Structure-activity relationships of 9-substituted-9-dihydroerythromycin- based motilin agonists: Optimizing for potency and safety

Article abstract of DOI:10.1021/jm901107f

A series of 9-dihydro-9-acetamido-N-desmethyl-N-isopropyl erythromycin A analogues and related derivatives was generated as motilin agonists. The compounds were optimized for potency while showing both minimal antibacterial activity and hERG inhibition. As the substituent on the amide was increased in lipophilicity the potency and hERG inhibition increased, while polar groups lowered potency, without significantly impacting hERG inhibition. The N-methyl acetamide 7a showed the optimal in vitro profile and was probed further by varying the chain length to the macrocycle as well as changing the macrocycle scaffold. 7a remained the compound with the best in vitro properties. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901107f

J. Med. Chem. 2009, 52, 6851–6859 6851
DOI: 10.1021/jm901107f
Structure-Activity Relationships of 9-Substituted-9-Dihydroerythromycin-Based Motilin Agonists:
Optimizing for Potency and Safety
Simon J. Shaw,*,† Yue Chen,† Hao Zheng,† Hong Fu,† Mark A. Burlingame,† Saul Marquez,† Yong Li,† Mark Claypool,‡
Christopher W. Carreras,^,‡ William Crumb,§ Dwight J. Hardy, David C. Myles,† and Yaoquan Liu†
†Departments of Chemistry and ‡ Pharmacology, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545,
§ Zenas Technologies LLC, 4609 Fairfield Street, Metairie, Louisiana 70006, and Clinical Microbiology Laboratories, University of Rochester
Medical Center, Rochester, New York 14642-8710. ^ Current address: Ardelyx Inc., 34175 Ardenwood Boulevard, Fremont, CA 94555.
Received July 26, 2009
A series of 9-dihydro-9-acetamido-N-desmethyl-N-isopropyl erythromycin A analogues and related
derivatives was generated as motilin agonists. The compounds were optimized for potency while
showing both minimal antibacterial activity and hERG inhibition. As the substituent on the amide was
increased in lipophilicity the potency and hERG inhibition increased, while polar groups lowered
potency, without significantly impacting hERG inhibition. The N-methyl acetamide 7a showed the
optimal in vitro profile and was probed further by varying the chain length to the macrocycle as well as
changing the macrocycle scaffold. 7a remained the compound with the best in vitro properties.
Introduction
phenomenon known as tachyphylaxis that is mediated
through a receptor desensitization mechanism.22,23 While 2
waspotentin invitro muscle contractility assays19 and promo-
ted gastric emptying following a single administration,24 it
was ineffective when administered as repeated doses.25-28 In
vitro assays for tachyphylaxis were developed and indicated
that agonist potency and tachyphylaxis were separable,22,29,30
validating the potential for finding motilides that retain
activity through multiple administrations.
In addition to agonist potency, tachyphylaxis, and anti-
biotic activity, we were also conscious of the ability of
macrolides to block the hERG potassium channel found in
the heart, potentially causing a fatal cardiac arrhythmia.31-34
We therefore included hERG assessment as a regular part of
the in vitro characterization of newly synthesized motilides.
During our initial investigation of the properties of simple
derivatives of 1, we learned that 9-dihydroerythromycin deri-
vatives can be potent motilin agonists with dramatically
reduced antibiotic activity.35 In this class, the (9S)-9-dihy-
dro-N-desmethyl-N-isopropylerythromycin 3 showed good
potency without causing tachyphylaxis. However, 3 retains
a weak antibacterial activity and blocks the hERG channel
with an IC50 of less than 30 μM. Here wedescribe the effects of
alkylating the 9-hydroxyl group as a route to lowering both
the hERG inhibition and antibacterial activity without caus-
ing tachyphylaxis or having a deleterious effect on the motilin
agonist activity.
The 22-amino acid hormone motilin plays a critical role in
thenormalregulation of gastrointestinal(GIa) motility.1,2 The
activity of motilin is mediated through a G-protein coupled
receptor found on smooth muscle and enteric neurons in the
GI tract.3-6 The macrolide erythromycin A (EryA) 1 is an
agonist of the motilin receptor (EC50 ∼1 uM) (Figure 1);1,7 it
competes with labeled motilin for receptor sites in membrane
preparations,8 causes Ca2þ currents in whole cell systems
expressing the receptor,9,10 induces contractions in isolated
GI smooth muscle,8,11 and is an effective prokinetic in animal
models which measure gastric emptying12 and motility.7,13
Indeed, both motilin and 1 stimulate gastric motility in
patients with gastroparesis,14,15 and clinical studies indicate
that 1 has a stronger effect on gastric emptying than metoclo-
pramide, domperidone, or cisapride and is capable of provid-
ing symptomatic relief to gastroparesis patients.16,17 Never-
theless, the antibiotic activityof1 is undesirable in a prokinetic
agent that could require chronic use. Nonantibiotic erythro-
mycin derived motilin agonists (often referred to as
“motilides”) have therefore been proposed for the treatment
of GI motility disorders such as gastresophageal reflux disease
(GERD) and gastroparesis.18
A number of efforts have been made to generate nonanti-
biotic, bioavailable motilides.19-21 Of these, the motilide
ABT-2292 displayed high potencyinvitrobut failedinclinical
studiesduetolossof efficacy uponrepeated administration-a
Results and Discussion
*To whom correspondence should be addressed. Phone: (650)624-
1423. Fax: (650)624-1423. E-mail: sshaw@rigel.com; simonjshaw@
gmail.com. Current address: Rigel, Inc., 1180 Veterans Boulevard,
South San Francisco, CA 94080.
a Abbreviations: GI, gastrointestinal; EryA, erythromycin A; hERG,
human ether-a-go-go related gene; GERD, gastresophageal reflux dis-
ease; TMSE, trimethylsilylethyl; TBAF, tetrabutylammonium fluoride;
TBDMS, tert-butyldimethylsilyl; EDCI, 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide; ELSD, evaporative light scattering detector;
MIC, minimal growth inhibitory concentration.
In our initial attempts to develop an erythromycin-based
motilin agonist, we had attempted to obtain rapid access to
compounds without antibiotic activity and improved acid
stability.35 The (9S)-9-dihydroerythromycins were an ideal
starting point because reduction of the 9-ketone improved the
acid stability, increased motilin agonist potency, and greatly
reduced antibiotic activity. It was found that the residual
r
2009 American Chemical Society
Published on Web 10/12/2009
pubs.acs.org/jmc

6852 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Shaw et al.
Figure 1. 1 Erythromycin A, 2 ABT-229, 3 9-dihydro-N-desmethyl-N-isopropylerythromycin A.
Scheme 1. Synthesis of 3a
(TBAF) and methoxylamine coupled to the free acid. For
the 2-hydroxyethylamide 7h, the coupling was carried out
with the hydroxyl protected as the tert-butyldimenthysilyl
(TBDMS) ether,40 which was later removed with TBAF.
Investigation of the 1H NMR spectrum of each compound
showed a 0.2-0.3 ppm downfield shift of the H-9 proton
relative to 3, suggesting that alkylation had occurred at this
position. To confirm this observation, extensive NMR char-
acterization was carried out with 7a. The HMBC spectrum
clearly shows a correlation between H-9 and the methylene
carbon of the acetamide, as well as a correlation between the
methylene protons of the acetamide and C-9 (see Supporting
Information, Figure 7). In this way a series of 9-substituted
acetamides was synthesized to probe the impact of the sub-
stituent on the nitrogen of the acetamide on the potency and
other properties of the molecules (Scheme 4).
The compounds were initially tested for motilin agonist
potency and antibacterial activity. Compounds that showed
strong potency (EC50 <150 nM) and low antibacterial activ-
ity were further tested for tachyphylaxis and inhibition of the
hERG channel (Table 1).
These experiments show that the 9-O-acetamides are,
in general, a potent class of motilin agonists, with lower
antibiotic activity than 3. The dimethyl acetamide 4b is a
significantly weaker agonist than all the other compounds,
suggesting a possible role for a free N-H bond or a steric
requirement around the amide nitrogen. There is a trend
toward lower potency as the lipophilicity of the side chain
is increased (e.g., 7a vs 7e, 7f, and 7i vs 7h), however,
increasing lipophilicity also increases the hERG inhibition.
a (i) NaBH4, iPrOH Et2O, 0 ꢀC to rt, 3 h; (ii) I2, NaOAc, MeOH-H2O,
3
50 ꢀC, 4 h; (iii) ICH(CH3)2, iPr2NEt, CH3CN, 70 ꢀC, 24 h.
antibiotic activity could be eliminated by replacing a methyl
group on the desosamine with sterically larger alkyl group of
which the isopropyl gave the optimal properties in terms of
potency and ease of synthesis. Thus, (9S)-9-dihydro-N-des-
methyl-N-isopropylerythromycin 3 was available in three
steps (Scheme 1).
The 9-hydroxyl can be regioselectively alkylated under basic
conditions.36 It was considered that this would be an ideal
position to introduce functional groups to tune both the hERG
and antibacterial properties and potentially to modulate po-
tency. We began our studies by investigating the feasibility of
an acetamide addition. Both 2-chloroacetamide and 2-bromo-
N, N-dimethylacetamide are commercially available. Thus,
treatment of 3 with potassium tert-butoxide in the presence
of either alkylating agent resulted in clean formation of the
corresponding 9-O-acetamides, 4a and 4b (Scheme 2).
None of the mono-N-substituted substituted acetamides
were commercially available, but they were readily synthe-
sized from bromoacetyl bromide 5 and the corresponding
amine in the presence of sodium bicarbonate (Scheme 3).37,38
Treatment of 3 under similar alkylating conditions as in
Scheme 2 furnished each of the 9-O-acetamide derivatives
7a-7m with two exceptions. In the case of the methoxyamide
7g, the trimethylsilylethyl (TMSE) ester was coupled,39 which
could be deprotected using tetrabutylammonium fluoride
Scheme 3. Synthesis of Bromoacetamides 6a-6ma
a (i) H2NR, NaHCO3, THF, 0 ꢀC to rt, 16 h.
Scheme 2. Synthesis of 9-O-Acetamides 4a and 4ba
a (i) tBuOK, ClCH2CONH2, DME, 0 ꢀC, 4 h; (ii) tBuOK, BrCH2CON(CH3)2, DME, 0 ꢀC, 2 h.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6853
Scheme 4. Synthesis of 9-O-Acetamides 7a-7ma
Table 1. In Vitro Assay Results for Compounds 4a, 4b, and 7a-7m
hERG %
inhibition
Streptococcus
potency
EC50 tachyphylaxis
compd (nM) %@ 4th dose
pneumoniae
ATCC 6301
MIC (μg/mL)
@30 @300
μM
μM
1
1200
7
97 ( 1
22 ( 10
85 ( 3
92 ( 3
95 ( 8
89 ( 8
89 ( 11
nda
0.0025
64
32
27
98
80
10
8
90
100
100
51
2
3
260
52
4a
4b
7a
7b
7c
7d
7e
7f
7g
7h
7i
>128
64
660
58
29
37
>128
>128
128
7
67
24
26
30
23
64
33
29
33
23
67
40
16
75
89
140
160
31
nd
128
128
82
60
82 ( 4
75 ( 1
nd
28
92
128
nd
100
82
120
50
87 ( 8
nd
128
128
68
93
7j
120
79
88 ( 9
nd
nd
>128
128
128
67
7k
7l
100
88
69
25
7m
63 ( 3
>128
60
a nd = not determined.
Table 2. In Vitro Assay Results for Compounds 8a-8c, 9, and 10
hERG %
inhibition
Streptococcus
a (i) tBuOK, 6a-6m, DME, rt, 2 h.
potency
EC50 tachyphylaxis
compd (nM) %@ 4th dose
pneumoniae
ATCC 6301
MIC (μg/mL)
@30 @300
μM
It is interesting to note the series of aromatic side chains 7k, 7l,
and 7m in which the introduction of heteroatoms into the ring
attenuates thehERGinhibitionwhilealsoimprovingpotency.
In general, as the compounds become more potent, there is
more tachyphylaxis observed.
Of these compounds, the N-methyl acetamide 7a stands out
for its combination of potency and lack of hERG inhibition. To
probe the methyl amide further, a series of N-methyl amides was
synthesized to study the effect of increasing the distance between
the amide and the macrolide as well as the effect of introducing
rigidity in this region. An alkylation procedure similar to that
described above was used. As before, the required alkylating
agents could be readily synthesized from the commercially
available ω-halo carboxylic acids and methylamine under stan-
dard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
conditions.41 A series of five compounds was synthesized in this
way (8a-8c, 9, and 10, Figure 2, Table 2).
μM
7a
8a
8b
8c
9
58
84
89 ( 8
79 ( 14
nda
>128
128
128
128
128
nd
7
13
3
37
56
270
150
45
61
nd
nd
13
63
nd
2100
1500
100
nd
10
nd
a nd = not determined.
The location of the amide is important for potency, suggest-
ing that the amide is making a specific interaction with the
receptor. As the chain length is increased, the potency de-
creases (e.g., 7a vs 8b). However, the five-carbon linker in 8c is
approximately 2-fold more potent than the four carbon linked
compound 8b. This effect may result from the increased
flexibility of the five carbon side-chain in that it may allow
Figure 2. Methylamides with increased linkers, 8a-8c, 9, 10.

6854 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Shaw et al.
Scheme 5. Synthesis N-Cyclobutyl and EryB Methylacetamides 13, 14a
a (i) tBuOK, 6a, DME, rt, 2 h.
the amide to be positioned more favorably. Further investiga-
tion would be necessary to determine the significance of this
effect. Both 9 and 10 are significantly less potent than their
corresponding flexible compounds 8b and 8c, respectively. In
addition, increasing the number of carbons in the side chain
increases the amount of hERG inhibition observed, however,
the increase is not as pronounced as when the carbons are
introduced onto the nitrogen (e.g., 8a vs 7f).
In the in vitro assays, the N-methyl amide 7a gave the most
desirable combination of properties. Thus, it was decided to
investigate transferring this group to other scaffolds. We
selected the 9-dihydro-N-desmethyl-N-cyclobutylerythromy-
cin A 11 and 9-dihydroerythromycin B 12 scaffolds. Again,
alkylation proceeds under the standard conditions to obtain
the N-cyclobutyl and erythromycin B derivatives 13 and 14,
respectively (Scheme 5).
activity. The nature of the substituent on the acetamide
impacts both the potency and the inhibition of the hERG
channel. In general, as the lipophilicity of the amide is
increased, the potency is increased as well as the hERG
inhibition. Introducing polar groups moderates the hERG
inhibition but also lowers the potency of the molecules as
motilin agonists. Introducing carbons between the macrolide
core and the amide group does not have as profound an effect
on the hERG inhibition as does adding them to the amide.
However, the potency of the compounds is lowered, suggest-
ing that the amide may have a specific interaction with the
motilin receptor. It appears that this interaction may require
the amide N-H as the dimethylamide is 10-fold lower in
potency relative to the corresponding monomethylamide
(4b vs 7a).
The methylacetamide 7a has an optimal in vitro profile that
predicts both prokinetic efficacy and safety. It is 20-fold more
potent than 1 as a motilin agonist and shows no measurable
tachyphylaxis. Furthermore, it has no detectable antimicro-
bial activity when tested against over 200 strains of clinically
relevant bacteria (data not shown). Importantly, 7a does not
interact with the hERG channel at concentrations necessary
for complete agonism of the motilin receptor. The motilin
agonist EC50:hERG blockade IC50 ratio of 7a, used as an in
vitro predictor of cardiovascular safety, is more than 200-fold
improved relative to erythromycin A 1 (Table 4).
The compounds were tested in the in vitro screens (Table 3).
The N-cyclobutyl derivative 12 showsa 10-fold loss inpotency
relative to 7a and increased inhibition of the hERG channel.
By contrast, the erythromycin B derivative14 is very similar to
7a in terms of potency, however, the hERG inhibition is
increased (Table 3).
Table 3. In Vitro Assay Results for Compounds 13 and 14
hERG %
inhibition
Streptococcus
potency
EC50 tachyphylaxis
compd (nM) %@ 4th dose
pneumoniae
ATCC 6301
MIC (μg/mL)
@30 @300
μM
μM
Table 4. Predicted Cardiovascular Safety Window of Benchmark
Compounds
7a
13
14
58
510
90
89 ( 8
nda
128
>128
>128
7
12
21
37
53
67
motilin agonist hERG current
inhibition
IC50 (uM)
fold
improvement
vs 1
86 ( 4
potency
compd EC50 (nM)
hERG IC50/
agonist EC50
a nd = not determined
Conclusion
EryA 1
1200
260
58
39
10
32.5
38
3
7a
1.6
The 9-O-acetamides are potent motilin agonists. Further-
more, introduction of the acetamide eliminates antibiotic
411
7086
218

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6855
Table 5. 1H NMR Resonances for Macrolide Portion of Compounds 3, 4a, 4b, 7a-7ia
3
4a
4b
7a
7b
7c
7d
7e
7f
7g
7h
7i
H-2
H-3
2.80
4.22
1.89
3.73
1.61
1.34
2.14
2.81
1.98
3.73
4.88
1.92
1.49
0.88
4.59
3.21
2.62
1.61
1.39
3.58
2.21
2.91
5.02
2.39
1.60
3.04
4.03
2.64
4.00
1.82
3.52
1.93
1.24
2.54
3.22
2.15
3.88
4.85
1.87
1.48
0.88
4.58
3.20
2.61
1.63
1.34
3.58
2.19
2.89
5.03
2.38
1.59
3.07
4.00
7.38
3.33
4.18
4.02
2.67
4.02
1.94
3.56
1.86
1.30
2.47
3.00
2.07
3.70
5.02
1.98
1.44
0.92
4.63
3.26
2.65
1.67
1.42
3.61
2.24
2.92
5.01
2.42
1.61
3.05
4.03
2.65
4.00
1.81
3.56
1.86
1.24
2.45
3.17
2.15
3.84
4.83
1.84
1.47
0.87
4.58
3.20
2.60
1.61
1.43
3.57
2.18
2.87
5.05
2.39
1.56
3.01
3.97
7.60
3.33
4.12
4.12
2.67
4.06
1.82
3.56
1.61
1.35
2.46
3.16
2.12
3.86
4.82
1.88
1.48
0.88
4.56
3.21
2.61
1.86
1.25
3.54
2.19
2.88
5.02
2.38
1.55
3.01
4.03
7.44
3.33
4.18
3.98
2.72
4.05
1.84
3.58
1.62
1.25
2.41
3.12
2.14
3.84
4.83
1.90
1.51
0.88
4.56
3.21
2.59
1.77
1.38
3.57
2.19
2.89
5.02
2.39
1.57
3.02
4.00
7.17
3.33
4.13
3.89
2.70
4.03
1.84
3.57
1.63
1.23
2.46
3.18
2.16
3.85
4.81
1.88
1.52
0.90
4.56
3.22
2.60
1.88
1.39
3.55
2.21
2.90
5.03
2.39
1.57
3.03
4.00
8.24
3.34
4.29
4.07
2.71
4.02
1.81
3.57
1.77
1.25
2.39
3.10
2.11
3.80
4.83
1.90
1.49
0.88
4.57
3.21
2.60
1.62
1.37
3.55
2.20
2.89
5.04
2.40
1.57
3.03
3.99
7.56
3.34
4.20
3.89
2.72
4.25
1.85
3.88
1.81
1.26
2.43
3.13
2.15
3.58
4.84
1.88
1.50
0.90
4.55
3.21
2.59
1.62
1.39
3.54
2.20
2.90
5.02
2.39
1.56
3.02
4.00
7.48
3.34
4.22
3.95
2.64
4.04
1.85
3.59
1.97
1.25
2.54
3.23
2.16
3.88
4.83
1.90
1.53
0.92
4.59
3.27
2.66
1.66
1.42
3.60
2.34
2.93
5.06
2.42
1.58
3.08
4.01
10.76
3.36
4.24
4.20
2.68
3.98
1.80
3.55
1.62
1.25
2.44
3.16
2.12
3.76
4.85
1.86
1.46
0.86
4.58
3.20
2.61
1.89
1.35
3.57
2.19
2.88
5.07
2.40
1.58
3.03
4.01
7.85
3.33
4.19
4.04
2.69
4.08
1.85
3.57
1.64
1.26
2.59
3.17
2.17
3.86
4.86
1.93
1.51
0.91
4.58
3.23
2.63
1.89
1.40
3.57
2.22
2.91
5.03
2.41
1.58
2.85
4.02
7.54
3.36
4.28
3.96
H-4
H-5
H-7a
H-7b
H-8
H-9
H-10
H-11
H-13
H-14a
H-14b
H-15
H-10
H-20
H-30
H-40a
H-40b
H-50
NCH3
NCH
H-100
H-200a
H-200b
H-400
H-500
NH
OCH3
H-90a
H-90b
3.34
3.37
4.49
3.99
a H-16, H-17, H-18, H-19, H-20, H-21, H-60, NCH(CH3)2, H-600, and H-700 are between 1.25 and 1.00 ppm.
This compound was taken forward into pharmacokinetic and
gastric emptying studies, which will be reported in due course.
A (17.20 g, 23.4 mmol, 1.0 equiv) and sodium acetate (9.75 g,
119.0 mmol, 5.0 equiv) in methanol-water (4:1 v/v, 400 mL) was
heated to 50 ꢀC. Iodine (7.25 g, 28.6 mmol, 1.2 equiv) was added
in two aliquots over 30 min. During the reaction, NaOH (3M,
7.9 mL) was added in small portions to maintain the pH > 8.
Completion of the reaction was determined by TLC (typically
4 h). The majority of the solvent was removed under vacuum,
and the residue extracted with EtOAc (3 ꢀ 100 mL). The com-
bined organics were dried (Na2SO4) and concentrated under
reduced pressure to obtain the demethylated product (15.6 g) as
a yellow solid, which was used for next step without further
purification. The crude 9-dihydro-N-desmethylerythromycin
(2.50 g, 3.41 mmol), diisopropylethylamine (6.1 mL, 10 equiv),
and 2-iodopropane (10.2 mL, 30 equiv) were dissolved in
CH3CN (50 mL) and heated to 70 ꢀC for 24 h. Water (100 mL)
and NaHCO3 (150 mL) were added and the organics extracted
with EtOAc (3 ꢀ 150 mL). The combined organics were dried
(MgSO4) and concentrated under reduced pressure. Column
chromatography (silica, 25% acetone-hexane, 1% triethyl-
amine) yielded 3 (1.80 g, 75% yield for 2 steps) as a white solid;
1H NMR δ (Table 5). 13C NMR δ 177.6, 103.0, 96.2, 83.7, 83.2,
79.3, 79.3, 78.3, 75.6, 75.1, 73.1, 70.8, 70.7, 69.7, 66.4, 62.7, 60.8,
52.9, 49.8, 45.2, 42.3, 37.1, 35.2, 34.8, 33.5, 32.4, 31.4, 25.9, 22.1,
22.0, 21.7, 21.5, 20.8, 20.2, 18.7, 16.8, 15.3, 14.8, 11.6, 9.6. m/z:
765 [M þH]þ.
Experimental Section
Unless otherwise noted, 1H and 13C NMR spectra were
recorded in CDCl3 at 300 K using a Bruker DRX 400 MHz
spectrometer, where possible spectra were assigned using COSY,
HSQC, and HMBC experiments. Infrared spectra were obtained
using a Perkin-Elmer Spectrum One FTIR with an attenuated
total reflectance accessory containing a zinc selenide plate. High
resolution mass spectra were obtained by flow injection with
manual peak-matching using an Applied Biosystems Mariner
TOF spectrometer with a turbo-ion spray source. All final
compounds were >95% pure by LC-MS when detecting by
evaporative light scattering detection (ELSD) using a linear
gradient of 15% (5 mM NH4OAc in CH3CN-MeOH [4:1]) in
5 mM NH4OAc in water to 100% (5 mM NH4OAc in CH3CN-
MeOH [4:1]) over 10 min using a 3 um, 4.6 mm ꢀ 150 mm Varian
Metasil Basic or Phenomenex Luna C-18(2) column. All number-
ing is consistent with that shown in Figure 1.
Synthesis of (9S)-9-Dihydro-N-desmethyl-N-isopropylery-
thromycin A 3. Erythromycin A 1 (20.0 g, 27.3 mmol, 1.0 equiv)
was dissolved in 2-propanol-ether (1:1 v/v, 400 mL) and cooled
to 0 ꢀC. Sodium borohydride (2.1 g, 54.5 mmol, 2.0 equiv) was
added in two aliquots. The mixture was warmed to room
temperature and stirred for 3 h. The excess borohydride was
destroyed by the addition of phosphate buffer (1.0M, pH 6.0,
approximately 50 mL) before adding triethanolamine (80 mL)
and stirring at room temperature for 2 h. The organics were
extracted with EtOAc (4 ꢀ 300 mL), dried (MgSO4), and con-
centrated under reduced pressure. Column chromatography
(33% acetone-hexane, 1% triethylamine) yielded 9-dihydroer-
ythromycin (17.2 g, 86%). A mixture of 9-dihydroerythromycin
General Procedure for the Synthesis of the Bromoacetamides
6a-6m. To a mixture of bromoacetyl bromide (1.0 equiv) and
sodium bicarbonate (2.0 equiv) in tetrahydrofuran at -78 ꢀC
was added the amine (1.5 equiv). The solution was allowed to
warm to room temperature over 14 h. The mixture was parti-
tioned between EtOAc and NaHCO3. The organics were
washed with brine, dried (MgSO4), and concentrated under
reduced pressure to yield the bromoacetamide, which was used
without further purification.

6856 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Shaw et al.
Table 6. 13C NMR Resonances for Macrolide Portion of Compounds
7a-7ha
(found [M þ H]þ, 849.5673; C43H80N2O14 requires [M þ H]þ
849.5682).
7a: Using the general procedure on a 0.131 mmol scale yielded
7a (0.067 g, 61%) as a white solid. IR (film) 3394, 2973, 2937,
7a
7b
7c
7d
7e
7f
7g
7h
C-1
C-2
177.4 177.5 177.6 177.7 177.6 177.6 177.5 177.8
1
1713, 1674, 1547, 1362, 1179, 1087, 1056, 1029, 997 cm-1. H
44.0
77.3
43.6
84.4
75.8
38.3
32.2
94.3
32.8
70.0
74.4
77.7
22.2
11.4
12.9
9.3
44.1
77.5
43.2
84.4
75.8
37.9
31.9
94.0
32.5
70.0
74.3
77.5
22.1
11.3
13.2
9.3
44.2
77.7
42.7
84.1
75.7
37.5
31.7
94.0
32.2
69.8
74.1
77.5
22.0
11.3
13.5
9.2
44.2
77.5
43.2
84.4
75.9
37.8
32.0
94.2
32.5
69.9
74.5
77.6
22.6
11.3
13.3
9.2
44.2
77.8
44.2
84.1
75.7
37.7
32.0
94.1
32.3
69.4
74.1
77.3
22.0
11.3
13.4
9.1
44.2
78.0
44.1
84.2
75.8
37.5
31.6
93.9
32.1
69.4
74.1
77.3
22.0
11.3
13.6
9.2
44.1
77.3
43.7
85.2
76.0
38.5
32.3
94.9
33.0
70.0
74.9
77.6
22.4
11.4
12.9
9.5
44.2
77.3
43.5
84.2
75.7
37.8
32.3
93.6
32.4
69.8
74.4
77.3
22.4
11.2
13.2
9.1
NMR δ (Table 5) 2.75 (3H, d, J 5.0 Hz, NCH3). 13C NMR δ
(Table 6) 25.6. m/z: 836 [M þ H]þ, 678 (found [M þ H]þ,
835.5498; C42H78N2O14 requires [M þ H]þ 835.5526).
C-3
C-4
C-5
C-6
7b: Using the general procedure on a 0.131 mmol scale yielded
7b (0.072 g, 65%) as a white solid. IR (film) 3421, 2973, 2936,
C-7
C-8
1732, 1661, 1539, 1451, 1378, 1179, 1084, 1054, 1029, 998 cm-1
.
1H NMR δ (Table 5) 3.26 (2H, m, NCH2CH3), 1.22 (3H, s,
NCH2CH3). 13C NMR δ (Table 6) 33.8, 15.0. m/z: 850 [MþH]þ,
692 (found [MþH]þ, 849.5698; C43H80N2O14 requires [MþH]þ
849.5682).
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-19
C-20
C-10
C-20
C-30
C-40
C-50
NCH3
NCH
C-100
C-200
C-300
C-400
C-500
C-600
OCH3
C-90
C-900
7c:Using the general procedure on a 0.131 mmol scale yielded
7c (0.051 g, 45%) as a white solid. IR (film) 3458, 2971, 1737,
1
1660, 1538, 1455, 1366, 1230, 1176, 1085, 1054, 998 cm-1. H
NMR δ (Table 5) 4.07 (1H, m, NHCH(CH3)2), 1.17 (6H, m,
NHCH(CH3)2). 13C NMR δ (Table 6) 40.9, 21.3. m/z: 864 [Mþ
H]þ, 706 (found [M þ H]þ, 863.5818; C44H82N2O14 requires
[M þ H]þ 863.5839).
16.8
14.5
16.8
14.7
16.8
15.4
16.9
14.9
16.7
15.1
16.8
15.5
16.9
14.5
16.9
15.1
7d: Using the general procedure on a 0.038 mmol scale yielded
7d (0.019 g, 45%) as a white solid. IR (film) 3438, 2972, 2936,
1731, 1691, 1535, 1452, 1380, 1163, 1085, 1055, 997, 900, 864
cm-1. 1H NMR δ (Table 5) 3.88 (2H, m, NHCH2CF3). m/z: 904
[M þ H]þ, 746 (found [M þ H]þ, 903.5385; C43H77N2O14F3
requires [M þ H]þ 903.5400).
102.1 102.3 102.3 102.4 102.3 102.4 102.5 102.1
70.0
62.1
32.8
69.5
31.0
52.7
94.6
34.5
72.8
77.3
65.6
17.5
49.3
72.8
70.0
62.2
32.9
69.4
31.0
52.6
94.8
34.6
72.1
77.8
65.6
17.7
49.3
72.8
70.1
62.3
32.9
69.4
31.0
53.0
95.0
34.6
72.8
77.8
65.6
17.9
49.3
71.5
70.0
62.2
32.9
69.4
31.0
52.7
94.9
34.6
71.9
77.7
65.7
17.6
49.3
72.8
70.4
62.2
32.9
68.8
31.0
52.6
94.9
34.6
72.8
77.3
65.7
17.8
49.3
71.9
70.1
62.4
32.9
70.0
31.0
52.6
95.1
34.7
72.8
77.5
65.6
17.9
49.3
71.3
70.2
62.3
33.0
69.7
31.2
53.0
94.9
34.6
72.7
77.7
65.8
17.7
49.4
72.9
70.0
62.1
32.8
69.5
31.1
52.7
94.7
34.5
72.8
77.7
65.8
17.6
49.3
72.1
7e: Using the general procedure on a 0.131 mmol scale yielded
7e (0.063 g, 56%) as a white solid. 1H NMR δ (Table 5) 2.71 (1H,
m, NHCH), 0.73 (2H,m, 2H of CH2CH2), 0.57 (2H, m, 2H of
CH2CH2). 13C NMR δ (Table 6) 22.1, 6.0 (2C). m/z: 862 [M þ
H]þ, 703 (found [M þ H]þ, 861.5695; C44H80N2O14 requires
[M þ H]þ 861.5682).
7f: Using the general procedure on a 0.131 mmol scale yielded
7f (0.058 g, 51%) as a white solid. IR (film) 3422, 2972, 2937,
1732, 1660, 1537, 1451, 1379, 1179, 1085, 1054, 1028, 998, 900,
863 cm-1. 1H NMR δ (Table 5) 4.38 (1H, app sextet, J 8.0 Hz,
NCH), 2.28 (2H, m, 1 ꢀ H-2cyb and 1 ꢀ H-4cyb), 1.94 (2H, m,
1 ꢀ H-2cyb and 1 ꢀ H-4cyb), 1.69 (2H, m, 2 ꢀ H-3cyb). 13C
NMR δ (Table 6) 44.1, 30.8, 30.7, 15.2. m/z: 876 [MþH]þ, 718
(found [M þ H]þ, 874.5833; C45H82N2O14 requires [M þ H]þ
874.5839).
170.4 169.5 168.7 170.7 171.2 168.7 166.7 170.7
a C-18, C-21, C-60, NCH(CH3)2 and C-700 are between 22.0 and
19.0 ppm.
General Procedure of the Synthesis of the 9-O-Acetamides. To
a solution of 9-dihydro-N-desmethyl-N-isopropylerythromycin
A 3 (0.100 g, 0.313 mmol, 1.0 equiv) in dimethoxyethane (1.0 mL)
was added potassium tert-butoxide (0.20 mL of a 1.0 M solution in
tetrahydrofuran, 0.196 mmol, 1.5 equiv). The solution was stirred
at room temperature for 10 min before adding a solution of
the haloacetamide (0.157 mmol in 0.5 mL of dimethoxyethane,
1.2 equiv). The solution was stirred at room temperature for 2 h
before adding NaHCO3 (20 mL). The organics were extracted with
EtOAc (3 ꢀ 20 mL), combined, dried (MgSO4), and concentrated
under reduced pressure. Column chromatography (silica, 30 f
60% acetone-hexane, 0.1% triethylamine) yielded the 9-O-acet-
amide as a white solid.
7g: Using the general procedure on a 0.085 mmol scale with
bromoacetic acid 2-(trimethylsilyl)ethyl ether yielded the 9-O-
acetic acid-2-(trimethylsilyl)ether ester (0.045 g, 57%), which
was dissolved in dimethylformamide (1.0 mL) and cooled to
0 ꢀC before adding TBAF (0.015 g, 0.059 mmol, 1.2 equiv).
The solution was stirred at 0 ꢀC for 5 h before EDCI (0.014 g,
0.074 mmol, 1.5 equiv), hydroxybenzotriazole (0.013 g, 0.098
mmol, 2.0 equiv), and methoxylamine hydrochloride (0.008 g,
0.098 mmol, 2.0 equiv). The solution was stirred at room
temperature for 18 h before diluting with EtOAc (15 mL) and
washing with NaHCO3 (15 mL) and brine (15 mL). The organics
were dried (Na2SO4) and concentrated under reduced pressure.
Column chromatography (silica, 30 f 50% acetone-hexane,
1% triethylamine) yielded 7g (0.009 g, 22%) as a white solid. IR
(film) 3457, 2971, 1733, 1684, 1456, 1380, 1179, 1083, 999, 899
4a: Using the general procedure on a 0.362 mmol scale yielded
4a (0.220 g, 73%) as a white solid; H NMR δ (Table 5). 13C
1
NMR δ 177.7, 173.2, 102.6, 95.1, 94.7, 85.4, 78.0, 77.7, 76.3,
74.6, 73.3, 72.3, 70.7, 70.4, 69.9, 66.1, 62.5, 53.1, 49.7, 44.4, 44.0,
38.8, 35.0, 33.3, 33.1, 31.5, 22.7, 22.3, 21.9, 21.6, 21.5, 20.8, 19.9,
18.2, 17.4, 15.2, 13.4, 11.8, 9.7. m/z: 822 [M þ H]þ, 663. m/z:
822.0 [M þ H]þ (found [M þ H]þ, 821.5385; C41H77N2O14
requires [M þH]þ 821.5369).
cm-1 1H NMR δ (Table 5) 3.76 (3H, s, CH3). 13C NMR
.
δ (Table 6) 64.1. m/z: 852 [M þ H]þ, 754 (found [M þ H]þ,
851.5490; C42H78N2O15 requires [M þ H]þ 851.5475).
7h: Using the general procedure on a 0.131 mmol scale yielded
9-O-(2-O-tert-butyldimethylsilyl)ethanolylacetamide (0.101 g,
79%) as a white solid, which was dissolved in tetrahydrofuran
(1.0 mL) and cooled to 0 ꢀC. TBAF (0.020 g, 0.114 mmol,
1.1 equiv) was added and the solution stirred at 0 ꢀC for 2.5 h
before adding NaHCO3 (15 mL). The organics were extracted
with EtOAc (3 ꢀ 15 mL), combined, washed with brine (25 mL),
dried (Na2SO4), and concentrated under reduced pressure.
Column chromatography (silica, 55% acetone-hexane, 1%
4b: Using the general procedure on a 0.362 mmol scale yielded
4b (0.212 g, 69%) as a white solid; 1H NMR δ (Table 5) 2.97 (3H,
s, 1 ꢀ NCH3), 2.87 (3H, s, 1 ꢀ NCH3). 13C NMR δ 176.9, 170.1,
102.4, 95.3, 93.3, 85.5, 78.2, 78.1, 77.9, 75.2, 74.0, 73.4, 70.5,
69.8, 69.6, 68.2, 65.8, 62.6, 53.0, 49.7, 44.4, 43.8, 36.9, 35.8,
35.6, 35.0, 33.3, 32.7, 32.1, 31.5, 31.4, 23.3, 22.8, 21.9, 21.7, 21.5,
20.8, 19.0, 18.1, 17.4, 16.5, 13.7, 12.0, 9.5. m/z: 850 [M þ H]þ

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6857
Table 7. 1H NMR Resonances for Macrolide Portion of Compounds 7j-7m, 8a-8c, 9, 10, 13, and 14a
7j
7k
7l
7m
8a
8b
8c
9
10
13
14
H-2
H-3
2.72
4.05
1.77
3.56
1.80
1.27
2.39
3.10
2.12
3.84
4.77
1.90
1.49
0.87
4.52
3.19
2.57
1.61
1.37
3.52
2.18
2.88
4.99
2.37
1.55
3.01
4.03
7.55
3.32
4.24
3.89
2.66
4.02
1.82
3.56
1.61
1.37
2.55
3.16
2.14
3.83
4.79
1.86
1.43
0.87
4.55
3.21
2.56
1.86
1.22
3.53
2.19
2.88
4.96
2.35
1.48
2.96
3.97
7.88
3.32
4.27
4.06
2.72
4.00
1.82
3.56
1.85
1.27
2.52
3.21
2.20
3.89
4.90
1.93
1.40
0.92
4.59
3.25
2.70
1.64
1.38
3.54
2.23
2.93
5.04
2.39
1.54
2.95
3.99
9.73
3.34
4.53
4.01
2.71
4.00
1.80
3.55
1.84
1.23
2.51
3.19
2.25
3.87
4.91
1.96
1.52
0.93
4.59
3.24
2.60
1.62
1.40
3.54
2.21
2.89
5.05
2.39
1.55
2.94
4.00
9.86
3.34
4.51
4.00
2.67
3.92
1.80
3.54
1.78
1.20
2.40
3.08
2.05
3.77
4.92
1.94
1.44
0.90
4.64
3.23
2.63
1.63
1.38
3.62
2.22
2.96
5.12
2.42
1.58
3.04
4.02
6.47
3.36
3.66
3.52
2.68
3.94
1.79
3.60
1.72
1.13
2.40
3.05
2.02
3.72
4.94
1.92
1.46
0.89
4.66
3.22
2.64
1.65
1.40
3.62
2.21
2.90
5.09
2.41
1.58
3.04
4.00
5.88
3.36
3.47
3.40
2.66
3.94
1.79
3.61
1.66
1.18
2.40
3.02
2.02
3.73
4.93
1.90
1.44
0.89
4.66
3.22
2.64
1.65
1.35
3.61
2.21
2.89
5.08
2.40
1.60
3.04
4.00
5.75
3.35
3.74
3.33
2.69
3.99
1.80
3.52
1.91
1.21
2.66
3.25
2.09
3.79
5.01
1.91
1.44
0.88
4.62
3.23
2.57
1.60
1.38
3.55
2.20
2.88
5.15
2.39
1.54
2.91
3.92
6.86
3.34
4.92
4.41
2.62
4.00
1.82
3.58
1.91
1.22
2.61
3.23
2.12
3.86
4.91
1.91
1.46
0.91
4.65
3.25
2.63
1.63
1.42
3.60
2.22
2.91
5.08
2.38
1.57
3.00
3.95
6.18
3.35
4.90
4.45
2.69
3.99
1.70
3.53
1.95
1.26
2.50
3.18
1.96
3.86
4.82
1.86
1.56
0.87
4.56
3.22
2.51
1.72
1.46
3.53
2.02
3.07
5.04
2.38
1.63
3.00
3.96
7.56
3.32
4.13
4.08
2.57
4.05
1.87
3.54
1.96
1.36
2.48
3.26
2.02
3.62
5.00
1.80
1.39
0.80
4.52
3.17
2.57
1.59
1.39
3.54
2.15
2.84
4.92
2.34
1.51
2.96
3.93
7.85
3.29
4.11
3.90
H-4
H-5
H-7a
H-7b
H-8
H-9
H-10
H-11
H-13
H-14a
H-14b
H-15
H-10
H-20
H-30
H-40a
H-40b
H-50
NCH3
NCH
H-100
H-200a
H-200b
H-400
H-500
NH
OCH3
H-90a
H-90b
a H-16, H-17, H-18, H-19, H-20, H-21, H-60, NCH(CH3)2, H-600, and H-700 are between 1.25 and 1.00 ppm.
triethylamine) yielded 7h (0.063 mg, 70%) as a white solid. IR
(film) 3423, 2972, 2936, 1732, 1661, 1542, 1451, 1379, 1179,
1082, 1053, 997, 899, 863 cm-1. 1H NMR δ (Table 5) 3.68 (2H,
br t, J 5.0 Hz, CH2CH2OH), 3.50-3.43 (1H, m, 1H of
CH2CH2OH), 3.37-3.31 (1H, m, 1H of CH2CH2OH). 13C
NMR δ (Table 6) 62.0, 42.0. m/z: 866 [M þ H]þ, 708 (found
[MþH]þ, 865.5655; C43H80N2O15 requires [MþH]þ 865.5632).
7i: Using the general procedure on a 0.131 mmol scale yielded
(found [M þ H]þ, 899.5563; C45H78N4O14 requires [M þ H]þ
899.5587).
7m: Using the general procedure on a 0.089 mmol scale
yielded 7m (0.014 g, 18%) as a white solid. IR (film) 3452,
2972, 2935, 1726, 1570, 1507, 1454, 1389, 1309, 1179, 1084, 1054,
1028, 997, 900 cm-1. 1H NMR δ (Table 7) 8.90 (1H, d, J 1.0 Hz,
ArH-2), 8.62 (1H, d, J 5.5 Hz, ArH-5), 8.19 (1H, dd, J 5.5, 1.5
Hz, ArH-4). 13C NMR δ (Table 8) 158.4, 158.3, 156.9, 110.6.
m/z: 922 [M þ Na]þ, 900 [M þ H]þ, 742 (found [M þ H]þ,
899.5552; C45H78N4O14 requires [M þ H]þ 899.5587).
1
7i as a white solid. H NMR δ (Table 5) 3.47 (2H, m, 2H of
OCH2CH2O), 3.41 (2H, m, 2H of OCH2CH2O), 3.36 (3H, s,
OCH3). m/z: 880 [M þH]þ, 722.
8a: Using the general procedure on a 0.107 mmol scale yielded
8a (0.014 g, 15%) as a white solid. IR (film) 3445, 2971, 2934,
1732, 1656, 1552, 1451, 1378, 1178, 1081, 1053, 1035, 998,
900 cm-1. 1H NMR δ (Table 7) 2.77 (3H, d, J 5.0 Hz, NHCH3),
2.31 (1H, m, 1H of OCH2CH2CH2CON), 2.21 (1H, m, 1H of
OCH2CH2CH2CON), 1.91 (2H, m, OCH2CH2CH2CON). 13C
NMR δ (Table 8) 33.1, 26.2 (2C). m/z: 864 [MþH]þ, 706 (found
[MþH]þ, 863.5814; C44H82N2O14 requires [MþH]þ 863.5839).
8b: Using the general procedure on a 0.224 mmol scale yielded
8b (0.051 g, 26%) as a white solid. IR (film) 3434, 2971, 2936,
7j: Using the general procedure on a 0.131 mmol scale yielded
7j (0.086 g, 73%) as a white solid. 1H NMR δ (Table 7) 3.97 (1H,
m, NHCH), 3.93 (1H, 2H of CH(CH2CH2)2O), 3.43 (1H, t,
J 11.0 Hz, 2H of CH(CH2CH2)2O), 1.82 (2H, m, 2H of
CH(CH2CH2)2O), 1.59 (2H, m, 2H of CH(CH2CH2)2O). 13C
NMR δ (Table 8) 45.2, 66.9, 32.8. m/z: 906 [M þ H]þ, 748
(found [M þ H]þ, 905.5957; C46H84N2O15 requires [M þ H]þ
905.5946).
7k: Using the general procedure on a 0.131 mmol scale yielded
7k (0.083 g, 70%) as a white solid. IR (film) 3431, 2972, 2936,
1731, 1663, 1536, 1454, 1379, 1266, 1179, 1084, 1052, 1028, 997,
957, 899 cm-1. 1H NMR δ (Table 7) 7.33-7.23 (5H, m, ArH),
4.43 (2H, t, J 6.5 Hz, PhCH2), 8.26 (1H, m, ArH-5). 13C NMR δ
(Table 8) 138.2, 128.5, 127.6, 127.6, 42.9. m/z: 912 [MþH]þ, 754
(found [M þ H]þ, 911.5813; C48H82N2O14 requires [M þ H]þ
911.5839).
7l: Using the general procedure on a 0.079 mmol scale yielded
7ll (0.025 g, 35%) as a white solid. IR (film) 3446, 2972, 2934,
1730, 1538, 1455, 1413, 1379, 1279, 1178, 1083, 1056, 998,
900 cm-1. 1H NMR δ (Table 7) 9.53 (1H, d, J 1.0 Hz, ArH-3),
8.34 (1H, d, J 2.5 Hz, ArH-6), 8.26 (1H, m, ArH-5). 13C NMR δ
(Table 8) 147.8, 142.0, 140.3, 137.5. m/z: 900 [M þ H]þ, 742
1733, 1650, 1552, 1451, 1379, 1178, 1082, 1029, 999, 900 cm-1
.
1H NMR δ (Table 7) 2.79 (3H, d, J 5.0 Hz, NHCH3), 2.18 (2H,
m, CH2CH2CON), 1.68-1.59 (4H, m, OCH2CH2CH2CH2-
CON). 13C NMR δ (Table 8) 36.3, 29.3, 26.2, 23.1. m/z: 878
[M þ H]þ, 720 (found [M þ H]þ, 877.5978; C45H84N2O14
requires [M þ H]þ 877.5995).
8c: Using the general procedure on a 0.128 mmol scale yielded
8c (0.067 g, 59%) as a white solid. IR (film) 3417, 2935, 1732,
1650, 1553, 1452, 1378, 1178, 1082, 1055, 1035, 998, 900 cm-1
.
1H NMR δ (Table 7) 2.78 (3H, d, J 5.0 Hz, NHCH3), 2.14 (2H,
m, CH2CH2CON), 1.63-1.52 (5H, m, 5H of OCH2CH2CH2-
CH2CH2CON), 1.34 (1H, m, 1H of OCH2CH2CH2CH2-
CH2CON). 13C NMR δ (Table 8) 36.4, 29.6, 26.2, 25.8, 25.5.

6858 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Shaw et al.
Table 8. 13C NMR Resonances for Macrolide Portion of Compounds
7j-7m, 8a-8c, 9, and 10a
(3H, d, J 4.5 Hz, NCH3), 1.68 (1H, m, H-12). 13C NMR δ 178.0,
171.1, 102.7, 95.2, 93.8, 85.4, 78.1, 77.8, 76.1, 75.0, 73.2, 71.1,
70.4, 69.7, 65.9, 62.6, 53.0, 49.7, 44.0, 39.2, 37.6, 35.1, 33.2, 31.4,
25.9, 24.7, 21.9, 21.7, 21.5, 20.8, 20.4, 18.0, 13.1, 12.0, 10.2, 10.1,
9.6. m/z: 842 [M þ Na]þ, 820 [M þ H]þ, 662 (found [M þ H]þ,
819.5572; C42H78N2O13 requires [M þ H]þ 819.5577).
7j
7k
7l
7m
8a
8b
8c
9
10
C-1
C-2
177.6 177.4 177.5 177.4 177.1 177.1 177.0 177.7 177.1
44.3 44.0 44.2 44.2 44.5 44.4 44.3 44.5 44.2
77.6 77.3 77.8 77.8 77.9 77.9 77.6 77.5 77.1
42.5 43.2 43.4 43.5 43.9 43.8 43.9 44.2 44.0
84.1 84.5 84.5 84.3 84.6 84.0 83.7 85.0 84.8
75.9 75.7 76.1 76.1 75.3 75.1 75.1 75.2 75.4
37.3 37.9 38.0 37.9 38.0 37.8 38.0 37.6 38.4
31.9 31.8 31.2 31.2 31.4 31.6 31.3 30.6 30.3
94.1 94.0 93.9 93.7 92.6 92.8 92.9 91.8 91.9
32.0 32.4 32.4 32.3 32.3 32.3 32.3 32.3 32.5
65.9 70.0 69.3 69.1 70.2 70.0 70.0 70.2 70.1
74.2 74.2 74.1 74.1 74.5 74.1 74.0 73.9 74.0
77.3 77.3 77.6 77.5 77.3 76.7 77.2 77.0 77.1
21.9 21.5 22.3 22.3 22.5 22.6 22.2 22.2 22.5
11.2 11.4 11.5 11.5 11.5 11.6 11.5 11.3 11.7
13.6 13.2 13.2 13.2 13.3 13.4 13.2 13.3 12.8
C-3
C-4
In Vitro Assays. Motilin agonist potency and tachyphylaxis
were measured in a rabbit smooth muscle contractility assays as
previously reported.29,30 EC50 values are the concentration 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 administra-
tion and washout. hERG inhibition was measured at 37 ꢀC using
a stably transfected HEK cell line at expressing the hERG
mRNA.32 For routine screening, compounds were tested in
replicate at 30 and 300 μM. For measurement of the IC50 of 1,
3, and 7a, duplicate measurements were made at 1, 3, 10, 50, 100,
and 300 μM.
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-19
C-20
C-10
C-20
C-30
C-40
C-50
Antibacterial activity was assessed by determining the mini-
mal growth inhibitory concentration (MIC) of each com-
pound.42 For routine screening, the highly erythromycin-
sensitive strain Streptococcus pneumoniae ATCC6301 was used.
For 7a, a panel of ∼200 strains were used and were representa-
tive of those commonly found in the gut flora, and as well as
those which are commonly know to develop resistance to
antibiotics. The strains were isolated from a variety of clinical
specimens and are part of a collection maintained at the Clinical
Microbiology Laboratories at the University of Rochester
Medical Center and included approximately 10 isolates each
of the following strains: Enterococcus faecalis, Enterococcus
faecium, Micrococcus luteus, Staphylococcus aureus, Staphylo-
coccus coagulase, Streptococcus bovis, Streptococcus pneumo-
niae, Streptococcus pyogenes, Corynebacterium jeikeium,
Corynebacterium species (not jeikeium), Lactobacillus species,
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,
Hemophilus influenzae, Moraxella catarrhalis, Bacteroides
fragilis, Clostridium difficile, Clostridium perfringens, and Pro-
pionbacterium acnes.
9.2
9.3
9.2
9.1
9.1
9.1
9.1
9.0
9.3
16.8 16.8 17.1 17.2 16.5 16.4 16.4 16.6 16.6
15.6 15.0 15.5 15.5 15.1 14.9 14.8 15.3 14.7
102.4 102.4 102.3 102.2 102.1 101.9 101.9 101.9 102.0
70.0 70.0 70.1 70.1 70.2 70.2 70.2 70.1 70.1
62.4 62.2 62.3 62.2 62.0 62.0 62.0 62.0 62.0
32.7 32.9 32.9 32.9 32.9 32.9 33.0 32.9 32.9
69.3 69.4 69.4 67.5 69.5 69.4 69.4 69.5 69.4
NCH3 30.9 31.0 31.2 31.1 31.2 31.2 31.2 31.2 31.2
NCH
C-100
C-200
C-300
C-400
C-500
C-600
52.6 52.6 52.9 52.7 52.7 52.7 52.7 52.7 52.8
95.1 95.0 95.0 94.9 94.9 94.8 94.8 94.6 94.5
34.6 34.5 34.5 34.5 34.7 34.7 34.7 34.5 34.6
72.7 72.8 72.8 72.8 72.9 72.9 72.9 72.9 73.0
77.8 77.7 77.7 77.6 77.9 77.9 77.9 77.7 77.7
65.6 65.6 65.8 65.8 65.8 65.8 65.8 65.7 65.7
17.9 17.7 17.7 17.6 18.0 18.0 17.9 17.2 17.7
OCH3 49.3 49.3 49.3 49.3 49.3 49.3 49.3 49.3 49.3
C-90
71.5 72.0 70.9 70.9 71.2 71.5 71.7 71.8 71.9
169.1 169.8 168.8 170.0 173.3 173.3 173.6 167.9 167.9
C-900
a C-18, C-21, C-60, NCH(CH3)2, and C-700 are between 22.0 and
19.0 ppm.
Acknowledgment. We thank John R. Carney and Chau Q.
Tran for mass spectral analysis and Pieter B. Timmermans
and Robert Johnson for useful discussions.
m/z: 892 [M þ H]þ, 734 (found [M þ H]þ, 891.6127; C46H86-
N2O14 requires [M þ H]þ 891.6152).
Supporting Information Available: A full NMR data set is
included for compound 7a along with LC-MS and IR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
9: Using the general procedure on a 0.082 mmol scale yielded
9 (0.052 g, 69%) as a white solid. 1H NMR δ (Table 7) 7.81 (1H,
d, J 7.5 Hz, ArH-4 or ArH-6), 7.69 (1H, s, ArH-2), 7.38-7.30
(2H, m, ArH-4 or ArH-6 and ArH-5), 2.99 (3H, d, J 5.0 Hz,
NCH3). 13C NMR δ (Table 8) 138.2, 135.0, 129.6, 128.5, 127.1,
124.5, 26.8. m/z: 912 [MþH]þ, 754 (found [MþH]þ, 911.5838;
C48H82N2O14 requires [M þH]þ 911.5839).
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10 (0.027 g, 47%) as a white solid. IR (film) 3461, 2972, 1732,
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1
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14: Using the standard coupling procedure with 12 on a 0.66
mmol scale yielded 14 as a white solid. 1H NMR δ (Table 7) 2.70

Article
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