Synthesis and antibacterial activity of novel 10,11-epoxy acylide erythromycin derivatives

Article abstract of DOI:10.1016/j.cclet.2013.02.015

A series of novel acylide derivatives have been synthesized from clarithromycin A via a facile procedure. The C-3 modifications involved replacing the natural C-3 cladinosyl group in clarithromycin core with different aryl-piperzine sidechain via chemical synthesis. Meanwhile a distinctive intermediate with 10,11-epoxy moiety was obtained. The structure and stereochemistry of this novel structure were confirmed via NMR and X-ray crystallography. Potential anti-bacterial activities against both Gram-positive and Gram-negative bacteria were reported. Because of existence of C10,11-epoxide, these derivatives can be used as intermediates for further structural modification.

Full text of DOI:10.1016/j.cclet.2013.02.015

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Chinese Chemical Letters xxx (2013) xxx–xxx
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and antibacterial activity of novel 10,11-epoxy acylide erythromycin
derivatives
Ying Nie a, Yin Sun b, Qi-Dong You b,
*
a Experimental Center of Pharmaceutical Science, China Pharmaceutical University, Nanjing 210009, China
b Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of novel acylide derivatives have been synthesized from clarithromycin A via a facile procedure.
The C-3 modifications involved replacing the natural C-3 cladinosyl group in clarithromycin core with
different aryl-piperzine sidechain via chemical synthesis. Meanwhile a distinctive intermediate with
10,11-epoxy moiety was obtained. The structure and stereochemistry of this novel structure were
confirmed via NMR and X-ray crystallography. Potential anti-bacterial activities against both Gram-
positive and Gram-negative bacteria were reported. Because of existence of C10,11-epoxide, these
derivatives can be used as intermediates for further structural modification.
Received 21 January 2013
Received in revised form 3 February 2013
Accepted 4 February 2013
Available online xxx
Keywords:
Epoxide acylide erythromycin derivatives
Synthesis
ß 2013 Qi-Dong You. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Single-crystal X-ray diffraction
Resistance antibacterial activity
1. Introduction
C3-cladinosyl group of erythromycin A is replaced by an O-acyl
group, are reported to be active against many bacterial respiratory
Macrolide antibiotics, including erythromycin and clarithro-
mycin, are a class of preferred drug class for the safe and effective
treatment of respiratory tract infections. They are potent against
several key respiratory pathogens including Staphylococcus aur-
eaus, Streptococcus pnuemoniae, etc. The 14-membered macrolide
antibiotic erythromycin is unstable under acidic condition [1,2].
Therefore, the second-generation macrolides have been developed,
such as clarithromycin, azithromycin, and roxithromycin. Over the
past decade the resistance to these second-generation marolides
has emerged. There are two main mechanisms of macrolide
resistance. One mechanism is target site modification via
methylation of the ribosomal target in bacteria. This type of
resistance is referred to as the MLSB phenotype [3,4]. The other
mechanism is antibiotic efflux caused by two classes of pumps,
ATP-binding-cassette (ABC) transporter superfamily and the major
facilitator superfamily (MFS) [3]. To overcome the resistance much
attention has been focused on the modification of C-3, C-6, C-9, C-
11 and C-12.
pathogens, including erythromycin-resistant strains [5].
The acylides, such as FMA0122 1 and TEA0929 2, exhibit
markedly enhanced activity against MLSB-resistant strains [6]. U.S.
Patent No. 2002/0103140 describes a new class of 6-O-alykl-2-nor-
2-sustituted ketolide derivatives [6]. In this invention the
compound example 3 demonstrates even better antibacterial
activity against Streptococcus pneumoniae than Cethromycin (ABT-
773), in which 2-methyl is replaced by sec-l,2-diols. The novel
analogs modified with C-2 position exhibit improved aqueous
solubility. As reported in the patent, the compound example 3 is
prepared through epoxidation of the double bond at C-2 position.
N
O
N
O
O
N
O
N
O
O
HO
O
OCH3
O
HO
O
HO
O
O
H
N
H
N
O
O
O
O
O
O
O
O
O
Ketolides which are developed in recent structural studies,
including Telithromycin and Cethromycin (ABT-773), represent
improved activity against erythromycin- and penicillin-resistant
isolates of Streptococcus pneumoniae. In addition, acylides, in which
O
O
O
O
O
O
O
O
N
OH
N
O
OH
O
2
1
3
To address the bacterial resistance and solubility problems, we
wish to produce a series of 3-O-acyl erythromycin derivatives with
well hydrophilicity. We have obtained a series of novel acylides in
which the aryl-piperzine sidechain is attached to the macrolide
* Corresponding author.
E-mail address: youqidong@gmail.com (Q.-D. You).
1001-8417/$ – see front matter ß 2013 Qi-Dong You. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.02.015
Please cite this article in press as: Y. Nie, et al., Synthesis and antibacterial activity of novel 10,11-epoxy acylide erythromycin
derivatives, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.02.015

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CCLET-2481; No. of Pages 3
2
Y. Nie et al. / Chinese Chemical Letters xxx (2013) xxx–xxx
core via
a C3-carbamate linkage with 10,11-epoxy moiety
simultaneously.
2. Experimental
The synthesis route of the target compounds was outlined in
Scheme 1. The synthesis of 14 started with commercially available
clarithromycin. Clarithromycin was reacted with acetic anhydride
in methylene chloride with the presence of pyridine and 4-
dimethylamino pyridine (DMAP) to give 20,400-diacetyl erythromy-
cin A derivative 4. Compound 4 was treated by bis(trichlor-
omethanol)carbonate (BTC) under base condition (pyridine) in
methylene chloride to produce 11,12-cabamate compound 5.
Treatment of 5 with 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) in
acetone yielded the corresponding enone 6 at room temperature
and under refluxing. Compound 6 was protected by a carboben-
zyloxy group (Cbz) to afford 30-N-benzyloxycarbonyl erythromycin
A derivative 7. Compound 7 was converted to the target C-3
desugarized compound 8 by diluted hydrochloric acid to remove
C3-cladinosyl group. Treatment of compound 8 with meta-
chloroperoxybenzoic acid resulted in epoxidation of the C10,11
alkene. The product isolated was the C10,11-epoxide 9.
The NMR spectra were in accordance with the C10,11-epoxide
acylide structure 9. The epoxy formation was supported by a singlet
at 2.90 ppm in the 1H NMR spectrum, and the carbamate signal at
212 ppm in the 13C NMR spectrum due to the C-9 carbonyl proton.
Meanwhile, thesignalcoupledtotheC10,11 doublebond at 6.41 ppm
in 1H NMR experiment disappeared. MS and NMR data indicate that
compound 9 is C10,11-epoxide, and the stereo configuration of the
two chiral carbons in the cyclic formation are 10R, 11S based on the
evidence in Fig. 1 from the X-ray crystallography.
Removal of the benzyloxycarbonyl and acetyl group of
compound 9 by catalytic hydrogenation furnished N-demethyl
erythromycin A derivative 10. Compound 10 underwent reductive
N-demethylation in ethanol with formaldehyde in the presence of
formic acid under mild reflux to afford compound 11.
Acetylation of 11 with acetic anhydride as before gave 12.
Alcohol 12 was reacted with acryloyl chloride, Et3N and DMAP in
CH2Cl2 at 0 8C to afford acrylate ester 13 [7].
Finally, the resulting acrylate ester 13 was reacted with phenyl
piperazine derivatives, and was removed the acetyl group to give
Fig. 1. The single-crystal X-ray diffraction data of compound 9: (A) Crystal packing
of compound 9; (B) Molecule structure of compound 9 without hydrogen.
N
AcO
N
O
N
N
HO
AcO
O
AcO
O
O
OCH3
OCH3
OCH3
OCH3
HO
HO
HO
HO
O
a
O
O
O
O
O
O
OCH3
b
O
O
OCH3
O
c
OCH3
OAc
O
OCH3
HO
HO
O
OH
OAc
OAc
O
O
O
O
O
O
O
O
O
O
O
O
O
4
O
1
O
6
O
5
COOCH2Ph
COOCH2Ph
f
COOCH2Ph
g
AcO
OCH3
NH
O
N
AcO
OCH3
N
AcO
OCH3
N
O
O
O
O
HO
OCH3
O
e
O
OCH3
O
O
O
O
d
O
HO
O
O
O
O
HO
HO
OAc
O
O
OH
O
OH
OH
O
O
O
O
O
7
10
9
8
R
N
HO
OCH3
N
14a phenyl
O
AcO
N
O
N
O
O
AcO
OCH3
O
HO
OCH3
O
14b p-methoxyphenyl
14c p-methylphenyl
14d -trifluoromethylphenyl
14e o-fluorophenyl
14f o-methylphenyl
14g o-methoxyphenyl
14h 2-pyridine
OCH3
O
O
O
O
h
O
O
k
HO
O
j
O
HO
O
HO
i
HO
O
N
N R
O
O
O
OH
O
O
OH
O
O
O
O
O
O
14i 2,4-dimethylphenyl
14j 2,5-dimethylphenyl
14a-j
13
12
11
Scheme 1. Reagents and conditions: (a) Ac2O, pyridine, DMAP, CH2Cl2, r.t. for 5 h; (b) BTC, pyridine, CH2Cl2, 0 8C for 4 h, r.t. for 10 h; (c) DBU, acetone, reflux, 8 h; (d) benzyl
chloroformate, NaHCO3, 1,4-dioxane, 55 8C, 3 h; (e) HCl, C2H5OH, 40 8C, 2 d; (f) mCPBA, CH2Cl2, 30 8C, 5 h; (g) H2, Pd-C, HAc, C2H5OH, H2O, N.T.P., 2 d; (h) HCHO, HCOOH,
C2H5OH, reflux, 3 h; (i) Ac2O, TEA, CH2Cl2, r.t. for 5 h; (j) acryloyl chloride, Et3N, DMAP, CH2Cl2, r.t. for 6 h; (k) (i) CH2Cl2, reflux, 3 d, (ii) CH3OH, reflux, 2 d.
Please cite this article in press as: Y. Nie, et al., Synthesis and antibacterial activity of novel 10,11-epoxy acylide erythromycin
derivatives, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.02.015

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Y. Nie et al. / Chinese Chemical Letters xxx (2013) xxx–xxx
3
Table 1
In vitro antibacterial activity of C10,11-epoxide acylide derivatives.
Organism
MIC (mg/mL)
14d
14f
128
14i
64
14j
128
Azithromycin
Clarithromycin
Erythromycin
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus aureus MRSA
Staphylococcus epidermidis MRSE
Escherichia coli
32
64
0.25
0.03125
128
4
4
>128
2
128
128
128
128
64
64
64
64
64
128
128
64
32
64
64
32
16
32
128
128
128
>128
>128
4
>128
128
64
2
32
>128
128
128
16
Salmonella typhi
128
128
>128
>128
>128
2
16
Klebsiella pneumonia
128
64
>128
64
2
32
Hemophilus influenzae
Escherichia coli ESBL
0.03125
1
128
>128
>128
>128
32
8
>128
>128
>128
>128
Klebsiella pneumonia ESBL
[4] J.C. Pechere, Macrolide resistance mechanisms in Gram-positive cocci, Int. J.
Antimicrob. Agents 18 (2001) S25–S28.
[5] T. Tanikawa, T. Asaka, M. Kashimura, et al., Synthesis and antibacterial activity of a
novel series of acylides: 3-O-(3-pyridyl) acetylerythromycin A derivatives, J. Med.
Chem. 46 (2003) 2706–2715.
target compounds 14a–j. The structures of the target compounds
synthesized herein were fully confirmed by melting points,
1HNMR, IR, ESI-MS, and elemental analysis [8].
[6] T. Ly, S. Yat, M. Zhenkun, 6-O-alkyl-2-nor-2-substituted ketolide derivatives, U.S.
Patent. 0103140 (2002).
3. Results and discussion
[7] A.K. Ghosh, K. Jae-Hun, An enantioselective synthesis of the C1 C9 segment of
antitumor macrolide peloruside A, Tetrahedron Lett. 44 (2003) 3967–3969.
[8] Selected characteristic data for the compounds. 9: Yellow powder; mp 91–93 8C, IR
(KBr, cmꢀ1): y 1706, 1729, 1748; 1H NMR (300 MHz, CDCl3): d 7.28–7.40 (m, 5H,
Ph-H), 5.03 (dd, 1 H, J = 2.3 Hz and 9.2 Hz, H13), 4.69 (dd, 1 H, J = 2.3 Hz and 8.2 Hz,
The 10,11-epoxy acylide erythromycin derivatives and the
reference compounds, clarithromycin and azithromycin were tested
against different representative pathogens. Various pathogens were
tested in order to identify the potency of these acylide analogs.
The in vitro antibacterial activity is reported as minimum
inhibitory concentrations (MICs). As shown in Table 1, most of
these compounds are as effective as erythromycin to the Gram-
positive bacteria. Meanwhile, several compounds displayed
improved activity to Gram-negative bacteria. To readily interpret
the structure–activity relationships of the C-3 position substituent,
the C6-carbamoyl side chain was initially held constant, while
various C3-carbamates were examined. Basic groups were intro-
duced in these C3-carbamate derivatives to increase their water
solubility. However, attempts to improve activity by structural
modification of acylides with aryl-piperzine sidechains shortening
or lengthening the pyridylalkyl chain, as in compounds aryl-
piperzine sidechain had a detrimental effect against either Gram-
positive bacteria or Gram-negative bacteria compared to clari-
thronycin, telithromycin, and azithromycin.
0
0
H
2 ), 3.10 (s, 3H, C6-OCH3), 1.88 (s, 3H, C2 -OCOCH3), 1.66 (s, 3H, C10-CH3); ESI-MS
m/z (%): 772 (M+Na+). X-ray analysis: C39H59NO13
,
Mr = 749.90, crystal size
0.586 mm ꢁ 0.173 mm ꢁ 0.049 mm, orthorhombic in p21/n with a = 14.024(3),
3
˚
˚
b = 28.998(6), c = 10.915(2) A, a, b, g = 908, V = 3185.0(9) A , Dcalu = 1.151
Mg/m3, and Z = 4, absorption coefficient 0.087 mmꢀ1, computing structure solu-
tion SHELXL-97, theta range for data collection 1.61 to 25.508, limiting indices
ꢀ16 ꢂ h ꢂ 10, ꢀ35 ꢂ k ꢂ 19, ꢀ13 ꢂ l ꢂ 12, reflection collected 23690, refinement
method full-matrix least-squares, final R indices [I > 2 sigma (I)] R1 = 0.1016,
wR2 = 0.2461, R indices (all data) R1 = 0.2601, wR2 = 0.3102, goodness-of-fit on
F2 0.894, largest difference peak 0.360 eA , largest difference hole ꢀ0.236 eA
ꢀ3
ꢀ3
.
˚
˚
14a: White crystal; mp 127–129 8C, IR (KBr, cmꢀ1): y 1165, 1706, 1741; 1H NMR
(300 MHz, CDCl3): d 7.09–7.28 (m, 5H, Ph-H), 5.01 (dd, 1 H, J = 2.4 Hz and 10.4 Hz,
H13), 3.23 (s, 3H, C6-OCH3), 3.18 (m, 4H, 2ꢁCH2 in piperazinyl), 2.65 (m, 6H, 2ꢁCH2
in piperazinyl and CH2N), 2.39 (s, 6H, N(CH3)2), 1.64 (s, 3H, C10-OCH3); ESI-MS m/z
(%): 803 (M+H+). 14b: White crystal; mp 117–119 8C, IR (KBr, cmꢀ1): y 1164, 1512,
1741; 1H NMR (300 MHz, CDCl3): d 6.81–7.27 (m, 4H, Ph-H), 5.01 (dd, 1 H, J = 2.3 Hz
and 10.3 Hz, H13), 3.76 (s, 3H, Ar-OCH3), 3.12 (s, 3H, C6-OCH3), 3.07 (m, 4H, 2ꢁCH2
in piperazinyl), 2.68 (m, 6H, 2ꢁCH2 in piperazinyl and CH2N), 2.46 (s, 6H, N(CH3)2),
1.62 (s, 3H, C10-CH3); ESI-MS m/z (%): 833 (M+H+). 14c: White crystal; mp 105-
106 8C, IR (KBr, cmꢀ1): y 1164, 1741; 1H NMR (300 MHz, CDCl3): d 6.81–7.27 (m,
4H, Ph-H), 5.01 (dd, 1 H, J = 2.7 Hz and 10.5 Hz, H13), 3.22 (s, 3H, C6-OCH3), 3.15 (m,
4H, 2ꢁCH2 in piperazinyl), 2.63 (m, 6H, 2ꢁCH2 in piperazinyl and CH2N), 2.38 (s,
6H, N(CH3)2), 2.17 (s, 3H, Ar-CH3), 1.65 (s, 3H, C10-OCH3); ESI-MS m/z (%): 817
(M+H+). 14d: White crystal; mp 112–113 8C, IR (KBr, cmꢀ1): y 1164, 1742; 1H NMR
(300 MHz, CDCl3): d 7.03–7.37 (m, 4H, Ph-H), 5.01 (dd, 1 H, J = 6.5 Hz and 14.0 Hz,
4. Conclusion
H
13), 3.24 (s, 3H, C6-OCH3), 3.11 (m, 4H, 2ꢁCH2 in piperazinyl), 2.66 (m, 6H, 2ꢁCH2
In summary, we have discovered a novel series of acylide
antibiotics that employ a C-3 carbamate for attachment of the
arylpiperzine sidechain with 10,11-epoxy moiety. Meanwhile, we
synthesized a unique 10,11-epoxy acylide skeleton structure, and
verified its spatial configuration through X-ray crystallography.
Compounds with various carbamate groups at the C-3 position
were studied for their antibacterial activity. Part of these
compounds showed improved activity against Gram-negative
bacteria compared with erythromycin and clathromycin.
in piperazinyl and CH2N), 2.40 (s, 6H, N(CH3)2), 1.65 (s, 3H, C10-OCH3); ESI-MS m/z
(%): 871 (M+H+). 14e: White crystal; mp 114–116 8C, IR (KBr, cmꢀ1): y 1164, 1742;
1H NMR (300 MHz, CDCl3): d 6.90–7.45 (m, 4H, Ph-H), 5.00 (dd, 1 H, J = 2.4 Hz and
10.3 Hz, H13), 3.23 (s, 3H, C6-OCH3), 3.14 (m, 4H, 2ꢁCH2 in piperazinyl), 2.82 (m,
6H, 2ꢁCH2 in piperazinyl and CH2N), 2.29 (s, 6H, N(CH3)2), 1.64 (s, 3H, C10-OCH3);
ESI-MS m/z (%): 821 (M+H+). 14f: White crystal; mp 124–125 8C, IR (KBr, cmꢀ1): y
1163, 1742; 1H NMR (300 MHz, CDCl3): d 6.95–7.18 (m, 4H, Ph-H), 5.00 (dd, 1 H,
J = 2.2 Hz and 10.2 Hz, H13), 3.24 (s, 3H, C6-OCH3), 3.19 (m, 4H, 2ꢁCH2 in piper-
azinyl), 2.83 (m, 6H, 2ꢁCH2 in piperazinyl and CH2N), 2.29 (s, 9H, Ph-CH3 and
N(CH3)2), 1.63 (s, 3H, C10-OCH3); ESI-MS m/z (%): 817 (M+H+). 14g: White crystal;
mp 134–136 8C, IR (KBr, cmꢀ1): y 1164, 1741; 1H NMR (300 MHz, CDCl3): d 6.87–
7.02 (m, 4H, Ph-H), 5.00 (dd, 1 H, J = 2.7 Hz and 10.1 Hz, H13), 3.86 (s, 3H, Ph-OCH3),
3.23 (s, 3H, C6-OCH3), 3.15 (m, 4H, 2ꢁCH2 in piperazinyl), 2.69 (m, 6H, 2ꢁCH2 in
piperazinyl and CH2N), 2.31 (s, 6H, N(CH3)2), 1.64 (s, 3H, C10-OCH3); ESI-MS m/z
(%): 833 (M+H+). 14h: White crystal; mp 99–107 8C, IR (KBr, cmꢀ1): y 1165, 1741;
1H NMR (300 MHz, CDCl3): d 8.18 (d, 1 H, Pyr-H6), 7.47 (t, 1 H, Pyr-H4), 6.63 (t, 2H,
Pyr-H3, Pyr-H5), 5.00 (dd, 1 H, J = 3.7 Hz and 8.9 Hz, H13), 3.22 (s, 3H, C6-OCH3),
3.18 (m, 4H, 4H, 2ꢁCH2 in piperazinyl), 2.61 (m, 6H, 2ꢁCH2 in piperazinyl and
CH2N), 2.41 (s, 6H, N(CH3)2), 1.65 (s, 3H, C10-OCH3); ESI-MS m/z (%): 842 (M+H+).
14i: White crystal; mp 100–102 8C, IR (KBr, cmꢀ1): y 1164, 1742; 1H NMR
Acknowledgments
This work was supported by Jiangsu Hengrui Pharmaceutical
Company. The X-ray crystallography was performed at Shanghai
Institute of Organic Chemistry, China Academy of Science.
References
(300 MHz, CDCl3): d 6.90–6.99 (m, 3H, Ph-H), 5.00 (dd,
1 H, J = 2.7 Hz and
10.1 Hz, H13), 3.23 (s, 3H, C6-OCH3), 3.11 (m, 4H, 2ꢁCH2 in piperazinyl), 2.66
(m, 6H, 2ꢁCH2 in piperazinyl and CH2N), 2.26 (s, 6H, N(CH3)2), 2.25 (s, 3H, Ph-CH3),
2.22 (s, 3H, Ph-CH3), 1.65 (s, 3H, C10-OCH3); ESI-MS m/z (%): 832 (M+H+). 14j:
White crystal; mp 114–115 8C, IR (KBr, cmꢀ1): y 1165, 1741; 1H NMR (300 MHz,
CDCl3): d 6.80–7.07 (m, 3H, Ph-H), 5.00 (dd, 1 H, J = 2.7 Hz and 10.1 Hz, H13), 3.22 (s,
3H, C6-OCH3), 3.14 (m, 4H, 2ꢁCH2 in piperazinyl), 2.65 (m, 6H, 2ꢁCH2 in piper-
azinyl and CH2N), 2.31 (s, 6H, N(CH3)2), 2.24 (s, 3H, Ph-CH3), 2.21 (s, 3H, Ph-CH3),
1.65 (s, 3H, C10-OCH3); ESI-MS m/z (%): 832 (M+H+).
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hemiketal of A, J. Antibiot. 26 (1973) 569–574.
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Please cite this article in press as: Y. Nie, et al., Synthesis and antibacterial activity of novel 10,11-epoxy acylide erythromycin
derivatives, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.02.015