9-Dihydroerythromycins as non-antibiotic motilin receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.030

A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.

Full text of DOI:10.1016/j.bmcl.2010.08.030

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5658–5661
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
9-Dihydroerythromycins as non-antibiotic motilin receptor agonists
Yaoquan Liu a, , Yong Li a, Yue Chen a, Hao Zheng a, Mark Claypool b, David C. Myles a,
⇑
Christopher W. Carreras b
a Department of Chemistry, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA
b Department of Pharmacology, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have
been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG
channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary
or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as
non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 14 July 2010
Revised 4 August 2010
Accepted 5 August 2010
Available online 9 August 2010
Keywords:
Dihydroerythromycin
Motilin receptor agonist
MIC
Tachyphylaxis
hERG
The motilin receptor is a G-protein-coupled receptor (GPCR)
found in the gastrointestinal smooth muscle and enteric neuronal
tissues. Motilin, a 22-amino acid peptide hormone, binds to the
receptor and stimulates coordinated gastric motility.1–3 The motilin
receptor is a target for the treatment of gastrointestinal (GI) disor-
ders such as gastroesophageal reflux disease (GERD) and gastropa-
resis. It has been shown that IV infusion of motilin or oral
administration of the macrolide antibiotic erythromycin A (EryA)
stimulates GI motility in man.4,5 However, while EryA may be used
to treat gastroparesis, its antibiotic activity makes it unsuitable for
chronic administration.
Attempts to optimize the motilin agonist activity of EryA while
engineering out the antibiotic activity have resulted in compounds
derived from the erythromycin enol ether, ABT-229 and GM-611 (
Fig. 1).6,7 ABT-229 was withdrawn from phase II clinical trials, due
to a lack of efficacy upon repeated administration, a phenomenon
known as tachyphylaxis.8
We also aimed to identify non-antibiotic motilin receptor ago-
nists that were effective upon repeated administration. We were
conscious of the acid instability of EryA, which results in cycliza-
tion of 6-hydroxyl onto the 9-ketone to form a reactive 6,9-enol
ether that is 50–100 fold more potent as a motilin agonist. Further,
N
O
N
N
O
HO
O
HO
O
HO
O
HO
HO
O
O
O
O
HO
O
HO
MeO
O
O
O
O
O
O
O
O
O
O
O
O
OH
OCH3
OH
OCH3
OCH3
EryA
ABT-229
GM-611
Figure 1. Chemical structures of erythromycin A (EryA), ABT-229 and GM-611.
⇑
Corresponding author. Present address: Genetic Chemistry, Inc., 2440 Embar-
cadero Way, Palo Alto, CA 94303, USA. Tel.: +1 (650) 644 8949; fax: +1 (650) 856
7950.
E-mail addresses: gyliu@comcast.net, gliu@geneticchemistry.com (Y. Liu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.030

Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5658–5661
5659
upon continued exposure to acid, the enol ether decomposes by
reaction with the 12-hydroxyl to form an inactive spiroketal.9 By
contrast, reduction of the 9-ketone results in the acid-stable 9-
dihydroerythromycin 1. Compound 1 is a weaker antibiotic but a
potent motilin agonist comparable to EryA.10 We decided to inves-
tigate 9-dihydroerythromycin A as a motilin agonist scaffold. In
contrast to the enol ether, which shows little antibiotic activity, 1
retains moderate antibiotic activity. Thus, we focused our efforts
on developing SAR around the dimethylamine of the desosamine,
which is crucial for antibacterial potency.11
9-Dihydro-N-desmethylerythromycin A was synthesized by so-
dium borohydride reduction of EryA to the 9(S)-isomer 1,12 fol-
lowed by reaction with iodine in the presence of sodium acetate
to yield the N-desmethyl compound 213 (Fig. 2). N-Alkyl derivatives
were available through alkylation of 2 with the variety of alkyl ha-
lides in the presence of Hünig’s base (compounds 3–13 and 18–
20)14 or with the corresponding epoxide in methanol at 40 °C
(14–17). In two cases the remaining N-methyl group of 2 was re-
moved again by treatment with iodine to obtain secondary amines
21 and 22.
OH
OH
R1
O
R1
O
OH
O
OH
O
N
O
NH
O
OH
OH
HO
NaBH4
MeOH
I2, NaOAc
HO
Ery
O
O
O
O
MeOH, 50 o
C
O
O
OH
OMe
OH
OMe
1
2
R2X, DIPEA
ACN, 80 o
C
OH
OH
OH
R2
R1
O
R1
O
OH
O
R2
OH
O
HN
N
O
I2, NaOAc
MeOH, 50 o
OH
HO
HO
O
O
O
O
O
C
O
O
OH
OMe
OH
OMe
Ery A series R1=OH, R2=
Ery A series R1=OH, R2=
21 22
3
4
8
5
9
6
7
10
O
OH
11
12
HO
13
OH
OH
OMe
OH
OH
OH
14
18
15
19
16
20
17
*
O
NH2
NH2
F
Ery B series R1=H, R2=
23
24
HO
OH
OH
25
26
27
Figure 2. Desosamine modification of 9-dihydroerythromycins.

5660
Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5658–5661
Table 1
EryA sensitive strain Streptococcus pneumoniae ATCC 6301 and in
our tachyphylaxis assay15 (Table 1).
Assay results for Ery A and 9-dihydroerythromycin A analogues
The results show that there are several compounds that met our
desired profile of motilin agonist potency of less than 350 nM.
There appears to be a steric requirement about the desosamine
nitrogen for compounds with small alkyl groups—the isopropyl 3,
isobutyl 4, cyclopropylmethyl 6 and cyclobutyl 8—show good po-
tency while the larger 2-methylcycloproplymethyl 7 and cyclopen-
tyl 10 are less potent. The potency is balanced by antibacterial
activity where the smaller groups are more potent. However the
N-cyclobutyl compound 8 does meet our profile showing good po-
Compounds
Agonist EC50 (nM)
MIC (
l
g/mL)
Tachyphylaxis
(% @ 4th dose)
EryA
ABT-229
3
1200
7
260
363
2400
310
2000
350
5700
>5000
210
7700
700
360
0.03
64
32
nd
nd
32
64
128
32
128
8
nd
nd
128
>128
>128
nd
nd
nd
nd
>128
16
97
1
22 10
76
81
nd
nd
nd
90
nd
nd
86
nd
nd
95
93
89
nd
nd
nd
nd
nd
nd
6
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
5
6
tency with acceptable antibacterial activity (P128
minimal tachyphylaxis (P90% at 4th dose).
lg/mL) and
It is interesting to note that moving from N-cyclobutyl to N-oxe-
tanyl as in 9 results in a significant drop in potency. It appears that
in general increasing polarity of the desosamine group is deleteri-
ous to potency. The ethylamino compound 19 and the acetamide
20 show little motilin agonist activity, while the secondary amino
compounds 21 and 22 are inactive. In the case of the hydroxy com-
pounds the effects are more subtle, however, tertiary hydroxyl
compound 16 shows the optimal profile of potency and off target
effects. Indeed this compound meets the criteria we had set.
In the case of the EryB derivatives as expected all the com-
pounds met our criteria in terms of lack of antibacterial activity
(Table 2). The five compounds synthesized 23–27 are analogous
to the EryA series compounds 3, 8, 14–16, respectively and show
similar or improved potency against the motilin receptor, however,
which many of these compounds a significant amount of tachyphy-
laxis was observed with only one compound 26 meeting the de-
sired profile.
460
100
5
2500
3800
7300
>5000
>5000
>5000
Motilin agonist potency and tachyphylaxis were measured in a rabbit smooth
muscle contractility assays as previously reported.15,18 EC50 values are the con-
centration that caused 50% of the maximal possible contraction. Tachyphylaxis is
reported as the % contractility response obtained from an EC90 drug concentration
following three cycles of administration and washout. Antibacterial activity was
assessed by determining the minimal growth inhibitory concentration19 (MIC) with
the highly erythromycin-sensitive strain Streptococcus pneumoniae ATCC6301.
nd = Not determined.
Table 2
Assay results for 9-dihydroerythromycin B analogues
Compounds
Agonist EC50 (nM)
MIC (lg/mL)
Tachyphylaxis
(% @ 4th dose)
Table 3
hERG inhibition for EryA, ABT-229 and
selected compounds as a percentage of inhibi-
tion at 30 lM
23
24
25
26
27
106
300
180
280
100
128
>128
>128
>128
>128
79
71 12
70
89
56
7
5
Compounds
hERG
EryA
ABT-229
3
8
14
16
26
50
98
80
38
16
25
38
Using a similar synthetic route a subset of these derivatives was
generated in the erythromycin B (EryB) scaffold, which lacks the
12-hydroxyl group. In this way it might be possible to take advan-
tage of the lower antibacterial activity of this scaffold (Fig. 2).
All the compounds were initially screened in a muscle strip con-
tractility assay to test for motilin agonist activity. Compounds of
interest were further examined for antibiotic activity against the
hERG inhibition was measured at 37 °C using
a stably transfected HEK cell line at express-
ing the hERG mRNA.20
OH
OH
OH
HO
OH
OH
HO
OH
NH2
O
R
O
I2, NaOMe
alkylation
OH
OH
2
O
O
O
O
O
O
O
O
O
O
OH
OH
OMe
OMe
28
O
N
R =
N
N
29
30
31
Figure 3.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5658–5661
5661
With these results in hand, it was decided to investigate cyclic
References and notes
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pholinyl compounds 29–31 were obtained. Unfortunately, these
compounds showed little or no motilin agonist activity.
From the compounds synthesized there were several that met
the profile which we had set, that is to say, a potent motilin agonist
without showing both tachyphylaxis and significant antibiotic
activity. However, we were conscious of the ability of macrolides
to inhibit the hERG, a potassium ion channel in the heart that
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nitrogen becomes more polar the hERG inhibition decreases. Four
compounds 8, 14, 16 and 26 showed hERG inhibition, which was
lower than EryA.
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activity from its antibiotic activity. In general the EryB series shows
less antibiotic activity but a more significant amount of tachyphy-
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biotic activity. These compounds do not show significant tachy-
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Acknowledgments
We wish to thank John R. Carney and Chau Q. Tran for mass
spectral analysis and Pieter B. Timmermans and Robert Johnson
for useful discussions.