
pyridine), 7.74-7.65 (m, 1 H, pyridine), 7.24 (dd, J = 7.9, 5.1 Hz, 1 H,
pyridine), 5.16 (dt, J = 10.9 Hz, J = 1.9 Hz, 1 H, H-13), 5.08 (d, J = 11.0 Hz,
1
2
1
H, H-3), 4.88 (s, 1 H, H-11), 3.95 (dd, J = 7.3, 1.4 Hz, 1 H, H-1'), 3.82 (s, 3
H, 9-O-CH ), 3.75-3.64 (m, 4 H, H-5, H-8, 3-O-CH C≡C), 3.45-3.35 (m, 1 H,
3
2
H-5'), 3.16 (dd, J = 10.4, 6.8 Hz, 1 H, H-2'), 3.10-3.00 (m, 5 H, 6-O-CH3,
-
N-CH ), 2.93-2.83 (m, 1 H, H-2), 2.67 (t, J = 6.3 Hz, 2 H, 3-O-CO-CH -),
2 2
2
.53-2.40 (m, 2 H, , H-10, H-3'), 2.27 (s, 6 H, -N(CH ) ), 2.15-2.07 (m, 1 H,
3 2
H-4), 1.95-1.85 (m, 1 H, H-14eq), 1.65-1.55 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H,
1
2-CH ), 1.44-1.37 (m, 1 H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.32-1.27 (m, 1 H, H-7b),
3 3
1
.23 (d, J = 6.8 Hz, 3 H, 5'-CH ), 1.17 (d, J = 6.0 Hz, 3 H, 10-CH ), 1.14-1.05 (m, 6
3
3
H, 2-CH , 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.86 (t, J = 7.3 Hz, 3 H, 15-CH );
3
3
3
3
1
3
C NMR (CDCl , 175 MHz), δ: 173.9, 172.4, 164.5, 154.7, 152.3, 148.6, 138.5,
3
1
6
1
4
9
23.0, 120.2, 103.8, 90.8, 84.9, 83.2, 81.9, 80.4, 78.5, 78.0, 75.7, 70.3, 69.6, 66.0,
1.4, 49.8, 44.0, 43.2, 40.3, 39.1, 37.3, 36.5, 34.8, 32.7, 28.4, 25.6, 22.2, 21.2, 19.4,
8.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.4. 3-O-Descladinosyl-3-O-[3'-allylamino-propionyl]-6-O-methylerythromycin A
-O-methyl oxime-11, 12-cyclic carbonate (17)
To a solution of 14 (1.0147 g, 1.45 mmol) and ethyldiisopropylamine (1.26 mL,
.24 mmol) in MeOH (20 mL) was added allylamine hydrochloride (0.41 g, 4.35
7
mmol). The reaction mixture was stirred at 60 ℃ for 5 h and then the solvent was
removed under reduced pressure. The mixture was extracted with dichloromethane
1
7

ACCEPTED MANUSCRIPT
and the organic phase was washed successively with water and brine. The organic
solvent was removed in vacuum. The residue was purified by column chromatography
on silica gel (CH Cl /C H OH/NH •H O, 10:0.3:0.1) to afford 17 (0.058 g, 0.077
2
2
2
5
3
2
mmol, 5.3%).
HRMS (ESI) (M+H) m/z 756.4626, Calcd for C H N O 756.4641. H
+
1
3
8
66
3
12
NMR (CDCl , 400 MHz), δ: 5.88 (ddt, J = 16.6, 11.4, 5.9 Hz, 1 H, allyl),
3
5
.25-5.15 (m, 2 H, allyl), 5.09 (t, J = 11.8 Hz, 2 H, H-3, H-13), 4.88 (s, 1 H,
H-11), 3.94 (d, J = 7.2 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.75-3.62 (m, 2
3
H, H-5, H-8), 3.45-3.35 (m, 1 H, H-5'), 3.27 (d, J = 6.0 Hz, 2 H,
CH =CH-CH ), 3.16 (dd, J = 10.0, 7.3 Hz, 1H, H-2'), 3.02 (s, 3 H, 6-O-CH ),
2
2
3
2
2
1
1
1
4
.95-2.83 (m, 3 H, -N-CH , H-2), 2.63 (t, J = 6.5 Hz, 2 H, 3-O-CO-CH -),
2 2
.51-2.40 (m, 2 H, H-10, H-3'), 2.27 (s, 6 H, -N(CH ) ), 2.15-2.05 (m, 1 H, H-4),
3
2
.95-1.85 (m, 1 H, H-14eq), 1.65-1.55 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H,
2-CH ), 1.44-1.36 (m, 1 H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.32-1.27 (m, 1 H, H-7b),
3
3
.22 (t, J = 7.3 Hz, 6 H, 5'-CH , 10-CH ), 1.10 (dd, J = 7.0, 3.2 Hz, 6 H, 2-CH ,
3
3
3
13
-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.86 (t, J = 7.3 Hz, 3 H, 15-CH ); C NMR
3
3
3
(
CDCl , 175 MHz), δ: 173.9, 172.5, 164.5, 154.7, 136.5, 116.2, 103.7, 85.0, 83.2,
3
8
3
4
3
1.6, 78.5, 77.9, 75.7, 70.4, 69.6, 65.9, 61.4, 52.4, 49.9, 44.3, 43.2, 40.3, 37.3, 36.5,
5.0, 32.7, 28.4, 25.6, 22.2, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.5.
-O-Descladinosyl-3-O-[3'-(3"-(3'''-pyridyl)allylamino)-propionyl]-6-O-methylerythr
omycin A 9-O-methyl oxime-11, 12-cyclic carbonate (18)
To a solution of 17 (0.5611 g, 0.742 mmol), palladium acetate (33 mg, 0.148
mmol) and tri(o-tolyl)phosphine (90 mg, 0.297 mmol) in acetonitrile (10 mL) were
added 3-bromopyridine (0.215 mL, 2.23 mmol) and triethylamine (0.206 mL, 1.48
mmol). The reaction mixture was flushed with argon and sealed in a pressure tube.
The reaction mixture was stirred at 60 ºC for 1 h and thereafter at 90 ºC for 24 h. The
reaction mixture was extracted with ethyl acetate, washed with water and brine and
concentrated in vacuum. The crude mixture was purified by column chromatography
1
8

ACCEPTED MANUSCRIPT
on silica gel (CH Cl /C H OH/NH •H O, 10:0.5:0.1) to afford 18 (0.188 g, 0.226
2
2
2
5
3
2
mmol, 30.5 %).
HRMS (ESI) (M+H) m/z 833.4907, Calcd for C H N O 833.4907. H
+
1
4
3
69
4
12
NMR (CDCl , 700 MHz), δ: 8.60 (s, 1 H, H-pyridine), 8.53 (d, J = 4.7 Hz, 1 H,
3
H-pyridine), 7.70 (d, J = 7.8 Hz, 1 H, H-pyridine), 7.26 (dd, J = 7.9, 4.8 Hz, 1
H, H-pyridine), 6.62-6.31 (m, 2 H, H-allyl), 5.18 (dd, J = 10.6, 2.5 Hz, 1 H,
H-13), 5.09 (d, J = 11.0 Hz, 1 H, H-3), 4.89 (s, 1 H, H-11), 3.96 (d, J = 7.2 Hz,
1
1
2
H, H-1'), 3.83 (s, 3 H, 9-O-CH ), 3.74-3.64 (m, 2 H, H-5, H-8), 3.44-3.36 (m,
3
H, H-5'), 3.21-3.14 (m, 1H, H-2'), 3.03-2.95 (m, 5 H, 6-O-CH , -NH-CH ),
3
2
.93-2.85 (m, 1 H, H-2), 2.69 (s, 2 H, 3-O-CO-CH -), 2.53-2.45 (m, 2 H, ,
2
H-10, H-3'), 2.34-2.22 (m, 6 H, -N(CH ) ), 2.15-2.09 (m, 1 H, H-4), 1.95-1.87
3
2
(
m, 1 H, H-14eq), 1.60-1.54 (m, 2 H, H-4'eq, H-14ax), 1.50 (s, 3 H, 12-CH ), 1.34 (s,
3
3
1
H, 6-CH ), 1.33-1.26 (m, 2 H, H-7a, H-7b), 1.24 (d, J = 6.8 Hz, 3 H, 10-CH ),
3 3
.21-1.19 (m, 3 H, 5'-CH ), 1.15-1.07 (m, 6 H, 2-CH , 4-CH ), 0.95 (d, J = 7.0 Hz, 3
3
3
3
1
3
H, 8-CH ), 0.87 (t, J = 7.4 Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ: 173.9,
3
3
3
1
7
2
4
3
72.2, 164.5, 154.6, 148.2, 132.8, 123.5, 123.4, 123.3, 103.7, 84.9, 83.2, 81.7, 78.5,
8.1, 75.7, 70.5, 69.6, 66.1, 61.4, 51.5, 49.9, 44.4, 43.1, 40.3, 37.2, 36.5, 34.9, 32.7,
8.5, 25.6, 22.2, 21.1, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.6.
-O-Descladinosyl-3-O-[3'-(3''-(3'''-pyridyl)-propylamino)propionyl]-6-O-methyleryt
hromycin A 9-O-methyl oxime-11, 12-cyclic carbonate (28a)
To a solution of 14 (0.356 g, 0.509 mmol) and ethyldiisopropylamine (0.444 mL,
2
.545 mmol) in MeOH (20 mL) was added 23a (0.2079 g, 1.53 mmol). The reaction
mixture was stirred at 60 ℃ for 5 h and then the solvent was removed under reduced
pressure. The mixture was extracted with dichloromethane and the organic phase was
washed successively with water and brine. The organic solvent was removed in
vacuum. The residue was purified by column chromatography on silica gel
(
CH Cl /C H OH/NH •H O, 10:0.2:0.1) to afford 28a (0.112 g, 0.134 mmol,
2 2 2 5 3 2
2
6.3%).
1
9

ACCEPTED MANUSCRIPT
+
1
HRMS (ESI) (M+H) m/z 835.5055, Calcd for C H N O 835.5063. H
4
3
71
4
12
NMR (CDCl , 400 MHz), δ: 8.45 (s, 2 H, H-pyridine), 7.50 (d, J = 7.7 Hz, 1 H,
3
H-pyridine), 7.21 (dd, J = 8.0, 4.6 Hz, 1 H, H-pyridine), 5.16 (d, J = 10.7 Hz,
1
7
3
2
H, H-13), 5.07 (d, J = 11.0 Hz, 1 H, H-3), 4.88 (s, 1 H, H-11), 3.93 (d, J =
.1 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.76-3.62 (m, 2 H, H-5, H-8),
3
.42-3.30 (m, 1 H, H-5'), 3.16 (t, J = 8.7 Hz, 1H, H-2'), 3.02 (s, 3 H, 6-O-CH ),
3
.95-2.83 (m, 3 H, H-2, -CO-CH -CH NH-), 2.70-2.59 (m, 6 H,
2
2
-
CO-CH -CH NH-, -NH-CH -CH -CH -), 2.52-2.35 (m, 2 H, , H-10, H-3'),
2 2 2 2 2
2
.26 (s, 6 H, -N(CH ) ), 2.15-2.05 (m, 1 H, H-4), 1.95-1.75 (m, 3 H, H-14eq,
3 2
-
NH-CH -CH -CH -), 1.65-1.55 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ),
2 2 2 3
1
.44-1.38 (m, 1 H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.32-1.27 (m, 1 H, H-7b), 1.25-1.17
3
(
m, 7 H, 5'-CH , 10-CH , H-4'ax), 1.14-1.06 (m, 6 H, 2-CH , 4-CH ), 0.93 (d, J = 7.0
3 3 3 3
13
Hz, 3 H, 8-CH ), 0.86 (t, J = 7.4 Hz, 3 H, 15-CH ); C NMR (CDCl , 100 MHz), δ:
3
3
3
1
7
3
4
3
73.9, 172.5, 164.5, 154.7, 149.9, 147.5, 137.1, 135.7, 123.3, 103.7, 85.0, 83.2, 81.5,
8.5, 77.9, 75.7, 70.4, 69.6, 66.0, 61.4, 49.9, 49.1, 45.0, 43.2, 40.3, 37.3, 36.5, 35.0,
2.7, 31.4, 30.6, 28.4, 25.6, 22.2, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.7.
-O-Descladinosyl-3-O-[3'-(3''-(3'''-carboxy-1'''-cyclopropyl-1''',4'''-dihydro-4'''-oxo-
quinol-6'''-yl)-propylamino)propionyl]-6-O-methylerythromycin
A
9-O-methyl
oxime-11, 12-cyclic carbonate (28b)
To a solution of 14 (0.22 g, 0.315 mmol) and ethyldiisopropylamine (0.27 mL,
.55 mmol) in MeOH (20 mL) was added 23b (0.455 g, 1.59 mmol). The reaction
1
mixture was stirred at 60 ℃ for 6 h. Following the work-up for 28a, 28b was obtained
by column chromatography on silica gel (CH Cl /C H OH/NH •H O, 10:0.6:0.1) (88
2
2
2
5
3
2
mg, 0.089 mmol, 28.3%).
+
1
HRMS (ESI) (M+H) m/z 985.5364, Calcd for C H N O 985.5380. H
5
1
77
4
15
NMR (CDCl , 400 MHz), δ: 8.86 (s, 1 H, H-aryl), 8.30 (s, 1 H, H-aryl), 8.03 (d,
3
J = 8.7 Hz, 1 H, H-aryl), 7.71 (d, J = 8.7 Hz, 1 H, H-aryl), 5.15 (d, J = 8.4 Hz, 1
H, H-13), 5.05 (d, J = 10.3 Hz, 1 H, H-3), 4.87 (s, 1 H, H-11), 3.94 (d, J = 7.4
2
0

ACCEPTED MANUSCRIPT
Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.75-3.56 (m, 3 H, H-5, H-8,
3
H-cyclopropyl), 3.41-3.32 (m, 1 H, H-5'), 3.16 (t, J = 8.8 Hz, 1H, H-2'), 3.01 (s,
3
H, 6-O-CH ), 2.95-2.56 (m,
9
H, H-2, 3-O-CO-CH -CH -,
2 2
3
-
NH-CH -CH -CH -), 2.52-2.35 (m, 2 H, , H-10, H-3'), 2.25 (s, 6 H, -N(CH ) ),
2 2 2 3 2
2
.15-1.81 (m, 4 H, H-4, -NH-CH -CH -CH -, H-14eq), 1.69-1.54 (m, 2 H, H-4'eq,
2 2 2
H-14ax), 1.49 (s, 3 H, 12-CH ), 1.45-1.38 (m, 3 H, H-7a, H-cyclopropyl ), 1.34 (s, 3
3
H, 6-CH ), 1.33-1.27 (m, 2 H, H-cyclopropyl), 1.25-1.17 (m, 6 H, 5'-CH , 10-CH ),
3
3
3
1
.13-1.05 (m, 6 H, 2-CH , 4-CH ), 0.94 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.85 (q, J = 7.6
3 3 3
1
3
Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ: 178.5, 174.0, 172.5, 167.1, 164.6,
3
3
1
8
3
8
4
3
54.7, 147.7, 140.9, 139.5, 134.7, 128.4, 126.0, 125.7, 117.4, 108.5, 103.7, 85.0, 83.2,
1.5, 78.5, 77.8, 75.7, 70.4, 69.7, 66.0, 61.4, 49.9, 49.1, 45.0, 43.1, 40.3, 37.2, 36.5,
5.4, 35.0, 33.0, 32.7, 31.4, 28.4, 25.6, 22.1, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2,
.9, 8.2.
.5.8.
-O-Descladinosyl-3-O-[3'-(3''-(4'''-(1''''-imidazolyl)phen-1'''-yl)-propylamino)propio
nyl]-6-O-methylerythromycin A 9-O-methyl oxime-11, 12-cyclic carbonate (28c)
To a solution of 14 (0.181 g, 0.259 mmol) and ethyldiisopropylamine (0.226 mL,
1
.3 mmol) in MeOH (20 mL) was added 23c (0.1568 g, 0.78 mmol). The reaction
mixture was stirred at 60 ℃ for 5 h. Following the work-up for 28a, 28c was obtained
by column chromatography on silica gel (CH Cl /C H OH/NH •H O, 10:0.3:0.1)
2
2
2
5
3
2
(
103.7 mg, 0.115 mmol, 44.4%).
+
1
HRMS (ESI) (M+H) m/z 900.5335, Calcd for C H N O 900.5328. H
4
7
74
5
12
NMR (CDCl , 400 MHz), δ: 7.82 (s, 1 H, H-phenyl), 7.34-7.27 (m, 5 H,
3
H-phenyl, H-imidazolyl), 7.19 (s, 1 H, H-imidazolyl), 5.16 (d, J = 10.6 Hz, 1
H, H-13), 5.07 (d, J = 11.0 Hz, 1 H, H-3), 4.88 (s, 1 H, H-11), 3.94 (dd, J = 7.4,
2
3
2
3
.1 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.75-3.63 (m, 2 H, H-5, H-8),
3
.42-3.33 (m, 1 H, H-5'), 3.17 (t, J = 8.9 Hz, 1H, H-2'), 3.02 (s, 3 H, 6-O-CH ),
3
.98-2.85 (m, 3 H, H-2, 3-O-CO-CH -CH -), 2.74-2.61 (m, 6 H,
2
2
-O-CO-CH -CH -, -NH-CH -CH -CH -), 2.52-2.38 (m, 2 H, , H-10, H-3'),
2
2
2
2
2
2
1

ACCEPTED MANUSCRIPT
2
.26 (s, 6 H, -N(CH ) ), 2.15-2.07 (m, 1 H, H-4), 1.96-1.80 (m, 3 H, H-14eq,
3
2
-
NH-CH -CH -CH -), 1.66-1.54 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ),
2 2 2 3
1
.46-1.39 (m, 1 H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.31-1.27 (m, 1 H, H-7b), 1.25-1.18
3
(
m, 7 H, 5'-CH , 10-CH , H-4'ax), 1.14-1.07 (m, 6 H, 2-CH , 4-CH ), 0.94 (d, J = 7.0
3 3 3 3
13
Hz, 3 H, 8-CH ), 0.86 (dt, J = 8.5, 4.1 Hz, 3 H, 15-CH ); C NMR (CDCl , 100
3
3
3
MHz), δ: 174.0, 172.5, 164.5, 154.7, 141.5, 135.6, 135.4, 130.3, 129.7, 121.6, 118.3,
1
4
1
4
3
03.7, 85.0, 83.2, 81.5, 78.5, 77.9, 75.7, 70.4, 69.6, 66.0, 61.4, 49.9, 49.3, 45.1, 43.2,
0.3, 37.3, 36.5, 34.9, 33.0, 32.7, 31.6, 28.4, 25.6, 22.2, 21.2, 19.4, 18.8, 15.6, 15.3,
3.0, 10.2, 8.9.
.5.9.
-O-Descladinosyl-3-O-[3'-(3''-(1"',2"',3"',4"'-tetrahydroisoquinol-6'''-yl)-propylamin
o)propionyl]-6-O-methylerythromycin A 9-O-methyl oxime-11, 12-cyclic carbonate
28d)
To a solution of 14 (0.154 g, 0.22 mmol) and ethyldiisopropylamine (0.19 mL,
.1 mmol) in MeOH (15 mL) was added 23d (0.123 g, 0.66 mmol). The reaction
(
1
mixture was stirred at 60 ℃ for 5.5 h. Following the work-up for 28a, 28d was
obtained by column chromatography on silica gel (CH Cl /C H OH/NH •H O,
2
2
2
5
3
2
1
0:0.3:0.1) (94 mg, 0.106 mmol, 48.2%).
+
1
HRMS (ESI) (M+H) m/z 889.5548, Calcd for C H N O 889.5533. H
4
7
77
4
12
NMR (CDCl , 400 MHz), δ: 6.78-6.72 (m, 2 H, H-phenyl), 6.40 (d, J = 7.9 Hz,
3
1
H, H-phenyl), 5.16 (d, J = 10.7 Hz, 1 H, H-13), 5.06 (d, J = 10.9 Hz, 1 H,
H-3), 4.88 (s, 1 H, H-11), 3.93 (d, J = 6.7 Hz, 1 H, H-1'), 3.82 (s, 3 H,
9
5
6
-O-CH ), 3.75-3.65 (m, 2 H, H-5, H-8), 3.42-3.33 (m, 1 H, H-5'), 3.27 (t, J =
3
.5 Hz, 2 H, H-piperidine), 3.16 (t, J = 8.8 Hz, 1H, H-2'), 3.02 (s, 3 H,
-O-CH ), 2.93-2.83 (m, 3 H, H-2, 3-O-CO-CH -CH -), 2.73 (t, J = 6.7 Hz, 2 H,
3
2
2
H-piperidine), 2.67-2.57 (m, 4 H, 3-O-CO-CH -CH -, -NH-CH -CH -CH -),
2
2
2
2
2
2
2
4
.52-2.36 (m, 4 H, , H-10, H-3', -NH-CH -CH -CH -), 2.26 (s, 6 H, -N(CH ) ),
2 2 2 3 2
.12-2.07 (m, 1 H, H-4), 1.96-1.87 (m, 3 H, H-14eq, H-piperidine), 1.77-1.55 (m,
H, -NH-CH -CH -CH -, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ), 1.45-1.37 (m, 1
2
2
2
3
2
2

ACCEPTED MANUSCRIPT
H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.31-1.27 (m, 1 H, H-7b), 1.25-1.17 (m, 7 H, 5'-CH ,
3
3
1
0
1
7
3
4
3
0-CH , H-4'ax), 1.12-1.07 (m, 6 H, 2-CH , 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ),
3 3 3 3
1
3
.86 (t, J = 7.4 Hz, 3 H, 15-CH ); C NMR (CDCl , 100 MHz), δ: 174.0, 172.5,
3
3
64.5, 154.7, 142.9, 130.4, 129.4, 126.6, 121.5, 114.4, 103.7, 85.0, 83.2, 81.6, 78.5,
7.8, 75.7, 70.4, 69.6, 65.9, 61.4, 49.9, 49.6, 45.1, 43.2, 42.1, 40.3, 37.3, 36.5, 35.0,
2.7, 32.0, 28.4, 27.0, 25.6, 22.4, 22.2, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.10.
-O-Descladinosyl-3-O-[3'-(4''-(3'''-pyridyl)butylamino)propionyl]-6-O-methylerythr
omycin A 9-O-methyl oxime-11, 12-cyclic carbonate (29)
To a solution of 14 (0.36 g, 0.515 mmol) and ethyldiisopropylamine (0.45 mL,
2
.58 mmol) in MeOH (20 mL) was added 27 (0.2342 g, 1.56 mmol). The reaction
mixture was stirred at 60 ℃ for 5 h. Following the work-up for 28a, 29 was obtained
by column chromatography on silica gel (CH Cl /C H OH/NH •H O, 10:0.4:0.1)
2
2
2
5
3
2
(
121 mg, 0.143 mmol, 27.8%).
+
1
HRMS (ESI) (M+H) m/z 849.5213, Calcd for C H N O 849.5220. H
4
4
73
4
12
NMR (CDCl , 400 MHz), δ: 8.43 (s, 2 H, H-pyridyl), 7.49 (d, J = 7.8 Hz, 1 H,
3
H-pyridyl), 7.20 (t, J = 6.4 Hz, 1 H, H-pyridyl), 5.16 (d, J = 10.6 Hz, 1 H,
H-13), 5.06 (d, J = 10.9 Hz, 1 H, H-3), 4.88 (s, 1 H, H-11), 3.93 (d, J = 7.2 Hz,
1
1
3
H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.75-3.63 (m, 2 H, H-5, H-8), 3.40-3.33 (m,
3
H, H-5'), 3.16 (t, J = 8.8 Hz, 1H, H-2'), 3.02 (s, 3 H, 6-O-CH ), 2.95-2.85 (m,
3
H, H-2, 3-O-CO-CH -CH -), 2.67-2.57 (m, 6 H, 3-O-CO-CH -CH -,
2
2
2
2
-
-
NH-CH -CH -CH -CH ), 2.52-2.36 (m, 2 H, H-10, H-3'), 2.26 (s, 6 H,
2 2 2 2
N(CH ) ), 2.14-2.06 (m, 1 H, H-4), 1.95-1.84 (m, 1 H, H-14eq), 1.72-1.53 (m, 6
3
2
H, -NH-CH -CH -CH -CH , H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ), 1.45-1.36 (m,
2
2
2
2
3
1
1
0
1
7
H, H-7a), 1.34 (s, 3 H, 6-CH ), 1.32-1.27 (m, 1 H, H-7b), 1.25-1.15 (m, 7 H, 5'-CH ,
3 3
0-CH , H-4'ax), 1.13-1.05 (m, 6 H, 2-CH , 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ),
3
3
3
3
1
3
.86 (t, J = 7.4 Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ: 174.0, 172.5,
3
3
64.5, 154.7, 150.0, 147.4, 137.4, 135.8, 123.3, 103.7, 85.0, 83.2, 81.5, 78.5, 77.8,
5.7, 70.3, 69.6, 65.9, 61.4, 49.9, 49.7, 45.1, 43.1, 40.3, 37.2, 36.5, 35.0, 32.9, 32.7,
2
3

ACCEPTED MANUSCRIPT
2
4
3
9.6, 28.8, 28.4, 25.6, 22.1, 21.2, 19.4, 18.8, 15.6, 15.3, 13.0, 10.2, 8.9.
.5.11.
-O-Descladinosyl-3-O-[3'-(2"-(3'''-carboxy-7'''-chloro-1'''-cyclopropyl-1''',4'''-dihydr
o-4'''-oxo-quinol-6'''-ylamino)ethylamino)propionyl]-6-O-methylerythromycin
-O-methyl oxime-11, 12-cyclic carbonate (31)
To a solution of 14 (0.5 g, 0.68 mmol) and ethyldiisopropylamine (0.59 mL, 3.4
A
9
mmol) in MeOH (20 mL) was added 30 (0.613 g, 2 mmol). The reaction mixture was
stirred at 60 ℃ for 5 h. Following the work-up for 28a, 31 was obtained by column
chromatography on silica gel (CH Cl /MeOH/NH •H O, 10:3:0.5) (64 mg, 0.063
2
2
3
2
mmol, 9.3%).
HRMS (ESI) (M+H) m/z 1020.4926, Calcd for C H ClN O
15
+
5
0
75
5
1
1
1
5
020.4943. H NMR (CDCl , 700 MHz), δ: 8.74 (s, 1 H, H-quinolyl), 8.04 (s,
3
H, H-quinolyl),7.52 (s, 1 H, H-quinolyl), 5.36 (s, 1 H, -NH-CH -CH -NH-),
2
2
.15 (d, J = 10.6 Hz, 1 H, H-13), 5.07 (d, J = 11.0 Hz, 1 H, H-3), 4.87 (s, 1 H,
H-11), 3.96 (d, J = 11.9 Hz, 1 H, H-1'), 3.83 (s, 3 H, 9-O-CH ), 3.75-3.63 (m,
3
2
H, H-5, H-8), 3.56 (s, 1 H, H-cyclopropyl), 3.49-3.33 (m, 3 H, H-5',
-
-
(
NH-CH -CH -NH-), 3.24 (dd, J = 10.0, 6.8 Hz, 1H, H-2'), 3.11-2.96 (m, 4 H,
2
2
NH-CH -CH -NH-, -CO-CH -CH -NH-), 3.02 (s, 3 H, 6-O-CH ), 2.97-2.82
2
2
2
2
3
m, 1 H, H-2), 2.75-2.64 (m, 2 H, -CO-CH -CH -NH-), 2.52-2.44 (m, 2 H, ,
2
2
H-10, H-3’), 2.39 (s, 6 H, -N(CH ) ), 2.14-2.06 (m, 1 H, H-4), 1.95-1.85 (m, 1 H,
3
2
H-14eq), 1.76-1.56 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ), 1.45-1.37 (m, 3
3
H, H-7a, H-cyclopropyl), 1.34 (s, 3 H, 6-CH ), 1.27-1.18 (m, 10 H, H-7b, H-4'ax,
3
5
'-CH , 10-CH , H-cyclopropyl), 1.13-1.02 (m, 6 H, 2-CH , 4-CH ), 0.94 (d, J = 7.0
3 3 3 3
13
Hz, 3 H, 8-CH ), 0.85 (t, J = 7.3 Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ:
3
3
3
1
1
4
1
4
77.5, 173.9, 172.4, 167.3, 164.5, 154.7, 145.9, 143.0, 132.6, 127.7, 126.3, 118.1,
07.2, 104.4, 103.8, 85.0, 83.1, 82.2, 78.5, 78.1, 75.8, 70.2, 69.2, 65.9, 61.4, 49.9,
7.3, 44.3, 43.1, 40.1, 40.0, 37.2, 36.6, 35.4, 32.7, 29.7, 25.6, 22.2, 21.1, 19.4, 18.8,
5.6, 15.3, 13.0, 10.2, 9.0, 8.1.
.5.12. 3-O-Descladinosyl-3-O-[N-propargylcarbamoyl]-6-O-methylerythromycin A
2
4

ACCEPTED MANUSCRIPT
9
-O-methyl oxime-11, 12-cyclic carbonate (32)
To a solution of 11 (1.0613 g, 1.55 mmol) in CH Cl (20 mL) at room
2
2
temperature were added DMAP (0.378 g, 3.09 mmol) and CDI (0.752 g, 4.64
mmol). The resulting solution was allowed to stirred for 20 h at the same
temperature. The reaction was quenched with saturated NaHCO (30 mL) and
3
the aqueous layer was extracted with CH Cl (2 × 20 mL) saturated brine (2 ×
2
2
2
0 mL). The organic layer were washed with saturated NH Cl (3 × 20 mL) and
4
brine (3 × 20 mL), and then the organic solvent was removed in vacuum to afford
an intermediate 3-O-imidazolylcarbonyl-11 (1.0745 g, 1.38 mmol, 89.0%).
To a solution of the intermediate 3-O-imidazolylcarbonyl-11 (1.0745 g, 1.38
mmol) in DMF (10 mL) at 0 ℃ were added DBU (0.267 mL, 1.89 mmol) and
propargylamine (0.13 mL, 1.89 mmol). The resulting solution was stirred for 48 h at
room temperature. The reaction was quenched with water (20 mL) and the aqueous
layer was extracted with CH Cl (3 × 15 mL). The combined organic layers were
2
2
washed with brine and the organic solvent was removed in vacuum to afford 32
0.7521 g, 0.983 mmol, 71.2%).
(
4
3
.5.13.
-O-Descladinosyl-3-O-[N-(3'-(3"-pyridyl)propargyl)carbamoyl]-6-O-methylerythro
mycin A 9-O-methyl oxime-11, 12-cyclic carbonate (33a)
To a solution of 32 (0.7521 g, 0.983 mmol), CuI (18.7 mg, 0.0982 mmol),
(
PPh ) PdCl (34.5 mg, 0.049 mmol) and triethylamine (0.205 mL, 1.47 mmol) in
3 2 2
acetonitrile (20 mL) was added 3-bromopyridine (0.284 mL, 2.95 mmol). The
reaction mixture was recharged with argon and stirred at 80 ℃ for 3 h in a sealed tube.
The mixture was extracted with ethyl acetate and the organic layer was washed
successively with water and brine. The organic solvent was removed in vacuum and
dried to afford 2'-OAc-33a (0.5567 mg, 0.66 mmol, 67.1%).
A solution of 2'-OAc-33a (0.5567 mg, 0.66 mmol) in MeOH (20 mL) was
stirred at 65 ℃ for 3 h. The organic solvent was removed in vacuum and the residue
was purified by column chromatography on silica gel (CH Cl /C H OH/NH •H O,
2
2
2
5
3
2
2
5

ACCEPTED MANUSCRIPT
1
0:0.2:0.1) to afford 33a (0.2538 mg, 0.316 mmol, 47.9%).
+
1
HRMS (ESI) (M+H) m/z 803.4451, Calcd for C H N O 803.4437. H
4
1
63
4
12
NMR (CDCl , 400 MHz), δ: 8.63 (s, 1 H, H-pyridyl), 8.56 (d, J = 4.7 Hz, 1 H,
3
H-pyridyl), 7.69 (d, J = 7.9 Hz, 1 H, H-pyridyl), 7.29-7.24 (m, 1 H, H-pyridyl),
5
.40 (s, 1 H, 3-O-CO-NH-CH -), 5.17 (dt, J = 10.8, 3.3 Hz, 1 H, H-13), 4.90
2
(
d, J = 11.0 Hz, 1 H, H-3), 4.86 (s, 1 H, H-11), 4.39-4.15 (m, 2 H,
3
3
-O-CO-NH-CH -), 4.04 (d, J = 7.3 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ),
2 3
.77-3.65 (m, 2 H, H-5, H-8), 3.44-3.36 (m, 1 H, H-5'), 3.17 (dd, J = 10.1, 7.3
Hz, 1H, H-2'), 3.03 (s, 3 H, 6-O-CH ), 2.92-2.83 (m, 1 H, H-2), 2.52-2.42 (m,
3
2
H, , H-10, H-3'), 2.20 (s, 6 H, -N(CH ) ), 2.14-2.06 (m, 1 H, H-4), 1.97-1.86
3 2
(m, 1 H, H-14eq), 1.71-1.53 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH3),
1
6
4
.43-1.37 (m, 1 H, H-7a), 1.32 (s, 3 H, 6-CH ), 1.28-1.25 (m, 1 H, H-7b), 1.23 (d, J =
3
.7 Hz, 3 H, 10-CH ), 1.20-1.13 (m, 6 H, 5'-CH , 2-CH ), 1.10 (d, J = 7.5 Hz, 3 H,
3
3
3
13
-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.86 (t, J = 7.3 Hz, 3 H, 15-CH ); C NMR
3
3
3
(
CDCl , 175 MHz), δ: 173.9, 164.5, 156.0, 154.7, 152.4, 149.1, 138.7, 123.1, 119.5,
3
1
4
1
4
3
03.5, 88.5, 85.0, 83.2, 81.8, 80.1, 79.3, 78.4, 75.6, 70.5, 69.4, 66.0, 61.4, 49.8, 43.4,
0.3, 40.2, 37.3, 36.0, 32.7, 31.6, 28.5, 25.6, 22.2, 21.1, 19.3, 18.8, 15.6, 15.0, 13.0,
0.2, 8.9.
.5.14.
-O-Descladinosyl-3-O-[N-(3'-(1''-cyclopropyl-1'',4''-dihydro-4''-oxo-3''-carboxyquin
ol-6''-yl)propargyl)carbamoyl]-6-O-methylerythromycin A 9-O-methyl oxime-11,
1
2-cyclic carbonate (33b)
A solution of 1-cyclopropyl-1,4-dihydro-6-iodo-4-oxo-3-quinolinecarboxylic
acid (0.278 g, 0.784 mmol), CuI (7.46 mg, 0.039 mmol) and triethylamine (10
mL) in acetonitrile (10 mL) was stirred at room temperature for 20 min. Then 32
(
0.3 g, 0.392 mmol) and (PPh ) PdCl (13.8 mg, 0.0196 mmol) were added and the
3 2 2
mixture was recharged with argon and stirred at 50 ℃ for 24 h in a sealed tube.
Following the work-up for 33a, 33b was obtained by column chromatography on
silica gel (CH Cl /MeOH/NH H O, 10:3:0.5) (23.4 mg, 0.025 mmol, 26.3%).
2
2
3•
2
2
6

ACCEPTED MANUSCRIPT
+
HRMS (ESI) (M+H) m/z 953.4761, Calcd for C H N O 953.4754.
4
9
69
4
15
4
3
.5.15.
-O-Descladinosyl-3-O-[N-(3'-(3"-pyridyl)propyl)carbamoyl]-6-O-methylerythromyci
n A 9-O-methyl oxime-11, 12-cyclic carbonate (34a)
To a solution 33a (0.2538 g, 0.316 mmol) in MeOH (10 mL) were added
HCOONH (1.0152 g, 16.09 mmol), HCOOH (0.179 mL, 4.74 mmol), and 10%
4
Pd-C (0.0254 g). The reaction mixture was flushed with hydrogen to 1 atm.
The reaction mixture was stirred at 65 ℃ for 24 h. 10 % Pd-C was removed by
filtration before the solvent was evaporated. The residue was extracted with
CH Cl and the organic layer was washed successively with water and brine. The
2
2
organic solvent was removed in vacuum and the residue was purified by column
chromatography on silica gel (CH Cl /C H OH/NH •H O, 10:0.6:0.1) to afford 34a
2
2
2
5
3
2
(
45mg, 0.056 mmol, 17.7%).
+
1
HRMS (ESI) (M+H) m/z 807.4746, Calcd for C H N O 807.4750. H
4
1
67
4
12
NMR (CDCl , 400 MHz), δ: 8.49-8.43 (m, 2 H, H-pyridyl), 7.5 (d, J = 7.9 Hz,
3
1
H, H-pyridyl), 7.23 (dd, J = 7.8, 4.9 Hz, 1 H, H-pyridyl), 5.17 (d, J = 10.7
Hz, 1 H, H-13), 5.12 (t, J = 6.1 Hz, 1 H, -NH-), 4.91-4.84 (m, 2 H, H-3, H-11),
.99 (d, J = 7.2 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.76-3.64 (m, 2 H, H-5,
3
3
H-8), 3.38-3.11 (m, 4 H, H-5', H-2', -NH-CH -CH -CH ), 3.02 (s, 3 H,
2
2
2
6
-O-CH ), 2.90-2.82 (m, 1 H, H-2), 2.67 (t, J = 7.8 Hz, 2 H,
3
-
NH-CH -CH -CH -), 2.52-2.37 (m, 2 H, , H-10, H-3'), 2.27 (s, 6 H, -N(CH ) ),
2 2 2 3 2
2
1
1
5
0
1
7
2
.12-2.04 (m, 1 H, H-4), 1.92-1.82 (m, 3 H, H-14eq, -NH-CH -CH -CH -),
2 2 2
.62-1.53 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ), 1.44-1.36 (m, 1 H, H-7a),
3
.32 (s, 3 H, 6-CH ), 1.27-1.21 (m, 5 H, H-7b, H-4'ax, 10-CH ), 1.20-1.13 (m, 6 H,
3
3
'-CH , 2-CH ), 1.07 (d, J = 7.4 Hz, 3 H, 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ),
3
3
3
3
1
3
.86 (t, J = 7.3 Hz, 3 H, 15-CH ); C NMR (CDCl , 175 MHz), δ: 174.0, 164.5,
3
3
56.5, 154.7, 149.7, 147.7, 136.5, 135.7, 123.5, 103.4, 85.0, 83.2, 81.6, 78.4, 78.3,
5.5, 70.4, 69.4, 66.1, 61.4, 49.8, 43.4, 40.6, 40.3, 37.3, 36.0, 32.7, 31.8, 30.2, 28.6,
5.6, 22.2, 21.1, 19.3, 18.9, 15.6, 14.9, 13.0, 10.2, 9.0.
2
7

ACCEPTED MANUSCRIPT
4
3
.5.16.
-O-Descladinosyl-3-O-[N-(3'-(3''-carboxy-1''-cyclopropyl-1'',4''-dihydro-4''-oxo-qui
9-O-methyl oxime-11,
nol-6''-yl)propyl)carbamoyl]-6-O-methylerythromycin
2-cyclic carbonate (34b)
To a solution 33b (0.063 g, 0.066 mmol) in MeOH (10 mL) were added
A
1
HCOONH (0.252 g, 3.99 mmol), HCOOH (0.037 mL, 0.99 mmol), and 10%
4
Pd-C (60 mg). The reaction mixture was flushed with hydrogen to 1 atm. The
reaction mixture was stirred at 65 ℃ for 24 h. Following the work-up for 34a, 34b
was obtained by column chromatography on silica gel (CH Cl /MeOH/NH •H O,
2
2
3
2
1
0:3:0.5) (16.7 mg, 0.017 mmol, 25.8%).
+
1
HRMS (ESI) (M+H) m/z 957.5063, Calcd for C H N O 957.5067. H
4
9
73
4
15
NMR (CDCl , 400 MHz), δ: 8.87 (s, 1 H, H-quinolyl), 8.30 (s, 1 H,
3
H-quinolyl), 8.05 (d, J = 8.7 Hz, 1 H, H-quinolyl), 7.70 (d, J = 8.7 Hz, 1 H,
H-quinolyl), 5.25-5.13 (m, 2 H, H-13, -NH-), 4.91-4.81 (m, 2 H, H-3, H-11),
4
.01 (d, J = 7.4 Hz, 1 H, H-1'), 3.82 (s, 3 H, 9-O-CH ), 3.75-3.58 (m, 3 H, H-5,
3
H-8, H-cyclopropyl), 3.42-3.09 (m, 2 H, H-5', H-2'), 3.02 (s, 3 H, 6-O-CH ),
3
2
2
.91-2.81 (m, 3 H, H-2, -NH-CH -CH -CH ), 2.52-2.38 (m, 2 H, , H-10, H-3'),
2 2 2
.26 (s, 6 H, -N(CH ) ), 2.12-2.02 (m, 1 H, H-4), 1.99-1.83 (m, 3 H, H-14eq,
3
2
-
NH-CH -CH -CH -), 1.65-1.55 (m, 2 H, H-4'eq, H-14ax), 1.49 (s, 3 H, 12-CH ),
2 2 2 3
1
.44-1.36 (m, 3 H, H-7a, H-cyclopropyl), 1.32 (s, 3 H, 6-CH ), 1.28-1.19 (m, 6 H,
3
H-cyclopropyl, H-7b, 10-CH ), 1.18-1.14 (m, 6 H, 5'-CH , 2-CH ), 1.08 (d, J = 7.6
3
3
3
Hz, 3 H, 4-CH ), 0.93 (d, J = 7.0 Hz, 3 H, 8-CH ), 0.86 (t, J = 7.5 Hz, 3 H, 15-CH );
3
3
3
1
3
C NMR (CDCl , 175 MHz), δ: 178.5, 174.1, 167.0, 164.6, 156.5, 154.7, 147.9,
3
1
7
2
4
3
40.1, 139.6, 134.7, 128.4, 126.0, 125.6, 117.6, 108.6, 103.4, 85.1, 83.2, 81,5, 78.4,
5.5, 70.5, 69.5, 66.0, 61.4, 49.8, 43.4, 40.4, 40.3, 37.3, 36.0, 35.4, 32.7, 32.4, 31.6,
9.7, 25.6, 22.2, 21.1, 19.3, 18.9, 15.6, 14.9, 13.0, 10.2, 8.9, 8.3.
.5.17.
-O-Descladinosyl-3-O-[N-(3'-(3"-aminopyrid-5"-yl)propyl)carbamoyl]-6-O-methyle
rythromycin A 9-O-methyl oxime-11, 12-cyclic carbonate (34e)
2
8