The role of the 4″-hydroxyl on motilin agonist potency in the 9-dihydroerythromycin series

Article abstract of DOI:10.1016/j.bmcl.2011.04.078

The role of the erythromycin 4″-hydroxyl group has been explored on the motilin agonist potential in the 9-dihydroerythromycin series of motilides. The compounds show potencies 2- to 4-fold superior to the corresponding hydroxylated compounds. The relationship is maintained when the 9-hydroxyl is alkylated to generate the corresponding 4″-deoxy-9-O-acetamido-9- dihydroerythromycins. However, concomitant with this increase in potency is an increase in hERG inhibition.

Full text of DOI:10.1016/j.bmcl.2011.04.078

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3712–3714
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The role of the 400-hydroxyl on motilin agonist potency
in the 9-dihydroerythromycin series
Yaoquan Liu a, Christopher W. Carreras b, Mark Claypool b, David C. Myles a, Simon J. Shaw a,
⇑
a Department of Chemistry, Kosan Biosciences, Inc., 3832, Bay Center Place, Hayward, California 94545, USA
b Department of Pharmacology, Kosan Biosciences, Inc., 3832, Bay Center Place, Hayward, California 94545, USA
a r t i c l e i n f o
a b s t r a c t
The role of the erythromycin 400-hydroxyl group has been explored on the motilin agonist potential in the
9-dihydroerythromycin series of motilides. The compounds show potencies 2- to 4-fold superior to the
corresponding hydroxylated compounds. The relationship is maintained when the 9-hydroxyl is alkyl-
ated to generate the corresponding 400-deoxy-9-O-acetamido-9-dihydroerythromycins. However, con-
comitant with this increase in potency is an increase in hERG inhibition.
Article history:
Received 1 March 2011
Revised 17 April 2011
Accepted 19 April 2011
Available online 24 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Motilin agonist
Erythromycin
Macrolide
hERG
MIC
The G-protein coupled motilin receptor present in smooth cells
and neurons of the gastrointestinal (GI) tract has been the target of
several drug discovery programs. Motilin is a 22-amino acid
peptide, which plays an important role in the normal regulation
of gastric motility.1,2 Targeting the motilin receptor may be
effective in the treatment of GI disorders such as gastroesophageal
reflux disease (GERD) and gastroparesis. Indeed, it has been shown
that IV infusion of motilin or oral administration of the antibiotic
this moiety plays in the 9-dihydro series. To investigate, we began
by looking at the 9-dihydro-400-deoxy-N-desmethyl-N-alkyl erythr-
omycins 5.
Previously it was shown that the 9-dihydroerythromycin
scaffold was an effective motilin agonist scaffold,12 with the 9-hy-
droxy-N-des-methyl-N-isopropylerythromycin showing motilin
agonist potency of 260 nM. In this study we set out to compare
the potency of the 400-deoxy compounds with the corresponding
hydroxylated compounds in the 9-dihydroerythromycin series.
To effect deoxygenation a similar route was used to that de-
scribed by the Abbott group.6 Thus, the 20-hydroxyl was protected
by treatment with acetic anhydride. Treatment with thi-
ocarbonyldiimidazole (TCDI) resulted in formation of the 400-thio-
carbamate 6, which upon treatment with tributyltin hydride in
refluxing toluene resulted in deoxygenation. Deprotection of the
20-acetyl group by treatment with methanol resulted in 400-deoxy-
erythromycin 7. Reduction of the 9-hydroxyl followed by N-
demethylation was carried out in a similar manner to that reported
previously.9,12 Thus treatment with sodium borohydride followed
by reaction with iodine in the presence of sodium acetate resulted
in 8, which could be functionalized by reaction with the corre-
sponding alkyl iodides 5a–5d or epoxides 5e, 5f (Scheme 1). The
compounds were examined for their on-target motilin agonist
activity in our muscle strip contractility assay as used in our
previous reports9,10,12 as well as for their undesired antibacterial
activity against an erythromycin-sensitive strain of Streptococcus
pneumonia (ATCC 6301). All the compounds except the acetamido
compound 5d showed good motilin agonist activity, with the 400-
erythromycin A 1, a motilin receptor agonist (EC50 ꢀ1
l
M),1 stim-
ulates GI motility in humans3,4 with gastroparesis.
As
a prokinetic that may be required chronically the
antibacterial nature of 1 is undesired. Attempts to engineer out
the antibiotic activity while optimizing the motilin agonist activity
of 1 have resulted in more acid stable compounds (often referred to
as motilides) including ABT-229 2,5,6 GM-611 37,8 and the efforts of
our own group including 4 (Fig. 1).9,10
It was reported in the case of the Abbott series that
deoxygenation of the cladinose 4-hydroxyl–the 400-deoxy
compound–showed improved potency (up to 1000-fold) over the
400-hydroxylated compounds.6
By contrast, Koga reported that deoxygenation of 3 did not have
a significant effect on potency,11 although there was a slight
improvement. Given the variability in the role of the 400-hydroxyl,
and the potential for the 400-deoxy compounds to show
significantly increased potency, it was of interest to learn what role
⇑
Corresponding author. Tel.: +1 650 624 1423; fax: +1 650 624 1101 (S.J.S.).
E-mail addresses: simonjshaw@gmail.com, sshaw@rigel.com (S.J. Shaw).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.078

Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3712–3714
3713
Figure 1. Structures of Erythromycin A 1 and motilin agonists 2–4.
Scheme 1. Reagents and conditions: (i) Ac2O, CH2Cl2; (ii) TCDI, DMAP, CH2Cl2; (iii) Bu3SnH, AIBN, toluene, 80 °C; (iv) CH3OH, 60 °C; (v) NaBH4, MeOH; (vi) I2, NaOAc, CH3OH,
i
50 °C; (vii) RI, Pr2NEt, CH3CN, 80 °C; (viii) epoxide, MeOH, 40 °C.
deoxy compounds being approximately 2.5- to 4- fold more potent
Table 1
than the analogous 400-hydroxy compounds (Table 1).
Potency of the standard compounds
1
and 2, 5a–5f and their 400-hydroxy
equivalents12
As expected, modification of the desosamine results in
compounds that were significantly reduced in antibacterial
Compound
R
Potency
Corresponding 400- ATCC 6310
potency relative the erythromycin (0.0025 l
g mLꢁ1).9
EC50
hydroxy EC50
(nM)
MIC
In the case of 2, it was found that in the clinic the compound
was not efficacious, despite the in vitro potency. This is thought
to result from receptor desensitization, often observed in G-protein
coupled receptors, a phenomenon known as tachyphylaxis.13 Nei-
ther 5a nor 5c exhibited tachyphylaxis in our assay,14 where they
showed 88% and 94% contractility response on fourth dosing at
an EC90 drug concentration following three cycles of
administration and washout (2 and 3 show 9% and 22% response
respectively in the same assay).
Previous work has shown that alkylation of the 9-hydroxyl
resulted in molecules that were more potent than the correspond-
ing 9-hydroxy compounds.9,12 Given that deoxygenation of the
400-hydroxyl improved the potency of the 9-hydroxy compounds
we were interested to know if the increase in potency would hold
for the 9-O-acetamido series. Thus the acetamido compounds
9a–9c were synthesized from 5a using a similar procedure to our
previous work (Scheme 2).9 The compounds were consistently
more potent although the difference is slightly smaller than for
the 9-hydroxy series 5a–5f. Again, the compounds did not show
significant antibacterial potency (Table 2).
(nM)
(l
g mLꢁ1
)
1, Ery A
2, ABT-229
1200
7
0.03
64
5a
59
260
64
5b
5c
120
63
350
210
>128
2
5d
5e
5f
>5000
140
nd
nd
360
100
128
128
24
Macrolides are known to block the hERG potassium ion channel
found in the heart.15 Inhibition of hERG can lead to prolongation of
the QT interval and potentially fatal cardiac arrhythmia eg torsades

3714
Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3712–3714
Scheme 2. Reagents and conditions: (i) KOtBu, ClCH2CONH2, DME, 0 °C, 4 h; (ii) KOtBu, BrCH2CONHCH3, 0 °C, 4 h; (iii) KOtBu, BrCH2CON(CH3)2, 0 °C, 2 h.
Chugai compounds such as 3.11 However, the increased motilin
agonist potency is balanced by an increase in the inhibition of
the hERG potassium channel, a potential cardiovascular safety lia-
bility. In the case of 9b, it is 3.4-fold more potent as a motilin ago-
Table 2
Potency of the 9-O-acetamido compounds 9a–9c and their 400-hydroxy analogues9
Compound
R1, R2
Potency
EC50 (nM)
Analogous
400-hydroxy
EC50 (nM)
ATCC 6310
MIC (l )
g mLꢁ1
nist, however, it has a hERG IC50 of approximately 30
l
M compared
with the 400-hydroxy compound, which has a hERG IC50 of 411
l
M9
9a
9b
9c
H, H
CH3, H
CH3, CH3
36
17
190
52
58
660
>128
128
128
(a more than 13-fold difference). The result is that despite their
superior agonist potency, the 400-deoxy compounds examined in
this study have a lower therapeutic window than their hydroxyl-
ated analogues.
de pointes.16 Thus we investigated these compounds for their inhi-
bition of hERG (Table 3).
Acknowledgments
Consistent with our previous studies, the hERG inhibition is sig-
nificantly decreased at 30 lM in the case of the 9-acetamide com-
pounds 9a–9c relative to the corresponding 9-hydroxy compound
5a.9 Further, the hERG inhibition can be attenuated through the
substituent on the desosamine nitrogen. The more polar hydroxy-
We are grateful to John R. Carney and Chau Q. Tran for mass
spectral analysis, William Crumb, Jr., Zenas Technologies LLC, for
hERG determinations, Dwight J. Hardy, University of Rochester
Medical Center, for MIC determinations and Robert Johnson and Pi-
eter B. Timmermans for useful discussions.
ethyl compound 5c shows less inhibition at 30
lM than the cyclo-
butyl analogue 5b (68% vs 92% inhibition at 30
l
M for 5c and 5b,
References and notes
respectively). However at 300 lM all the compounds are very po-
tent inhibitors of hERG. This is in contrast to the 400-hydroxy ana-
1. Brown, J. C.; Cook, M. A.; Dryburgh, J. R. Can. J. Biochem. 1972, 51, 533.
2. Miller, P.; Gagnon, D.; Dickner, M.; Aubin, P.; St-Pierre, S.; Poitras, P. Peptides
1995, 16, 11.
logues of 9a–9c, which showed inhibitions of 29%, 37%, and 51%
at 300 l
M, respectively.9 The results suggest that the hERG po-
3. Peeters, T. L.; Muls, E.; Janssens, J.; Urbain, J. L.; Bex, M.; Van Cutsem, E.;
Depootere, I.; De Roo, M. Gastroenterology 1992, 102, 97.
4. Janssens, J.; Peeters, T. L.; Vantrappen, G.; Tack, J.; Urbain, J. L.; De Roo, M.;
Muls, E.; Bouillon, R. N. N. Engl. J. Med. 1990, 322, 1028.
5. Faghih, R.; Nellans, H. N.; Plattner, J. Drugs Future 1998, 23, 861.
6. 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.
7. Koga, H.; Takanashi, H.; Itoh, Z.; Omura, Z. Drugs Future 2002, 27, 255.
8. Koga, H.; Sato, T.; Tsuzuki, K.; Onoda, H.; Kuboniwa, H.; Takanashi, H. Bioorg.
Med. Chem. Lett. 1994, 4, 1347.
9. Shaw, S. J.; Chen, Y.; Zheng, H.; Fu, H.; Burlingame, M. A.; Marquez, S.; Li, Y.;
Claypool, M.; Carreras, C. W.; Crumb, W.; Hardy, D. J.; Myles, D. C.; Liu, Y. J. Med.
Chem. 2009, 52, 6851.
10. Liu, Y.; Li, Y.; Myles, D. C.; Claypool, M.; Carreras, C. W.; Shaw, S. J. Bioorg. Med.
Chem. 2010, 18, 7651.
tency is increased by approximately 10-fold when the 400-hydroxyl
group is removed (e.g., in the case of 9c, hERG inhibition of 50% at
30
lM
versus 400-hydroxyl analogue hERG inhibition 51% at
300
lM).
In summary the 400-hydroxyl has been investigated in the
9-dihydro series of motilides. The 400-deoxy compounds are 3- to
4-fold more potent motilin agonists. The trend holds for the more
potent 9-dihydro-9-O-acetamide compounds, which are again
more potent agonists in the 400-deoxy series by 1.5- to 3.5-fold.
The observation is similar to that observed by Koga in the case of
11. Koga, H.; Sato, T.; Tsuzuki, K.; Takanashi, H. Bioorg. Med. Chem. Lett. 1994, 4,
1649.
12. Liu, Y.; Li, Y.; Chen, Y.; Zheng, H.; Claypool, M.; Myles, D. C.; Carreras, C. W.
Bioorg. Med. Chem. Lett. 2010, 20, 5858.
Table 3
hERG inhibition for selected compounds as a percentage of inhibition
13. Thielmans, L.; Depoortere, I.; Perret, J.; Robberecht, P.; Liu, Y.; Thijs, T.;
Carreras, C. W.; Burgeon, E.; Peeters, T. L. J. Pharmacol. Exp. Ther. 2005, 313,
1379.
14. Carreras, C. W.; Claypool, M.; Santi, D. V.; Schuurkes, J. A.; Peeters, T. L.;
Johnson, R. G. Gastroenterology 2004, 126, A276.
15. Stanat, S. J. C.; Carlton, C. G.; Crumb, W. J.; Agrawal, K. C.; Clarkson, C. W. Mol.
Cell. Biochem. 2003, 254, 1.
16. Ray, W. A.; Murray, K. T.; Meredith, S.; Narasimhulu, S. S.; Hall, K.; Stein, C. M.
N. Engl. J. Med. 2004, 351, 1089.
Compound
hERG% inhibition
@30
lM
@300 lM
5a
5b
5c
5e
5f
9a
9b
9c
74
92
68
89
86
28
57
50
nd
100
nd
100
100
86
97
100