Design, synthesis and antibacterial evaluation of novel 15-membered 11a-azahomoclarithromycin derivatives with the 1, 2, 3-triazole side chain

Article abstract of DOI:10.1016/j.ejmech.2019.07.022

Macrolides are widely prescribed in clinic to treat various respiratory tract infections. However, due to their inappropriate use, the prevalence of macrolide-resistant strains among clinical isolates has become a concern for public health. Therefore, nov

Full text of DOI:10.1016/j.ejmech.2019.07.022

Accepted Manuscript
Design, synthesis and antibacterial evaluation of novel 15-membered 11a-
azahomoclarithromycin derivatives with the 1, 2, 3-triazole side chain
Yinhui Qin, Yuetai Teng, Ruixin Ma, Fangchao Bi, Zhiyang Liu, Panpan Zhang,
Shutao Ma
PII:
S0223-5234(19)30643-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.022
EJMECH 11519
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 April 2019
Revised Date: 17 June 2019
Accepted Date: 7 July 2019
Please cite this article as: Y. Qin, Y. Teng, R. Ma, F. Bi, Z. Liu, P. Zhang, S. Ma, Design, synthesis and
antibacterial evaluation of novel 15-membered 11a-azahomoclarithromycin derivatives with the 1, 2,
3-triazole side chain, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.07.022.
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Design, synthesis and antibacterial evaluation of novel 15-membered
11a-azahomoclarithromycin derivatives with the 1, 2, 3-triazole side chain
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Yinhui Qin , Yuetai Teng , Ruixin Ma , Fangchao Bi , Zhiyang Liu , Panpan Zhang , Shutao Ma
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Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012,
China
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The Affiliated Hospital of Qingdao University, Qingdao 266003, China
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Running title: 15-membered 11a-azahomoclarithromycin derivatives
* Corresponding author.
E-mail addresses: mashutao@sdu.edu.cn (S. Ma)
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ABSTRACT
Macrolides are widely prescribed in clinic to treat various respiratory tract infections. However, due
to their inappropriate use, the prevalence of macrolide-resistant strains among clinical isolates has
become a concern for public health. Therefore, novel macrolides skeleton structures against resistant
pathogens are badly needed. Thus, three series of novel 15-membered 11a-azahomoclarithromycin
derivatives (the series A-C) with the 1, 2, 3-triazole side chain were designed and synthesized through
creatively opening the ring of clarithromycin (CAM), expanding the ring properly and introducing a
suitable side chain of 1, 2, 3-triazole at the C12 and C13 positions, and evaluated for their
antibacterial activity. The antibacterial results indicated that compounds 38b, 38l and 38v possessed
strong antibacterial activity against Staphylococcus aureus ATCC25923 (0.25 µg/mL) and Bacillus
subtilis ATCC9372 (0.25 µg/mL). Furthermore, compounds 9e and 38g were found to exhibit
promising potent activity (8 µg/mL) against Streptococcus pneumonia AB11 expressing the ermB and
mefA genes. In addition, the determination of minimum bactericidal concentration (MBC) indicated
that the most promising compounds 38b, 38l, 38v, 9e and 38g were excellent bacteriostatic agents.
The bactericidal curve showed that 9e exhibited antibacterial activity through bacteriostatic
mechanism. Finally, 38b, 38l and 38v were confirmed to be non-toxic to MCF-7 breast cancer cells
up to a concentration of 32 µg/mL in preliminary cytotoxicity assay. In summary, 38b, 38l, 38v, 9e
and 38g can be served as lead compounds to provide a new perspective for further structural
optimization.
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Keywords: macrolide antibiotics, antibacterial activity, resistant bacterial strains, structure-activity
relationships
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1. Introduction
Macrolides [1, 2] (Fig. 1) are widely used in the treatment of respiratory tract infections because
of their potent activity, low toxicity and high safety. Since the first macrolide antibiotic erythromycin
A was discovered, the development of macrolide antibiotics has gone through three generations.
Erythromycin A, as the first generation macrolide antibiotic, was found from a Philippine soil sample
in 1949 [3]. It is effective for various infections, including skin and soft tissue infections, sexually
transmitted diseases and respiratory tract infections, but its clinical use is relatively limited due to its
bioavailability and gastrointestinal side effects [4]. Therefore, it is urgent to study a new generation of
macrolide antibiotics with excellent pharmacokinetic properties. Clarithromycin (CAM) [5, 6] and
azithromycin (AZM) [7] launched in 1991, which belong to the second generation macrolide
antibiotics. Their pharmacokinetic properties are more significantly improved than that of
erythromycin A, including broader antibacterial spectrum, higher intracellular concentration, longer
half-life and higher bioavailability of oral administration [8, 9]. However, some bacterial strains have
gradually become resistant strains to the second generation macrolide antibiotics. Their mechanism of
drug resistance mainly includes two kinds of ribosomal methylation (mediated by erm
methyltransferase) and efflux pump (mediated by mef-gene) [10-12]. Those leads to new generation
of macrolide scaffolds that are badly needed [13]. Therefore, the third generation macrolides:
telithromycin [14], cethromycin [15] and solithromycin [16, 17] have been rapidly developed, which
belong to ketolides derivatives. Telithromycin, for example, is the first ketolide macrolide approved
by the US FDA in 2004 for mainly treating infections such as chronic bronchitis, pharyngitis and
community-acquired pneumonia (CAP) [18]. Besides, cethromycin and solithromycin as promising
clinical candidates, are known to be effective for the treatment of CAP caused by
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macrolide-lincosamide-streptogramin B (MLS ) resistant bacteria. Compared with the previous two
B
generations of macrolides, the third generation of macrolides are extremely effective against
erythromycin-resistant pathogens [14, 15, 19]. For example, telithromycin showed strong activity
against erythromycin-resistant Streptococcus pneumoniae and Haemophilus influenza, cethromycin
exhibited excellent potency against macrolide-resistant respiratory tract pathogens and solithromycin
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exhibited promising activity against Staphylococcus and Enterococcus spp. They share common
structural characteristics, such as a 3-ketone group and an aryl alkyl side chain. In particular, the aryl
alkyl side chains play an important role in generating anti-resistant bacteria activity because the aryl
alkyl side chains allow the drugs to bind tightly to bacterial ribosomes [20-25]. Although the third
generation macrolides have excellent antimicrobial activity, researchers have been still making great
efforts to modify the structure of macrolides to overcome the problems of bacterial resistance. Ian B.
Seiple1 et al [26] designed and sythesized some 14-membered and 15-membered azaketolide
derivatives by replacing the C3-cladinose residue of CAM by a ketone group, introducing a nitrogen
atom and some active triazole side chains into the macrocycle. The azaketolide derivatives with
various macrocyclic scaffolds exhibited extremely strong activity against erythromycin-resistant
strains.
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<Insert Fig. 1>
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It has been reported that in order to seek newer macrolides with novel structural skeletons, the
C13 position of erythromycin A was modified by chemical biosynthesis [27, 28]. The resulting C13
derivatives enhanced the antibacterial activity against drug-resistant strains, which indicated that the
C13 position of erythromycin A plays an important role in combating the drug-resistant strains.
However, the number of the C13 derivatives that were obtained by chemical biosynthesis did not meet
the research needs due to the lack of reactivity of the C13 position. In view of this, Tomoaki Miura et
al [29] established a relevant macrolactone skeleton reconstruction methodology by chemical
synthesis. Subsequently, Tomohiro Sugimoto et al [30] reported a related macrolactone skeleton
reconstruction method using CAM as raw material.
The above described methodologies and feasible synthetic methods give us some inspiration,
which also prompted our new research. Therefore, our design strategy is outlined as follows (Fig. 2).
The C-C bond between the C11 and C12 positions of CAM was oxidatively cleaved, an active triazole
side was inserted into the resulting macrolactone skeleton, and macrolactonization of the acyclic
skeleton produced the desired novel 15-membered 11a-azahomoclarithromycin derivatives with an
active triazole side chains at the C12 and C13 positions, one type of side chains substituted at C12
position giving series B, and the other type of side chains substituted at C13 position affording series
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A and C. The above two types of side chains were expected to be spatially close and combine with the
new bacterial ribosomal base U790, which may generate hydrogen bond interaction, π-π stacking
interaction, electrostatic interaction and hydrophobic interaction [20, 31]. Thus, we designed series A,
B and C based on the following ideas. Firstly, the main group 2'-OH was retained to interact with
A2058 and A2059 in domain V of 23S rRNA, producing strong antibacterial activity against
susceptible bacteria [32]. Secondly, the stereo conformation of CAM skeleton was changed to form
favorable conformation to increase the binding force to bacterial ribosomes. Spatially, some groups on
the 11a-azahomoclarithromycin skeleton approach and bind to new nucleotide binding sites in domain
V of 23S rRNA to give additional binding forces. These changes in stereochemistry are to enhance the
antibacterial activity against drug-resistant bacteria. Finally, the introduced side chains with
appropriate length, flexibility and extension direction in space were easily to interact with the U790 of
23S rRNA [20, 31].
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2. Chemistry
2.1. Synthesis of 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series A)
The 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin 9a-l (Series A) as the C-13
substituted derivatives, the synthetic route of which is shown in Scheme 1. CAM 1 was treated with
sodium borohydride to give the 9(S)-dihydro-clarithromycin 2 [33], and subsequent selective
protection of 9, 2', 4''-hydroxyl groups of 2 by triethylchlorosilane provided 11, 12-diol 3 [34]. 3 was
oxidized by lead tetraacetate to afford an acyclic aldehyde intermediate 4. The intermediate 6a-l was
obtained from 4 in 2 steps: (1) reductive amination [35] of 4 with various substituted aminoalcohols
5a-l gave the secondary amine; (2) methylation of the intermediate secondary amine at the 11-a with
formaldehyde. 6a-l was successfully prepared in "one-pot" method from 3 due to the instability of 4
and the resulting secondary amine. However, due to many byproducts produced during the
preparation 6a-l, led to the purification of 6a-l was difficult. Consequently, only the crude
intermediate 6a-l could be obtained by column chromatography. Saponification of 6a-l formed
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carboxylic acid 7a-l, which was followed macrolactonization [36] to give the desired 15-membered
macrolides 8a-l. Finally, 9a-l (series A) were successfully prepared by deprotection of the silane
groups from 8a-l by using hydrogen fluoride-pyridine. In summary, the synthesis of 9a-l involves two
procedures: ring opening procedure (from starting materials 1 to intermediate 4) and cyclization
procedure (from intermediate 4 to target compounds 9a-l).
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The synthetic route of the key intermediates 5a-l is shown in Scheme 2. Under weak acid
conditions, R-epichlorohydrin 10 was stereoselectively ring-opened with sodium azide to yield an
azide intermediate 11 [37]. Subsequently, 11 was treated with a variety of substituted phenylacetylene
12 in the presence of L-ascorbic acid sodium salt and copper sulfate to form chloroethanol 13 [38].
Cyclization reaction of 13 with sodium hydroxide solution gave epoxy 14, which was
stereoselectively ring-opened with aqueous ammonia to provide various substituted aminoalcohols 5a,
5c, 5e, 5g, 5i and 5k. Followed as the general procedure described above, the other corresponding
enantiomers 5b, 5d, 5f, 5h, 5j and 5l were also prepared from S-epichlorohydrin 15 as raw starting
material. The two groups of synthesized intermediates consist of 5a-l.
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2.2. Synthesis of 15-membered 11a-aza-12-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series B)
The 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin 23a-l (series B) as the C-12
substituted derivatives, the synthetic route of which is shown in Scheme 3. The target compounds
23a-l (series B) were prepared from 4 by application of the same cyclization procedure as 9a-l (series
A).
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The synthetic route of intermediates 19a-l is shown in Scheme 4. The azide 26 was given from
Boc-L-serine methyl ester 24 in 2 steps: (1) mesylation of 24 with methanesulfonyl chloride formed
the mesylate; (2) azidation of the resulting mesylate with sodium azide. Subsequently, 26 was treated
with various substituted phenylacetylene 12 in the presence of L-ascorbic acid sodium salt and copper
sulfate to produce ester 27. Then 27 was reduced by sodium borohydride [39] to yield alcohol 28,
which was followed deprotection of the Boc group from 28 using hydrochloric acid to give the
various substituted aminoalcohols 19a, 19c, 19e, 19g, 19i and 19k. According to the general
procedure, the other corresponding enantiomers 19b, 19d, 19f, 19h, 19j and 19l were also given from
Boc-D-serine methyl ester 29 as raw starting material. The two groups of synthesized intermediates
consist of 19a-l.
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2.3. Synthesis of 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series C)
The 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin 38a-v (series C) as the C-13
substituted derivatives, the synthetic route of which is shown in Scheme 5. The target compounds
38a-v (series C) were prepared from 4 by application of the same cyclization procedure as 9a-l (series
A).
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Furthermore, the synthetic route of intermediates 34a-v is shown in Scheme 6. Diazotization of
39 with sodium nitrite under acidic conditions, and then treatment with sodium azide formed the azide
40. Subsequently, 40 was reacted with methyl propiolate 41 in the presence of L-ascorbic acid sodium
salt and copper sulfate to obtain ester 42. 42 was subjected to reduction reaction with lithium
aluminum hydride to give alcohol 43, which then was treated with phosphorus tribromide [40] to
yield bromine 44. Introduction of epoxy moiety (R)-glycidol 45 to bromine 44 was achieved to
produce the epoxy 46 by utilizing the intermolecular nucleophilic substitution reaction [41]. 46 was
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stereoselectively ring-opened with aqueous ammonia to give various substituted aminoalcohols 34a,
34c, 34e, 34g, 34i, 34k, 34m, 34o, 34q, 34s and 34u as side chains [34]. The other corresponding
enantiomers 34b, 34d, 34f, 34h, 34j, 34l, 34n, 34p, 34r, 34t and 34v were provided by using the
general procedure described above. The two groups of synthesized intermediates consist of 34a-v.
However, we failed to synthesize 2-azidopyridine 40 according to the synthetic procedure when 39
was 2-aminopyridine (Scheme 7). Therefore, 40 was prepared by another feasible synthetic method.
The 2-hydrazinopyridine 49 was subjected to azidation with tert-butyl nitrite to generate 40 on the
scale of 200 mg [42]. The corresponding substituted ethanolamine 34k and 34l were prepared from 40
according to the above synthetic procedure (Scheme 6).
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2.4. Analysis and determination of the absolute configuration of compounds
By two consecutive reductive ammoniation, hydrolysis, cyclization and deprotection, the
compounds in series A, B and C were synthesized from 4 and 5a-l, 4 and 19a-l, 4 and 34a-v,
respectively. None of the above reaction processes involved the cleavage and formation of chemical
bonds linking the chiral carbon (C12 or C13). Therefore, the stereo configurations of C12 or C13 in
the compounds are determined by the configurations of the 5a-l, 19a-l, and 34a-v. The intermediates
5a-l and 34a-v belong to 1-substituted ethanolamines, and 19a-l belong to 2-substituted
ethanolamines. Finally, the representative intermediates 5a, 5b, 19a and 19b were selected to
determine their stereo configurations using Mosher method [43, 44] and circular dichroism spectrum.
2.4.1. Analysis and determination of the absolute configuration of intermediates by Mosher
method
According to the Mosher method reported in literatures [43, 44], the absolute configurations of
intermediate 5a possessing a secondary alcohol moiety were clarified by measuring the NMR spectra
of their methoxy(trifluoromethyl)-phenylacetic (MTPA) esters. 5a was reacted with (S)-(+)-MTPA
chloride in the presence of pyridine to yield (S)-MTPA ester 50. Meanwhile, the (R)-MTPA ester 51
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was also prepared according to this same synthetic method. Based on the H NMR spectral data of
MTPA esters (50 and 51), the chemical shift values were assigned to each other and the Δδ values (Δδ
= Δδs – Δδ ) were calculated by Mosher method (Table 1). The results show Δδ >0 and Δδ >0, but
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Finally, the stereochemistry of 5a was finally determined to be S-configuration.
<Insert Table 1>
2.4.2. Analysis and determination of the absolute configuration of intermediates by circular
dichroism (CD) spectrum
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The commercially available materials N-Boc-L-serine methyl ester 24 and N-Boc-D-serine
methyl ester 29 are a pair of enantiomers, and their CD spectra show a mirror symmetry relationship.
24 and 29 were used as control, the CD spectra data of 5a, 5b, 19a and 19b were analyzed.
Consequently, their configurations were determined to be S-, R-, S-, and R-configurations,
respectively (Fig. 3). Here, we can clearly see that the conclusion about the configuration of 5a is the
same by using Mosher method and circular dichroism spectrum.
<Insert Fig. 3>
Based on the above analysis, the configuration of C13 in compound 9a should be S-configuration,
while those of other chiral carbons remain unchanged. Similarly, the configurations of C12 or C13 in
other compounds were determined using the same method as that of 9a.
3. Results and discussion
3.1. In vitro antibacterial activity of 11a-azahomoclarithromycin derivatives
The in vitro antibacterial activity of the 15-membered 11a-azahomoclarithromycin derivatives with
1, 2, 3-triazoles were evaluated against various sensitive and resistant bacterial strains. All the tested
bacterial strains include Staphylococcus aureus ATCC25923 (erythromycin-susceptible strain),
Streptococcus pyogenes 1 (erythromycin-susceptible strain isolated clinically), Escherichia coli
ATCC25922
(penicillin-susceptible
strain),
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(penicillin-susceptible strain, not characterized), Bacillus subtilis ATCC9372 (penicillin-susceptible
strain), Staphylococcus aureus ATCC31007 (penicillin-resistant strain), Staphylococcus aureus
ATCC43300 (methicillin-resistant strain), Streptococcus pneumonia B1 (erythromycin-resistant strain
expressing the ermB gene), Streptococcus pneumoniae AB11 (erythromycin-resistant strain
expressing the ermB and mefA genes) and Streptococcus pyogenes 2 (erythromycin-resistant strain
isolated clinically). In all the above assays, AZM and CAM were used as controls. Determination of
the in vitro antibacterial activity by minimum inhibitory concentration (MIC) was conducted using the
broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines
[45].
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3.1.1. In vitro antibacterial activity of the series A (9a-9l)
The antibacterial activity of the series A is showed in Table 2. The MIC results indicated that 9a,
9b, 9f, 9h and 9i had better activity against S. aureus ATCC25923, S. pyogenes 1 and B. subtilis
ATCC9372 than the others in the series A. Among them, 9i exhibited the best activity against the
three tested bacterial strains of S. aureus ATCC25923, S. pyogenes 1 and B. subtilis ATCC9372 with
0.5, 0.25 and 0.5 µg/mL, respectively. Besides, 9e showed stronger activity against the tested resistant
strains than CAM, exhibiting 16-32 fold increase in antibacterial activity against S. aureus
ATCC31007, S. aureus ATCC43300, S. pneumoniae AB11 and S. pyogenes 2. In series A, 9e had the
strongest antibacterial activity against all the resistant bacteria, and its activity was well balanced.
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Based on the above MIC data, the structure-activity relationships (SARs) of the series A against
susceptible bacteria are summarized as follows. In series A, 9a exhibited excellent antibacterial
activity, but 9c-9l showed a slight decrease in the antibacterial activity. This shows that the
antibacterial activity of compounds without substituents on the phenyl group of the 1, 2, 3-triazole is
stronger than those of the compounds with substituents. We infer that the introduction of substituent
can affect the stereo structure of 11a-azahomoclarithromycin skeleton binding to bacterial ribosomes,
thus weakening the binding ability of 2'-OH to A2058 and A2059 in domain V of 23S rRNA [32].
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Therefore, the antibacterial activity of 9a is stronger than that of 9c-9l. However, 9i exhibited
excellent activity against sensitive bacteria. It is well known that the introduction of F atom can
improve the liposolubility of compounds, which may explain why 9i has excellent activity. Besides,
the antibacterial activity of 9a and 9b is similar, which indicates that when the same substituent is on
the phenyl group, its extension direction in space has little effect on the antibacterial activity.
Similarly, 9c and 9d, 9i and 9j also conform to the above SARs.
On the other hand, the SARs of the series A against resistant bacteria are summarized as follows.
Introduction of a long-chain aliphatic alkyl (4 carbon atoms) into the phenyl group of 9e and 9f
showed extremely significantly improved activity against the resistant bacteria. However, introduction
of the other substituents into the phenyl group of 9a-9d and 9g-9l did not increase significantly
activity against the resistant bacteria. This indicates that the length of the aliphatic alkyl on the phenyl
group has important effect on the antibacterial activity. The length of the aliphatic alkyl is too short
(less than 3 atoms) has a negative impact on the antibacterial activity. It is possible that the 1, 2,
3-triazole side chain with appropriate length and flexibility more easily generates the π-π stacking
interaction, electrostatic interaction and hydrophobic interaction with the U790 of 23S rRNA [20, 31].
So, 9e and 9f generate additional binding force with new binding site U790, thereby leading to
stronger antibacterial activity. The antibacterial activity of 9e is slightly stronger than that of 9f, which
indicates that the 1, 2, 3-triazole side chain with S-configuration is better than that with
R-configuration in the antibacterial activity. In series A, 9i and 9j, 9k and 9l have the same SARs as
9e and 9f. The results indicate that the extension direction of 1, 2, 3-triazole side chains in space
possesses some effect on the antibacterial activity.
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3.1.2. In vitro antibacterial activity of the series B (23a-23l)
The antibacterial activity of the series B is showed in Table 3. Compared with CAM, the series B
almost lose their activity against most susceptible bacteria. However, 23e, 23f and 23l showed
stronger activity against the tested resistant strains than CAM, especially 23e exhibiting 8-16-fold
increase in activity against S. aureus ATCC31007, S. pneumonia AB11 and S. pyogenes 2. 23e had
relatively balanced antibacterial activity against S. aureus ATCC31007 (16 µg/mL), S. pneumonia
AB11 (16 µg/mL) and S. pyogenes 2 (16 µg/mL).
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<Insert Table 3>
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We systematically summarized the SARs of series B. 23e displayed significantly improved
activity against S. aureus ATCC31007, S. pneumonia AB11 and S. pyogenes 2, much better than the
others in the series B. It suggests that introduction of a long-chain aliphatic alkane (4 carbon atoms)
into the phenyl group can enhance the antibacterial activity compared with other substituents in the
series B. We infer that the long-chain aliphatic alkane with 4 carbon atoms can make the 1, 2,
3-triazole side chain more easily to combine with the binding site U790 of 23S rRNA through the π-π
stacking interaction, electrostatic interaction and hydrophobic interaction [20, 31]. Consequently, the
additional binding ability to bacterial ribosomes of drug-resistant bacteria greatly improve in
antibacterial activity. Besides, the MIC values of 23a and 23b are basically equal, which indicates that
the stretching direction of the 1, 2, 3-triazole side chain in space do not affect the antibacterial activity
against resistant bacteria. In series B, 23c and 23d, 23i and 23j have the similar SARs to 23a and 23b.
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
3.1.3. In vitro antibacterial activity of the series C (38a-38v)
The antibacterial activity of the series C is showed in Table 4. In Series C, 38b, 38l and 38v
displayed the stronger activity against S. aureus ATCC25923 and B. subtilis ATCC9372 than the
others with the MIC values 0.25 µg/mL, better than or equal to CAM. Moreover, the antibacterial
activity of 38d and 38f against most susceptible bacteria was comparable to that of CAM, but they
exhibited excellent antibacterial activity against B. subtilis ATCC9372 with an MIC value of 0.25
µg/mL.
On the other hand, 38g and 38i had not only significantly improved activity against all the
resistant bacteria, showing 8-16-fold more potent antibacterial activity than CAM, but also extremely
balanced antibacterial activity against the drug-resistant bacteria with the MIC values of 8-32 µg/mL.
Besides, 38e, 38j, 38o, 38q and 38s exhibited slightly stronger anti-resistant antibacterial activity with
MIC values of 32-64 µg/mL than CAM.
2
2
7
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<Insert Table 4>
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The SARs of the series C against susceptible bacteria are shown below. 38b exhibits stronger
antibacterial activity against S. aureus ATCC25923, S. pyogenes 1, P. aeruginosa ATCC27853, B.
subtilis ATCC9372 and E. coli ATCC25922 than that of 38a, and the ratio (MIC /MIC ) of which is 2
S
R
~ 8. It shows that the 1, 2, 3-triazole side chain with R-configuration is better than that with
S-configuration in the antibacterial activity. In series C, 38c and 38d, 38e and 38f, 38g and 38h, 38i
and 38j, 38k and 38l, 38m and 38n, 38o and 38p, 38u and 38v possess the same SARs as 38a and
38b. Obviously, we can draw the conclusion that the extension direction of the 1, 2, 3-triazoles side
chain in space has a significant effect on the antibacterial activity. In particular, the R-configuration
side chain probably has more favorable conformation to interact with bacterial ribosomes than the
S-configuration side chain, which may explain that compounds with the R-configuration side chains
are stronger than compounds with S-configuration side chains in the antibacterial activity.
1
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1
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1
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2
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2
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The SARs of the series C against resistant bacteria are also summarized as below. The
antibacterial activity of 38c, 38e, 38g and 38i increases with the extension of the aliphatic alkane
group, which demonstrates that the length of the introduced aliphatic alkane group on the phenyl
group is closely related to its activity. Specially, the aliphatic alkane group in compound 38g is 3
carbon atoms, which leads to the strongest antibacterial activity. This may be because the aliphatic
alkane group with 3 carbon atoms produces stronger binding force with the binding site U790 than the
others [20, 31]. Besides, the conformations of the 1, 2, 3-triazole side chains also have a certain
influence on the antibacterial activity. 38e exhibits more potent antibacterial activity against resistant
bacteria than that of 38f, which demonstrates that the S-configuration side chain is better than the
R-configuration side chain in the antibacterial activity. 38g and 38h, 38i and 38j, 38o and 38p, 38q
and 38r, 38s and 38t have also similar SARs as described above.
In general, the series A and series C belong to the C-13 substituted derivatives, possessing the 1, 2,
3-triazole side chains with one atom and three atoms between the 1, 2, 3-triazole and the
11a-azahomoclarithromycin skeleton, respectively. While the series B belongs to the C-12 substituted
derivatives, possessing the 1, 2, 3-triazole side chains with one atom between the 1, 2, 3-triazole and
the 11a-azahomoclarithromycin skeleton. As for the antibacterial activity against susceptible bacteria,
the order of antibacterial activity of the three series is the series C > the series A > the series B. The
results show that the compounds with the 1, 2, 3-triazole side chains at the C-13 position exhibit
stronger activity than those with the 1, 2, 3-triazole side chains at the C-12 position. In addition, the
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length between the 1, 2, 3-triazole and the 11a-azahomoclarithromycin skeleton has a positive effect
on the antibacterial activity against susceptible bacteria. On the other hand, most of the derivatives in
the series A and B exerted similar activity against resistant bacteria, while only 9e and 9f (the C-13
substituted derivatives) are much stronger activity against S. aureus ATCC43300 and S. pneumoniae
B1 than 23e and 23f (the C-12 substituted derivatives). As for the series A and C, their antibacterial
activity is similar, which shows clearly that the length between the 1, 2, 3-triazole and the
11a-azahomoclarithromycin skeleton does not have much effect on the antibacterial activity against
resistant bacteria.
3.2. Bactericidal or bacteriostatic action
1
1
1
1
1
1
1
1
To confirmed whether the above promising compounds were bactericidal or bacteriostatic, we
determined the minimal bactericidal concentration (MBC) values of 38b, 38l and 38v against
susceptible bacteria (S. aureus ATCC25923), and 9e and 38g against resistant bacteria (S. pneumoniae
AB11) (Table 5). The results indicate that 38b, 38l and 38v have all the MBC/MIC values of more
than or equal to 512, exhibiting bacteriostatic action against S. aureus ATCC25923. In contrast, 9e
and 38g have all the MBC/MIC values of 8, showing bacteriostatic behavior against S. pneumoniae
AB11. The above results demonstrate that the tested compounds against sensitive and resistant
bacterial strains are essentially achieved through bacteriostatic action.
1
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<Insert Table 5>
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7
3.3. Kinetics of the bactericidal activity
In order to further study and clarify the sterilization of the promising compounds in each time
period, 9e with certain bactericidal activity was chose to determine its time-bactericidal curve (Fig. 4).
The results indicate that at the concentrations of 1, 2 and 4 MIC, 9e exhibits certain bactericidal effect
(0-9 h) and excellent bacteriostatic effect (9-24 h) against S. pneumoniae AB11, respectively. Thus, 9e
have a concentration-dependent inhibition on bacterial production, which also demonstrate that it is
an excellent bacteriostatic agent from the microscopic point of view.
2
2
8
9
<Insert Fig. 4>
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3.4. Cytotoxic evaluation on mammalian cells
38b, 38l and 38v with the MIC values of 0.25~0.5 µg/mL against most susceptible bacteria (such
as S. aureus ATCC25923, S. pyogenes 1 and B. subtilis ATCC 9372) were selected to evaluate their
cytotoxicity
on
MCF-7
breast
cancer
cells
using
3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 5). The results show that
38l has non-toxic to MCF-7 breast cancer cells up to a concentration of 32 µg/mL, while 38b and 38v
are slight toxic. After incubation at a concentration of 64 or 128 µg/mL for 24 h, the tested cells
percent viable is approximately 60%, which indicate that 38b, 38l and 38v have moderate toxicity to
MCF-7 breast cancer cells at this concentration. Therefore, 38b, 38l and 38v exhibit no cytotoxicity at
its effective antibacterial concentration.
1
1
1
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3
<Insert Fig. 5>
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4. Conclusion
Three series of novel 15-membered 11a-azahomoclarithromycin derivatives (the series A-C) with
the 1, 2, 3-triazole side chains were designed and synthesized through creatively opening the ring of
CAM, expanding the ring properly and introducing a suitable side chain of 1, 2, 3-triazole at the C12
and C13 positions, and evaluated for their antibacterial activity against drug-sensitive and -resistant
strains. The antibacterial results indicated that the 15-membered 11a-azahomoclarithromycin
derivatives exhibited excellent activity against sensitive bacterial strains and greatly improved activity
against resistant bacterial strains. Among them, 38b, 38l and 38v displayed the most potent activity
against sensitive bacterial strains S. aureus ATCC25923 (0.25 µg/mL) and B. subtilis ATCC9372
(0.25 µg/mL), while 9e and 38g exerted the strongest activity against resistant bacterial strains S.
pneumoniae AB11 expressing the ermB and mefA genes (8 µg/mL). In addition, the determination of
MBC indicated that the most promising compounds 38b, 38l, 38v, 9e and 38g were excellent
bacteriostatic agents. Further, the bactericidal curve demonstrated that A5 exhibited antibacterial
activity through bacteriostatic mechanism. Finally, 38b, 38l and 38v were confirmed to be non-toxic
to MCF-7 breast cancer cells up to a concentration of 32 µg/mL in preliminary cytotoxicity assay.
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Therefore, 38b, 38l, 38v, 9e and 38g can be served as lead compounds to provide a new perspective
for further structural optimization.
5. Experimental section
5.1. Synthetic procedures
All reagents and solvents were commercially available and could be used directly without
purification unless were specifically stated. TLC was used to monitor the reactions process. Column
1
13
chromatography was used to separate and purify intermediates and compounds. H NMR and
C
NMR spectra were recorded at 600 MHz and 150 MHz, respectively. And CDCl , d -DMSO and
3
6
1
1
1
1
1
1
1
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C D N were used as solvents in those spectra. Mass spectra and HRMS were measured by the API
5 5
4000 instrument and Orbitrap analyzer, respectively. The melting points of derivatives were
performed by an uncorrected RY-1 melting point apparatus.
5.1.1. Synthesis of 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series A)
5.1.1.1. (R)-1-chloro-3-(4-phenyl-1H-1, 2, 3-triazol-1-yl)propan-2-ol (13)
To a solution of R-epichlorohydrin 10 (1 g, 10.9 mmol) in AcOH (10 mL) and H O (10 mL) was
2
o
added NaN (3.7 g, 56.9 mmol) at room temperature. The reaction mixture was warmed to 30 C and
3
stirred for 5 h. EtOAc (20 mL) was added, the organic phase was separated and aqueous layer was
extracted with EtOAc (20 mL×3). The combined organic layers were dried over Na SO . The solvent
2
4
was removed in vacuo and the residue was purified by silica gel column (PE/ EtOAc = 6:1) to afford
azide 11 (1.27 g, 86%) as a light yellow oil.
To a solution of azide 11 (0.5 g, 3.70 mmol) in 75% CH OH (20 mL) was added sequentially
3
phenylacetylene 12 (0.38 g, 3.70 mmol), CuSO (37 mg), and L-ascorbic acid sodium salt (111 mg).
4
o
The reaction was stirred at 35~40 C for 24 h. The reaction mixture was concentrated and the residue
was diluted with DCM (20 mL) and H O (20 mL). The organic phase was separated and aqueous
2
layer was extracted with DCM (20 mL×2). The combined organic layers were dried over Na SO . The
2
4
solvent was removed in vacuo and the residue was purified by silica gel column (DCM/CH OH =
3
30:1) to give chlorohydrin 13 (0.81 g, 92%) as a white solid. Mp: 102–104 °C, TLC R = 0.32
f
1
(DCM/CH OH = 30:1); H NMR (600 MHz, CDCl , δ ppm): 7.84 (s, 1H), 7.72 – 7.70 (m, 2H), 7.40
3
3
(t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 4.69 (dd, J = 13.9, 3.2 Hz, 1H), 4.51 (dd, J = 13.9, 7.1 Hz,
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1H), 4.45 – 4.41 (m, 1H), 3.63 (d, J = 5.5 Hz, 2H); MS (ESI) m/z calcd for C H ClN O [M + H] :
11 12 3
238.1, found: 238.3.
5.1.1.2. (S)-1-amino-3-(4-phenyl-1H-1, 2, 3-triazol-1-yl)propan-2-ol (5a)
To a solution of chlorohydrin 13 (0.81 g, 3.4 mmol) in CH CN (2 mL) was added NaOH solution
3
(10 mL, 10 M). The reaction was stirred at room temperature for 2 h. DCM (20 mL) was added, the
organic phase was separated and aqueous layer was extracted with DCM (20 mL×2). The combined
organic layers were concentrated to afford epoxy 14 as a white solid. The epoxy 14 was used directly
for the next step without purification.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
To a solution of epoxy 14 in CH CN (2 mL) was added 25% aqueous ammonia (20 mL) at room
3
temperature. The reaction was stirred at room temperature for 3 h. DCM (20 mL) was added, the
organic phase was separated and aqueous layer was extracted with DCM (20 mL×3). The combined
organic layers were dried over Na SO . The solvent was removed in vacuo and the residue was
2
4
purified by silica gel column (DCM/CH OH/NH ·H O = 10:1:0.1) to afford the substituted
3
3
2
ethanolamine 5a (0.42 g, 56% in two steps from 13) as a white solid. Mp: 131–133 °C, TLC R = 0.11
f
1
(DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz, DMSO-d , δ ppm): 8.49 (s, 1H), 7.86 –
3
3
2
6
7.85 (m, 2H), 7.46 – 7.43 (m, 2H), 7.34 – 7.31 (m, 1H), 5.13 (s, 1H), 4.52 (dd, J = 13.8, 4.0 Hz, 1H),
4.30 (dd, J = 13.8, 7.7 Hz, 1H), 3.80 (d, J = 5.2 Hz, 1H), 2.58 – 2.52 (m, 2H), 1.59 (s, 2H); MS (ESI)
+
m/z calcd for C H N O [M + H] : 219.1, found: 219.4.
11
14
4
5.1.1.3. 9-dihydro-6-O-methylerythromycin A (2)
NaBH (6 g, 158.6 mmol) was added to a solution of CAM 1 (20 g, 26.8 mmol) in anhydrous THF
4
o
(150 mL) and CH OH (300 mL) in three batches within 1 h at 0 C. The reaction mixture was stirred
3
at room temperature for 24 h under N . The reaction mixture was concentrated, then DCM (200 mL)
2
and saturated NH Cl solution (200 mL) were added to the resulting residue. The organic phase was
4
separated and aqueous layer was extracted with DCM (100 mL×2). The combined organic layers were
dried over Na SO . The solvent was removed in vacuo and the residue was purified by silica gel
2
4
column (DCM/CH OH/NH ·H O = 30:1:0.1~20:1:0.1~10:1:0.1) to afford 9(S)-OH-2 (10.5 g, 52%)
3
3
2
1
as a white solid. Mp: 208–210 °C, TLC R = 0.45 (DCM/CH OH/NH ·H O = 10:1:0.1); H NMR
f
3
3
2
(600 MHz, CDCl , δ ppm): 5.73 (d, J = 9.8 Hz, 1H), 5.30 (s, 1H), 5.22 (dd, J = 11.2, 2.2 Hz, 1H),
3
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4.98 (dd, J = 20.3, 4.7 Hz, 1H), 4.51 (d, J = 7.1 Hz, 1H), 4.34 (d, J = 1.6 Hz, 1H), 4.05 – 4.03 (m, 1H),
3.83 – 3.72 (m, 3H), 3.53 – 3.45 (m, 3H), 3.37 (s, 3H), 3.33 (s, 3H), 3.29 – 3.21 (m, 2H), 3.04 – 2.95
(m, 3H), 2.39 – 2.33 (m, 7H), 2.19 – 2.13 (m, 2H), 2.02 – 1.84 (m, 4H), 1.53 – 1.44 (m, 4H), 1.38 (s,
3H), 1.32 – 1.22 (m, 15H), 1.13 – 1.08 (m, 9H), 0.85 (t, J = 7.4 Hz, 3H); MS (ESI) m/z calcd for
+
C H NO [M + H] : 750.5, found: 750.8.
38
71
13
5.1.1.4. 2', 4'', 9(S)-triethylsilane-6-O-methylerythromycin A (3)
Imidazole (7.2 g, 105.8 mmol) and 2 (8 g, 10.7 mmol) were dissolved in anhydrous DMF (100
o
mL) at room temperature. TESCl (5.5 g, 36.5mmol) was added dropwise to the mixture at 0 C. The
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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reaction mixture was stirred at room temperature for 18 h under N . EtOAc (100 mL) and n-hexane
2
(100 mL) were added, the organic phase was separated and washed with distilled water (100 mL). The
organic layer was dried over Na SO . The solvent was removed in vacuo and the residue was purified
2
4
by silica gel column (PE/acetone = 80:1~50:1~30:1~10:1) to give silane protected product 3 (7.9 g,
1
68%) as a white foam. Mp: 86–89 °C, TLC R = 0.76 (PE/acetone = 3:1); H NMR (600 MHz, CDCl ,
f
3
δ ppm): 5.09 (dd, J = 11.2, 2.3 Hz, 1H), 4.86 (d, J = 4.6 Hz, 1H), 4.48 (s, 1H), 4.32 (d, J = 7.0 Hz,
1H), 4.26 – 4.24 (m, 1H), 3.89 (d, J = 9.7 Hz, 1H), 3.80 (s, 1H), 3.64 (d, J = 9.0 Hz, 1H), 3.51 – 3.48
(m, 1H), 3.36 – 3.32 (m, 4H), 3.29 (s, 3H), 3.25 – 3.21 (m, 2H), 3.13 (dd, J = 9.7, 7.0 Hz, 1H), 2.88 –
2.86 (m, 1H), 2.54 – 2.50 (m, 1H), 2.39 (d, J = 14.8 Hz, 1H), 2.19 (s, 6H), 2.10 –2.09 (m, 1H), 1.97 –
1.95 (m, 1H), 1.85 – 1.82 (m, 1H), 1.69 (d, J = 14.3 Hz, 1H), 1.64 – 1.61 (m, 1H), 1.52 (d, J = 5.0 Hz,
1H), 1.49 – 1.46 (m, 4H), 1.29 – 0.96 (m, 44H), 0.96 – 0.89 (m, 10H), 0.84 (t, J = 7.4 Hz, 3H), 0.71 –
2+
0.54 (m, 18H); MS (ESI) m/z calcd for C H NO Si [M + 2H] /2: 546.9, found: 547.4.
56
113
13
3
5.1.1.5. Ring-opening intermediate with carboxylic acid (7a)
To a solution of silane protected product 3 (375 mg, 0.34 mmol) in CHCl (20 mL) was added
3
o
o
Pb(OAc) (183mg, 0.41 mmol) at 0 C. The reaction mixture was stirred at 0 C for 0.5 h under N .
4
2
The aldehyde intermediate 4 was prepared and used directly for the next step without purification.
5a (150 mg, 0.69 mmol) and NaBH(OAc) (218 mg, 1.03mmol) were added to the solution of 4 at
3
room temperature. The reaction mixture was stirred at room temperature for 4 h under N , then 37%
2
aqueous formaldehyde solution (167 mg, 2.06 mmol) and NaBH(OAc) (218 mg, 1.03mmol) were
3
added. The reaction mixture was stirred at room temperature for 4 h. A saturated NaHCO (20 mL)
3
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was added,
the organic phase was separated and aqueous layer was extracted with DCM (20 mL×2). The
combined organic layers were dried over Na SO . The solvent was removed in vacuo and the residue
2
4
was purified by silica gel column (DCM/CH OH/NH ·H O = 30:1:0.1) to afford crude product of
3
3
2
ester 6a as a colorless oil.
To a solution of intermediate 6a in THF-C H OH-H O (20 mL, THF/C H OH/H O = 3:1:1)
2
5
2
2
5
2
was added LiOH (41mg, 1.71 mmol) at room temperature. The reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was concentrated in vacuo and the residue was purified by
silica gel column (DCM/CH OH/NH ·H O = 15:1:0.1~10:1:0.1) to afford carboxylic acid 7a (185 mg,
3
3
2
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1
1
1
1
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2
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3
44% in four steps from intermediate 3) as a white solid. Mp: 96–98 °C, TLC R = 0.42
f
1
(DCM/CH OH/NH ·H O = 15:1:0.1); H NMR (600 MHz, CDCl , δ ppm): 8.04 (s, 1H), 7.84 (dd, J =
3
3
2
3
8.2, 1.1 Hz, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.34 – 7.32 (m, 1H), 4.80 (d, J = 4.7 Hz, 1H), 4.58 (dd, J =
14.0, 4.1 Hz, 1H), 4.47 – 4.42 (m, 2H), 4.39 (d, J = 3.6 Hz, 1H), 4.28 – 4.25 (m, 1H), 3.93 (dd, J =
6.4, 2.4 Hz, 1H), 3.74 – 3.72 (m, 1H), 3.61 (m, 1H), 3.30 (s, 3H), 3.24 – 3.16 (m, 7H), 3.06 (d, J =
11.3 Hz, 1H), 2.76 (s, 1H), 2.54 (s, 3H), 2.49 (s, 1H), 2.39 – 2.22 (m, 5H), 2.20 (s, 6H), 1.78 (m, 1H),
1.60 – 1.57 (m, 2H), 1.47 (dd, J = 14.9, 4.9 Hz, 1H), 1.28 (s, 3H), 1.26 (m, 1H), 1.22 (d, J = 6.3 Hz,
3H), 1.18 – 1.13 (m, 10H), 1.08 – 1.07 (m, 6H), 0.99 – 0.90 (m, 30H), 0.68 – 0.57 (m, 18H); MS (ESI)
2
+
m/z calcd for C H N O Si [M + 2H] /2: 611.9, found: 612.3.
6
3
119
5
12
3
5.1.1.6. 15-membered 11a-azahomoclarithromycin intermediate (8a)
To a solution of intermediate 7a (185 mg, 0.15 mmol) in THF (3 mL) was added sequentially
triethylamine (153 mg, 1.51 mmol), 2, 4, 6-trichlorobenzoyl chloride (111 mg, 0.46 mmol) at room
temperature. The reaction mixture was stirred at room temperature for 4 h under N . The solution was
2
added to a refluxed solution of DMAP (466 mg, 3.78 mmol) in CH CN (30 mL) for 0.5 h. The
3
reaction mixture was concentrated in vacuo and the residue was purified by silica gel column
(PE/acetone = 15:1~10:1) to afford cyclic intermediate 8a (90 mg, 49%) as a white solid. Mp: 96–
1
98 °C, TLC R = 0.53 (PE/acetone = 3:1); H NMR (600 MHz, CDCl , δ ppm): 7.83 – 7.81 (m, 2H),
f
3
7.77 (s, 1H), 7.44 – 7.41 (m, 2H), 7.35 (t, J = 7.4 Hz, 1H), 5.25 (s, 1H), 4.81 (d, J = 4.2 Hz, 1H), 4.73
– 4.70 (m, 1H), 4.58 (dd, J = 14.2, 6.6 Hz, 1H), 4.39 (d, J = 6.9 Hz, 1H), 4.26 – 4.23 (m, 1H), 4.06 (s,
1H), 3.65 (d, J = 8.0 Hz, 1H), 3.54 (m, 1H), 3.34 (s, 1H), 3.27 (s, 3H), 3.20 – 3.13 (m, 5H), 2.76 –
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2.70 (m, 2H), 2.59 – 2.44 (m, 3H), 2.34 – 2.26 (m, 4H), 2.19 (s, 6H), 2.13 – 2.10 (m, 1H), 1.98 (m,
1H), 1.93 – 1.92 (m, 1H), 1.83 – 1.81 (m, 1H), 1.63 – 1.57 (m, 2H), 1.43 (dd, J = 14.8, 4.6 Hz, 1H),
1.31 (s, 3H), 1.25 – 1.21 (m, 4H), 1.16 – 1.07 (m, 13H), 0.99 – 0.92 (m, 33H), 0.68 – 0.56 (m, 18H);
2+
MS (ESI) m/z calcd for C H N O Si [M + 2H] /2: 602.9, found: 603.2.
6
3
117
5
11
3
5.1.1.7. 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin (9a)
To a solution of cyclic intermediate 8a (90 mg, 0.07 mmol) in THF (5 mL) was added hydrogen
fluoride–pyridine (46 mg, 0.30 mmol, 65%) at room temperature. The reaction mixture was stirred at
room temperature for 18 h. A saturated solution of NaHCO was added until the pH >7 and the
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
mixture was stirred for 10 min. DCM (20 mL) was added, the organic phase was separated and
aqueous layer was extracted with DCM (20 mL×2). The combined organic layers were dried over
Na SO . The solvent was removed in vacuo and the residue was purified by silica gel column
2
4
(DCM/CH OH/NH ·H O = 10:1:0.1) to afford compound 9a (31 mg, 48%) as a white solid. Mp:
3
3
2
1
126–129 °C, TLC R = 0.40 (DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz, CDCl , δ
f
3
3
2
3
ppm): 7.84 – 7.82 (m, 2H), 7.78 (s, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H), 4.86 (d, J =
4.7 Hz, 1H), 4.59 (dd, J = 14.5, 4.6 Hz, 1H), 4.51 (dd, J = 14.5, 5.4 Hz, 1H), 4.39 (d, J = 7.2 Hz, 1H),
4.32 (s, 1H), 4.06 – 4.03 (m, 1H), 3.72 (d, J = 8.1 Hz, 1H), 3.49 (dd, J = 14.6, 5.5 Hz, 2H), 3.27 (s,
3H), 3.23 – 3.21 (m, 5H), 2.99 – 2.93 (m, 2H), 2.71 (dd, J = 13.3, 11.4 Hz, 1H), 2.57 – 2.36 (m, 5H),
2.36 – 2.30 (m, 9H), 2.26 – 2.23 (m, 2H), 2.18 – 2.14 (m, 1H), 1.88 – 1.86 (m, 1H), 1.75 – 1.72 (m,
2H), 1.44 (dd, J = 15.2, 4.9 Hz, 1H), 1.32 (s, 3H), 1.29 (d, J = 6.3 Hz, 3H), 1.23 – 1.18 (m, 10H), 1.08
13
(d, J = 7.3 Hz, 3H), 0.85 (d, J = 7.1 Hz, 3H), 0.73 (d, J = 6.7 Hz, 3H); C NMR (150 MHz, CDCl , δ
3
ppm): 176.26, 148.00, 130.38, 128.90, 128.90, 128.30, 125.72, 125.72, 120.39, 103.52, 96.20, 80.22,
79.10, 78.94, 78.10, 72.62, 71.07, 68.85, 67.91, 65.42, 65.27, 62.96, 59.09, 51.74, 50.13, 49.44, 45.37,
44.24, 41.41, 40.41, 36.65, 34.94, 32.54, 30.95, 29.71, 29.26, 22.17, 21.55, 21.39, 18.26, 14.66, 10.63;
+
HRMS (ESI) m/z calcd for C H N O [M + H] : 862.5463, found: 862.5723.
4
5
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According to the synthetic procedure described above, the following compounds (9b-9l) were also
prepared:
5.1.1.8. Compound (9b)
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White solid, yield 59%, mp: 126–129 °C, TLC R = 0.40 (DCM/CH OH/NH ·H O = 10:1:0.1); H
f 3 3 2
NMR (600 MHz, CDCl , δ ppm): 7.83 – 7.73 (m, 3H), 7.52 – 7.40 (m, 2H), 7.35 (t, J = 7.3 Hz, 1H),
3
5.16 (s, 1H), 4.80 (s, 1H), 4.72 – 4.60 (m, 2H), 4.46 – 4.41 (m, 1H), 4.05 – 4.02 (m, 1H), 3.90 (s, 1H),
3.81 (s, 1H), 3.63 – 3.40 (m, 2H), 3.26 (s, 3H), 3.22 – 2.91 (m, 6H), 2.77 – 2.48 (m, 5H), 2.42 – 1.83
(m, 12H), 1.81 –1.61 (m, 4H), 1.59 – 1.09 (m, 20H), 1.09 (d, J = 7.4 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H);
13
C NMR (150 MHz, CDCl , δ ppm): 172.53, 146.83, 129.49, 127.82, 127.82, 127.18, 124.73, 124.73,
3
119.35, 102.36, 95.53, 79.33, 79.06, 78.42, 77.03, 76.88, 71.56, 70.86, 70.06, 67.73, 64.85, 64.69,
64.40, 50.40, 49.17, 48.42, 48.39, 44.38, 39.40, 37.21, 35.98, 34.11, 31.80, 29.41, 28.68, 28.31, 20.60,
+
20.51, 20.43, 20.35, 20.27, 17.57, 9.96, 9.11; HRMS (ESI) m/z calcd for C H N O [M + H] :
45
75
5
11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
862.5463, found: 862.5709.
1
13
Physical characteristics, H NMR, C NMR, MS and HRMS for other target compounds of series
A, were reported in the supporting information.
5.1.2. Synthesis of 15-membered 11a-aza-12-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series B)
5.1.2.1. (S)-methyl 2-[(tert-butoxycarbonyl)amino]-3-(4-phenyl-1H-1,2,3-triazol-1-yl)propanoate (26)
To a solution of N-Boc-L-serine methyl ester 24 (2 g, 9.1 mmol) in anhydrous DCM (50 mL)
was added Et N (2 g, 19.8 mmol) at room temperature. A solution of MsCl (1.15 g, 10 mmol) in
3
o
anhydrous DCM (10 mL) was added dropwise to the mixture at 0 C under N . The reaction mixture
2
o
was stirred at 0 C for 0.5 h under N . H O (50 mL) was added, the organic phase was separated and
2
2
aqueous layer was extracted with DCM (30 mL×2). The combined layers were concentrated to form
methanesulfonate 25 as a colorless oil. The crude intermediate 25 was used directly for the next step
without purification.
To a solution of methanesulfonate 25 in DMF (60 mL) was added NaN (1.4 g, 21.5 mmol) at
3
o
room temperature. The reaction mixture was stirred at 50 C for 0.5 h. EtOAc (60 mL) and H O (60
2
mL) were added, the organic phase was separated and aqueous layer was extracted with EtOAc (60
mL×2). The combined organic layers were dried over Na SO . The solvent was concentrated in vacuo
2
4
to afford the azide 26 (8.74g) as a colorless oil.
To a solution of azide 26 (4.37 g) in 75% CH OH (20 mL) was added sequentially
3
phenylacetylene 12 (0.21 g, 2.06 mmol), CuSO (41 mg), and L-ascorbic acid sodium salt (123 mg) at
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room temperature. The reaction mixture was stirred at 35~40 C for 48 h. The reaction mixture was
concentrated and the residue was diluted with DCM (20 mL) and H O (20 mL). The organic phase
2
was separated and aqueous layer was extracted with DCM (20 mL×2). The combined organic layers
were dried over Na SO . The solvent was removed in vacuo and the residue was purified by silica gel
2
4
column (PE/EtOAc = 4:1~1:1) to afford 1, 2, 3-triazole intermediate 27 (0.37 g, 23% in three steps
1
from 24) as a white solid. Mp: 118–120 °C, TLC R = 0.58 (DCM/MeOH = 30:1); H NMR (600
f
MHz, CDCl , δ ppm): 7.82 – 7.81 (m, 2H), 7.73 (s, 1H), 7.44 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 7.4 Hz,
3
1H), 5.42 (d, J = 6.6 Hz, 1H), 4.92 (dd, J = 14.0, 3.8 Hz, 1H), 4.86 (dd, J = 14.0, 4.4 Hz, 1H), 4.76 (d,
+
J = 3.3 Hz, 1H), 3.81 (s, 3H), 1.45 (s, 9H); MS (ESI) m/z calcd for C H N O [M + H] : 347.2,
17
22
4
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
found: 347.4.
5.1.2.2. (S)-2-amino-3-(4-phenyl-1H-1, 2, 3-triazol-1-yl)propan-1-ol (19a)
To a solution of 1, 2, 3-triazol intermediate 27 (0.37 g, 1.07 mmol) in CH OH (20 mL) was
3
o
o
added NaBH (121 mg, 3.20 mmol) at 0 C. The reaction mixture was stirred at 0 C for 2 h. The
4
reaction mixture was concentrated and the residue was diluted with DCM (20 mL) and H O (20 mL).
2
The organic phase was separated and aqueous layer was extracted with DCM (20 mL×2). The
combined organic layers were concentrated to afford alcohol 28 as a white solid.
To a solution of alcohol 28 in EtOAc (10 mL) was added concentrated HCl (4 mL, 48 mmol) at
room temperature. The reaction mixture was stirred at room temperature for 4 h. A saturated solution
of NaHCO was added until the pH >7 and the mixture was stirred for 10 min. The resulting mixture
3
was concentrated in vacuo and the residue was purified by silica gel column (DCM/CH OH/NH ·H O
3
3
2
= 10:1:0.1) to afford substituted ethanolamine 19a (0.14 g, 60% in two steps from 27) as a white solid.
1
Mp: 121–123 °C, TLC R = 0.36 (DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz,
f
3
3
2
DMSO-d , δ ppm): 8.53 (s, 1H), 7.85 – 7.83 (m, 2H), 7.46 – 7.43 (m, 2H), 7.34 – 7.31 (m, 1H), 4.48
6
(dd, J = 13.6, 4.9 Hz, 1H), 4.23 (dd, J = 13.6, 7.8 Hz, 1H), 3.35 – 3.31 (m, 2H), 3.14 – 3.11 (m, 1H),
+
1.91 (s, 1H); MS (ESI) m/z calcd for C H N O [M + H] : 219.1, found: 219.4.
11
14
4
5.1.2.3. Ring-opening intermediate with carboxylic acid (21a)
To a solution of silane protected product 3 (350 mg, 0.32 mmol) in CHCl (20 mL) was added
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Pb(OAc) (171mg, 0.39 mmol) at 0 C. The reaction mixture was stirred at 0 C for 0.5 h under N .
4
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The aldehyde intermediate 4 was prepared and used directly for the next step without purification.
19a (140 mg, 0.64 mmol) and NaBH(OAc) (204 mg, 0.96 mmol) were added to the solution of 4
3
at room temperature. The reaction mixture was stirred at room temperature for 4 h under N , then 37%
2
aqueous formaldehyde solution (156 mg, 1.92 mmol) and NaBH(OAc) (204 mg, 0.96 mmol) were
3
added. The reaction mixture was stirred at room temperature for 4 h. The saturated NaHCO (20 mL)
3
was added, the organic phase was separated and aqueous layer was extracted with DCM (20 mL×2).
The combined organic layers were dried over Na SO . The solvent was removed in vacuo and the
2
4
residue was purified by silica gel column (DCM/CH OH/NH ·H O = 30:1:0.1) to afford crude
3
3
2
product of ester 20a as a colorless oil.
To a solution of intermediate 20a in THF-C H OH-H O (20 mL, THF/C H OH/H O = 3:1:1) was
1
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added LiOH (38mg, 1.59 mmol) at room temperature. The reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was concentrated in vacuo and the residue was purified by
silica gel column (DCM/CH OH/NH ·H O = 15:1:0.1~10:1:0.1) to afford carboxylic acid 21a (200
3
3
2
mg, 51% in four steps from 3) as a white solid. Mp: 78–81 °C, TLC Rf = 0.42
1
(DCM/CH OH/NH ·H O = 15:1:0.1); H NMR (600 MHz, CDCl , δ ppm): 7.83 – 7.81 (m, 2H), 7.77
3
3
2
3
(s, 1H), 7.44 – 7.41 (m, 2H), 7.35 (t, J = 7.4 Hz, 1H), 5.25 (s, 1H), 4.81 (d, J = 4.2 Hz, 1H), 4.73 –
4.70 (m, 1H), 4.58 (dd, J = 14.2, 6.6 Hz, 1H), 4.39 (d, J = 6.9 Hz, 1H), 4.26 – 4.23 (m, 1H), 4.06 (s,
1H), 3.65 (d, J = 8.0 Hz, 1H), 3.54 (s, 1H), 3.34 (s, 1H), 3.27 (s, 3H), 3.20 – 3.13 (m, 5H), 2.76 – 2.70
(m, 2H), 2.59 – 2.44 (m, 3H), 2.34 – 2.26 (m, 4H), 2.19 (s, 6H), 2.13 – 2.10 (m, 1H), 1.98 (m, 1H),
1.92 (m, 1H), 1.83 – 1.81 (m, 1H), 1.62 – 1.57 (m, 2H), 1.43 (dd, J = 14.8, 4.6 Hz, 1H), 1.31 (s, 3H),
1.25 – 1.21 (m, 4H), 1.16 – 1.07 (m, 13H), 0.99 – 0.92 (m, 33H), 0.68 – 0.56 (m, 18H); MS (ESI) m/z
2
+
calcd for C H N O Si [M + 2H] /2: 611.9, found: 611.8.
6
3
119
5
12
3
5.1.2.4. 15-membered 11a-azahomoclarithromycin intermediate (22a)
To a solution of intermediate 21a (200 mg, 0.16 mmol) in THF (3 mL) was added sequentially
triethylamine (166 mg, 1.64 mmol), 2, 4, 6-trichlorobenzoyl chloride (120 mg, 0.49 mmol) at room
temperature. The reaction mixture was stirred at room temperature for 4 h under N . The solution was
2
added to a refluxed solution of DMAP (504 mg, 4.09 mmol) in CH CN (30 mL) for 0.5 h. The
3
reaction mixture was concentrated in vacuo and the residue was purified by silica gel column
(PE/acetone = 15:1~10:1) to afford cyclic intermediate 22a (70 mg, 36%) as a white solid. Mp: 60–
23

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63 °C, TLC R = 0.53 (PE/acetone = 3:1); H NMR (600 MHz, CDCl , δ ppm): 7.84 – 7.78 (m, 3H),
f
3
7.44 – 7.40 (m, 2H), 7.35 – 7.32 (m, 1H), 4.70 (s, 1H), 4.47 – 4.40 (m, 2H), 4.27 – 4.26 (m, 1H), 4.17
– 3.97 (m, 3H), 3.74 – 3.71 (m, 1H), 3.59 – 3.58 (m, 1H), 3.50 – 3.49 (m, 1H), 3.29 – 3.25 (m, 3H),
3.22 – 3.14 (m, 5H), 2.63 (s, 2H), 2.45 – 2.28 (m, 7H), 2.20 (s, 6H), 2.13 (m, 1H), 2.02 (m, 1H), 1.77
– 1.76 (m, 2H), 1.52 – 1.48 (m, 2H), 1.43 – 1.39 (m, 1H), 1.26 (s, 3H), 1.25 – 1.24 (m, 1H), 1.20 (d, J
= 5.5 Hz, 3H), 1.16 – 1.11 (m, 10H), 1.09 (d, J = 7.2 Hz, 3H), 1.00 – 0.92 (m, 33H), 0.67 – 0.55 (m,
2+
18H); MS (ESI) m/z calcd for C H N O Si [M + 2H] /2: 602.9, found: 603.3.
6
3
117
5
11
3
5.1.2.5. 15-membered 11a-aza-12-(1, 2, 3-triazoles)homoclarithromycin (23a)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
To a solution of cyclic intermediate 22a (70 mg, 0.06 mmol) in THF (5 mL) was added hydrogen
fluoride–pyridine (26 mg, 0.17 mmol, 65%) at room temperature. The reaction mixture was stirred at
room temperature for 18 h. A saturated solution of NaHCO was added until the pH >7 and the
3
mixture was stirred for 10 min. DCM (20 mL) was added, the organic phase was separated and
aqueous layer was extracted with DCM (20 mL×2). The combined organic layers were dried over
Na SO . The solvent was removed in vacuo and the residue was purified by silica gel column
2
4
(DCM/CH OH/NH ·H O = 10:1:0.1) to afford compound 23a (30 mg, 60%) as a white solid. Mp:
3
3
2
1
108–110 °C, TLC R = 0.40 (DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz, CDCl , δ
f
3
3
2
3
ppm): 7.88 (s, 1H), 7.83 (dd, J = 10.2, 3.1 Hz, 2H), 7.44 (dd, J = 10.0, 5.4 Hz, 2H), 7.35 (t, J = 7.4 Hz,
1H), 4.78 (d, J = 4.4 Hz, 1H), 4.48 – 4.39 (m, 3H), 4.22 (d, J = 10.1 Hz, 1H), 4.10 – 3.95 (m, 3H),
3.79 (d, J = 5.9 Hz, 1H), 3.50 – 3.46 (m, 2H), 3.29 – 3.14 (m, 7H), 3.01 (t, J = 9.5 Hz, 1H), 2.95 –
2.84 (m, 2H), 2.55 – 2.21 (m, 14H), 2.15 – 1.96 (m, 3H), 1.86 – 1.83 (m, 1H), 1.81 – 1.75 (m, 1H),
1.71 – 1.63 (m, 1H), 1.52 (s, 1H), 1.31 (s, 3H), 1.30 – 1.21 (m, 10H), 1.18 (d, J = 7.3 Hz, 3H), 1.07 (d,
13
J = 6.9 Hz, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 5.0 Hz, 3H); C NMR (150 MHz, CDCl , δ
3
ppm): 175.50, 147.94, 130.55, 128.86, 128.86, 128.17, 125.71, 125.71, 120.37, 103.12, 95.54, 80.72,
79.65, 78.82, 78.40, 78.00, 72.77, 70.90, 68.94, 67.99, 65.55, 65.50, 62.96, 62.01, 50.33, 49.45, 47.92,
44.11, 40.36, 39.73, 36.68, 35.06, 34.82, 34.50, 29.71, 28.86, 25.62, 21.62, 21.37, 20.85, 18.17, 13.38,
+
10.93; HRMS (ESI) m/z calcd for C H N O [M + H] : 862.5463, found: 862.5537.
4
5
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According to the synthetic procedure described above, the following compounds (23b-23l) were
also prepared:
24

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5.1.2.6. Compound (23b)
White solid, yield 80%, mp: 120–122 °C, TLC R = 0.40 (DCM/CH OH/NH ·H O = 10:1:0.1); H
1
f
3
3
2
NMR (600 MHz, CDCl , δ ppm): 7.84 (d, J = 9.3 Hz, 3H), 7.45 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.4 Hz,
3
1H), 4.90 (d, J = 4.5 Hz, 1H), 4.52 – 4.42 (m, 3H), 4.35 (dd, J = 13.9, 6.3 Hz, 1H), 4.10 – 4.00 (m,
2H), 3.89 (dd, J = 11.9, 4.0 Hz, 1H), 3.77 (d, J = 7.1 Hz, 1H), 3.56 – 3.45 (m, 3H), 3.32 (s, 3H), 3.27
– 3.17 (m, 4H), 3.05 (t, J = 9.7 Hz, 1H), 2.94 – 2.87 (m, 1H), 2.68 (dd, J = 12.2, 5.8 Hz, 1H), 2.48 (s,
3H), 2.45 – 2.18 (m, 5H), 2.32 (s, 6H), 2.08 – 1.83 (m, 3H), 1.79 – 1.74 (m, 1H), 1.59 (dd, J = 15.2,
4.9 Hz, 2H), 1.36 (s, 3H), 1.31 (d, J = 6.2 Hz, 3H), 1.24 – 1.21 (m, 7H), 1.19 (d, J = 7.3 Hz, 3H), 1.10
13
(d, J = 7.4 Hz, 3H), 0.92 (dd, J = 15.4, 6.6 Hz, 6H); C NMR (150 MHz, CDCl , δ ppm): 176.09,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
147.77, 130.52, 128.86, 128.86, 128.19, 125.72, 125.72, 120.45, 102.90, 95.89, 80.48, 79.49, 79.00,
78.17, 78.00, 72.77, 71.05, 68.75, 65.68, 65.42, 62.58, 62.02, 50.60, 49.45, 44.67, 41.25, 40.38, 36.24,
35.02, 31.52, 29.05, 21.59, 21.43, 21.00, 18.36, 14.35, 10.17; HRMS (ESI) m/z calcd for C H N O
45
75
5
11
+
[M + H] : 862.5463, found: 862.5610.
1
13
Physical characteristics, H NMR, C NMR, MS and HRMS for other target compounds of series
B, were reported in the supporting information.
5.1.3. Synthesis of 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin derivatives
(Series C)
5.1.3.1. 4-(bromomethyl)-1-phenyl-1H-1, 2, 3-triazole (44)
To a solution of aniline 39 (2 g, 21.49 mmol) in H O (30 mL) was added sequentially
2
o
concentrated HCl (9 mL, 108 mmol), NaNO (1.78g, 25.80 mmol) at 0 C. The reaction mixture was
2
o
stirred at 0 C for 0.5 h. A solution of NaN (2.37 g, 36.46 mmol) in H O (10 mL) was added
3
2
o
o
dropwise to the mixture at 0 C. This reaction mixture was stirred at 0 C for 0.5 h and stirred at room
temperature for 4 h. Then DCM (50 mL) was added, the organic phase was separated and aqueous
layer was extracted with DCM (30 mL×2). The combined organic layers were concentrated to afford
azide intermediate 40 (2.48g, 97%) as a reddish brown oil.
To a solution of azide 40 (2.48 g, 20.83 mmol) in 75% CH OH (20 mL) was added sequentially
3
methyl propiolate 41 (1.76 g, 20.95 mmol), CuSO (235 mg), and L-ascorbic acid sodium salt (705
4
o
mg). The reaction mixture was stirred at 35~40 C for 48 h. The mixture was concentrated and the
residue was diluted with DCM (20 mL) and H O (20 mL). The organic phase was separated and
2
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aqueous layer was extracted with DCM (20 mL×2). The combined organic layers were dried over
Na SO . The solvent was removed in vacuo and the residue was purified by silica gel column
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4
(DCM/CH OH = 30:1) to afford methyl ester intermediate 42 (2.09 g, 49%) as a light yellow solid.
3
The methyl ester intermediate 42 (2.09 g, 10.29 mmol) was added slowly to a suspension of
o
LiAlH (1.18 g, 31.09 mmol) in tetrahydrofuran (20 mL) at 0 C. The reaction mixture was stirred at 0
4
o
C for 1 h. The reaction was quenched by adding H O (1.5g) and CH OH (20 mL) slowly, then
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filtered, concentrated to afford the residue. DCM (20 mL) and H O (20 mL) were added, the organic
2
phase was separated and aqueous layer was extracted with DCM (20 mL×2). The combined organic
layers were dried over Na SO . The solvent was concentrated to afford primary alcohol intermediate
2
4
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43 (1.7 g, 94%) as a light yellow solid.
To a solution of primary alcohol intermediate 43 (1.7 g, 9.71 mmol) in anhydrous DCM (20 mL)
was added PBr (7.89 g, 29.13 mmol) at room temperature. The reaction mixture was stirred at room
3
temperature overnight. The reaction mixture was quenched by adding H O (20 mL) slowly, adjusted
2
the pH to 7-8 with NaHCO . The organic phase was separated and aqueous layer was extracted with
3
DCM (20 mL×2). The combined organic layers were dried over Na SO . The solvent was removed in
2
4
vacuo and the residue was purified by silica gel column (PE/EtOAc = 4:1) to afford bromine
intermediate 44 (1.11 g, 48%) as a white solid. Mp: 126–130 °C, TLC R = 0.70 (PE/EtOAc = 2:1);
f
1
H NMR (600 MHz, CDCl , δ ppm): 8.02 (s, 1H), 7.74 – 7.72 (m, 2H), 7.56 – 7.53 (m, 2H), 7.48 –
3
+
7.45 (m, 1H), 4.66 (s, 2H); MS (ESI) m/z calcd for C H BrN [M +2 + H] : 240.0, found: 240.2.
9
8
3
5.1.3.2. (S)-1-amino-3-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)propan-2-ol (34a)
To a solution of (R)-glycidol 45 (187 mg, 2.53 mmol) in anhydrous THF (10 mL) was added 80%
o
o
NaH (316 mg, 10.53 mmol) at 0 C. The reaction mixture was stirred at 0 C for 0.5 h, then a solution
of bromine intermediate 44 (500 mg, 2.11 mmol) in anhydrous THF (10 mL) was added dropwise at 0
o
C under N . The reaction was stirred at room temperature overnight under N . H O (20 mL) and
2
2
2
DCM (20 mL) were added, the organic phase was separated and aqueous layer was extracted with
DCM (20 mL×2). The combined organic layers were concentrated in vacuo and the residue was
purified by silica gel column (PE/EtOAc = 2:1 ~1:1) to afford epoxy 46 (340 mg, 70%) as a white
solid.
To a solution of epoxy 46 (340 mg, 1.47 mmol) in CH CN (2 mL) was added 25% aqueous
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ammonia (15 mL) at room temperature. The reaction was stirred at room temperature for 3 h. DCM
(20 mL) was added, the organic phase was separated and aqueous layer was extracted with DCM (20
mL×3). The combined organic layers were concentrated in vacuo and the residue was purified by
silica gel column (DCM/CH OH/NH ·H O = 10:1:0.1) to afford substituted ethanolamine 34a (260
3
3
2
1
mg, 71%) as a white solid. Mp: 107–109 °C, TLC R = 0.11 (DCM/CH OH/NH ·H O = 10:1:0.1); H
f
3
3
2
NMR (600 MHz, CDCl , δ ppm): 8.01 (s, 1H), 7.74 – 7.73 (m, 2H), 7.54 – 7.52 (m, 2H), 7.46 – 7.44
3
(t, J = 7.5 Hz, 1H), 4.77 (s, 2H), 3.81 (s, 1H), 3.66 – 3.64 (dd, J = 9.6, 3.1 Hz, 1H), 3.59 – 3.56 (m,
+
1H), 2.81 (d, J = 63.1 Hz, 2H); MS (ESI) m/z calcd for C H N O [M + H] : 249.1, found: 249.3.
12
16
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5.1.3.3. Ring-opening intermediate with carboxylic acid (36a)
To a solution of silane protected product 3 (352 mg, 0.32 mmol) in CHCl (20 mL) was added
3
o
o
Pb(OAc) (171mg, 0.39 mmol) at 0 C. The reaction mixture was stirred at 0 C for 0.5 h under N .
4
2
The aldehyde intermediate 4 was prepared and used directly for the next step without purification.
34a (160 mg, 0.64 mmol) and NaBH(OAc) (205 mg, 0.97 mmol) were added to the solution of 4
3
at room temperature. The reaction mixture was stirred at room temperature for 4 h under N , then 37%
2
aqueous formaldehyde solution (157 mg, 1.93 mmol) and NaBH(OAc) (205 mg, 0.97 mmol) were
3
added. The reaction mixture was stirred at room temperature for 4 h. The saturated NaHCO (20 mL)
3
was added and the aqueous phase was extracted with DCM (20 mL×2). The combined organic layers
were dried over Na SO . The solvent was removed in vacuo and the residue was purified by silica gel
2
4
column (DCM/CH OH/NH ·H O = 30:1:0.1) to afford crude product of ester 35a as a colorless oil.
3
3
2
To a solution of ester intermediate 35a in THF-C H OH-H O (20 mL, THF/C H OH/H O = 3:1:1)
2
5
2
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5
2
was added LiOH (39mg, 1.63 mmol) at room temperature. The reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was concentrated in vacuo and the residue was purified by
silica gel column (DCM/CH OH/NH ·H O = 15:1:0.1~10:1:0.1) to afford carboxylic acid 36a (250
3
3
2
mg, 62% in four steps from intermediate 3) as a white solid. Mp: 63–66 °C, TLC R = 0.53
f
1
(DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz, CDCl , δ ppm): 8.06 (s, 1H), 7.76 – 7.75
3
3
2
3
(m, 2H), 7.54 – 7.51 (m, 2H), 7.46 – 7.43 (m, 1H), 4.82 (d, J = 4.7 Hz, 1H), 4.76 (s, 2H), 4.46 (d, J =
7.0 Hz, 1H), 4.28 – 4.25 (m, 1H), 4.22 – 4.21 (m, 1H), 3.95 – 3.94 (m, 1H), 3.74 – 3.72 (m, 1H), 3.67
(dd, J = 9.9, 5.1 Hz, 1H), 3.61 (dd, J = 10.2, 5.2 Hz, 1H), 3.57 (dd, J = 9.8, 6.1 Hz, 1H), 3.31 (s, 3H),
3.26 (d, J = 7.1 Hz, 4H), 3.22 (dd, J = 13.5, 7.6 Hz, 2H), 3.09 (d, J = 10.9 Hz, 1H), 3.01 – 2.97 (m,
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1H), 2.78 (d, J = 12.6 Hz, 1H), 2.67 – 2.64 (m, 1H), 2.60 (s, 3H), 2.50 – 2.46 (m, 3H), 2.36 (d, J =
14.8 Hz, 2H), 2.20 (s, 6H), 1.81 (dd, J = 13.1, 6.8 Hz, 1H), 1.60 – 1.57 (m, 2H), 1.47 (dd, J = 14.9,
4.9 Hz, 1H), 1.30 (s, 3H), 1.28 – 1.27 (m, 1H), 1.22 (d, J = 6.3 Hz, 3H), 1.17 – 1.15 (m, 10H), 1.14 (d,
J = 7.2 Hz, 3H), 1.10 (d, J = 7.4 Hz, 3H), 0.97 – 0.92 (m, 30H), 0.67 – 0.57 (m, 18H); MS (ESI) m/z
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+
calcd for C H N O Si [M + 2H] /2: 626.9, found: 627.3.
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5.1.3.4. 15-membered 11a-azahomoclarithromycin intermediate (37a)
To a solution of intermediate 36a (230 mg, 0.18 mmol) in THF (4 mL) was added sequentially
triethylamine (185 mg, 1.83 mmol), 2, 4, 6-trichlorobenzoyl chloride (135 mg, 0.55 mmol) at room
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temperature. The reaction mixture was stirred at room temperature for 4 h under N . The solution was
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added to a refluxed solution of DMAP (561 mg, 4.55 mmol) in CH CN (40 mL) for 0.5 h. The
3
reaction mixture was concentrated in vacuo and the residue was purified by silica gel column
(PE/acetone = 15:1~10:1) to afford cyclic intermediate 37a (140 mg, 62%) as a white solid. Mp: 62–
1
64 °C, TLC R = 0.44 (PE/acetone = 3:1); H NMR (600 MHz, CDCl , δ ppm): 7.96 (s, 1H), 7.74 (d,
f
3
J = 7.8 Hz, 2H), 7.54 – 7.52 (m, 2H), 7.46 (dd, J = 10.8, 4.1 Hz, 1H), 5.09 (d, J = 4.6 Hz, 1H), 4.85 (d,
J = 4.6 Hz, 1H), 4.75 (dd, J = 19.2, 11.6 Hz, 2H), 4.41 (d, J = 6.8 Hz, 1H), 4.29 – 4.26 (m, 1H), 4.13
(s, 1H), 3.72 (dd, J = 10.2, 5.0 Hz, 1H), 3.66 (d, J = 6.3 Hz, 2H), 3.56 (dd, J = 9.9, 5.8 Hz, 1H), 3.40
(s, 1H), 3.30 (s, 3H), 3.22 – 3.20 (m, 5H), 2.80 (dd, J = 13.7, 7.4 Hz, 1H), 2.72 (s, 1H), 2.47 (d, J =
5.6 Hz, 3H), 2.35 – 2.32 (m, 1H), 2.25 (s, 3H), 2.20 (s, 6H), 2.11 (dd, J = 11.8, 8.3 Hz, 1H), 1.99 (m,
1H), 1.92 – 1.88 (m, 1H), 1.85 – 1.83 (m, 1H), 1.63 – 1.59 (m, 2H), 1.47 (dd, J = 14.9, 4.9 Hz, 1H),
1.30 (s, 3H), 1.25 (s, 1H), 1.23 (d, J = 6.3 Hz, 3H), 1.17 – 1.15 (m, 10H), 1.11 (d, J = 7.3 Hz, 3H),
2+
0.98 – 0.92 (m, 33H), 0.68 – 0.59 (m, 18H); MS (ESI) m/z calcd for C H N O Si [M + 2H] /2:
64
119
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3
617.9, found: 618.4.
5.1.3.5. 15-membered 11a-aza-13-(1, 2, 3-triazoles)homoclarithromycin (38a)
To a solution of cyclic intermediate 37a (120 mg, 0.10 mmol) in THF (5 mL) was added hydrogen
fluoride–pyridine (44 mg, 0.29 mmol, 65%) at room temperature. The reaction mixture was stirred at
room temperature for 18 h. Then the pH of mixture was adjusted to 7-8 with saturated NaHCO (20
3
mL), and the aqueous phase was extracted with DCM (20 mL×3). The combined organic layers were
dried over Na SO . The solvent was removed in vacuo and the residue was purified by silica gel
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column (DCM/CH OH/NH ·H O = 10:1:0.1) to afford compound 38a (50 mg, 58%) as a white solid.
3
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Mp: 99–101 °C, TLC R = 0.26 (DCM/CH OH/NH ·H O = 10:1:0.1); H NMR (600 MHz, CDCl , δ
f
3
3
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ppm): 7.99 (s, 1H), 7.75 – 7.74 (m, 2H), 7.55 – 7.52 (m, 2H), 7.47 – 7.44 (m, 1H), 5.36 – 5.34 (m,
1H), 4.88 (d, J = 4.7 Hz, 1H), 4.75 – 4.69 (m, 2H), 4.41 (d, J = 7.2 Hz, 1H), 4.33 (d, J = 5.3 Hz, 1H),
4.08 – 4.03 (m, 1H), 3.72 (d, J = 7.6 Hz, 1H), 3.64 – 3.57 (m, 3H), 3.50 – 3.47 (m, 1H), 3.29 – 3.24
(m, 8H), 3.00 (d, J = 8.7 Hz, 1H), 2.94 – 2.90 (m, 1H), 2.86 (t, J = 12.4 Hz, 1H), 2.39 (d, J = 6.6 Hz,
5H), 2.34 – 2.18 (m, 11H), 2.06 – 1.94 (m, 2H), 1.78 – 1.70 (m, 2H), 1.46 (dd, J = 15.3, 5.0 Hz, 1H),
1.32 (s, 3H), 1.30 (d, J = 6.3 Hz, 3H), 1.26 – 1.21 (m, 7H), 1.16 (d, J = 7.6 Hz, 3H), 1.06 (d, J = 7.3
13
Hz, 3H), 0.83 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 6.7 Hz, 3H); C NMR (150 MHz, CDCl , δ ppm):
3
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176.48, 145.57, 137.03, 129.82, 129.82, 128.82, 120.82, 120.54, 120.54, 103.34, 96.04, 80.01, 79.54,
78.38, 78.13, 72.70, 71.26, 70.80, 68.65, 68.06, 65.42, 65.09, 64.73, 62.89, 58.45, 50.06, 49.47, 45.33,
44.25, 42.00, 41.59, 40.46, 37.11, 34.95, 32.72, 30.53, 29.97, 27.03, 22.72, 21.60, 21.37, 18.35, 15.24,
+
10.38; HRMS (ESI) m/z calcd for C H N O [M + H] : 892.5569, found: 892.5642.
4
6
77
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According to the synthetic procedure described above, the following compounds (38b-38v) were
also prepared:
5.1.3.6. Compound (38b)
White solid, yield 63%, mp: 174–176 °C, TLC R = 0.26 (DCM/CH OH/NH ·H O = 10:1:0.1); H
1
f
3
3
2
NMR (600 MHz, CDCl , δ ppm): 8.01 (s, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.55 (t, J = 7.9 Hz, 2H), 7.47
3
– 7.44 (m, 1H), 5.01 (s, 1H), 4.89 (s, 1H), 4.74 – 4.67 (m, 2H), 4.44 (d, J = 7.2 Hz, 2H), 4.10 – 4.06
(m, 1H), 3.88 (s, 1H), 3.77 – 3.58 (m, 3H), 3.52 – 3.42 (m, 2H), 3.39 – 3.12 (m, 8H), 3.04 – 2.96 (m,
2H), 2.89 (s, 1H), 2.71 – 2.21 (m, 14H), 2.18 – 2.10 (m, 2H), 2.00 – 1.91 (m, 2H), 1.84 – 1.80 (m,
1H), 1.67 – 1.65 (m, 1H), 1.50 (d, J = 10.8 Hz, 1H), 1.35 – 1.22 (m, 13H), 1.17 (d, J = 7.8 Hz, 3H),
13
1.09 (d, J = 5.5 Hz, 3H), 0.97 – 0.83 (m, 6H); C NMR (150 MHz, CDCl , δ ppm): 176.48, 145.57,
3
137.03, 129.82, 129.82, 128.82, 120.82, 120.54, 120.54, 103.34, 96.04, 80.01, 79.54, 78.38, 78.13,
72.70, 71.26, 70.80, 68.65, 68.06, 65.42, 65.09, 64.73, 62.89, 58.45, 50.06, 49.47, 45.33, 44.25, 42.00,
41.59, 40.46, 37.11, 34.95, 32.72, 30.53, 29.97, 27.03, 22.72, 21.60, 21.37, 18.35, 15.24, 10.38.
+
HRMS (ESI) m/z calcd for C H N O [M + H] : 892.5569, found: 892.5632.
4
6
77
5
12
1
13
Physical characteristics, H NMR, C NMR, MS and HRMS for other target compounds of series
C, were reported in the supporting information.
29