Design, synthesis and in vivo activity of 9-(S)-dihydroerythromycin derivatives as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2006.08.076

The synthesis of a new class of 9-(S)-dihydroerythromycin derivatives and their anti-inflammatory activity on in vivo PMA assay are described. Modifying the desosamine sugar on the C-3′ amino group, it was possible to differentiate between anti-biotic and

Full text of DOI:10.1016/j.bmcl.2006.08.076

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5801–5804
Design, synthesis and in vivo activity of 9-(S)-dihydroerythromycin
derivatives as potent anti-inflammatory agents
A. Mereu,a,* E. Moriggi,a M. Napoletano,a C. Regazzoni,a S. Manfredini,b
T. P. Mercuriob and F. Pellacinia
aInpharzam Ricerche S. A.; Via ai So¨i, BP 328, 6807 Taverne, Switzerland
bUniversity of Ferrara, Department of Pharmaceutical Sciences, Ferrara, Italy
Received 25 July 2006; revised 15 August 2006; accepted 15 August 2006
Available online 18 September 2006
Abstract—The synthesis of a new class of 9-(S)-dihydroerythromycin derivatives and their anti-inflammatory activity on in vivo
PMA assay are described. Modifying the desosamine sugar on the C-30 amino group, it was possible to differentiate between
anti-biotic and anti-flammatory action. The compounds are completely devoid of anti-microbial effects but their anti-inflammatory
properties are enhanced. These results strongly suggest the potential of macrolides as a new class of anti-inflammatory agents.
Ó 2006 Elsevier Ltd. All rights reserved.
Macrolide anti-biotics have been used effectively and
safely for the treatment of respiratory tract infections
for more than 40 years. The first macrolide, erythromy-
cin A (Scheme 1), was introduced in the 1950s and has
enjoyed widespread clinical use especially in patients
with allergic reactions to penicillin.1
on the inflammatory cascade and avoid anti-microbial
activity.
In this report, we describe the preliminary results
regarding a study aimed at the design and synthesis of
a new class of erythromycin derivatives provides with
potent in vivo anti-inflammatory properties. Several
compounds were prepared and tested in an iterative
approach in order to enhance the anti-inflammatory
activity but at the same time eradicating the anti-bacte-
rial effect.
There are growing evidences that macrolide anti-biotics
may have beneficial effects in chronic inflammatory air-
way diseases such as asthma, diffuse panbronchiolitis
(DPB) and chronic sinusitis that are independent of
their anti-bacterial effects.2–4 However, the anti-inflam-
matory activities seem to be limited to the 14-membered
ring macrolides like erythromycin and clarithromycin,
and 15-membered ring macrolides like azithromycin,
but are not shared by 16-membered ring macrolide like
josamycin.5
During this study, we have found that erythromycin
anti-bacterial activity can be reduced by structural
modifications at different functional groups:
(1) C-30 dimethylamino modification,
(2) Cladinose removal,6
(3) C-9 carbonyl modification.
Great caution must be used for administering anti-mi-
crobial drugs as anti-inflammatory agents for chronic
treatment in order to avoid selecting microbial resis-
tance. A better approach would involve chemical modi-
fication of the basic structure in order to enhance effects
The dimethyl amino group is directly involved in the
anti-bacterial mechanism and its modification drastical-
ly reduces this effect.7
One of the major problems of erythromycin is its insta-
bility in the acidic environment of the stomach. The acid
degradation products generated by intramolecular hem-
iketal formation between the hydroxyls at C-6 and C-12
with the C-9 carbonyl are responsible for its poor bioav-
ailibility and gastrointestinal (GI) side effects.8
Keywords: Macrolide; Erythromycin; Erythronoid; Anti-inflammato-
ry, amide; Amine; Desosamine; Cladinose; PMA induced ear edema;
CD1 mice.
*
Corresponding author. Tel.: +41 919354520; fax: +41 919354512;
e-mail: andrea.mereu@zambongroup.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.076

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A. Mereu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5801–5804
Scheme 1. Reagents and conditions: (a) NaBH4 (1 equiv), 5% H2O/THF, 0 °C, 1 h, 72%, 95:5 isomer ratio; (b) DEAD (1 equiv), CH2Cl2, 16 h, 98%.
Among all the possible C-9 carbonyl modifications in
erythromycins, we selected the reduction to secondary
alcohol.9 C-9 carbonyl reduction increased metabolic
stability and played an important role in reducing the
anti-bacterial effect, which was completely eliminated
by the concomitant modification of the dimethylamino
group.7
ethyl-alcohol 3 and it was chosen for its synthesis
(Scheme 1).
Reductive amination of desmethyldihydroerythromycin
3 with benzaldehyde gave product 8 (Scheme 2). Similar-
ly intermediate 5 was obtained reacting 2 with an alloc-
protected thiazole-diamino-aldehyde 4. Alloc removal
under standard conditions13 gave product 6, which
was further treated in acid media to remove cladinose
sugar, yielding the cladinose-free analogue 7 (Scheme
2). Acid promoted cladinose removal gave a class of ery-
thronoids that were found to be inactive in many anti-
bacterial tests even without further structural
modifications.6
A study on the diastereoselective reduction of C-9 car-
boxyl group was performed. The diastereoselection
was induced by the presence of 18 stereogenic centers
in erythromycin and its 3D-conformation. The highest
isomer ratio was obtained using polar, aprotic solvents
such as THF, rather than protic solvents such as MeOH
or H2O. On the other hand, the global yield was quan-
titative in protic solvents but much lower in THF. The
best condition consisted of using a 5% H2O/THF solu-
tion at 0 °C to give the 9-(S)-dihydroerythromycin 2 in
good yield and isomer ratio (Scheme 1).
Macrolide 10 and its cladinose-free analogue 11 have
been prepared from 3 by a three-step procedure (Scheme
2). Michael addition of 3 to refluxing acrylonitrile, fol-
lowed by catalytic hydrogenation gave amine 9. This lat-
ter was in turn subjected to reductive amination with
thiazolecarboxaldehyde to give product 10 in satisfacto-
ry yield (Scheme 3).
Functionalization of the desosamine sugar by introduc-
tion of a C-30 substituent was envisaged as a suitable
modification. The amino group was demethylated fol-
lowing two protocols. The first one, an Iodine-promoted
demethylation in MeOH, could be performed either
refluxing10 or irradiating with a 400 W lamp11 bringing
to 50–60% of isolated yield. The second one, a DEAD
mediated reaction,12 gave quantitative yield of the desm-
We finally devised another class of compounds featuring
a substituent connected to the desosamine sugar
through an amide bond in C-30 position. This modifica-
tion completely suppressed the unwanted anti-bacterial
effect.
Scheme 2. Reagents and conditions: (a) benzaldehyde (1.1 equiv), Me4NBH(OAc)3 (2 equiv), AcOH (1.5 equiv), dichloroethane, 67%; (b) 4 (1 equiv),
Me4NBH(OAc)3 (2 equiv), AcOH (1.5 equiv), dichloroethane, 55%; (c) pyrrolidine (2.5 equiv), tetrakis(triphenylphospin)palladium (0.05 equiv),
CHCl3, 2 h, 56%; (d) HCl 2 N in MeOH, 50–80%; (e) acrylonitrile, reflux, 6 h; 60%; (f) H2, 5 atm, 5% Pd/C, MeOH, 95%; (g) thiazolecarboxaldehyde
(1 equiv), Me4NBH(OAc)3 (2 equiv), AcOH (1.5 equiv), dichloroethane, 87%.

A. Mereu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5801–5804
5803
Scheme 3. Reagents and conditions: (a) long-chain acid (1.5 equiv), DCC-resin (2 equiv), dichloroethane, 40 °C, 16 h, 50–60%; (b) pyrrolidine (2
equiv), tetrakis(triphenylphospine)palladium (0.05 equiv), CH2Cl2, 60–80%; (c) acetyl chloride (1 equiv), NEt3 (1.2 equiv), CH2Cl2, 1 h, 90%; (d) HCl
1 N in MeOH, 90%; (e) 2-Cl-acetyl (1.1 equiv), DCC-resin (2 equiv), CH2Cl2, 40 h, 66%; (f) dimethylaminoethylenamine or benzylamine (1.1 equiv)
THF, 40 °C, 1 h, 75–80%.
Amide 16 was prepared from 3 by simple acetylation.14
DCC-resin supported coupling of 3 with the corre-
Table 1. Inhibition of ear edema in CD1 mice after topically
application (500 lg/ear) of erythromycin derivatives17
Macrolide derivatives Subclass Cladinose Inhibitiona SE
sponding aromatic long-chain acids gave the alloc-pro-
code no.
(%)
( )b
tected amines that, after Pd° catalyzed deprotections,13
yielded compounds 14 and 15. Starting from the chloro
acetamide intermediate 18, compounds 19 and 20 were
synthesized by nucleophilic substitution with the corre-
sponding amines. Cladinose-free macrolides 17 and 21
were prepared reacting compounds 16 and 19 in metha-
nolic HCl solution.
Erythromycin
Amine
Amine
Amine
Amine
Amine
Amine
Amide
Amide
Amide
Amide
Amide
Amide
Amide
+
+
ꢀ
+
+
ꢀ
+
+
+
ꢀ
+
+
ꢀ
42
43
45
58
74
53
87
77
79
66
18
71
31
6
7
11
5
8
5
10
11
14
15
16
17
19
20
21
2
9
6
5
The prepared macrolides were preliminary screened
through a first level of tests regarding cytotoxicity15
and in vitro ROS inhibition.16 The compounds whose
synthesis are described above passed this first selection
and their anti-inflammatory activity was evaluated in
vivo by topical administration of 0.5 mg/ear on PMA-
induced ear edema in mice using erythromycin as
standard (Table 1).17
5
5
13
6
5
a Values are means of five experiments.
b Standard error.
All the compounds were soluble, nontoxic and, except
for compounds 8 and 10, no effect was observed on sev-
eral bacterial strains.18
The anti-bacterial action was greatly reduced or elimi-
nated as the size of the C-30 substituent increased.
The best results were achieved with the amide subclass,
which never showed toxicity or anti-microbial effect.
Their anti-inflammatory properties were improved rang-
ing from 66% inhibition of cladinose-free acetamide 17
Most of these compounds are as effective as erythromy-
cin (i.e., aminomethylthiazoles 6, 7, and 11 or benzyl-
amine 8). The best anti-inflammatory activity on
amino subclass was obtained by aminomethylthiazole 10.

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A. Mereu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5801–5804
Mohning, K. M.; Bush, E. N.; Wegner, C. D.; Greer, J.
J. Med. Chem. 2004, 47, 1085.
to 86% of the benzylamine 14. Only dimethyl amines 19
and 21 showed a low activity.
11. Lartey, P. A.; Nellans, H. N.; Faghih, R.; Petersen, A.;
Edwards, C. M.; Freiberg, L.; Quigley, S.; Marsh, K.;
Klein, L. L.; Plattner, J. J. J. Med. Chem. 1995, 38, 1793.
12. (a) Smissman, E. E.; Makriyannis, A. J. Org. Chem. 1973,
33, 1652; (b) Denis, A.; Renou, C. Tetrahedron Lett. 2002,
43, 4171.
13. (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis; John Wiley and Sons: New York, 1999,
pp. 526–528; (b) Kocienski, P. J. Protecting Groups;
Thieme: Stuttgart, 1994, pp. 199–202.
In general cladinose removal did not increase the anti-in-
flammatory effect, but these compounds showed a better
physical–chemical profile and no anti-microbial action.
In summary, we have developed a new class of 9-(S)-
dihydroerythromycin derivatives in order to obtain a
new class of derivatives endowed with anti-inflammato-
ry activity but devoid of anti-bacterial effects.
14. Amide 16 was also prepared by Polonovsky protocol
treating the corresponding dimethylamino-N-oxide with
acetic anhydride in ethyl acetate at rt for 4 h followed by
hydrolysis of 20-O-acetyl functionality with K2CO3 in
aqueous methanol media at 60 °C. Ku, Y.-Y.; Riley, D.;
Patel, H.; Yang, C.; Liu, J.-H. Bioorg. Med. Chem. Lett.
1997, 7, 1203.
These results led us to further evaluate this class of com-
pounds. In consideration of bioavailability, permeation,
and in vivo toxicity studies, acetamido macrolide 17 was
selected as drug candidate for preclinical and clinical
development studies.
15. The cytotoxicity of macrolide compounds was evaluated
in vitro in a human hepatocellular carcinoma cell line
(Hep G2) by measuring the cellular metabolic function.
16. The ability of macrolide compounds to modulate the
oxidase activity of phagocytic cells (reactive oxygen
species production) was tested in vitro in human whole
blood.
References and notes
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17. Zunic, M.; Bahr, G. M.; Mudde, G. C.; Meingassner, J.
G.; Lam, C. J. Invest. Dermatol. 1998, 111, 77, PMA
Method: The PMA-induced edema in ear tissue was
evaluated in five CD1 mice per compound. The com-
pounds were solubilized in trans-phase delivery system
(TPDS), a vehicle containing benzyl alcohol 10%, acetone
40%, and isopropanol 50%. The test item was topically
applied at 0.5 mg/ear to the inner surface of one ear and
30 min later 15 ll of 0.01% PMA dissolved in acetone was
applied to the same area of the test item application. Six
hours later, mice were sacrificed by CO2 inhalation. The
edema was evaluated by weighting a constant portion of
the treated ear pinna, in comparison with the untreated
controlateral one.
18. The ‘in vitro’ antibacterial effect of macrolides was
evaluated on different bacterial strains: Streptococcus
pneumoniae (ATCC 49619), Staphylococcus aureus
(ATCC 29213 or ATCC 6538), Enterococcus faecalis
(ATCC 29212), and Streptococcus pyogenes (ATCC
19615).
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