A structure-activity relationship of non-peptide macrocyclic histone deacetylase inhibitors and their anti-proliferative and anti-inflammatory activities

Article abstract of DOI:10.1016/j.bmc.2015.10.045

Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from 14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative activities and compound 26f (IC₅₀ 47.2 nM) with superior anti-inflammatory (IC₅₀ 88 nM) activity relative to SAHA.

Full text of DOI:10.1016/j.bmc.2015.10.045

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A structure–activity relationship of non-peptide macrocyclic histone
deacetylase inhibitors and their anti-proliferative and
anti-inflammatory activities
Subhasish Tapadar a,y, Shaghayegh Fathi a,y, Idris Raji a,y, Wilson Omesiete a, James R. Kornacki b,c
,
Sandra C. Mwakwari a, Masanori Miyata d, Kazunori Mitsutake d, Jian-Dong Li d, Milan Mrksich b,c
,
Adegboyega K. Oyelere a,
⇑
a School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
b Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
c Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
d Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for
cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been
reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from
14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc
chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate
the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and
investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative
and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative
activities and compound 26f (IC50 47.2 nM) with superior anti-inflammatory (IC50 88 nM) activity relative
to SAHA.
Received 27 August 2015
Revised 21 October 2015
Accepted 31 October 2015
Available online xxxx
Keywords:
Histone deacetylase (HDAC)
Histone deacetylase inhibitors (HDACi)
Non-peptide macrocyclic HDACi
Anti-cancer
Ó 2015 Elsevier Ltd. All rights reserved.
Structure activity relationship study (SAR)
Anti-inflammation
NTHi
NF-jB
1. Introduction
suberoylanilide hydroxamic acid (SAHA)6,7 and FK2288 approved
for the treatment of cutaneous T-cell lymphoma, Panobinostat
Histone deacetylases (HDACs) are an emerging therapeutic tar-
get for cancer and other diseases such as malaria and leishmania.1,2
Together with histone acetyltransferases (HATs), they regulate the
chromatin structure by controlling the acetylation state of histone
and Chidamide, approved for multiple myeloma,9,10 and Belinostat
recently granted accelerated approval for peripheral T cell lym-
phoma.11 Macrocyclic HDACi (Fig. 1b) possess the most complex
cap group moieties capable of optimal interactions with amino
acid residues near the entrance of the HDAC active site, an attri-
bute that is essential for the modulation of the biological activities
of these agents. Although they possess potent HDAC inhibition
activity (nanomolar range), the interest in their clinical develop-
ment has been hampered by the disadvantages associated with
the peptide group(s) present in the macrocycles and the difficulty
in the synthesis of strained ring frameworks for structure activity
relationship (SAR) studies.12
In previous studies, we have shown that macrocycles derived
from two 14-membered macrolide rings—clarithromycin (5e and
5f) and TE-802 (26e and 26f), and a 15-membered azalide ring—
azithromycin (17e and 17f), are excellent mimetics for the peptide
backbone of macrocyclic HDACi.13–15 The replacement of the amide
proteins as well as non-histone proteins such as tubulin, ERa, p53,
HSP90, NF-YA, and GATA-1.3 Histone deacetylase inhibitors
(HDACi) have been shown to cause growth arrest, differentiation,
and apoptosis in a variety of cancer cell lines.4 To date, several
classes of small molecule HDAC inhibitors—fitting a three-motif
pharmacophoric model, namely, a zinc binding group (ZBG), a
hydrophobic linker, and a recognition cap group5—have been
reported. Morever, many HDACi have been approved for
hematological malignancies (Fig. 1a). The approved HDACi are
⇑
Corresponding author. Tel.: +1 404 894 4047; fax: +1 404 894 2291.
E-mail address: aoyelere@gatech.edu (A.K. Oyelere).
These authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmc.2015.10.045
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

2
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
H
N
O
S
O
O
O
H
N
a
NHOH
N
H
NHOH
NHOH
N
H
O
O
Panobinostat
Belinostat
SAHA
N
O
N
O
HO
HO
HO
HO
OH
OH
HO
N
N
HO
O
O
O
O
NHOH
HN
O
O
NHOH
O
O
O
O
O
N
O
N
N
n
O
O
17e (n = 4)
17f (n = 5)
O
OH
OH
b
A
O
N
O
N
HO
HO
O
HO
O
HO
O
N
O
O
NHOH
N
O
N
N
N
N
N
N
O
n
NHOH
O
O
O
n
O
O
O
26e (n = 4)
26f
5e (n = 4)
5f (n = 5)
O
(n = 5)
O
O
O
OH
Figure 1. Representative HDACi: (a) Approved hydroxamic acid based HDACi, (b) selected examples of non-peptide (5e–f, 17e–f, 26e–f) macrocyclic HDACi.
moiety, by its bioisostere, triazole unit, increased the HDAC inhibi-
tory potency of matched compounds by almost 8-fold [17f
(HDAC1/2 IC50 13.9 nM vs A (HDAC1/2 IC50 107.1 nM)].14 Drawing
inspiration from the naturally occurring HDACi such as TSA (tri-
chostatin A) we have hitherto studied only the para-substitution
pattern of the aryl-triazole cap group of these non-peptide macro-
cyclic HDACi.14,15
To further understand the depth of the SAR of this class of HDAC
inhibitors, we investigated and disclosed herein the consequence
of (i) ortho-, meta, and para-substitution pattern of the triazole
cap group; (ii) varied methylene linker lengths; and (iii) point of
attachment of aryl-triazole cap group, on their biological activities.
We observed that the new compounds reported here possess anti-
HDAC, anti-proliferative and anti-inflammatory activities that are
highly dependent of the identity of the macrolide, the point of
attachment of the HDAC inhibition group and the methylene linker
lengths.
TBS-group afforded the desired compounds 24a–b, 25a–b, and
26a-d (Scheme 2).
Introduction of the ethynylbenzyl moiety to cladinose sugar of
clarithromycin 27 and azithromycin 28 was achieved in four
steps.20b Acetic anhydride treatment of clarithromycin 27 and
azithromycin 28 in dichloromethane gave selective 20-O-
acetylclarithromycin 29 and 20-O-acetylazithromycin 30. Corey-
Kim oxidation of 29 and 30 followed by Corey-Chaykovsky
epoxidation of intermediates 400-oxo-20-O-acetylclarithromycin
31 and 400-oxo-20-O-acetylazithromycin 32 yielded epoxy com-
pounds 33 and 34. Diastereoselective opening of epoxides 33
and 34 with 4-ethynylbenzyl-N-methyamine 35 in methanol,
followed by a concomitant acetyl group deprotection, gave key
intermediates 36 and 37 respectively. The alkynyl intermediates
36 and 37 were subjected to AAC reaction with various
TBS-protected azido hydroxamates 51a–f followed by removal
of TBS-group to give target molecules 38a–f and 39a–f in
moderate to good yields (Scheme 3).
We also synthesized macrocyclic-nonpeptide-hydroxamic acids
with dimethylamino methyl group placed in the C400 position of
cladinose sugar (Scheme 4), a substitution which may impact extra
acid-stability to the cladinose sugar glycosidic bond.20c Also, we
were interested in probing if this small change influenced the
HDAC inhibitory and antiproliferative activity profile. Syntheses
of the target compounds 49a–b and 50a–b were achieved from
intermediates 4 and 14 following the same route as described for
compounds 38a–f and 39a–f. The previously synthesized
compounds 5f, 17f, and 26f are included here as controls for each
macrolide group.
2. Chemistry
The syntheses of the target compounds are achieved following
the reaction routes shown in Schemes 1–4. The crucial ethynylben-
zyl moieties were installed at either the desosamine sugar
(azithromycin and clarithromycin) or N10 position of azithromycin
to furnish the requisite alkynyl-macrolides 4, 7 12, 13 and 14
following the literature procedure.14–17 Subsequently, copper(I)
catalyzed azide–alkyne-cycloaddition (AAC)18 reaction between
TBS-protected azidohydroxamates 51a–f and compounds 4, 7
and 12–14, followed by removal of TBS-group19 afforded the final
compounds 5a–d, 8a–f, 15a–b, 16a–b, and 17a–d (Scheme 1).
The triketolide derived hydroxamic acid compounds were syn-
thesized in two steps starting from previously reported intermedi-
ate 30-desmethyltricyclic ketolide 18.15,20a Reductive amination
reactions between 18 and 2-ethynylbenzaldehyde 19, 3-ethynyl-
benzaldehyde 20, 4-ethynylbenzaldehyde 6 yielded 30-(2-ethynyl-
benzyl)tricyclic ketolide 21, 30-(3-ethynylbenzyl)tricyclic ketolide
22, and 30-(4-ethynylbenzyl)tricyclic ketolide 23 in 53%, 83%, and
65% respectively. AAC reaction between TBS-protected azidohy-
droxamates 51a–f and compounds 21–23 followed by removal of
3. Results and discussion
All the new and six previously synthesized controls (5e, 5f, 17e,
17f, 26e, 26f)14,15 compounds were tested against recombinant
class I (HDAC1 and HDAC8) and class IIb (HDAC6) enzymes to eval-
uate their anti-HDAC activities. HDAC activity was determined by
the label-free mass spectrometry-based SAMDI assay.21 As antici-
pated from previous observations, most of these new analogs were
less active against HDAC8 except for compounds 5f (IC50 713 nM,
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S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
X
MsO
R'
N
N
O
HO
HO
OCH3
HO
HO
HO
OR
HO
N
OHC
HO
HO
OH
HO
N
6
3
a
O
O
O
O
d
O
O
O
O
O
O
O
O
from 1
2
from
O
O
O
O
O
O
O
O
OH
OH
OH
7
1. X = -CO-; R = CH3; R' = H
2. X = -NH-CH2-; R = H; R' = CH3
9: X = -N(CH3)-CH2-; R = R' = H
4
NHOTBS
NHOTBS
N3
N3
n
O
n
b,c
b, c
O
51a-d
51a-f
MsO
O
O
a
n
NHOH
N
10 (ortho)
N
N
from 9
11 (meta)
HO
HO
OCH3
HO
N
3 (para)
O
n
O
O
O
NHOH
N
N
N
HO
HO
OH
HO
O
O
N
N
N
O
HO
HO
OH
HO
O
O
N
O
O
O
OH
O
O
O
O
O
5a - d: n = 1 - 4
O
O
O
O
OH
N
O
HO
HO
OH
N
HO
12 (ortho)
13 (meta)
14 (para)
O
OH
15a
(ortho); n = 5
NHOTBS
N3
n
O
15b (ortho); n = 6
16a (meta); n =5
O
N
8a - f; n = 1 - 6
O
N
51a-f
16b (meta); n = 6
17a - d (para); n = 1- 4
N
O
O
O
b, c
O
n
NHOH
O
OH
Scheme 1. Reagents and conditions: (a) Hünig’s base, DMSO, 85 °C, 3 h; (b) CuI (15 mol %), Hünig’s base, THF–DMSO (1:1), 40 °C, 12 h; (c) CsF, MeOH, rt, 30 min; (d)
NaBH3CN, AcOH, DMF, 70 °C, 7 h.
OHC
O
n
N
N3
N
N
N
O
O
NHOTBS
NH
O
N
O
19 (ortho)
20 (meta)
6 (para)
HO
O
HO
O
HO
O
N
51a f
-
NHOH
N
N
n
O
O
O
b, c
a
N
O
N
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
24a (ortho); n = 5
24b (ortho); n = 6
21 (ortho)
22 (meta)
18
25a
(meta); n =5
23
(para)
25b (meta); n = 6
26a - d (para); n = 1- 4
Scheme 2. Reagents and conditions: (a) borane–pyridine complex, AcOH, MeOH, rt, 9 h. (b) CuI (15 mol %), Hünig’s base, THF–DMSO (1:1), 40 °C, 12 h; (c) CsF, MeOH, rt,
30 min.
Table 1), 16a and 17f (IC50s 604 nM and 314 nM, respectively,
Table 2), and 25b (IC50 310 nM, Table 4).
HDAC1 (5e–f, 8e–f, 15a–b, 16a–b, 17e–f, 25a–b, 26e–f, 38e–f,
39e–f, 49a–b, 50a–b) and HDAC6 (5e–f, 8e–f, 15a–b, 16a–b,
17e–f, 25a–b, 26e–f, 38e–f, 39e–f, 49a–b, 50a–b) inhibitory
potency (Tables 1–4). Conversely, compounds having one to four
methylene spacers were either inactive or very poorly active
(5a–d, 8a–d, 17a–d, 26a–d, 38a–d, and 39a–d) (Tables 1–4).
Introduction of the dimethylamino methyl group at cladinose
sugar C4 position had a modest effect on HDAC1 inhibitory activi-
ties in both clarithromycin (49a–b) and azithromycin (50a–b) ser-
ies when compared to their analogs 5e–f and 17e–f. However,
HDAC6 inhibition was affected substantially in the case of the clar-
ithromycin compounds, with a 5–10 fold drop in potency (49a: IC50
32.9 nM; 49b: IC50 31.4 nM compared to 5e: IC50 3.61 nM, 5f: IC50
Analysis of the HDACs 1 and 6 inhibitory effects of these com-
pounds revealed an interesting trend that is largely dependent
on the macrolide template, the substitution pattern on the aryl
cap group and the length of the methylene spacer-group separat-
ing the triazolyl group from the hydroxamate moiety. For com-
pounds in the same macrolide series, an increase in the length of
the methylene spacer (C1–C6) resulted in gradual increase in
HDAC1 and HDAC6 inhibitory potencies. In most cases, the maxi-
mum change was observed at five methylene spacers and potency
plateaued at six methylene spacers. In general, compounds having
five or six methylene spacers showed low to mid nanomolar
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

4
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
X
X
X
X
R'
HO
HO
OR
AcO
N
R'
R'
N
HO
HO
OR
AcO
N
R'
HO
HO
OR
AcO
HO
HO
OR
HO
HN
N
35
O
O
O
d
b
c
a
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
OH
27. X = -CO-; R = R' = CH3
28. X = -N(CH3)-CH2-; R = H; R' = CH3
31. X = -CO-; R = R' = CH3
32. X = -N(CH3)-CH2-; R = H; R' = CH3
33. X = -CO-; R = R' = CH3
34. X = -N(CH3)-CH2-; R = H; R' = CH3
29. X = -CO-; R = R' = CH3;
30. X = -N(CH3)-CH2-; R = H; R' = CH3
X
X
R'
N
R'
HO
HO
O
n
OR
HO
HO
HO
OR
HO
N
N3
NHOTBS
51a-f
O
O
O
O
O
O
e, f
O
O
O
O
O
O
O
O
HO
HO
N
N
N
N
N
O
NHOH
n
n
= 1 - 6
38a-f. X = -CO-; R = R' = CH3
39a-f. X = -N(CH3)-CH2-; R = H; R' = CH3
36. X = -CO-; R = R' = CH3
37. X = -N(CH3)-CH2-; R = H; R' = CH3
Scheme 3. Reagents and conditions: (a) CH2Cl2, Ac2O, rt, 3 h; (b) NCS, DMS, TEA, CH2Cl2, ꢀ15 °C, 4.5 h; (c) (CH3)3SO+Iꢀ, NaH, DMSO, THF, rt, 4 h; (d) KI, MeOH, 60 °C, 6 h; (e)
CuI (15 mol %), Hünig’s base, THF, rt, 12 h; (f) CsF, MeOH, rt, 2 h.
X
X
X
HO
HO
HO
HO
HO
HO
OR
AcO
N
OR
HO
N
OR
AcO
N
a or b
O
O
d
O
O
O
O
c
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
42. X = -CO-; R =CH3; R = CH3
40. X = -CO-; R =CH3; R = CH3
41. X = -N(CH3)-CH2-; R = H
4. X = -CO-; R =CH3; R = CH3
43
. X = -N(CH3)-CH2-; R = H
14
. X = -N(CH3)-CH2-; R = H
X
X
X
O
n
N3
HO
HO
HO
HO
HO
HO
NHOTBS
OR
HO
N
OR
N
OR
HO
N
AcO
HN
51e-f
O
46
O
O
O
O
O
O
O
O
g, h
O
NHOH
O
O
e, f
N
N
n
O
O
N
O
O
O
O
N
N
O
n = 5, 6
O
O
O
HO
O
HO
44. X = -CO-; R =CH3; R = CH3
45. X = -N(CH3)-CH2-; R = H
49a-b. X = -CO-; R =CH3; R = CH3
50a-b. X = -N(CH3)-CH2-; R = H
47
. X = -CO-; R =CH3; R = CH3
48. X = -N(CH3)-CH2-; R = H
Scheme 4. Reagents and conditions: (a) for 40: acetic anhydride, CH2Cl2, rt, 3 h; (b) for 41: acetic anhydride, CH2Cl2, 40 °C, 48 h; (c) NCS, DMS, TEA, CH2Cl2, ꢀ15 °C, 6 h; (d)
(CH3)3SO+Iꢀ, NaH, DMSO, THF, rt, 4 h; (e) KI, MeOH, 60 °C, 6 h; (f) MeOH, 90 °C, 3 days; (g) CuI (15 mol %), Hünig’s base, THF, rt, 12 h; (h) CsF, MeOH, rt, 2 h.
6.67 nM). Attachment of the aryl-triazolyl cap group to cladinose
sugar (38a–f and 39a–f) also influenced HDAC1 and HDAC6 inhibi-
tory activities. In the clarithromycin series, compound 38c, in
which the hydroxamic acid moiety was separated from aryl–tria-
zole cap by three methylene groups, showed a five-fold decrease
in HDAC6 (IC50 361 nM) inhibitory potency compared to the anal-
ogous desosamine modified compound 5c (IC50 70.9 nM) and was
6.67 nM, respectively). Moreover, the HDAC1 inhibitory activity
of 38f was eight-fold higher than that of 5f (IC50 23.9 nM vs IC50
207 nM, respectively). In azithromycin series, the only noticeable
change was exhibited by compound 39f which showed a four-fold
loss in HDAC6 inhibition potency, relative to the analogous control
compound 17f (IC50 31.3 nM vs 7.29 nM, respectively).
Docking studies on compounds 8a–f (azithromycin-derived
analogs with aryl-triazolyl cap group attached to N10 position of
azithromycin), using AutoDock Vina as previously reported,14,15,20d
predicted that compounds with 4–6 methylene spacers would
have HDAC1 and HDAC6 inhibitory activities (Supplementary
information). HDAC inhibition data presented in Table 3 validated
completely inactive against HDAC1 (27% at 10
lM for 38c vs
9.91 M IC50 value for 5c). Interestingly for 38f, in which the
l
hydroxamic acid moiety was separated from the aryl–triazole cap
by six methylene groups, the HDAC6 inhibitory activity increased
by two fold compared to control compound 5f (IC50 2.85 nM vs
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 1
representative members of each group against two transformed
cell lines—lung (A549) and breast (MCF-7) cancers—and one nor-
mal cell line (Vero—monkey kidney epithelial cell). We selected
compounds with diverse anti-HDAC activities, ranging from those
with weak HDAC inhibition activities to those which potently inhi-
bit the HDAC isoforms tested. SAHA and the previously disclosed
control compounds 5f, 17f and 26f are all cytotoxic to the two can-
cer cell lines (Table 5). We observed that compounds 8d, 15b, 17c
and 24b are devoid of antiproliferative activities up to the maxi-
mum tested concentration. This observation may not be surprising
since these compounds either lack or poorly inhibit HDAC1. Sur-
prisingly however, compounds 25a and 39f, despite their good
anti-HDAC1 activities, are non-cytotoxic against A549 and MCF-7
cells. This result is in contrast to the effect of 16b which, despite
having a similar anti-HDAC1 activity as 25a and 39f, is robustly
anti-proliferative against the two cancer cell lines. The reason for
the lack of whole cell effect of 25a and 39f is not obvious from this
study. However, the triketolide-derived compound 25b, an analog
of 25a with a single extra methylene linker but not much different
anti-HDAC1 activity, was cytotoxic to both cancer cell lines with a
strong preference for the MCF-7 cell (IC50 ꢁ900 nM). All other
strongly HDAC inhibiting compounds tested (8f, 16b, 25b, 38f,
49b and 50b) possess varying degree of anti-proliferative activities
against A549 and MCF-7 cell lines. For analogous compounds
(same methylene-linker length), the macrolide type and the points
of attachment to the macrolide templates are strong determinants
of potency. Specifically, the azithromycin-derived desosamine
functionalized compound 17f, despite its weaker anti-HDAC1
activity, is slightly more cytotoxic than the analogous N-10 modi-
fied compound 8f and the cladinose sugar amine functionalized
50b. However, amine functionalization of the cladinose sugar
enhanced the cytotoxicity of the clarithromycin compound 49b
relative to analogous azithromycin 50b. In fact, the cladinose ring
is the optimum point of attachment of the HDAC inhibiting moiety
to the clarithromycin template as the resulting HDACi 38f is the
most potent among the compounds tested for anti-proliferative
activity. In contrast, the analogous azithromycin compound 39f is
inactive (Table 5).
HDAC1, HDAC6, and HDAC8 inhibition activities (IC50 in nM) of clarithromycin
derived hydroxamic acid compounds
Compound
n
HDAC1
HDAC6
>10
919 45
70.9 12.6
120 19
3.61 1.01
6.67 1.23
5350 190
3560 370
361 67
269 100
5.89 2.73
2.85 1.07
32.9 2.4
31.4 5.5
HDAC8
>10 lM
2450 600
986 81
877 125
1180 310
713 748
7120 2130
10200 1600
2530 880
1820 400
985 325
1840 460
3200 820
2210 300
5a
5b
5c
5d
5e
5f
38a
38b
38c
38d
38e
38f
49a
49b
1
2
3
4
5
6
1
2
3
4
5
6
5
6
>10
>10
l
l
M
M
lM
9910 3000
4870 500
533 123
207 84
>10
>10
>10
l
l
l
M
M
M
3690 860
652 130
23.9 3.3
201 17
33.3 8.1
the docking prediction as compound 8f emerged the best in this
series with low nonamolar HDAC1 and single digit nanomolar
HDAC6 inhibition activities.
To further delineate the SAR of this class of HDACi, with synthe-
sized compounds 15a–b, 16a–b, 24a–b and 25a–b, azithromycin-
and triketolide-derived analogs having ortho- and meta-substitution
patterns at the aryltriazolyl cap group. For the azithromycin series,
the ortho-substituted compounds 15a–b, and meta-substituted
compound 16b have attenuated anti-HDAC activities relative to
the corresponding para-substituted compounds 17e–f. The meta-
substituted, five methylene-linked compound 16a is slightly more
potent than the corresponding para-substituted compound 17e
against HDAC1 while the trend is reversed against HDAC6.
However, the two compounds have identical HDAC8 inhibition
activities (Table 2). The triketolide-based ortho- and meta-
substituted compounds 24a–b and 25a–b are mostly less active
against the corresponding para-substituted compounds 26e–f with
the ortho-substituted compounds 24a–b devoid of HDAC1 inhibi-
tion activity. An exception within this series is compound 25b
which is approximately 2- and 5-fold more potent than the
analogous 26e against HDAC6 and HDAC8 respectively (Table 4).
To obtain preliminary information about tumor cell selectivity,
we tested these compounds against the non-transformed Vero cell
3.1. Cell growth inhibitory assay
Table 3
N
10-modified
To verify if the anti-HDAC activities of these macrolide-derived
hydroxamates translate to anti-proliferative activities, we tested
HDAC1, HDAC6, and HDAC8 Inhibition Activities (IC50 in nM) of
azithromycin derived hydroxamic acid compounds
Compound
n
HDAC1
HDAC6
>10
1230 300
620 110
24.5 7.4
10.3 3.8
4.72 .20
HDAC8
8a
8b
8c
8d
8e
8f
1
2
3
4
5
6
>10
>10
>10
lM
lM
lM
lM
>10
>10
l
l
M
M
Table 2
2770 580
HDAC1, HDAC6, and HDAC8 inhibition activities (IC50 in nM) of azithromycin derived
hydroxamic acid compounds
2550 550
145 20
16.1 2.5
>10
lM
2370 890
1700 190
Compound
n
HDAC1
HDAC6
HDAC8
15a
15b
16a
16b
17a
17b
17c
17d
17e
17f
39a
39b
39c
39d
39e
39f
5
6
5
6
1
2
3
4
5
6
1
2
3
4
5
6
5
6
613 57
923 238
109 12
101 15
178 30
4300 2160
1280 400
604 161
30.9 12.4
43.8 1.8
41.0 4.6
5660 320
2170 200
106 13
145 42
14.3 .6
7.29 .56
Table 4
1440 140
HDAC1, HDAC6, and HDAC8 inhibition activities (IC50 in nM) of TE-802 derived
hydroxamic acid compounds
>10
>10
>10
lM
lM
lM
>10
>10
>10
lM
lM
lM
Compound
n
HDAC1
HDAC6
HDAC8
1650 456
316 61
68.6 3.3
5380 650
644 244
314 90
24a
24b
25a
25b
26a
26b
26c
26d
26e
26f
5
6
5
6
1
2
3
4
5
6
>10
>10
l
l
M
M
248 20
1190 300
2450 380
3230 960
310 47
2850 550
>10 lM
2240 440
3210 890
2030 200
1500 260
75.4 6.9
17.5 2.2
3.76 .17
109 12
54.5 11.1
>10
>10
>10
lM
lM
lM
>10
lM
4510 660
2230 560
1180 210
709 141
786 176
817 308
1090 240
749 98
2030 300
181 24
>10
>10
>10
lM
lM
lM
>10
l
M
1730 220
194 24
77.2 4.0
8.90 .34
6.99 .18
531 79
203 35
50.4 8.4
160 66
79.5 54
95.3 12.3
31.9 1.2
31.3 1.3
14.7 1.8
18.9 3.4
269 72
73.1 4.5
20.8 5.7
50a
50b
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6
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
implicated in the regulation of inflammation and HDACi25 such
as SAHA,26 trichostatin A (TSA),27 butyrates,28 and MS-275,29 atten-
Table 5
Anti-proliferative activity of selected HDAC inhibitors (IC50 values in l
M)a
uate cellular inflammation processes through inhibition of NF-jB
Compound
A549
MCF-7
Vero
activation and or blockage of pro-inflammatory cytokine release.30
In addition to their antibiotic activities, the macrolide templates
(azithromycin, and clarithromycin) for the HDACi described herein
have intrinsic anti-inflammatory and immunostimulatory activi-
ties. To test if these macrolide-derived HDACi preserve these useful
properties and additively or synergistically enhance the latent
anti-inflammatory activity of HDACi, we evaluated their anti-
inflammatory activities in BEAS-2B cell infected with nontypeable
5f
8d
8f
2.29 0.73
NI
8.28 0.96
NI
4.09 0.46
NI
2.32 0.53
NI
2.86 0.10
NI
6.30 0.58
NI
2.37 0.32
NI
4.08 1.03
NI
4.90 0.34
NT
6.66 0.21
NT
8.35 1.34
NT
5.90 0.18
NT
NT
>20
NT
1.55 0.12
NT
15b
16b
17c
17f
24b
25a
25b
26f
38f
39f
49b
50b
SAHA
NI
NI
7.48 0.23
2.19 0.10
0.99 0.08
NI
3.58 0.79
6.80 0.50
5.00 0.24
0.86 0.18
1.98 0.15
0.69 0.05
NI
1.43 0.17
5.92 1.82
3.27 0.05
Haemophilus influenza (NTHi) using NF-j
B luciferase assay31 NTHi
is a Gram-negative bacterium which causes infection in the human
respiratory tract.32,33 Upon infection by NTHi, transcriptional regu-
2.08 0.14
5.73 0.49
1.03 0.09
lator, NF-jB, in human epithelial cell is strongly activated by
translocating from cytoplasm to nucleus and consequently up-reg-
ulating certain pro-inflammatory cytokines such as IL-1b, IL-6, and
a
Each value is obtained from a duplicate of three simultaneous experiments.
TNF-
activity, we treated them with NTHi infected BEAS-2B cells at
M. We observed that compounds lacking or those with weak
anti-HDAC activities did not suppress NF- B activation while those
compounds with potent anti-HDAC activities suppressed NF-
activation to varying degrees which closely correlate with their
HDAC inhibition potency (Supporting information, Fig. S1).
We then determined the IC50 values of selected compounds
a. To pre-screen these compounds for their effect on NF-jB
NI = no inhibition, NT = not tested.
1
l
j
jB
Table 6
Anti-inflammatory activity (NF-
jB inhibition) of selected HDAC inhibitors
*
Compound
IC50 (nM)
Imax (%)
5e
5f
8f
785
243
244
609
280
NA
47.2
785
197
368
260
575
88
45.1
35.9
31.4
43.3
41.4
71
35.3
50.7
35.6
46.1
33.4
43.4
37.4
which suppressed NF-jB activation in the pre-screening. The
tested compounds were selected from each of the three macrolide
templates reported here and we used SAHA as a positive control.
We observed that these compounds suppressed the NTHi-induced
16b
17f
25a
26f
38e
38f
39e
49b
50b
SAHA
NF-
The only exception is 25a, which despite suppressing NF-
vation at 1 M, showed no dose-dependent effect. Based on IC50
j
B activation with IC50 ranging from low to high nanomolar.
jB acti-
l
,
the TE-802-derived 26f is the most potent among these com-
pounds and it is 2-fold more potent than SAHA (Table 6). In addi-
tion to 26f, compounds 5f, 8f, 38f and 49b have lower Imax value
compared to SAHA, implying that at maximum concentration of
*
1 lM, these compounds suppressed NF-jB activity more than
Imax (%) at 1 lM.
SAHA. Interestingly, the starting macrolide templates did not exhi-
bit any anti-inflammatory activity in this assay as their relative
percentage luciferase activity was indistinguishable from no drug
treatment in presence of NTHi (100%). To further confirm the
mechanism of anti-inflammatory activities of this class of macro-
lide HDACi, we performed Q-PCR analysis to determine the effect
of these HDACi on the expression levels of mRNAs of inflammatory
cytokines known to be NTHi-inducible.40,41 We observed that rep-
resentative macrolide-derived HDACi 8f, 26f and 38f more signifi-
line. The control compound SAHA is not tumor cell selective as it is
about 3–5 folds more cytotoxic to the Vero cell compared to the
two tumor cell lines. In contrast, the macrolide-derived com-
pounds are either significantly less cytotoxic or equally cytotoxic
to the transformed and normal cells tested. Compound 25b has a
selectivity edge over others, being 2.7-fold and 23-fold more selec-
tive for A549 and MCF-7 respectively (Table 5).
cantly suppressed NTHi-induced TNF-
expression relative to SAHA in BEAS-2B cells (Fig. 2). Collectively,
these data further suggest that the suppression of NF- B activation
a, IL-1a, IL-1b mRNA
3.2. Anti-inflammatory activity assay
j
Inflammation is a salient factor in cancer, particularly lung can-
cer and many other chronic lung diseases.22–24 HDACs have been
induced by these compounds is derived from their HDAC inhibition
activities.
Figure 2. HDACi suppress NTHi-induced expression of cytokines. BEAS-2B cells were pretreated with HDAC inhibitor (8f, 26f, 38f; 1
lM) or SAHA (1 lM) or DEX (0.1 lM) for
2 h followed by 1.5 h stimulation with NTHi, and cytokines mRNA (TNF- , IL-1
a
a
and IL-1b) expressions were analyzed. Data are mean STD (n = 3); **p <0.01, ***p <0.005. Data
are representative of three independent experiments. CON = BEAS-2B cells treated with DMEM control; NTHi = BEAS-2B cells treated with NTHi.
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S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The crude was purified by column chromatography (Silica gel, 15%
ethyl acetate in hexane) to afford the target compound 51a
(438 mg, 35%) as colorless oil. 1H NMR (CDCl3, 400 MHz) d (ppm)
8.29 (br s, 1H), 3.97 (s, 2H), 0.97 (s, 9H), 0.16 (s, 6H).
4. Conclusion
We have synthesized diverse series of non-peptide macrocyclic
hydroxamic acid based HDAC inhibitors derived from three macro-
lide skeletons to further explore the SAR of this class of HDACi. Sev-
eral of these compounds exhibited nanomolar anti-HDAC activity
against recombinant HDAC1 and HDAC6 enzymes. Among the
new compounds tested for whole cell activity, compounds 16b,
38f, and 49b potently inhibited lung cancer cell line (A549) while
16b, 25b, 38f, and 49b potently inhibited breast cell line (MCF-
7). Unlike SAHA which is much more toxic to the non-transformed
Vero cells, these macrolide-derived compounds are either
significantly less cytotoxic to the Vero cells or equally cytotoxic
to the transformed and normal cells tested with compound 25b
being the most tumor cell line selective. Also, many of these
compounds exhibited anti-inflammatory activity in NTHi infected
BSAS-2B cells and a lead compound (26f) is 2-fold more potent
than SAHA.
5.1.2. 3-Azido-N-((tert-butyldimethylsilyl)oxy)propanamide
(51b)
3-azidopropanoic acid 5437 (413 mg, 3.59 mmol) and O-(tert-
butyldimethylsilyl)hydroxylamine 59 (1.38 g, 4.31 mmol) were
subjected to same reaction condition as described for the synthesis
of 51a, afforded 51b as colorless oil (351 mg, 40%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.78 (br s, 1H), 3.62 (t, J = 6.9 Hz, 2H), 2.30 (t,
J = 6.9 HZ, 2H), 0.93 (s, 9H), 0.16 (s, 6H).
5.1.3. 4-Azido-N-((tert-butyldimethylsilyl)oxy)butanamide
(51c)
4-Azidobutanoic acid 5538 (539 mg, 4.18 mmol) and O-(tert-
butyldimethylsilyl)hydroxylamine 59 (1.61 g, 5.02 mmol) were
subjected to same reaction condition as described for the synthesis
of 51a, afforded 51c as colorless oil (745 mg, 68%). 1H NMR (CDCl3,
400 MHz) d (ppm)7.79 (br s, 1H), 3.33 (t, J = 6.9 Hz, 2H), 2.17 (t,
J = 6.9 Hz, 2H), 1.89 (q, J = 6.9 Hz and 4.3 Hz, 2H), 0.93 (s, 9H),
0.16 (s, 6H).
5. Experimental
5.1. Materials and methods
All commercially available starting materials were used without
further purification. Clarithromycin and azithromycin were pur-
chased from Greenfield Chemicals. 2-Ethynylbenzyl alcohol, 3-bro-
mobenzaldehyde, and 4-ethynylbenzyl alcohol were purchased
from Sigma–Aldrich. Reaction solvents were either high perfor-
mance liquid chromatography (HPLC) grade or American Chemical
Society (ACS) grade and used without further purification. Analtech
silica gel plates (60 F254) were used for analytical TLC, and Analtech
5.1.4. 5-Azido-N-((tert-butyldimethylsilyl)oxy)pentanamide
(51d)
5-Azidopentanoic acid 5638 (427 mg, 2.98 mmol) and O-(tert-
butyldimethylsilyl)hydroxylamine 59 (877 g, 5.96 mmol) were
subjected to same reaction condition as described for the synthesis
of 51a, afforded 51d as colorless oil (284 mg, 35%). 1H NMR (CDCl3,
400 MHz) d (ppm) 8.22 (br s, 1H), 3.25 (t, J = 6.9 Hz, 2H), 2.27 (br s,
2H), 1.63 (m, 4H), 0.91 (s, 9H), 0.16 (s, 6H).
preparative TLC plates (UV 254, 2000 lm) were used for purifica-
tion. UV light and anisaldehyde/iodine stain were used to visualize
the spots. 200–400 Mesh silica gel was used in column chromatog-
raphy. Nuclear magnetic resonance (NMR) spectra were recorded
on a Varian-Gemini 400 MHz or Bruker 500 MHz magnetic reso-
nance spectrometer. 1H NMR Spectra were recorded in parts per
million (ppm) relative to the residual peaks of CHCl3 (7.24 ppm)
in CDCl3 or CHD2OD (4.78 ppm) in CD3OD or DMSO-d5 (2.49 ppm)
in DMSO-d6. 13C spectra were recorded relative to the central peak
of the CDCl3 triplet (77.0 ppm) or CD3OD septet (49.3 ppm) or
DMSO-d6 septet (39.7 ppm) and were recorded with complete
hetero-decoupling. Original ‘fid’ files were processed using
MestReNova LITE (version 5.2.5–5780) program. High-resolution
mass spectra were recorded at the Georgia Institute of Technology
mass spectrometry facility in Atlanta. 30-Desmethylclarithromycin
(1), 30-desmethylazithromycin (9), 30-desmethyltricyclic ketolide
(18), 2-ethynylbenzyl methanesulfonate (10), 3-ethynylbenzyl
methanesulfonate (11), 4-ethynylbenzyl methanesulfonate (3),
4-ethynylbenzaldehyde (6), 30-O-acetylclarithromycin (29) were
synthesized as we previously reported.14,15,34
5.1.5. 6-Azido-N-((tert-butyldimethylsilyl)oxy)hexanamide
(51e)
6-Azidoheptanoic acid 5738 (1.08 g, 6.87 mmol) and O-(tert-
butyldimethylsilyl)hydroxylamine 59 (2.20 g, 6.87 mmol) were
subjected to same reaction condition as described for the synthesis
of 51a, afforded 51f as colorless oil (1.62 g, 82%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.64 (br s, 1H), 3.24 (t, J = 6.9 Hz, 2H), 2.35 (br
s, 2H), 1.61 (dq, J = 21.9 and 7.2 Hz, 4H), 1.39 (dd, J = 15.1 and
8.0 Hz, 2H), (0.93 (s, 9H), 0.16 (s, 6H).
5.1.6. 7-Azido-N-((tert-butyldimethylsilyl)oxy)heptanamide
(51f)
7-Azidoheptanoic acid 5838 (1.01 g, 5.90 mmol) and O-(tert-
butyldimethylsilyl)hydroxylamine 59 (1.64 g, 8.93 mmol) were
subjected to same reaction condition as described for the synthesis
of 51a, afforded 51e as colorless oil (1.28 g, 72%). 1H NMR (CDCl3,
400 MHz) d (ppm) 8.03 (br s, 1H), 3.22 (t, J = 6.9 Hz, 2H), 2.22 (br
s, 2H), 1.62 (m, 4H), 1.22 (m, 4H), 0.93 (s, 9H), 0.16 (s, 6H). 13C
NMR (CDCl3, 100 MHz) d (ppm) 171.8, 51.5, 33.2, 28.6, 26.6, 25.8,
25.4, 18.4, -5.1. HRMS (ESI) m/z Calcd for C13H20O2N4Si [M+H+]:
301.2054, found 301.2050.
5.1.1. 2-Azido-N-((tert-butyldimethylsilyl)oxy)acetamide (51a)
2-Azidoacetic acid 5335 (540 mg, 5.34 mmol) was dissolved in
anhydrous dichloromethane (20 mL). The solution was cooled to
0 °C and then TBTU (2.06 g, 6.41 mmol) was added and the solution
was stirred for another 15 min at 0 °C. After that O-(tert-
butyldimethylsilyl)hydroxylamine 5936 (1.28 g, 6.95 mmol) dis-
solved in 5 mL of anhydrous dichloromethane containing Hünig’s
base (2 mL, 10.69 mmol) was added and the resulting reaction mix-
ture was stirred at room temperature for 12 h. Reaction was
quenched by adding water (5 mL) and the organic layer was sepa-
rated. The aqueous layer was extracted twice with dichloromethane
(10 mL) and the combined organic layer was washed with saturated
aqueous NaHCO3 solution (5 mL), water (10 mL), brine (10 mL),
5.1.7. (Clarithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxyac-
etamide (5a)
30-N(Desmethyl)-30-N-(4-ethynylbenzyl)clarithromycin
4
(0.15 g, 0.18 mmol) and 2-azido-N-((tert-butyldimethylsilyl)oxy)
acetamide 51a (0.073 g, 0.318 mmol) are dissolved in anhydrous
THF and purged with argon for 10 min. DIPEA (0.06 ml, 0.35 mmol)
and CuI (0.017 g, 0.088 mmol) were then added to the mixture and
purged further for another 20 min. The resulting suspension was
stirred at room temperature for 12 h. Reaction was quenched with
a solution of 4:1 satd. Aqueous NH4Cl/NH4OH and extracted with a
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8
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
mixture of 10% MeOH in DCM. Combined organic layer was dried
over anhydrous Na2SO4 and concentrated in vacuo. The crude
was subjected to next reaction without further purification.
The crude was dissolved in anhydrous methanol (2 mL) along-
side caesium fluoride (0.04 g, 0.27 mmol) and left to stir under Ar
until TLC showed complete conversion (1.5 h). Water was added
to quench the reaction and the aqueous layer extracted with
DCM. Combined organic layer was dried over Na2SO4 and concen-
trated in vacuo. Crude obtained was purified by preparative chro-
matography (Silica gel, 12:1:0.1 DCM–MeOH–NH4OH) to give
target compound 5a as light yellow solid (0.068 g, 40%). 1H NMR
(400 MHz, CD3OD) d (ppm) 8.39 (s, 1H), 7.84 (d, J = 8.1 Hz, 2H),
7.50 (d, J = 8.2 Hz, 2H), 5.12 (d, J = 3.7 Hz, 2H), 4.53 (d, J = 7.2 Hz,
1H), 4.08 (dq, J = 12.6, 6.9 Hz, 3H), 3.80–3.68 (m, 5H), 3.42 (d,
J = 7.5 Hz, 1H), 3.30 (dt, J = 3.3, 1.6 Hz, 4H), 3.17 (s, 3H), 3.15–
3.07 (m, 2H), 3.04 (s, 1H), 3.01 (d, J = 9.5 Hz, 2H), 2.94–2.86 (m,
1H), 2.59 (d, J = 9.0 Hz, 1H), 2.44 (s, 3H), 2.38 (d, J = 15.2 Hz, 1H),
2.15 (s, 1H), 2.04–2.00 (m, 2H), 1.98–1.82 (m, 4H), 1.67 (d,
J = 13.0 Hz, 1H), 1.52 (ddd, J = 18.2, 13.3, 6.1 Hz, 3H), 1.38 (d,
J = 17.4 Hz, 4H), 1.24 (dq, J = 12.9, 8.0 Hz, 13H), 1.13 (dt, J = 12.5,
8.6 Hz, 16H), 0.85 (dd, J = 8.4, 6.3 Hz, 3H). 13CNMR (126 MHz,
CDCl3) d (ppm) 175.8, 147.2, 130.0, 125.8, 122.2, 102.5, 96.1,
81.2, 78.5, 78.3, 74.2, 72.7, 70.8, 69.1, 68.2, 65.7, 64.3, 57.7, 50.6,
49.4, 45.1, 39.2, 39.1, 37.3, 36.7, 34.9, 29.7, 22.7, 21.3, 21.0, 19.9,
18.7, 18.0, 16.0, 12.3, 10.6, 9.3. HRMS (ESI) m/z Calcd for
1.80 (dd, J = 26.9, 14.6 Hz, 3H), 1.68 (d, J = 13.9 Hz, 2H), 1.58–1.43
(m, 3H), 1.43–1.36 (m, 5H), 1.31–1.20 (m, 14H), 1.16 (d,
J = 7.1 Hz, 5H), 1.14–0.99 (m, 23H), 0.96–0.85 (m, 1H), 0.87–0.77
(m, 5H). 13C NMR (126 MHz, CDCl3) d (ppm) 176.0, 147.6, 129.8,
125.8, 120.3, 102.8, 96.0, 81.2, 78.4, 78.0, 74.4, 72.7, 70.9, 69.2,
68.7, 65.9, 64.0, 57.7, 50.8, 49.3, 45.4, 39.3, 37.4, 36.9, 35.1, 29.8,
29.7, 21.6, 21.0, 19.7, 18.9, 18.4, 16.0, 12.4, 10.7, 9.3. HRMS (ESI)
m/z Calcd for C50H83N5O15 [M+H+]: 992.5807, found 992.5839.
5.1.10. (Clarithromycin-30-(N-(4-triazolylbenzyl)))-N-
hydroxypentanamide (5d)
Reaction of 30-N(desmethyl)-30-N-(4-ethynylbenzyl)clarithro-
mycin
4 (0.15 g, 0.18 mmol) with 5-azido-N-((tert-butyldim-
ethylsilyl)oxy)pentanamide 51d (0.072 g, 0.265 mmol) followed
by TBS deprotection with caesium fluoride as described for the
synthesis of compound 5a, gave 5d as a light yellow solid
(0.068 g, 45%). 1H NMR (500 MHz, CDCl3) d (ppm) 7.86 (s, 1H),
7.75 (s, 2H), 7.35 (s, 2H), 5.03 (dd, J = 11.1, 1.6 Hz, 1H), 4.87 (d,
J = 3.7 Hz, 1H), 4.41 (t, J = 9.4 Hz, 1H), 4.35 (s, 2H), 3.99–3.89 (m,
2H), 3.83 (d, J = 10.5 Hz, 1H), 3.78–3.68 (m, 2H), 3.62 (d,
J = 6.9 Hz, 1H), 3.54–3.41 (m, 2H), 3.38–3.29 (m, 1H), 3.17 (d,
J = 20.3 Hz, 1H), 3.12 (s, 3H), 3.04–2.91 (m, 5H), 2.91–2.81 (m,
1H), 2.64 (s, 1H), 2.56 (dd, J = 10.3, 7.4 Hz, 1H), 2.24 (d,
J = 40.5 Hz, 6H), 1.88 (dt, J = 26.6, 13.4 Hz, 4H), 1.85–1.73 (m, 2H),
1.71–1.56 (m, 3H), 1.56–1.41 (m, 2H), 1.42–1.33 (m, 3H), 1.33–
1.27 (m, 2H), 1.30–1.18 (m, 8H), 1.16 (d, J = 7.0 Hz, 3H), 1.13–
1.00 (m, 15H), 0.82 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d (ppm) 175.8, 170.3, 147.3, 129.6, 125.7, 120.1, 102.6, 95.9, 80.9,
78.3, 74.2, 72.5, 70.7, 69.0, 68.4, 65.6, 63.8, 57.6, 50.5, 49.8, 49.3,
45.9, 45.1, 45.0, 39.2, 39.0, 37.2, 36.8, 34.7, 31.7, 29.8, 29.6, 29.3,
22.1, 21.4, 21.2, 20.9, 19.8, 18.6, 17.9, 15.9, 12.2, 10.5, 9.1, 8.5.
HRMS (ESI) m/z Calcd for C51H83N5O15Na [M+Na+]: 1028.5783,
found 1028.5795.
C
48H78N5O15 [M+H+]: 964.5494, found 964.5541.
5.1.8. (Clarithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxypro-
panamide (5b)
Reaction of 30-N(desmethyl)-30-N-(4-ethynylbenzyl)clarithro-
mycin
4
(0.15 g, 0.18 mmol) with 3-azido-N-((tert-butyldi-
methylsilyl)oxy)propanamide 51b (0.065 g, 0.265 mmol) followed
by TBS deprotection with caesium fluoride as described for the
synthesis of compound 5a, gave 5b as a light yellow solid
(0.067 g, 39%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.25 (s, 1H),
7.78 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 5.13 (dd, J = 11.1,
2.1 Hz, 1H), 4.74 (t, J = 6.5 Hz, 2H), 4.51 (d, J = 7.2 Hz, 1H), 4.11
(dd, J = 9.4, 6.3 Hz, 1H), 3.93 (d, J = 13.2 Hz, 1H), 3.79–3.69 (m,
5H), 3.68–3.62 (m, 1H), 3.38–3.32 (m, 2H), 3.30 (dt, J = 3.3,
1.6 Hz, 3H), 3.18 (s, 3H), 3.11 (d, J = 7.8 Hz, 1H), 3.03 (d,
J = 7.3 Hz, 4H), 3.01 (d, J = 9.5 Hz, 2H), 2.94–2.84 (m, 2H), 2.77 (t,
J = 6.3 Hz, 2H), 2.64–2.53 (m, 1H), 2.40 (s, 1H), 2.34 (s, 4H), 1.96–
1.78 (m, 5H), 1.67 (d, J = 13.0 Hz, 1H), 1.60–1.47 (m, 2H), 1.39 (d,
J = 14.0 Hz, 4H), 1.25 (q, J = 7.8 Hz, 5H), 1.21 (d, J = 6.0 Hz, 7H),
1.18–1.08 (m, 17H), 0.85 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) d (ppm) 175.8, 167.1, 147.1, 129.7, 129.4, 127.3, 125.7,
121.3, 102.7, 96.0, 81.1, 78.4, 78.3, 77.8, 76.7, 74.3, 72.6, 70.8,
69.1, 69.0, 68.4, 65.7, 63.8, 57.6, 50.6, 49.4, 46.2, 45.2, 45.1, 39.3,
39.1, 37.3, 36.9, 34.8, 33.1, 29.9, 29.7, 21.4, 21.2, 21.0, 19.8, 18.6,
18.0, 16.0, 12.3, 10.6, 9.2. HRMS (ESI) m/z Calcd for C49H79N5O15Na
[M+Na+]: 1000.5454, found 1000.5479.
5.1.11. (N10-(4-Ethynylbenzyl))azithromycin (7)
N
10-Desmethylazithromycin
2 (2.72 g, 3.55 mmol) and 4-
ethynylbenzaldehyde 639 (2.31 g, 17.75 mmol) were dissolved in
anhydrous DMF (40 mL). Acetic acid (2.0 mL, 35.50 mmol) was
added and the solution was stirred for 30 min and then sodium
cyanoborohydride (465 mg, 7.10 mmol) was added to the reaction
mixture. The mixture was then stirred at 70 °C for 7 h after which it
was cooled and the pH of the solution was raised to 8 by adding
saturated aqueous NaHCO3 solution. The crude mixture was
diluted into CH2Cl2 and was washed water, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The crude was purified
by column chromatography (Silica gel, 5:1:1 EtoAc/hexane/triethy-
lamine) to afford the title compound 7 as white solid (603 mg,
20%). 1H NMR (500 MHz, MeOH-d4) d (ppm) 7.41 (s, 4H), 5.01 (m,
2H), 4.60 (d, J = 6.8 Hz, 1H), 4.20 (ddd, J = 24.8, 13.7, 6.6 Hz, 2H),
3.92 (d, J = 10.9 Hz, 1H), 3.73 (ddd, J = 37.9, 14.5, and 5.8 Hz, 4H),
3.45 (m, 1H), 3.38 (d, J = 11.2 Hz, 3H), 3.32 (m, 1H), 3.07 (dd,
J = 11.5, and 7.2 HZ, 1H), 2.99 (m, 1H), 2.88 (dd, J = 14.7, and
8.4 Hz, 2H), 2.79 (m, 1H), 2.47 (d, J = 15.1 Hz, 1H), 2.40 (s, 6H),
2.19 (m, 1H), 2.11 (m, 1H), 1.96 (d, J = 21.6 Hz, 1H), 1.74 (m, 4H),
1.62 (m, 2H), 1.43 (m, 1H), 1.32 (m, 4H), 1.27 (m, 11H), 1.22 (m,
4H), 1.18 (d, J = 7.4 Hz, 3H), 1.14 (dd, J = 8.0, and 4.1 Hz, 3H), 0.94
(m, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm)
178.7, 132.9, 130.7, 104.2, 97.6, 80.7, 79.4, 78.4, 76.9, 76.5, 74.5,
72.7, 69.4, 67.1, 65.6, 50.1, 46.6, 40.8, 36.3, 31.9, 23.2, 22.3, 21.9,
21.8, 19.1, 11.8, 10.8. HRMS (ESI) m+2/2z Calcd for C46 H78 O12 N2
[M+2H+]: 425.2772, found 425.2762.
5.1.9. (Clarithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxybu-
tanamide (5c)
Reaction of 30-N(desmethyl)-30-N-(4-ethynylbenzyl)clarithro-
mycin 4 (0.15 g,0.18 mmol) with 4-azido-N-((tert-butyldimethylsi-
lyl)oxy)butanamide 51c (0.082 g, 0.318 mmol) followed by TBS
deprotection with caesium fluoride as described for the synthesis
of compound 5a, gave 5c as a light yellow solid (0.075 g, 43%). 1H
NMR (500 MHz, CDCl3) d (ppm) 7.85 (s, 1H), 7.74 (d, 2H), 7.34 (d,
2H), 5.03 (d, J = 11.2 Hz, 1H), 4.88 (d, J = 4.1 Hz, 1H), 4.41 (d,
J = 6.9 Hz, 4H), 4.01–3.89 (m, 3H), 3.81 (d, J = 10.8 Hz, 1H), 3.77–
3.69 (m, 4H), 3.67–3.55 (m, 3H), 3.46 (dd, J = 8.1, 5.3 Hz, 3H),
3.37–3.27 (m, 2H), 3.18 (d, J = 13.1 Hz, 1H), 3.15 (d, J = 16.9 Hz,
5H), 3.06–2.92 (m, 8H), 2.89–2.80 (m, 2H), 2.57 (dd, J = 11.0,
6.5 Hz, 3H), 2.35–2.13 (m, 11H), 1.90 (dd, J = 14.4, 7.3 Hz, 3H),
5.1.12. (Azithromycin(-N10-(4-triazolylbenzyl)))-N-
hydroxyacetamide (8a)
In an oven dried round bottomed flask charged with a magnetic
stirring bar, (N10-(4-ethynylbenzyl))azithromycin
7 (120 mg,
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
0.141 mmol)
and
2-azido-N-((tert-butyldimethylsilyl)oxy)ac-
74.6, 71.4, 69.2, 68.6, 67.1, 66.5, 50.8, 50.1, 47.8, 46.5, 40.3, 36.2,
35.4, 31.4, 30.3, 27.4, 25.1, 24.1, 22.9, 22.7, 22.3, 21.8, 21.7, 19.1,
14.9, 14.5, 11.7, 11.5, 10.5, 9.5. HRMS (ESI) m/z Calcd for
etamide 51a (73 mg, 0.254 mmol) were placed under argon. To
the mixture were added 2 mL of degassed1:1 mixture of THF/
DMSO, CuI (4 mg, 0.02 mmol) and N,N0-diisopropylethylamine
C
50H85N6O14 [M+H+]: 993.6118, found 993.6125.
(20 lL, 0.09 mmol) in succession. The mixture was heated at
45 °C for 12 h. The reaction was cooled to room temperature and
diluted with 50 mL of ethyl acetate. Organic layer was washed with
4:1 mixture of satd. NH4Cl soln.–NH4OH soln. (2 ꢂ 10 mL), water
(10 mL), and brine (10 mL), dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. The crude was subjected to next reac-
tion without further purification.
5.1.15. (Azithromycin-(N10-(4-triazolylbenzyl)))-N-hydroxypen-
tanamide (8d)
Reaction of (N10-(4-ethynylbenzyl))azithromycin 7 (131 mg,
0.15 mmol) and 5-azido-N-((tert-butyldimethylsilyl)oxy)pen-
tanamide 51d (44 mg, 0.28 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 8a, gave
8d as white solid (60 mg, 40%). 1H NMR (500 MHz, MeOH-d4) d
(ppm) 8.24 (s, 1H), 7.69 (d, J = 7.6 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H),
4.92 (d, J = 10.2 Hz, 1H), 4.88 (d, J = 4.3 Hz, 1H), 4.55 (d, J = 6.3 Hz,
1H), 4.37 (t, J = 6.6 Hz, 2H), 4.08 (ddd, J = 23.2, 20.9, 10.7 Hz, 2H),
3.70 (dd, J = 29.4, 21.5 Hz, 3H), 3.54 (dd, J = 20.2, 11.4 Hz, 2H),
3.27 (s, 4H), 3.10 (dt, J = 14.4, 7.0 Hz, 1H), 2.96 (dd, J = 17.4,
9.9 Hz, 5H), 2.87 (d, J = 11.2 Hz, 1H), 2.79 (m, 1H), 2.49 (s, 7H),
2.36 (d, J = 15.1 Hz, 1H), 2.18 (s, 1H), 2.05 (m, 4H), 1.88 (d,
J = 6.5 Hz, 3H), 1.79 (m, 4H), 1.67 (s, 2H), 1.57 (m, 4H), 1.48 (m,
2H), 1.31 (m, 6H), 1.20 (d, J = 5.8 Hz, 6H), 1.16 (m, 10H), 1.09 (m,
6H), 0.80 (m, 6H). 13C NMR (126 MHz, MeOD) d (ppm) 178.6,
172.4, 169.5, 148.6, 133.8, 132.6, 131.6, 130.0, 126.3, 122.4,
103.6, 97.6, 80.8, 79.2, 76.8, 76.5, 74.6, 73.8, 71.8, 69.3, 68.9,
67.1, 66.2, 51.2, 50.1, 49.8, 49.78, 49.7, 49.6, 49.5, 49.4, 49.3,
49.2, 49.0, 48.8, 48.6, 47.8, 46.5, 40.3, 36.3, 35.4, 33.1, 31.8, 31.5,
30.9, 30.8, 30.3, 29.9, 25.1, 24.2, 23.8, 23.1, 22.7, 22.4, 21.8, 21.8,
20.4, 19.1, 16.1, 14.9, 14.6, 11.8, 11.6, 10.6, 9.8, 9.3. HRMS (ESI)
m/z Calcd for C51H87N6O14 [M+H+]: 1007.6275, found 1007.6278.
The crude was dissolved in methanol (2 mL) and caesium fluo-
ride (43 mg, 0.282 mmol) was added and the resulting reaction
was stirred at room temperature for 2 h. Reaction was quenched
by adding water and was extracted with ethyl acetate (100 mL)
and the organic layer was washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
was purified by preparative chromatography (Silica gel, 10:1:0.1
CH2Cl2–MeOH–concd NH4OH soln.) to give 8a as a white solid
(35 mg, 25%). 1H NMR (500 MHz, MeOH-d4) d (ppm) 8.25 (s, 1H),
7.69 (d, J = 7.5 Hz, 2H), 7.42 (d, J = 7.5 Hz, 2H), 4.90 (m, 2H), 4.55
(d, J = 6.1 HZ, 1H), 4.04 (m, 4H), 3.69 (m, 3H), 3.51 (m, 3H), 3.37
(m, 1H), 3.08 (td, J = 16.2, and 8.9 Hz, 2H), 2.97 (m, 4H); 2.72 (m,
1H), 2.85 (m, 3H), 2.50 (s, 6H), 2.34 (t, J = 12.6 Hz, 1H), 2.06 (m,
2H), 1.78 (m, 5H), 1.64 (m, 5H), 1.47 (m, 3H), 1.18 (m, 17H), 0.81
(t, J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm) 178.6, 148.8,
131.7, 126.8, 123.8, 103.5, 97.6, 80.8, 79.2, 77.0, 76.5, 74.6, 73.8,
72.3, 71.6, 71.4, 68.5, 67.0, 66.3, 62.4, 50.1, 47.8, 46.5, 40.2, 38.1,
36.3, 35.4, 32.9, 31.4, 29.8, 21.8, 20.4, 19.1, 14.8, 14.6, 14.4, 13.5,
11.7, 10.6, 9.6. HRMS (ESI) m+2/2z Calcd for C48H82N6O14 [M
+2H+]: 483.2939, found 483.2933.
5.1.16. (Azithromycin-(N10-(4-triazolylbenzyl)))-N-hydroxyhex-
anamide (8e)
5.1.13. (Azithromycin-(N10-(4-triazolylbenzyl)))-N-hydroxypro-
Reaction of (N10-(4-ethynylbenzyl))azithromycin 7 (155 mg,
panamide (8b)
0.18 mmol) and
6-azido-N-((tert-butyldimethylsilyl)oxy)hex-
Reaction of (N10-(4-ethynylbenzyl))azithromycin
7
(85 mg,
anamide 51e (94 mg, 0.33 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 8a, gave
8e as white solid (70 mg, 38%). 1H NMR (500 MHz, MeOD) d
(ppm) 8.23 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H),
4.92 (dd, J = 10.2, 2.3 Hz, 1H), 4.88 (d, J = 4.7 Hz, 1H), 4.52 (d,
J = 7.1 Hz, 1H), 4.37 (t, J = 7.0 Hz, 2H), 4.10 (m, 2H), 3.65 (q,
J = 13.5 Hz, 3H), 3.56 (m, 2H), 3.49 (m, 1H), 3.29 (s, 3H), 3.22 (m,
6H), 2.98 (m, 2H), 2.91 (m, 1H), 2.80 (dd, J = 19.0, 12.8 Hz, 3H),
2.33 (m, 9H), 2.15 (m, 1H), 2.02 (t, J = 7.3 Hz, 3H), 1.90 (m, 4H),
1.71 (dd, J = 40.3, 26.2 Hz, 3H), 1.60 (dt, J = 15.1, 7.5 Hz, 4H), 1.53
(dt, J = 11.8, 6.9 Hz, 3H), 1.30 (ddd, J = 15.6, 12.5, 10.2 Hz, 6H),
1.22 (m, 8H), 1.15 (dd, J = 16.9, 8.2 Hz, 12H), 1.07 (m, 12H), 0.96
(m, 2H), 0.82 (tt, J = 24.9, 7.3 Hz, 6H). 13C NMR (126 MHz, MeOD)
d (ppm) 178.6, 172.7, 148.9, 131.3, 126.64, 122.2, 104.0, 97.6,
80.7, 79.3, 76.5, 74.5, 72.5, 69.2, 67.0, 65.7, 57.6, 51.4, 50.1, 49.8,
49.7, 49.6, 49.6, 49.4, 49.4, 49.4, 49.4, 49.3, 49.3, 49.2, 49.2, 49.2,
49.2, 49.2, 49.2, 49.2, 49.1, 48.9, 48.8, 48.6, 46.5, 40.7, 36.3, 33.6,
31.8, 31.0, 27.0, 26.2, 23.2, 22.3, 21.9, 21.7, 19.1, 17.5, 17.4, 17.2,
0.10 mmol) and 3-azido-N-((tert-butyldimethylsilyl)oxy)propana-
mide 51b (34 mg, 0.14 mmol) followed by TBS deprotection with
caesium fluoride as described for the synthesis of 8a, gave 8b as
white solid (38 mg, 40%). 1H NMR (500 MHz, MeOH-d4) d (ppm)
8.21 (s, 1H), 7.71 (d, J = 7.4 Hz, 2H), 7.44 (d, J = 7.5 Hz, 2H), 4.91
(m, 2H), 4.56 (d, J = 6.2 Hz, 1H), 4.12 (m, 1H), 3.99 (m, 2H), 3.74
(m, 3H), 3.51 (m, 2H), 3.18 (m, 5H), 3.07 (m, 4H), 2.96 (m, 2H);
2.82 (m, 1H), 2.62 (s, 6H), 2.36 (d, J = 15.1 Hz, 2H), 2.00 (dd,
J = 22.1 and 17.7 Hz, 1H), 1.86 (m, 5H), 1.68 (m, 5H), 1.52 (m,
3H), 1.32 (ddd, J = 17.7, 10.9, and 4.0 Hz, 3H), 1.14 (m, 17H), 1.03
(m, 7H), 0.78 (t, J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm)
178.6, 169.5, 148.5, 133.7, 132.5, 132.1, 130.0, 126.9, 123.0, 97.6,
80.9, 79.0, 76.5, 74.6, 73.8, 71.2, 69.2, 68.5, 67.1, 66.6, 47.8, 40.3,
39.9, 36.2, 35.4, 31.7, 31.2, 30.1, 30.6, 30.2, 29.5, 25.1, 24.2, 22.7,
21.8, 21.7, 19.2, 14.8, 14.5, 11.6, 11,5, 10.4, 9.4. HRMS (ESI) m/z
Calcd for C49H83N6O14 [M+H+]: 979.5962, found 979.5967.
5.1.14. (Azithromycin-(N10-(4-triazolylbenzyl)))-N-hydroxybu-
11.8, 10.7. HRMS (ESI) m/z Calcd for C
52H88N6O14 [M+H+]:
tanamide (8c)
1021.6431, found 1021.6444.
Reaction of (N10-(4-ethynylbenzyl))azithromycin
7
(93 mg,
0.11 mmol)
and
4-azido-N-((tert-butyldimethylsilyl)oxy)bu-
5.1.17. (Azithromycin-(N10-(4-triazolylbenzyl)))-N-hydroxyhep-
tanamide (8f)
tanamide 51c (51 mg, 0.20 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 8a, gave
8c as white solid (54 mg, 50%). 1H NMR (500 MHz, MeOH-d4) d
(ppm) 8.25 (s, 1H), 7.70 (d, J = 7.7 Hz, 2H), 7.41 (d, J = 7.5 Hz, 2H),
4.88 (m, 2H), 4.55 (d, J = 6.1 Hz, 2H), 4.39 (s, 3H), 4.06 (m, 4H),
3.69 (m, 4H), 3.49 (m, 2H), 3.01 (m, 10H), 2.80 (m, 2H), 2.56 (s,
6H), 2.36 (dt, J = 47.8 and 19.1 Hz, 3H), 2.03 (m, 7H), 1.84 (m,
7H), 1.62 (m, 3H), 1.47 (m, 3H), 1.28 (m, 8H), 1.15 (m, 17H), 1.01
(m, 7H), 0.80 (t, J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm)
178.6, 148.8, 131.8, 126.8, 122.5, 103.3, 97.6, 80.8, 79.1, 76.5,
Reaction of (N10-(4-ethynylbenzyl))azithromycin 7 (125 mg,
0.15 mmol) and 7-azido-N-((tert-butyldimethylsilyl)oxy)hep-
tanamide 51f (80 mg, 0.27 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 8a, gave
8f as white solid (75 mg, 45%). 1H NMR (500 MHz, MeOD) d
(ppm) 8.22 (s, 1H), 7.66 (t, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H),
4.92 (m, 1H), 4.87 (d, J = 4.7 Hz, 1H), 4.50 (d, J = 7.0 Hz, 1H), 4.35
(t, J = 7.1 Hz, 2H), 4.09 (m, 2H), 3.65 (t, J = 13.9 Hz, 3H), 3.55 (m,
1H), 3.28 (s, 3H), 3.21 (m, 5H), 2.97 (dd, J = 9.3, 5.0 Hz, 1H), 2.81
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

10
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(m, 4H), 2.32 (m, 7H), 2.12 (m, 1H), 2.01 (m, 3H), 1.88 (dd, J = 13.9,
6.9 Hz, 3H), 1.72 (dd, J = 43.3, 24.2 Hz, 4H), 1.53 (m, 4H), 1.32 (m,
7H), 1.21 (t, J = 6.4 Hz, 5H), 1.15 (m, 10H), 1.08 (dd, J = 15.7,
7.4 Hz, 9H), 0.79 (m, 6H). 13C NMR (126 MHz, MeOD) d (ppm)
178.4, 172.8, 148.7, 131.2, 126.4, 122.0, 103.8, 97.4, 80.5, 79.1,
76.3, 74.3, 72.3, 69.1, 66.9, 65.5, 57.4, 51.3, 49.9, 49.6, 49.4, 49.4,
49.3, 49.2, 49.1, 48.9, 48.8, 48.6, 48.4, 46.4, 40.5, 36.1, 33.5, 31.7,
31.0, 29.3, 27.0, 26.4, 23.0, 22.2, 21.7, 21.6, 18.9, 17.2, 17.1, 11.6,
10.6. HRMS (ESI) m/z Calcd for C53H91N6O14 [M+H+]: 1035.6588,
found 1035.6591.
yellow oil (490 mg; 69% overall yield). 1H NMR (CDCl3, 400 MHz)
d (ppm) 7.31 (4H, m), 4.61 (2H, s), 3.02 (1H, s).
5.1.21. 2-Ethynylbenzyl methanesulfonate (10)
(2-Ethynylphenyl)methanol 60 (400 mg, 3.03 mmol) was dis-
solved in anhydrous dichloromethane. Triethylamine (0.84 mL,
6.05 mmol) was added at room temperature and the reaction mix-
ture was stirred for 10 min. After cooling the reaction mixture to
ꢀ15 °C, methanesulfonyl chloride (0.28 mL, 3.63 mmol) was added
to the reaction mixture. After 40 min, the reaction was quenched
by adding saturated aqueous NaHCO3 solution (10 mL) and the
organic phase was separated. The aqueous phase was extracted
twice with dichloromethane (100 mL). The combined organic layer
was washed with water (20 mL), brine (20 mL), dried over anhy-
drous Na2SO4, filtered and concentrated in vacuo. The crude mesy-
lated product 10 was used in the next step without further
purification.
5.1.18. 2-Ethynylbenzaldehyde (19)
To
a solution of (2-ethynylphenyl)methanol 60 (510 mg,
3.82 mmol) in dichloromethane (20 mL) was added pyridinium
dichromate (2.87 g, 7.64 mmol) and stirred at room temperature
for 5 h. The reaction mixture was quenched with cold Et2O
(100 mL) and the suspension was filtered off and the filtrate was
concentrated in vacuo The crude was subjected to column chro-
matography (Silica gel, 1:2 ethyl acetate-hexane) to give the title
compound 19 as a brown-white solid (420 mg, 85%). 1H NMR
(CDCl3, 400 MHz) d (ppm) 10.54 (1H, s), 7.93 (1H, m), 7.55 (3H,
m), 3.46 (1H, s).
5.1.22. (30-N-(2-Ethynylbenzyl))azithromycin (12)
To a solution of 30-desmethylazithromycin 9 (1.2 g, 1.63 mmol)
in anhydrous DMSO (8 mL) was added N,N-diisopropylethylamine
(3 mL) and 2-ethynylbenzyl methanesulfonate 10 (450 mg,
2.12 mmol). The reaction mixture was heated with stirring under
argon at 85 °C for 3 h. The reaction mixture was cooled to room
temperature, diluted with ethyl acetate (100 mL) and washed with
saturated NaHCO3 (3 ꢂ 50 mL) and saturated brine (50 mL). The
organic layer was dried over Na2SO4 and concentrated in vacuo.
The crude product was purified by column chromatography (Silica
gel, 12:1:0.1 to 4:1:0.1 CH2Cl2–Acetone–Et3N) to give 12 as a
brown-white solid (600 mg, 44%). 1H NMR (CDCl3, 400 MHz) d
(ppm) 7.43 (d, J = 7.6 Hz, 1H), 7.26 (m, 2H), 7.16 (d, J = 7.2 Hz,
1H), 5.02 (d, J = 7.6 Hz, 1H), 4.72 (m, 1H), 4.62 (d, J = 9.6 Hz, 1H),
4.35 (d, J = 7.2 Hz, 1H), 4.14 (m, 1H), 3.98 (m, 1H), 3.57 (m, 5H),
3.22 (s, 2H), 2.92 (m, 4H), 2.64 (m, 2H), 2.46 (m, 2H), 2.34 (s,
3H), 2.19 (s, 3H), 2.09 (m, 1H), 1.83 (m, 6H), 1.42 (m, 3H), 1.04
(m, 30H). 13C NMR (CDCl3, 100 MHz) d (ppm) 178.8, 141.3, 133.2,
129.6, 128.9, 127.1, 122.2, 102.9, 94.5, 83.8, 82.1, 81.4, 78.2, 77.9,
76.8, 74.2, 73.8, 73.6, 72.8, 70.5, 70.1, 68.6, 65.5, 63.4, 56.5, 49.2,
45.2, 42.2, 42.1, 36.3, 36.1, 34.7, 29.5, 27.5, 26.7, 21.9, 21.5, 21.4,
21.2, 18.2, 16.2, 14.7, 11.2, 9.0, 7.4. HRMS (MALDI) m/z Calcd for
5.1.19. 3-Ethynylbenzaldehyde (20)
A solution of 3-bromobenzaldehyde 61 (1.0 g, 5.40 mmol),
ethynyltrimethylsilane (850 mg, 8.6 mmol), palladium (II) acetate
(15 mg,
0.065 mmol),
and
triphenylphosphine
(28 mg,
0.108 mmol) in anhydrous triethylamine (5 mL) was heated to
reflux under argon. After 15 min of reflux, a clear yellow solution
resulted and a white precipitate began to form. The reaction was
stopped after 4 h of refluxing, cooled, and filtered. The orange-
brown filtrate was mixed with 50 ml of saturated NaHCO3 and
extracted with dichloromethane (3 ꢂ 50 mL). The organic fractions
were combined, dried over anhydrous Na2SO4, and concentrated to
yield oil which was purified by silica gel column (EtOAc/Hexane
1:4) to give 0.86 g of silyl intermediate which was subsequently
treated with anhydrous K2CO3 (100 mg) in anhydrous methanol
(10 mL) under argon for 3 h at room temperature. Saturated
NaHCO3 (50 mL) was added to the reaction mixture and extracted
with dichloromethane (3 ꢂ 50 mL). The organic fractions were
combined, dried over anhydrous Na2SO4 and concentrated in
vacuo. Purification by column chromatography (Silica gel, CH2Cl2–
Hexane 2:3) afforded the target compound 20 as a brown-white
solid (430 mg, 62%). 1H NMR (CDCl3, 400 MHz) d (ppm) 10.00
(1H, s), 7.99 (1H, s), 7.87 (1H, m), 7.74 (1H, m), 7.50 (1H, m),
3.16 (1H, s).
C
46H77N2O12 [M+H+]: 849.5471, found 849.5530.
5.1.23. (30-N-(3-Ethynylbenzyl))azithromycin (13)
Reaction of 30-desmethylazithromycin 9 (1.42 g, 2.04 mmol) in
anhydrous DMSO (8 ml), N,N0-diisopropylethylamine (3 ml) and
3-ethynylbenzyl methanesulfonate 11 (560 mg, 2.65 mmol) as
described for the synthesis of 12, afforded compound 13 as a
brown-white solid (530 mg, 32%). 1H NMR (CDCl3, 400 MHz) d
(ppm) 7.41 (m, 2H), 7.22 (m, 2H), 5.14 (d, J = 6.0 Hz, 1H), 4.70 (d,
J = 8.0 Hz, 1H), 4.43 (d, J = 7.6 Hz, 1H), 4.25 (m, 1H), 4.05 (m, 1H),
3.55 (m, 5H), 3.19 (s, 3H), 3.16 (s, 2H), 3.00 (m, 2H), 2.64 (m,
4H), 2.32 (s, 3H), 2.23 (s, 3H), 1.89 (m, 7H), 1.23 (m, 33H). 13C
NMR (CDCl3, 100 MHz) d (ppm) 178.5, 141.5, 131.6, 130.2, 129.9,
127.3, 122.5, 102.8, 94.7, 83.8, 83.5, 78.1, 76.8, 74.3, 74.0, 73.5,
72.8, 70.8, 70.7, 70.0, 68.5, 65.5, 62.2, 57.4, 49.3, 45.1, 42.3, 41.7,
36.8, 36.4, 34.7, 30.0, 27.4, 26.7, 22.0, 21.6, 21.3, 21.2, 18.2, 16.2,
14.9, 11.2, 9.2, 7.5. HRMS (MALDI) m/z Calcd for C46H77N2O12 [M
+H+]: 849.5471, found 849.5385.
5.1.20. 3-Ethynylbenzyl alcohol (52)
Palladium(II) acetate (14 mg, 0.064 mmol) and triphenylphos-
phine (28 mg, 0.106 mmol) were added to a solution of 3-bro-
mobenzyl
alcohol
51
(1.0 g,
5.34 mmol)
and
ethynyltrimethylsilane (0.84 g, 8.55 mmol) in triethylamine
(5 mL). The reaction mixture was subjected to reflux under argon
for 4 h. The orange-brown reaction mixture was cooled, diluted
with excess ether (200 mL) and filtered. The filtrate was washed
saturated NaHCO3 (50 mL), water (50 mL), brine (50 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo. The
crude was subjected to next reaction without further purification.
The crude was dissolved in anhydrous methanol (10 mL) and
was treated with K2CO3 (100 mg) under argon at room tempera-
ture for 3 h. Saturated NaHCO3 (50 mL) was added to the reaction
mixture and extracted with CH2Cl2 (3 ꢂ 50 mL). The organic frac-
tions were combined, dried over Na2SO4, concentrated in vacuo
and the crude was purified by column chromatography (Silica
gel, 4:0.5 CH2Cl2–acetone) to give the target compound 52 as
5.1.24. (Azithromycin-30-(N-(2-triazolylbenzyl)))-N-hydroxyhe-
xanamide (15a)
To a solution of 30-(2-ethynylbenzyl)azithromycin 12 (50 mg,
0.06 mmol) and copper(I) iodide (8 mg, 0.04 mmol) in anhydrous
THF (5 ml) under argon was added N,N0-diisopropylethylamine
(0.6 mL), and stirred under argon at room temperature for
15 min. 6-azido-N-((tert-butyldimethylsilyl)oxy)hexanamide 51e
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
(34 mg, 0.12 mmol) was added to the reaction mixture, and stirring
continued for 3 h. The reaction mixture was diluted with 1:4 NH4-
OH/saturated NH4Cl (40 mL) and extracted with 20% MeOH/CH2Cl2
(3 ꢂ 20 mL). The organic layer was dried over Na2SO4 and concen-
trated in vacuo. The crude was subjected to the next reaction with-
out further purification.
tanamide 51f (0.035 g, 0.118 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 15a,
afforded 16b as a brown-white solid (28 mg, 46%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.84 (s, 1H), 7.74 (m, 2H), 7.34 (m, 2H), 5.15 (d,
J = 4.0 Hz, 1H), 4.73 (m, 3H), 4.08 (m, 2H), 3.85 (m, 1H), 3.39 (m,
7H), 2.64 (m, 4H), 2.26 (m, 16H), 1.85 (m, 4H), 1.40 (m, 34H),
0.92 (m, 6H). 13C NMR (CDCl3, 100 MHz) d (ppm) 178.3, 171.5,
147.5, 139.0, 130.6, 129.1, 128.9, 126.3, 124.8, 120.1, 102.7, 94.6,
83.3, 78.4, 78.3, 76.7, 74.0, 73.9, 73.3, 72.7, 70.5, 69.2, 68.6, 66.0,
63.6, 62.9, 58.0, 51.3, 50.2, 49.3, 45.4, 42.8, 41.8, 36.9, 35.7, 33.0,
29.9, 29.7, 28.6, 28.5, 26.3, 25.3, 21.8, 21.7, 21.4, 21.3, 17.7, 16.8,
14.5, 11.5, 8.8, 6.6. HRMS (MALDI) m/z Calcd for C53H91N6O14 [M
+H+]: 1035.6588, found 1035.6714.
The crude was dissolved in anhydrous methanol (2 mL) and
treated with caesium fluoride (20 mg, 0.12 mmol) at room temper-
ature for 2 h. Water was added to the reaction and the mixture was
extracted with ethyl acetate (2 ꢂ 50 mL). The organic fraction was
washed with brine (10 mL), dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The crude product was purified by
preparative chromatography (Silica gel, 12:1:0.1 CH2Cl2/MeOH/
concd NH4OH) to give 15a as a brown-white solid (40 mg, 67%).
1H NMR (CDCl3, 400 MHz) d (ppm) 7.72 (s, 1H), 7.50 (m, 1H),
7.41 (m, 1H), 7.28 (m, 2H), 5.08 (d, J = 4.8 Hz, 1H), 4.34 (m, 3H),
4.07 (m, 1H), 3.95 (m, 1H), 3.40 (m, 8H), 2.85 (m, 4H), 2.61 (m,
2H), 2.45 (m, 2H), 2.28 (s, 5H), 2.13 (s, 3H), 1.92 (m, 8H), 1.59
(m, 8H), 1.18 (m, 24H), 0.84 (m, 6H). 13C NMR (CDCl3, 100 MHz)
d (ppm) 178.4, 171.2, 146.4, 136.0, 131.1, 128.5, 127.6, 122.6,
102.6, 94.6, 83.6, 78.5, 78.4, 78.1, 76.7, 73.9, 73.8, 73.4, 72.7,
70.3, 69.3, 68.3, 66.2, 62.6, 61.0, 56.1, 51.2, 50.1, 49.2, 45.3, 42.8,
35.6, 29.9, 28.5, 27.0, 26.7, 26.2, 25.8, 25.0, 22.7, 21.8, 21.7, 21.4,
21.3, 17.7, 16.8, 14.4, 11.6, 8.9, 6.4. HRMS (MALDI) m/z Calcd for
5.1.28. (Azithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxyace-
tamide (17a)
(30-N-(4-Ethynylbenzyl))azithromycin 14 (0.10 g, 0.12 mmol)
and 2-Azido-N-((tert-butyldimethyl silyl)oxy)ethanamide 51a
(0.05 g, 0.22 mmol) were dissolved in degassed anhydrous THF
(5 mL)
. CuI (0.01 g, 0.06 mmol) and Hünig’s base (0.04 mL,
0.24 mmol) were added to the reaction and the resulting reaction
was stirred at room temperature for another 12 h. Then, the reac-
tion mixture was diluted with excess ethyl acetate (30 mL) and
was transferred to a separatory funnel and then the ethyl acetate
layer was washed with a solution (20 mL) of 4:1 mixture of satu-
rated aqueous NH4Cl solution /NH4OH solution, water (10 mL),
brine (10 mL), dried over anhydrous Na2SO4, filtered, and concen-
trated in vacuo. The crude was dissolved in 5 mL of anhydrous
methanol and to that the solution caesium fluoride (0.03 g,
0.18 mmol) was added and the reaction was stirred at room tem-
perature for 2 h. Afterward, water (10 mL) and ethyl acetate
(30 mL) were added to the reaction and the organic layer was sep-
arated and then the organic layer was washed with brine (10 mL),
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. .
The crude was purified by preparative chromatography (Silica gel,
5:1:1 EtOAc/MeOH/NH4OH) to give the title compound 17a as light
yellow solid (0.045 g, 40% yield). 1H NMR (400 MHz, CD3OD) d
(ppm) 8.47 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 7.0 Hz, 2H), 7.62 (d,
J = 7.4 Hz, 2H), 5.13 (d, J = 4.6 Hz, 2H), 4.66 (d, J = 7.2 Hz, 1H),
4.31–4.20 (m, 2H), 4.15 (d, J = 12.9 Hz, 1H), 3.86 (d, J = 12.7 Hz,
2H), 3.76–3.70 (m, 2H), 3.52 (d, J = 6.9 Hz, 2H), 3.43 (dd, J = 3.2,
1.6 Hz, 3H), 3.29 (s, 3H), 3.20–3.12 (m, 2H), 3.07 (s, 1H), 3.01–
2.97 (m, 1H), 2.88 (s, 3H), 2.55 (s, 3H), 2.30 (d, J = 14.2 Hz, 2H),
2.09–2.01 (m, 28H), 1.69 (dd, J = 15.2, 5.1 Hz, 2H), 1.56 (s, 3H),
1.44 (s, 3H), 1.32 (s, 3H), 1.17 (s, 3H), 1.03 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, CD3OD) d (ppm) 180.7, 177.2, 149.5, 141.0,
140.9, 131.7, 127.5, 124.5, 104.7, 102.3, 97.6, 85.1, 80.1, 78.9,
76.8, 76.7, 76.2, 75.5, 73.2, 70.2, 70.1, 67.5, 66.8, 65.4, 59.3, 52.0,
51.0, 50.1, 49.3, 47.5, 44.1, 43.5, 38.4, 37.7, 36.7, 33.8, 32.7, 31.6,
29.1, 27.7, 24.5, 22.8, 19.8, 18.2, 16.5, 12.3, 10.6, 8.9. HMRS (ESI)
m+2/2z Calcd for C48H82N6O14 [M+2H+]: 483.2939, found for
483.2935.
C
52H89N6O14 [M+H+]: 1021.6431, found 1021.6469.
5.1.25. (Azithromycin-30-(N-(2-triazolylbenzyl)))-N-hydroxyhe-
ptanamide (15b)
Reaction of 30-(2-ethynylbenzyl)azithromycin 12 (0.05 g,
0.059 mmol) and 7-azido-N-((tert-butyldimethylsilyl)oxy)hep-
tanamide 51f (0.035 g, 0.118 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 15a,
afforded 15b as a brown-white solid (41 mg, 68%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.73 (s, 1H), 7.44 (m, 2H), 7.30 (m, 2H), 5.23 (d,
J = 4.0 Hz, 1H), 4.35 (m, 3H), 4.04 (m, 1H), 3.95 (m, 1H), 3.41 (m,
8H), 2.83 (m, 4H), 2.62 (m, 2H), 2.46 (m, 2H), 2.25 (m, 8H), 1.92
(m, 8H), 1.56 (m, 10H), 1.19 (m, 24H), 0.84 (m, 6H). 13C NMR
(CDCl3, 100 MHz) d (ppm) 178.4, 171.4, 146.4, 136.2, 131.1,
130.1, 128.6, 127.8, 122.6, 102.6, 94.6, 83.7, 78.5, 78.4, 78.1, 76.7,
73.9, 73.8, 73.3, 72.7, 69.3, 68.2, 66.2, 62.7, 61.0, 56.0, 51.3, 50.1,
49.2, 45.3, 42.8, 35.7, 29.7, 28.6, 28.0, 27.0, 26.7, 26.4, 25.8, 25.3,
25.0, 21.8, 21.4, 21.3, 18.1, 17.7, 16.8, 14.4, 11.5, 8.9, 6.5. HRMS
(MALDI) m/z Calcd for C53H91N6O14 [M+H+]: 1035.6588, found
1035.6581.
5.1.26. (Azithromycin-30-(N-(3-triazolylbenzyl)))-N-hydroxyhe-
xanamide (16a)
Reaction of 30-N-(3-ethynylbenzyl)azithromycin 13 (0.05 g,
0.059 mmol) and 6-azido-N-((tert-butyldimethylsilyl)oxy)hex-
anamide 51e (0.034 g, 0.12 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 15a,
afforded 16a as a brown-white solid (28 mg, 47%). 1H NMR (CDCl3,
400 MHz) d (ppm) 8.10 (s, 1H), 7.82 (m, 2H), 7.36 (m, 2H), 5.10 (m,
1H), 4.37 (m, 3H), 4.03 (m, 2H), 3.41 (m, 8H), 2.78 (m, 4H), 2.28 (m,
16H), 1.89 (m, 4H), 1.40 (m, 32H), 0.91 (m, 6H). 13C NMR (CDCl3,
100 MHz) d (ppm) 178.2, 171.2, 147.3, 139.0, 130.9, 129.2, 126.8,
125.8, 125.9, 125.2, 120.6, 102.8, 94.7, 83.4, 78.4, 77.8, 76.8, 74.2,
72.8, 72.7, 70.3, 68.4, 66.0, 63.4, 56.0, 54.0, 51.2, 51.1, 50.1, 49.3,
42.2, 36.8, 35.6, 34.6, 32.8, 29.7, 29.3, 28.5, 26.2, 25.5, 24.5, 21.8,
21.6, 21.3, 18.0, 17.7, 16.8, 14.1, 12.0, 11.4, 8.9, 6.9. HRMS (MALDI)
m/z Calcd for C52H89N6O14 [M+H+]: 1021.6431, found 1021.6500.
5.1.29. Azithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxypro-
panamide (17b)
Reaction of (30-N-(4-ethynylbenzyl))azithromycin 14 (0.18 g,
0.22 mmol) and 3-Azido-N-((tert-butyldimethyl silyl)oxy)propana-
mide 51b (0.08 g, 0.33 mmol) followed by TBS deprotection with
caesium fluoride as described for the synthesis of 17a, gave 17b
as light yellow solid (170 mg, 79%). 1H NMR (400 MHz, CD3OD) d
(ppm) 8.39 (s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H),
5.14 (d, J = 4.5 Hz, 1H), 4.88 (s, 3H), 4.67 (d, J = 7.3 Hz, 1H), 4.23
(d, 2H), 4.15 (d, J = 13.2 Hz, 1H), 3.86 (d, J = 13.3 Hz, 2H),
3.77–3.71 (m, 2H), 3.62–3.52 (m, 2H), 3.46–3.41 (m, 3H), 3.30
(s, 3H), 3.16 (d, J = 9.5 Hz, 2H), 2.91 (s, 3H), 2.55 (s, 3H), 2.32
(s, 1H), 2.15–2.01 (m, 24H), 1.70 (d, J = 10.6 Hz, 2H), 1.57 (s, 3H),
5.1.27. (Azithromycin-30-(N-(3-triazolylbenzyl)))-N-hydroxyhe-
ptanamide (16b)
Reaction of (30-N-(3-ethynylbenzyl))azithromycin 13 (0.05 g,
0.059 mmol) and 7-azido-N-((tert-butyldimethylsilyl)oxy)hep-
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

12
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
1.48 (d, J = 6.9 Hz, 2H), 1.43 (s, 3H), 1.38–1.33 (m, 2H), 1.29 (s, 3H),
1.20 (s, 3H), 1.16 (s, 3H), 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CD3OD) d (ppm) 178.3, 175.0, 147.1, 138.1, 129.6, 125.4, 102.5,
95.4, 83.0, 78.4, 77.8, 76.5, 73.9, 73.2, 72.3, 70.7, 70.0, 69.8, 65.4,
64.7, 60.8, 57.1, 5.54, 51.2, 48.2, 48.0, 47.9, 47.2, 46.1, 45.7, 45.3,
45.2, 41.9, 41.1, 36.1, 35.6, 31.7, 29.4, 29.1, 25.5, 22.7, 22.4,
20.6, 16.1, 13.2, 12.9, 10.1, 8.5, 6.9. HRMS (ESI) m/z Calcd for
(210 mg, 58%). 1H NMR (CDCl3, 400 MHz)
d (ppm) 7.44 (d,
J = 7.2 Hz, 1H), 7.22 (m, 3H), 4.88 (d, J = 8.4 Hz, 1H), 4.22 (d,
J = 7.2 Hz, 1H), 4.15 (d, J = 8.8 Hz, 1H), 3.91 (m, 1H), 3.70 (m, 4H),
3.35 (m, 4H), 2.94 (m, 2H), 2.59 (m, 4H), 2.10 (s, 3H), 1.48 (m,
27H), 0.98 (d, J = 7.2 Hz, 3H), 0.79 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 100 MHz) d (ppm) 204.2, 181.2, 169.5, 156.0, 141.1,
133.3, 129.3, 128.9, 127.2, 122.1, 104.0, 82.1, 81.6, 81.5, 79.2,
78.4, 76.4, 70.4, 69.5, 65.2, 59.9, 56.7, 51.1, 49.5, 49.1, 48.1, 42.7,
42.3, 38.5, 36.3, 35.8, 29.5, 22.0, 21.2, 19.6, 19.1, 16.4, 14.4, 12.8,
10.8, 10.4. HRMS (MALDI) m/z Calcd for C41H60N3O9 [M+H+]:
738.4324, found 738.4348.
C
49H83N6O14 [M+H+]: 979.5962, found for 979.5958.
5.1.30. Azithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxybu-
tanamide (17c)
Reaction of (30-N-(4-ethynylbenzyl))azithromycin 14 (0.15 g,
0.17 mmol) and 4-Azido-N-((tert-butyldimethyl silyl)oxy)bu-
tanamide 51c (0.08 g, 0.32 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 17a, gave
17c as light yellow solid (110 mg, 62%). 1H NMR (400 MHz, CD3OD)
d (ppm) 8.21 (s, 1H), 7.66 (d, J = 7.5 Hz, 2H), 7.33 (d, J = 6.2 Hz, 2H),
4.35 (d, J = 7.1 Hz, 2H), 3.97–3.88 (m, 1H), 3.66–3.55 (m, 1H), 3.42
(s, 3H), 3.26 (d, J = 10.4, 7.0 Hz, 3H), 3.13–3.11 (m, 2H), 2.96 (s, 3H),
2.92–2.78 (m, 1H), 2.66 (dd, J = 12.4, 7.4 Hz, 3H), 2.59 (s, 3H), 2.29
(s, 3H), 2.18 (d, J = 15.0 Hz, 2H), 2.00 (dd, J = 20.2, 12.5 Hz, 2H), 1.81
(d, J = 7.6 Hz, 2H), 1.75 (s, 24H), 1.39 (dd, J = 15.2, 9.7 Hz, 2H), 1.26
(s, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.11 (d, J = 3.8 Hz, 3H), 1.06–1.00 (m,
1H), 0.96 (s, 3H), 0.88 (d, J = 7.4 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H),
0.71 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CD3OD) d (ppm)
180.5, 177.2, 172.2, 149.5, 131.7, 131.6, 127.5, 123.1, 104.7, 97.6,
85.2, 80.6, 80.1, 78.8, 76.7, 76.1, 75.6, 75.1, 73.1, 70.1, 65.4, 59.4,
51.5, 50.8, 50.5, 50.2, 50.1, 49.5, 47.6, 44.1, 38.2, 37.7, 36.7, 32.8,
31.6, 31.5, 31.3, 31.2 28.1, 27.8, 22.9, 22.9, 22.8, 22.4, 19.8, 18.2,
16.5, 12.3, 10.6, 9.0. HRMS (ESI) m/z Calcd for C50H85N6O14 [M
+H+]: 993.6118, found 993.6113.
5.1.33. (30-N-(3-Ethynylbenzyl))tricyclic ketolide (22)
Reaction of 30-desmethyltricyclic ketolide 18 (0.50 g, 0.8 mmol)
and 3-ethynylbenzaldehyde 20 (0.21 g, 1.6 mmol) in MeOH (9 mL)
and acetic acid (91.4 lL, 1.6 mmol) for 1 h at room temperature
followed by addition of borane-pyridine complex (0.2 ml,
1.6 mmol) within 3 h and then purification as described in the pro-
tocol for 21 afforded 22 as a brown solid (490 mg, 83%). 1H NMR
(CDCl3, 400 MHz) d (ppm) 7.15 (m, 2H), 7.06 (m, 2H), 4.72 (d,
J = 8.0 Hz, 1H), 4.08 (d, J = 7.2 Hz, 1H), 3.99 (d, J = 8.4 Hz, 1H),
3.53 (m, 4H), 3.76 (m, 1H), 3.20 (m, 3H), 2.78 (m, 2H), 2.78 (m,
2H), 2.44 (m, 4H), 1.93 (s, 3H), 1.34 (m, 28H), 0.82 (d, J = 7.2 Hz,
3H), 0.62 (t, J = 7.6 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d (ppm)
204.2. 181.1, 169.5, 156.0, 139.1, 132.2, 131.0, 129.1, 128.4,
122.2, 103.8, 83.5, 81.5, 79.2, 78.5, 76.5, 70.3, 69.5, 65.6, 59.9,
57.2, 51.1, 49.5, 48.0, 42.8, 42.3, 38.5, 36.8, 36.3, 29.5, 29.2, 22.0,
21.1, 19.6, 19.1, 16.4, 14.3, 12.8, 10.8, 10.4. HRMS (MALDI) m/z
Calcd for C41H60N3O9 [M+H+]: 738.4324, found 738.4329.
5.1.34. (Tricyclic ketolide-30-(N-(2-triazolylbenzyl)))-N-hydroxy-
hexanamide (24a)
5.1.31. Azithromycin-30-(N-(4-triazolylbenzyl)))-N-hydroxypen-
tanamide (17d)
To a round bottomed flask containing (30-N-(2-ethynylbenzyl))
tricyclic ketolide 21 (0.045 g, 0.061 mmol) and copper (I) iodide
(10 mg, 0.05 mmol) under argon, was added degassed THF (4 mL)
and Hunig’s base (0.5 mL). Stirring under argon and at room tem-
perature continued for 15 min after which 6-azido-N-((tert-
butyldimethylsilyl)oxy)hexanamide 51e (35 mg, 0.12 mmol) was
added to the reaction mixture, and stirred for an additional 24 h
at 40 °C. The reaction mixture was diluted with ethyl acetate
(50 mL) and the organic layer was washed with 1:4 NH4OH soln./
saturated aqueous NH4Cl soln. (2 ꢂ 4 mL), water (10 mL), and brine
(10 mL). The organic layer was dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The crude was subjected to next
reaction without further purification.
Reaction of (30-N-(4-ethynylbenzyl))azithromycin 14 (0.14 g,
0.16 mmol) and 5-azido-N-((tert-butyldimethyl silyl)oxy)pen-
tanamide 51d (0.07 g, 0.24 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 17a, gave
17d as light yellow solid (110 mg, 66%). 1H NMR (400 MHz, CD3OD)
d (ppm) 8.28 (s, 1H), 7.73 (d, J = 7.7 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H),
4.47 (d, J = 7.2 Hz, 1H), 4.41 (s, 2H), 4.12 (d, J = 17.0 Hz, 2H), 3.80 (d,
J = 13.0 Hz, 1H), 3.68 (s, 1H), 3.64–3.50 (m, 4H), 3.35–3.24 (m, 2H),
3.13 (s, 3H), 3.00–2.91 (m, 2H), 2.76 (dd, J = 7.5, 4.4 Hz, 2H), 2.65 (d,
J = 11.8 Hz, 1H), 2.39 (s, 3H), 2.32 (d, J = 15.0 Hz, 2H), 2.24 (s, 3H),
2.15–2.09 (m, 3H), 1.98–1.86 (m, 22H), 1.31 (s, 3H), 1.23 (d,
J = 5.9 Hz, 5H), 1.18–1.09 (m, 7H), 1.06 (s, 3H), 1.01 (d, J = 7.5 Hz,
4H), 0.91 (s, 2H), 0.84 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CD3-
OD) d (ppm) 180.5, 177.2, 149.5, 141.7, 131.6, 131.4, 127.4,
122.9, 104.8, 97.3, 85.3, 80.5, 80.2, 78.9, 76.3, 76.2, 76.1, 75.1,
73.3, 70.0, 67.3, 65.3, 59.6, 51.8, 50.8, 50.3, 50.1, 50.0, 49.8, 49.5,
49.3, 47.5, 44.3, 43.9, 38.2, 37.6, 36.8, 32.8, 31.5, 28.5, 28.3, 24.4,
23.1, 22.9, 22.5, 19.8, 18.2, 16.5,12.4, 10.7, 8.6. HRMS (ESI) m/z
Calcd for C51H87N6O14 [M+H+]: 1007.6275, found 1007.6272.
The crude was dissolved in anhydrous methanol (2 mL) and
caesium fluoride (18 mg, 0.12 mmol) was added and the resulting
reaction mixture was stirred at room temperature for 2 h. Reaction
was quenched by adding water (10 mL) and was extracted with
ethyl acetate (30 mL) and the organic layer was washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The crude was purified by preparative chromatography (Sil-
ica gel, 12:1:0.1 CH2Cl2/MeOH/concd NH4OH) to give 24a as a
brown-white solid (30 mg, 55%). 1H NMR (CDCl3, 400 MHz) d
(ppm) 7.78 (s, 1H), 7.52 (m, 1H), 7.28 (m, 3H), 4.88 (d, J = 8.4 Hz,
1H), 4.33 (m, 2H), 4.14 (m, 2H), 3.79 (m, 5H), 3.43 (m, 1H), 3.16
(m, 2H), 2.98 (m, 1H), 2.87 (m, 1H), 2.62 (m, 5H), 2.08 (s, 3H),
1.59 (m, 34H), 0.96 (d, J = 6.0 Hz, 3H), 0.78 (t, J = 7.6 Hz, 3H). 13C
NMR (CDCl3, 100 MHz) d (ppm) 204.4, 182.5, 170.3, 169.6, 156.1,
147.0, 136.0, 131.1, 130.5, 129.9, 128.3, 127.9, 122.3, 105.0,
103.5, 81.6, 78.6, 78.5, 76.5, 70.3, 69.3, 63.6, 59.9, 56.9, 51.1,
49.9, 49.2, 49.1, 47.9, 42.3, 38.4, 36.3, 35.3, 29.6, 29.3, 28.5, 25.6,
24.4, 21.1, 22.1, 19.6, 19.0, 16.4, 14.4, 12.9, 10.8, 10.4. HRMS
(MALDI) m/z Calcd for C47H72N7O11 [M+H+]: 910.5284, found
910.5245.
5.1.32. (30-N-(2-Ethynylbenzyl))tricyclic ketolide (21)
A
solution of 30-desmethyltricyclic ketolide 18 (0.30 g,
0.48 mmol) and 2-ethynylbenzaldehyde 19 (0.19 g, 1.45 mmol) in
MeOH (5 mL) and acetic acid (55.2 L, 0.96 mmol) was stirred for
l
30 min at room temperature. Borane-pyridine complex (0.12 mL,
0.96 mmol) was added and the reaction stirred was for another
3 h. The reaction was diluted with ethyl acetate (20 mL) and
washed with saturated aqueous NaHCO3 solution (20 mL) and
brine (20 mL). The organic layer was dried over Na2SO4, concen-
trated in vacuo and purified by preparative chromatography (Silica
gel, 12:1 CH2Cl2/MeOH) to afford compound 21 as a brown solid
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
13
5.1.35. (Tricyclic Ketolide-30-(N-(2-triazolylbenzyl)))-N-hydrox-
yheptanamide (24b)
and DMSO, CuI (6.4 mg, 0.03 mmol) and N,N0-diisopropylethy-
lamine (30 L, 0.15 mmol) in succession. The mixture was heated
l
Reaction of (30-N-(2-ethynylbenzyl))tricyclic Ketolide 21
(0.05 g, 0.067 mmol) and 7-azido-N-((tert-butyldimethylsilyl)oxy)
heptanamide 51f (0.04 g, 0.134 mmol) followed by TBS deprotec-
tion with caesium fluoride as described for the synthesis of 24a,
gave 24b as a brown-white solid (35 mg, 56%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.80 (s, 1H), 7.51 (m, 1H), 7.31 (m, 3H), 4.88
(d, J = 10.4 Hz, 1H), 4.34 (m, 2H), 4.20 (d, J = 7.2 Hz, 1H), 4.15 (d,
J = 8.4 Hz, 1H), 3.78 (m, 5H), 3.46 (m, 1H), 3.19 (m, 2H), 3.0 (m,
1H), 2.88 (m, 1H), 2.68 (m, 2H), 2.64 (s, 3H), 2.16 (s, 3H), 1.62
(m, 36H), 0.99 (d, J = 6.8 Hz, 3H), 0.79 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 100 MHz) d (ppm) 204.4, 182.3, 170.4, 169.6, 156.0,
147.0, 130.5, 129.9, 128.4, 122.3, 103.6, 81.6, 78.7, 78.5, 76.5,
70.2, 69.1, 64.1, 63.9, 59.9, 53.7, 51.3, 51.1, 50.1, 49.1, 47.9, 42.3,
42.0, 38.5, 36.4, 35.2, 32.7, 32.2, 29.7, 28.6, 27.7, 26.3, 25.5, 25.2,
24.8, 22.0, 21.1, 19.6, 19.0, 18.0, 16.3, 14.4, 12.9, 12.1, 10.8, 10.4.
HRMS (MALDI) m/z Calcd for C48H74N7O11 [M+H+]: 924.5441,
found 924.5422.
at 45 °C for 12 h. The reaction mixture was cooled to room temper-
ature and diluted with 50 mL of ethyl acetate. Organic layer was
washed with 4:1 mixture of satd. NH4Cl soln. and NH4OH soln.
(2 ꢂ 10 mL), water (10 mL), and brine (10 mL), dried over anhy-
drous Na2SO4, filtered and concentrated in vacuo. The crude was
used for the next reaction without further purification.
The crude was dissolved in methanol (3 mL) and to that solu-
tion caesium fluoride (68 mg, 0.442 mmol) was added and the
resulting mixture was stirred at room temperature for 2 h. The
reaction was quenched by adding water and extracted with ethyl
acetate (100 mL) and the organic layer was washed with brine
(10 mL), dried over anhydrous Na2SO4, filtered and concentrated
in vacuo. The crude was purified by preparative chromatography
(Silica gel, 10:1:0.1 CH2Cl2/MeOH/concd NH4OH soln.) to give
26a as a white solid (76 mg, 40%). 1H NMR (500 MHz, MeOH-d4)
d (ppm) 8.24 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz,
2H), 5.00 (s, 2H), 4.18 (m, 2H), 3.81 (dd, J = 44.1, 13.9 Hz, 2H),
3.69 (m, 2H), 3.50 (m, 4H), 3.26 (m, 1H), 3.09 (p, J = 7.5 Hz, 1H),
2.85(m, 2H); 2.72 (m, 1H), 2.58 (m, 5H), 2.21 (d, J = 15.7 Hz, 3H),
1.72 (m, 2H), 1.60 (m, 2H), 1.52 (m, 1H), 1.44 (m, 3H), 1.19 (m,
17 H), 1.01 (t, J = 13.0 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). 13C
(125 MHz, MeOH-d4) d (ppm) 184.6, 170.0, 163.4, 156.6, 147.3,
139.0, 129.4, 125.3, 122.3, 103.6, 82.2, 78.5, 77.7, 76.2, 70.7, 69.0,
57.3, 50.7, 49.8, 48.6, 48.3, 42.1, 41.9, 38.1, 36.4, 36.0, 30.9, 21.8,
20.1, 18.9, 17.8, 15.1, 13.5, 11.7, 9.8, 9.6. HRMS (ESI) m/z Calcd
for C43H64N7O11 [M+H+]: 854.4658, found 854.4656.
5.1.36. (Tricyclic ketolide-30-(N-(3-triazolylbenzyl)))-N-hydroxy-
hexanamide (25a)
Reaction of (30-N-(3-ethynylbenzyl))tricyclic Ketolide 22
(0.04 g, 0.05 mmol) and 6-azido-N-((tert-butyldimethylsilyl)oxy)
hexanamide 51e (0.03 g, 0.11 mmol) followed by TBS deprotection
with caesium fluoride as described for the synthesis of 24a, gave
25a as
a
brown-white solid (32 mg, 64%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.86 (s, 1H), 7.74 (m, 2H), 7.35 (m, 1H), 7.23
(m, 1H), 4.94 (d, J = 9.6 Hz, 1H), 4.37 (m, 2H), 4.31 (d, J = 6.4 Hz,
1H), 4.23 (d, J = 8.0 Hz, 1H), 3.98 (m, 1H), 3.63 (m, 5H), 2.13 (m,
4H), 2.60 (m, 5H), 2.20 (s, 3H), 1.68 (m, 34H), 1.04 (d, J = 6.4 Hz,
3H), 0.84 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d (ppm)
204.3, 182.1, 170.3, 169.6, 156.0, 147.5, 139.5, 130.7, 128.4,
126.0, 124.6, 120.2, 103.8, 81.6, 78.9, 78.5, 76.4, 70.3, 69.5, 65.4,
59.9, 57.6, 53.6, 51.1, 50.0, 49.3, 49.1, 47.9, 42.5, 42.3, 42.9, 38.5,
36.8, 29.6, 29.5, 25.6, 22.0, 21.1, 19.6, 19.1, 18.1, 16.2, 14.4, 12.8,
12.2, 10.8, 10.4. HRMS (MALDI) m/z Calcd for C47H72N7O11 [M
+H+]: 910.5284, found 910.5278.
5.1.39. (Tricyclic Ketolide-30-(N-(4-triazolylbenzyl)))-N-propan-
amide (26b)
Reaction of (30-N-(4-ethynylbenzyl))tricyclic ketolide 23
(0.20 g, 0.271 mmol) and 3-azido-N-((tert-butyldimethylsilyl)oxy)
propanamide 51b (0.11 g, 0.406 mmol) followed by TBS deprotec-
tion with caesium fluoride as described for the synthesis of 26a,
gave 26b as white solid (105 mg, 45%). 1H NMR (500 MHz,
MeOH-d4) d (ppm) 8.13 (s, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.35 (d,
J = 8.1 Hz, 2H), 4.65 (t, J = 6.2 Hz, 2H), 4.19 (dd, J = 16.2, and
7.7 Hz, 2H), 3.87 (m, 1H), 3.72 (m, 3H), 3.57 (m, 1H), 3.53 (m,
1H), 3.48 (m, 2H), 3.27 (m, 1H), 3.09 (m, 1H), 2.88 (dd, J = 14.3,
and 10.1 Hz, 2H), 2.69 (t, J = 10.8 Hz, 3H), 2.60 (m, 4H), 2.18 (d,
J = 14.1 Hz, 3H), 1.74 (m, 2H), 1.62 (t, J = 7.6 Hz, 2H), 1.54 (m,
1H), 1.47 (m, 3H), 1.21 (m, 17H), 1.03 (t, J = 12.4 Hz, 3H), 0.78 (t,
J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm) 196.6, 176.1,
161.6, 159.1, 148.2, 138.7, 130.9, 120.8, 116.8, 112.8, 95.2, 73.7,
70.0, 69.2, 67.8, 62.3, 60.6, 55.6, 51.7, 48.9, 42.3, 40.2, 39.8, 37.5,
33.7, 33.5, 29.6, 27.9, 27.5, 22.5, 13.4, 11.6, 10.5, 9.4. HRMS (ESI)
m/z Calcd for C44H66N7O11 [M+H+]: 868.4815, found 868.4812.
5.1.37. (Tricyclic Ketolide-30-(N-(3-triazolylbenzyl)))-N-hydroxy-
heptanamide (25b)
Reaction of (30-N-(3-ethynylbenzyl))tricyclic Ketolide 22
(0.05 g, 0.067 mmol) and 7-azido-N-((tert-butyldimethylsilyl)oxy)
heptanamide 51f (0.04 g, 0.134 mmol) followed by TBS deprotec-
tion with caesium fluoride as described for the synthesis of 24a,
gave 25b as a brown-white solid (34 mg, 55%). 1H NMR (CDCl3,
400 MHz) d (ppm) 7.74 (s, 1H), 7.71 (s, 1H),. 7.63 (d, J = 7.6 Hz,
1H), 7.30 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 4.89 (d,
J = 8.4 Hz, 1H), 4.32 (m, 2H), 4.25 (d, J = 7.2 Hz, 1H), 4.17 (d,
J = 8.4 Hz, 1H), 3.92 (m, 1H), 3.71 (m, 4H), 3.20 (m, 5H), 2.64 (m,
5H), 2.15 (s, 3H), 1.71 (m, 36H), 0.99 (d, J = 7.2 Hz, 3H), 0.79 (t,
J = 7.6 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d (ppm) 204.3, 181.9,
170.5, 169.6, 156.1, 147.6, 139.6, 130.7, 129.0, 128.6, 126.0,
124.7, 119.9, 103.8, 81.6, 78.9, 78.5, 76.5, 70.4, 69.5, 65.4, 59.9,
57.6, 51.2, 50.1, 49.4, 49.1, 47.9, 42.3, 38.6, 36.9, 36.4, 29.8, 29.7,
29.5, 27.9, 25.6, 24.9, 22.1, 21.0, 19.6, 19.1, 16.2, 14.4, 14.2, 12.9,
10.9, 10.4. HRMS (MALDI) m/z Calcd for C48H74N7O11 [M+H+]:
924.5441, found 924.5455.
5.1.40. (Tricyclic Ketolide-30-(N-(4-triazolylbenzyl)))-N-hydroxy-
butanamide (26c)
Reaction of (30-N-(4-ethynylbenzyl))tricyclic ketolide 23
(0.173 g, 0.230 mmol) and 4-azido-N-((tert-butyldimethylsilyl)
oxy)butanamide 51c (0.011 g, 0.420 mmol) followed by TBS depro-
tection with caesium fluoride as described for the synthesis of 26a,
gave 26c as white solid (106 mg, 52%). 1H NMR (500 MHz, MeOH-
d4) d (ppm) 8.23 (s, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 7.9 Hz,
2H), 4.41 (t, J = 6.7 Hz, 2H), 4.19 (dd, J = 15.6, and 7.7 Hz, 2H), 3.88
(m, 2H), 3.73 (q, J = 13.3 Hz, 3H), 3.59 (m, 1H), 3.50 (dd, J = 17.4 and
9.4 Hz, 2H), 3.28 (dd, J = 10.0 and 7.6 Hz, 1H), 3.09 (m, 1H), 2.89
(dd, J = 14.6 and 10.4 Hz, 2H), 2.69 (dd, J = 15.5, and 6.7 Hz, 1H),
2.60 (m, 4H), 2.17 (m, 5H), 2.08 (m, 2H), 1.75 (m, 2H), 1.62 (m,
1H), 1.55 (m, 1H), 1.48 (s, 3H), 1.22 (m, 17H), 1.02 (d, J = 6.9 Hz,
3H), 0.79 (t, J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm)
206.6, 186.1, 171.6, 158.2, 149.0, 141.0, 130.8, 126.8, 122.4,
105.2, 83.8, 80.1, 79.2, 77.9, 72.3, 70.7, 65.6, 61.7, 59.0, 52.4,
5.1.38. (Tricyclic Ketolide-30-(N-(4-triazolylbenzyl)))-N-hydroxy-
acetamide (26a)
In an oven dried round bottomed flask charged with a magnetic
stirring bar, (30-N-(4-ethynylbenzyl))tricyclic ketolide 2315
(0.165 g, 0.224 mmol) and 2-azido-N-((tert-butyldimethylsilyl)
oxy)acetamide 51a (0.09 g, 0.40 mmol) were placed under argon.
To the mixture were added 2 mL of degassed1:1 mixture of THF
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

14
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
51.9, 50.8, 50.2, 49.9, 48.0, 39.7, 37.9, 37.5, 32.6, 30.5, 27.4, 26.2,
23.4, 21.6, 20.5, 19.4, 16.6, 15.1, 13.2, 11.3, 11.1, 9.5. HRMS (ESI)
m/z Calcd for C45H68N7O11 [M+H+]: 882.4971, found 882.4971.
nium iodide (3.90 g, 17.7 mmol) was added over a period of 5 min.
When hydrogen evolution ceased and a clear solution obtained, a
solution of compound 31 (6.35 g, 8.06 mmol) in anhydrous THF
(10 mL) was added over a period of 10 min and left to stir for
2 h. Once TLC showed 100% conversion, THF was removed under
vacuum and ethyl acetate was added to the remaining solution.
The solution was washed severally with H2O to remove DMSO.
Organic layer was dried with NaSO4 and concentrated in vacuo.
The crude product was purified by column chromatography using
EtOAc/Acetone (5:1) to give compound 33 as a white solid (2.43 g,
38%). 1H NMR (400 MHz, CDCl3) d (ppm) 5.06 (dq, J = 6.2, 4.6 Hz,
2H), 4.77–4.59 (m, 4H), 3.95 (s, 1H), 3.76–3.66 (m, 3H), 3.55 (s,
1H), 3.45 (dd, J = 9.3, 6.0 Hz, 1H), 3.34–3.28 (m, 4H), 3.19 (s, 1H),
3.01 (d, J = 7.0 Hz, 1H), 3.01–2.95 (m, 4H), 2.94–2.90 (m, 1H),
2.86 (dd, J = 9.9, 7.1 Hz, 1H), 2.69–2.63 (m, 1H), 2.61 (dd, J = 9.8,
4.4 Hz, 2H), 2.53 (dd, J = 9.6, 7.1 Hz, 1H), 2.34 (s, 1H), 2.27–2.20
(m, 7H), 2.19–2.13 (m, 2H), 2.06–2.00 (m, 4H), 1.95–1.83 (m,
3H), 1.72–1.61 (m, 2H), 1.56 (d, J = 1.6 Hz, 1H), 1.53 (s, 1H), 1.49–
1.36 (m, 1H), 1.33 (d, J = 7.2 Hz, 3H), 1.28 (s, 1H), 1.26–1.12 (m,
11H), 1.14–0.99 (m, 16H), 0.92 (t, J = 8.2 Hz, 3H), 0.81 (dd, J = 9.8,
4.9 Hz, 3H). 13C NMR (101 MHz, CDCL3) d (ppm) 221.1, 175.3,
170.0, 100.1, 96.4, 96.3, 80.3, 78.9, 78.3, 78.2, 76.6, 76.1, 74.2,
73.7, 71.7, 69.1, 67.8, 64.3, 63.2, 60.4, 60.2, 50.4, 49.6, 46.6, 45.3,
44.8, 40.7, 38.6, 38.1, 37.2, 36.0, 30.8, 29.2, 21.6, 21.3, 21.0, 20.2,
19.7, 18.2, 18.0, 16.1, 15.9, 14.9, 14.2, 14.2, 12.4, 10.5, 9.1. HRMS
(MALDI) m/z Calcd for C41H77NO14 [M+H+]: 802.4947, found
802.4938.
5.1.41. Tricyclic Ketolide-30-(N-(4-triazolylbenzyl)))-N-hydrox-
ypentanamide (26d)
Reaction of (30-N-(4-ethynylbenzyl))tricyclic ketolide 23
(0.114 g, 0.150 mmol) and 5-azido-N-((tert-butyldimethylsilyl)
oxy)pentanamide 51d (0.044 g, 0.28 mmol) followed by TBS depro-
tection with caesium fluoride as described for the synthesis of 26a,
gave 26d as white solid (67 mg, 50%). 1H NMR (500 MHz, MeOH-
d4) d (ppm)8.23 (s, 1H), 7.70 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 7.9 Hz,
2H), 4.38 (d, J = 6.8 Hz, 2H), 4.20 (dd, J = 17.7, and 7.7 Hz, 2H),
3.94 (q, J = 6.7 Hz, 1H), 3.88 (d, J = 14.6 Hz, 1H), 3.81 (d,
J = 13.3 Hz, 1H), 3.71 (m, 2H), 3.58 (dd, J = 20.0, and 11.2 Hz, 2H),
3.50 (m, 1H), 3.30 (dd, J = 9.9, and 7.6 Hz, 1H), 3.11 (m, 1H), 2.89
(dd, J = 14.4, and 10.4 Hz, 2H), 2.74 (m. 1H), 2.60 (m, 3H), 2.23 (s,
3H), 2.07 (t, J = 7.1 Hz, 2H), 1.88 (m, 2H), 1.77 (m, 2H), 1.57 (m,
5H), 1.48 (s, 3H), 1.24 (m, 17H), 1.02 (d, J = 6.9 Hz, 3H), 0.79 (t,
J = 7.4 Hz, 3H). 13C (125 MHz, MeOH-d4) d (ppm) 206.6, 186.2,
172.4, 171.6, 158.2, 148.8, 131.0, 126.9, 122.4, 105.2, 83.8, 80.0,
79.2, 77.9, 72.2, 70.6, 65.6, 61.7, 58.8, 52.4, 51.2, 50.2, 49.9, 39.7,
37.9, 37.6, 33.1, 32.5, 30.8, 23.8, 23.4, 21.6, 20.5, 19.4, 16.6, 15.1,
13.3, 11.4, 11.1. HRMS (ESI) m/z Calcd for C46H70N7O11 [M+H+]:
896.5128, found 896.5121.
5.1.42. 400-Oxo-30-O-acetylclarithromycin (31)
A solution of N-chlorosuccinimide (2.00 g, 14.97 mmol) in
anhydrous DCM (30 mL) was stirred at ꢀ15 °C for 10 min. Dimethyl
sulfide (1.10 mL, 14.97 mmol) was then added drop wise to the
solution. After stirring for 20 minutes at the same temperature a
DCM solution (10 mL) of acetylated clarithromycin 29 (5.91 g,
7.49 mmol) was added over a period of 30 min to the suspension
and the resulting suspension was stirred at ꢀ15 °C for another
30 min, afterward TEA (2.09 mL, 14.97 mmol) was added. The
resulting solution was stirred at ꢀ10 °C for another 2 h. The reac-
tion was quenched by adding saturated aqueous NaHCO3 solution
(50 mL), the organic layer was separated. The aqueous layer was
extracted with DCM (2 ꢂ 25 mL) and the combined organic layer
was dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The crude compound 31 (6.35 g) was sufficiently pure to
be used for the next step without further purification. 1H NMR
(400 MHz, CDCl3) d (ppm) 5.11–5.02 (m, 1H), 4.97 (d, J = 10.7 Hz,
1H), 4.75 (dd, J = 10.3, 7.6 Hz, 1H), 4.39 (t, J = 6.1 Hz, 1H), 4.29 (q,
J = 6.7 Hz, 1H), 3.67–3.53 (m, 3H), 3.38 (td, J = 15.1, 5.0 Hz, 2H),
3.30 (d, J = 1.3 Hz, 4H), 3.15 (t, J = 9.1 Hz, 3H), 2.93–2.82 (m, 4H),
2.78 (dd, J = 16.9, 7.3 Hz, 1H), 2.68 (d, J = 1.3 Hz, 1H), 2.65 (d,
J = 2.7 Hz, 1H), 2.60 (d, J = 1.2 Hz, 1H), 2.56 (d, J = 1.3 Hz, 1H),
2.49–2.37 (m, 8H), 2.29–2.14 (m, 3H), 2.07–2.02 (m, 2H), 1.97 (t,
J = 8.3 Hz, 3H), 1.94–1.73 (m, 15H), 1.60–1.47 (m, 1H), 1.41–1.33
(m, 2H), 1.32–1.23 (m, 8H), 1.21 (d, J = 9.9 Hz, 3H), 1.12 (dd,
J = 11.2, 6.4 Hz, 7H), 1.07–0.93 (m, 10H), 0.78 (t, J = 9.6 Hz, 3H),
0.70 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) d (ppm) 221.0,
210.6, 175.6, 170.4, 99.6, 96.4, 80.0, 78.7, 78.1, 78.0, 76.7, 76.0,
74.2, 72.9, 72.7, 69.8, 69.2, 67.5, 61.7, 51.2, 50.2, 49.9, 45.1, 44.4,
39.0, 38.4, 38.1, 37.9, 37.6, 37.2, 30.8, 30.4, 29.5, 28.3, 26.5, 21.3,
21.3, 20.9, 20.5, 20.4, 20.0, 19.5, 18.9, 17.8, 16.2, 16.1, 15.5, 14.7,
13.0, 12.2, 10.4, 8.9. HRMS (MALDI) m/z Calcd for C40H70NO14 [M
+H+]: 788.4791, found 788.4781.
5.1.44. (400-(Methylamino)-N-(methyl)(4-ethynylbenzyl))
clarithromycin (36)
1-(4-Ethynylphenyl)-N-methylmethanamine
35
(1.52 g,
10.47 mmol) was added to a solution of compound 33 (2.80 g,
3.49 mmol) in MeOH (20 mL). The solution was heated at 60 °C
for 6 h. Excess MeOH was evaporated off and the crude was puri-
fied by column chromatography using Hexane/EtOAc/MeOH/NH4-
OH (2:1:0.6:0.1) to give compound 36 as a light yellow solid
(2.41 g, 73%). 1H NMR (400 MHz, CDCl3)
d (ppm) 7.33 (d,
J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 4.98–4.86 (m, 2H), 4.35–
4.28 (m, 1H), 4.06 (q, J = 5.9 Hz, 1H), 3.66 (d, J = 11.1 Hz, 2H),
3.57 (d, J = 7.0 Hz, 2H), 3.34 (s, 1H), 3.20 (s, 3H), 3.09 (dd, J = 10.0,
7.3 Hz, 2H), 3.02 (d, J = 10.1 Hz, 1H), 2.97 (d, J = 6.6 Hz, 1H), 2.94
(s, 2H), 2.91–2.85 (m, 2H), 2.81–2.72 (m, 1H), 2.47 (d, J = 7.4 Hz,
1H), 2.34 (dd, J = 15.6, 6.7 Hz, 1H), 2.24 (s, 1H), 2.20 (s, 6H), 2.10
(s, 3H), 2.04 (d, J = 3.6 Hz, 1H), 2.00 (s, 1H), 1.96 (s, 1H), 1.93–
1.87 (m, 1H), 1.84–1.70 (m, 3H), 1.57 (dd, J = 19.0, 11.8 Hz, 2H),
1.41–1.31 (m, 1H), 1.30 (d, J = 11.1 Hz, 3H), 1.15–1.07 (m, 10H),
1.05 (s, 4H), 0.97 (dd, J = 17.3, 7.0 Hz, 10H), 0.71 (dd, J = 13.8,
6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) d (ppm) 220.8, 175.7,
139.1, 132.1, 128.6, 121.2, 102.7, 96.4, 83.2, 80.9, 78.9, 78.0, 76.3,
76.1, 75.9, 74.2, 70.9, 70.6, 68.9, 68.2, 67.4, 65.4, 63.1, 56.9, 50.6,
49.5, 45.2, 44.8, 43.2, 40.2, 39.3, 38.8, 37.1, 31.3, 28.7, 21.7, 20.9,
20.2, 19.6, 19.1, 18.7, 18.0, 15.9, 15.3, 14.8, 14.6, 13.0, 12.2, 11.4,
10.6, 10.5, 9.3, 9.1. HRMS (ESI) m/z Calcd for [C49H81N2O13 [M
+H+]: 905.5739, found 905.5693.
5.1.45. (Clarithromycin-(400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxyacetamide (38a)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.15 g, 0.17 mmol) with 2-Azido-N-((tert-
butyldimethylsilyl)oxy)acetamide 51a (0.057 g, 0.249 mmol) fol-
lowed by TBS removal with caesium fluoride as described for the
synthesis of compound 5a, gave 38a as a light yellow solid
(0.057 g, 38%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.37 (d,
J = 5.4 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H),
5.50 (d, J = 5.4 Hz, 1H), 5.12 (d, J = 14.2 Hz, 2H), 5.00–4.95
(m, 1H), 4.43 (d, J = 7.3 Hz, 1H), 4.22 (d, J = 6.4 Hz, 1H), 3.85–3.65
5.1.43. 400-Epoxy-30-O-acetylclarithromycin (33)
NaH (60% in mineral oil) (0.73 g, 17.7 mmol) was added to an
oven dried three-neck round bottom flask and was washed with
petroleum ether. The flask was immediately flushed with Ar and
5 mL of anhydrous DMSO added through the septum. The mixture
was stirred at room temperature under Ar and trimethyloxosulfo-
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
15
(m, 5H), 3.61 (s, 1H), 3.50 (s, 1H), 3.32–3.25 (m, 5H), 3.13–3.00 (m, 5H),
2.96–2.85 (m, 1H), 2.76 (d, J = 10.3 Hz, 1H), 2.58 (s, 1H), 2.53 (s,
1H), 2.48 (s, 5H), 2.35 (s, 1H), 2.32–2.25 (m, 3H), 2.24 (s, 1H),
2.21 (s, 1H), 2.16 (d, J = 7.6 Hz, 1H), 2.14 (d, J = 5.4 Hz, 1H), 2.01
(s, 1H), 1.98–1.74 (m, 5H), 1.66 (d, J = 12.7 Hz, 1H), 1.57–1.44 (m,
1H), 1.40 (s, 3H), 1.31–1.15 (m, 17H), 1.12 (dt, J = 11.4, 4.9 Hz,
10H), 0.85 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d (ppm)
176.0, 147.3, 129.4, 125.8, 122.3, 102.4, 96.5, 81.1, 78.3, 78.2,
74.3, 70.8, 69.1, 68.0, 67.5, 65.4, 62.9, 53.5, 50.7, 49.6, 45.2,
45.0, 43.4, 40.3, 39.1, 37.3, 31.5, 29.7, 21.5, 21.0, 19.8, 18.9, 18.0,
16.0, 15.4, 14.9, 14.8, 13.1, 12.3, 11.5, 10.7, 10.6, 9.3. HRMS
(MALDI) m/z Calcd for C51H86N6O15 [M+H+]: 1021.6073, found
1021.6095.
5.1.48. (Clarithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxypentanamide (38d)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.15 g, 0.17 mmol) with 5-Azido-N-((tert-
butyldimethylsilyl)oxy)pentanamide 51d (0.068 g, 0.249 mmol)
followed by TBS removal with caesium fluoride as described for
the synthesis of compound 5a, gave 38d as a light yellow solid
(0.067 g, 44%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.30 (s, 1H),
7.76 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 5.12–5.03 (m, 1H),
4.47–4.36 (m, 3H), 4.19 (d, J = 6.4 Hz, 1H), 3.81–3.59 (m, 5H),
3.48 (s, 1H), 3.29–3.18 (m, 4H), 3.04 (dd, J = 10.3, 4.5 Hz, 2H),
3.00 (d, J = 9.6 Hz, 4H), 2.92–2.82 (m, 1H), 2.66 (s, 1H), 2.55 (d,
J = 7.3 Hz, 1H), 2.40 (s, 6H), 2.30 (s, 1H), 2.23 (s, 4H), 2.16 (d,
J = 9.6 Hz, 1H), 2.15–2.06 (m, 4H), 1.86 (ddd, J = 20.9, 14.7, 7.6 Hz,
6H), 1.75 (d, J = 13.2 Hz, 1H), 1.62 (d, J = 12.2 Hz, 3H), 1.50–1.41
(m, 1H), 1.35 (s, 3H), 1.26–1.12 (m, 16H), 1.09 (td, J = 12.8,
7.9 Hz, 11H), 0.81 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d
(ppm) 176.5, 147.8, 138.7, 130.3, 129.9, 126.3, 121.0, 103.1, 97.0,
81.6, 79.5, 79.2, 78.9, 78.7, 76.7, 74.9, 71.5, 71.2, 69.6, 68.7, 68.1,
66.0, 63.6, 57.3, 51.6, 51.2, 50.4, 50.1, 45.8, 45.5, 43.8, 40.9, 39.9,
39.5, 37.8, 32.0, 30.2, 29.9, 22.7, 22.2, 21.5, 20.8, 20.3, 19.4, 18.9,
18.6, 16.5, 15.9, 15.4, 15.3, 13.6, 12.9, 12.1, 11.2, 11.1, 10.0, 9.9.
HRMS (MALDI) m/z Calcd for C54H91N6O15 [M+H+]: 1063.6542,
found 1063.6588.
5.1.46. (Clarithromycin-400(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxypropanamide (38b)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.15 g,0.17 mmol) with 3-Azido-N-((tert-
butyldimethylsilyl)oxy)propanamide 51b (0.061 g, 0.249 mmol)
followed by TBS removal with caesium fluoride as described for
the synthesis of compound 5a, gave 38b as a light yellow solid
(0.07 g, 47%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.22 (s, 1H),
7.75 (d, J = 7.7 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 5.13–5.06 (m, 1H),
4.97–4.90 (m, 1H), 4.69 (d, J = 18.5 Hz, 2H), 4.40 (d, J = 7.0 Hz,
1H), 4.19 (d, J = 6.5 Hz, 1H), 3.81–3.64 (m, 5H), 3.47 (s, 1H), 3.30–
3.20 (m, 5H), 3.09–3.03 (m, 2H), 3.00 (d, J = 8.3 Hz, 3H), 2.94–
2.82 (m, 1H), 2.73 (t, J = 19.8 Hz, 3H), 2.56 (d, J = 7.6 Hz, 1H), 2.41
(s, 6H), 2.31 (s, 1H), 2.25 (d, J = 12.9 Hz, 4H), 2.18 (s, 1H), 2.16–
2.12 (m, 1H), 2.14–2.09 (m, 7H), 1.95–1.80 (m, 4H), 1.76 (d,
J = 12.9 Hz, 1H), 1.62 (d, J = 14.9 Hz, 1H), 1.53–1.41 (m, 1H), 1.36
(s, 3H), 1.27–1.13 (m, 16H), 1.09 (td, J = 13.0, 8.1 Hz, 11H), 0.83
(t, J = 12.v0, 5.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) d (ppm)
175.7, 146.9, 137.8, 129.2, 125.5, 121.0, 102.2, 96.2, 80.9, 80.9,
78.7, 78.4, 78.1, 77.9, 76.4, 75.9, 75.4, 74.1, 71.2, 70.9, 70.5, 68.8,
67.8, 67.3, 65.1, 62.6, 56.3, 50.8, 50.5, 50.5, 49.4, 46.0, 45.0, 44.7,
44.6, 43.1, 40.2, 39.1, 38.8, 37.0, 31.2, 29.9, 29.5, 25.4, 21.3, 20.8,
20.1, 19.8, 19.5, 18.9, 18.6, 18.1, 17.8, 15.8, 15.2, 14.7, 14.5, 12.9,
12.1, 11.3, 10.4, 10.3, 10.3, 9.3, 9.2. HRMS (MALDI) m/z Calcd for
5.1.49. (Clarithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxyhexanamide (38e)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.135 g, 0.149 mmol) with 6-Azido-N-((tert-
butyldimethylsilyl)oxy)hexanamide 51e (0.077 g, 0.268 mmol)
followed by TBS removal with caesium fluoride as described for
the synthesis of compound 5a, gave 38e as a light yellow solid
(0.053 g, 35%). 1H NMR (500 MHz, CDCl3) d (ppm) 7.86 (s, 1H),
7.77 (d, J = 7.4 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 5.06–4.95 (m, 2H),
4.43–4.30 (m, 2H), 4.19–4.11 (m, 1H), 3.81–3.71 (m, 2H), 3.65
(dd, J = 13.3, 6.4 Hz, 2H), 3.48–3.39 (m, 1H), 3.29 (s, 3H), 3.21
(dd, J = 12.4, 6.3 Hz, 3H), 3.06 (d, J = 6.5 Hz, 1H), 3.05–2.93 (m,
4H), 2.92–2.82 (m, 1H), 2.58–2.45 (m, 2H), 2.31 (dd, J = 28.6,
7.4 Hz, 6H), 2.20 (d, J = 14.3 Hz, 3H), 2.11 (dd, J = 35.3, 9.4 Hz,
5H), 2.00–1.83 (m, 5H), 1.75–1.57 (m, 6H), 1.57–1.49 (m, 2H),
1.41–1.27 (m, 7H), 1.28–1.11 (m, 16H), 1.13–1.01 (m, 11H), 0.90–
0.85 (m, 1H), 0.85–0.74 (m, 3H). 13C NMR (126 MHz, CDCl3) d
(ppm) 176.0, 147.4, 138.4, 129.8, 129.4, 125.8, 120.1, 102.7, 96.6,
96.5, 81.1, 81.0, 78.4, 78.2, 77.3, 77.1, 76.8, 76.6, 76.2, 76.1, 76.0,
74.3, 71.1, 70.7, 69.1, 68.3, 67.6, 65.4, 63.1, 57.0, 51.2, 51.0, 50.7,
50.1, 49.6, 45.3, 45.0, 43.2, 40.3, 39.4, 39.0, 37.2, 31.5, 29.8, 29.7,
29.3, 28.5, 26.2, 25.6, 24.9, 24.5, 21.7, 21.0, 19.7, 18.9, 18.1, 16.0,
16.0, 15.4, 14.9, 14.8, 13.1, 12.3, 11.5, 10.6, 9.3. HRMS (MALDI)
m/z Calcd for C55H93N6O15 [M+H+]: 1077.6699, found 1077.6692.
C
52H87N6O15 [M+H+]: 1035.6270, found 1035.6210.
5.1.47. (Clarithromycin-400(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxybutanamide (38c)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.15 g, 0.17 mmol) with 4-Azido-N-((tert-
butyldimethylsilyl)oxy)butanamide 51c (0.064 g, 0.245 mmol) fol-
lowed by TBS removal with caesium fluoride as described for the
synthesis of compound 5a, gave 38c as a light yellow solid
(0.060 g, 40%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.32 (s, 1H),
7.77 (d, J = 7.7 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 5.09 (d, J = 9.2 Hz,
1H), 4.46 (s, 2H), 4.39 (d, J = 7.2 Hz, 1H), 4.19 (d, J = 6.4 Hz, 1H),
3.82–3.61 (m, 5H), 3.48 (s, 1H), 3.29–3.21 (m, 4H), 3.05 (t,
J = 4.8 Hz, 2H), 3.00 (d, J = 9.9 Hz, 3H), 2.92–2.82 (m, 1H), 2.67 (s,
1H), 2.54 (s, 1H), 2.41 (s, 5H), 2.30 (s, 1H), 2.26–2.18 (m, 6H),
2.11 (t, J = 7.4 Hz, 3H), 1.95–1.79 (m, 4H), 1.75 (d, J = 13.0 Hz,
1H), 1.62 (d, J = 14.6 Hz, 1H), 1.52–1.40 (m, 1H), 1.36 (s, 3H), 1.18
(dt, J = 11.1, 7.0 Hz, 15H), 1.13–1.02 (m, 11H), 0.81 (t, J = 7.2 Hz,
3H). 13C NMR (126 MHz, CDCl3) d (ppm) 176.1, 171.4, 147.5,
138.4, 129.8, 129.5, 125.9, 120.8, 102.7, 102.4, 96.9, 96.6, 81.2,
81.2, 80.3, 79.1, 78.8, 78.5, 78.2, 76.3, 76.2, 75.7, 74.4, 71.5, 71.1,
70.8, 70.1, 69.2, 68.3, 68.1, 67.9, 67.7, 65.6, 63.1, 60.6, 56.9, 53.6,
51.2, 50.8, 49.7, 49.5, 45.4, 45.1, 44.9, 43.4, 40.5, 39.4, 39.1, 37.4,
31.6, 29.8, 29.5, 26.2, 21.7, 21.2, 21.1, 20.4, 20.1, 19.9, 19.3, 19.0,
18.5, 18.2, 16.1, 15.6, 15.0, 14.9, 14.3, 13.2, 12.5, 11.7, 10.8, 9.6,
9.5. HRMS (MALDI) m/z Calcd for C53H89N6O15 [M+H+]1049.6386,
found 1049.6376.
5.1.50. (Clarithromycin-400-(methylamino)-N(methyl)-4-benzyl-
triazolyl))-N-hydroxyheptanamide (38f)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
clarithromycin 36 (0.15 g, 0.17 mmol) with 7-Azido-N-((tert-
butyldimethylsilyl)oxy)heptanamide 51f (0.075 g, 0.249 mmol)
followed by TBS removal with caesium fluoride as described for
the synthesis of compound 5a, gave 38f as a light yellow solid
(0.060 g, 40%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.34 (s, 1H),
7.81 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 5.16–5.08 (m, 1H),
4.96 (d, J = 5.2 Hz, 1H), 4.45 (t, J = 6.5 Hz, 3H), 4.22 (d, J = 6.4 Hz,
1H), 3.85–3.64 (m, 5H), 3.52 (s, 1H), 3.33–3.23 (m, 9H),
3.12–2.98 (m, 5H), 2.90 (dd, J = 19.0, 11.7 Hz, 1H), 2.77 (s, 1H),
2.58 (s, 1H), 2.49 (s, 6H), 2.35 (s, 1H), 2.31–2.23 (m, 4H), 2.21 (s,
1H), 2.18–2.12 (m, 3H), 2.12–2.04 (m, 4H), 1.99–1.85 (m, 6H),
1.83 (d, J = 13.2 Hz, 2H), 1.69–1.53 (m, 7H), 1.44–1.31 (m, 12H),
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

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S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
1.28–1.18 (m, 15H), 1.12 (dt, J = 11.4, 4.9 Hz, 12H), 0.90 (s, 1H),
0.85 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) d (ppm) 176.2,
147.6, 138.4, 130.1, 129.7, 126.1, 120.3, 102.8, 96.8, 81.4, 78.6,
78.4, 74.6, 71.3, 71.0, 69.3, 68.4, 67.9, 65.7, 63.3, 57.1, 51.6, 51.0,
50.4, 49.9, 45.5, 45.2, 43.5, 40.7, 39.6, 39.2, 37.5, 31.8, 30.1, 30.0,
28.9, 28.8, 28.2, 26.6, 26.0, 25.6, 25.3, 21.9, 21.3, 20.6, 20.0, 19.5,
19.1, 18.3, 16.3, 15.7, 15.2, 15.0, 12.6, 10.9, 9.7. HRMS (MALDI)
m/z Calcd for C56H95N6O15 [M+H+]: 1091.6815, found 1091.6869.
45.4, 44.5, 41.4, 40.2, 36.2, 29.3, 26.9, 24.0, 22.6, 20.7, 16.2, 14.5,
11.6, 10.8, 8.5, 7.8. HRMS (ESI) m/2z Calcd for C40H73N2O13
[M+2H+]/2: 395.2590, found 395.2587.
5.1.53. 400-Epoxy-30-O-acetylazithromycin (34)
NaH (0.3 g, 7.18 mmol, 60% w/w) was added to an oven dried
three necked round bottom flask and was washed with petroleum
ether (ꢂ3). The flask was immediately flushed with argon and dry
DMSO (6 mL) was introduced through a septum. The mixture was
stirred at room temperature under Ar. Over a period of 5 min,
trimethyloxosulfonium iodide (1.58 g, 7.18 mmol) was added to
the reaction mixture. When hydrogen gas ceased to evolve, the
resulting yellow clear solution was treated with a solution of oxi-
dized azithromycin 32 (2.57 g, 3.26 mmol) in anhydrous THF
(5 mL) over 10 min, and left to stir for 2 h. TLC (4:3:1:0.1 Hex-
ane/EtOAc/MeOH/NH4OH) after 2 h showed 100% conversion to
the product. THF was removed under vacuum and EtOAc (50 mL)
was added to the remaining solution. The solution was washed
severely with water to remove DMSO. Organic layer was dried over
Na2SO4 and concentrated to give the product (2.48 g, 95% yield) as
white solid. 1H NMR (400 MHz, CDCl3) d 5.04 (d, J = 17.3 Hz, 2H),
4.83 (s, 1H), 4.68 (d, J = 11.2 Hz, 2H), 4.06 (s, 1H), 3.68 (s, 2H),
3.63–3.52 (m, 2H), 3.29 (p, J = 16.5 Hz, 7H), 2.86 (s, 2H), 2.72–
2.39 (m, 9H), 2.11 (dd, J = 21.0, 9.5 Hz, 4H), 1.98 (d, J = 14.8 Hz,
2H), 1.83 (s, 3H), 1.66–1.47 (m, 5H), 1.41 (s, 3H), 1.27 (dt,
J = 58.5, 7.3 Hz, 14H), 1.06–0.98 (m, 9H), 0.96 (s, 3H), 0.90–0.73
(m, 3H). 13C NMR (101 MHz, CDCl3) d 180.6, 167.0, 158.0, 127.9,
121.1, 104.1, 102.5, 95.8, 92.1, 74.2, 73.6, 64.7, 63.5, 60.3, 56.2,
50.0, 49.6, 46.4, 45.4, 43.3, 42.3, 41.2, 40.4, 37.1, 36.7, 31.9, 29.8,
28.4, 26.9, 25.5, 23.6, 22.6, 20.9, 18.3, 16.8, 15.4, 14.1, 13.9, 11.4,
8.9, 7.9. HRMS (ESI) m/z Calcd for C41H74N2O13 [M+H+]: 803.5264,
found 803.5262.
5.1.51. 30-O-Acetylazithromycin (30)
Acetic anhydride (0.8 ml, 8.34 mmol) was added to a solution of
azithromycin 28 (2.50 g, 3.34 mmol) in DCM (10 mL) at room tem-
perature. The resulting solution was stirred under Ar for 3 h. The
reaction was quenched by adding saturated NaHCO3 and the
organic layer was separated. The aqueous layer was extracted
twice with DCM (20 mL) and the combined organic layer was
washed with water, brine, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo to give the target compound as a white
solid (2.50 g, 95%). 1H NMR (400 MHz, CDCl3) d (ppm) 5.25 (dd,
J = 1.6, 0.9 Hz, 1H), 4.93 (s, 3H), 4.74–4.67 (m, 2H), 4.59 (d,
J = 10.2 Hz, 2H), 4.51 (d, J = 7.5 Hz, 2H), 4.18–4.13 (m, 2H), 4.01–
3.93 (m, 2H), 3.59 (s, 2H), 3.54 (d, J = 6.3 Hz, 2H), 3.30 (s, 3H),
3.23 (s, 2H), 2.96 (d, J = 9.0 Hz, 2H), 2.79–2.71 (m, 2H), 2.68–2.52
(m, 4H), 2.40 (d, J = 11.8 Hz, 2H), 2.28 (d, J = 13.8 Hz, 4H), 2.04–
2.01 (m, 1H), 1.98 (d, J = 2.3 Hz, 3H), 1.94–1.85 (m, 3H), 1.64 (d,
J = 13.7 Hz, 3H), 1.53 (dd, J = 15.3, 4.9 Hz, 2H), 1.28–1.23 (m, 7H),
1.20 (d, J = 6.4 Hz, 3H), 1.16 (d, J = 6.0 Hz, 3H), 1.11 (d, J = 8.3 Hz,
3H), 1.09 (s, 3H), 1.03 (d, J = 6.7 Hz, 3H), 0.99 (s, 3H). 13C NMR
(101 MHz, CDCl3) d (ppm) 178.2, 176.2, 169.8, 166.3, 100.6, 95.4,
83.8, 78.7, 78.1, 75.8, 74.9, 74.4, 73.5, 72.6, 71.8, 70.1, 68.2, 65.6,
63.7, 61.9, 49.3, 45.1, 42.0, 40.7, 36.5, 35.1, 30.4, 27.2, 26.3, 22.5,
22.0, 21.6, 21.2, 21.1, 18.5, 16.3, 15.4, 11.3, 9.2, 7.8. HRMS (ESI)
m + 2/2z Calcd for
C
40H76N2O13 [M+2H+]/2: 396.2668, found
396.2656.
5.1.54. 1-(4-Ethynylphenyl)-N-methylmethanamine (35)
Methylamine (9.32 mL, 18.63 mmol, 2 M in THF) was added to a
solution of 4-ethynylbenzyl methanesulfonate (3) (0.39 g,
1.86 mmol) in THF (20 mL) and left to stir at 50 °C for 12 h. Methy-
lamine was evaporated off and the residue dissolved in 1 M HCl.
This was then extracted multiple times with DCM. The aqueous
layer was basified with 1 M NaOH and extracted with DCM.
Organic layer was dried over anhydrous Na2SO4 and concentrated
in vacuo. The residue obtained was purified by column chromatog-
raphy to give compound 35 as a yellow liquid (0.15 g, 55%). 1H
NMR (400 MHz, CDCl3) d (ppm) 7.41–7.36 (d, 2H), 7.20 (d,
J = 8.4 Hz, 2H), 3.65 (d, J = 9.0 Hz, 2H), 3.03 (d, J = 3.2 Hz, 1H),
2.65 (s, 1H), 2.34 (s, 3H). 13C NMR (101 MHz, CDCl3) d (ppm)
140.5, 132.1, 128.1, 120.6, 83.7, 55.3, 35.6.
5.1.52. 400-Oxo-30-O-acetylazithromycin (32)
N-Iodosuccinimide (0.75 g, 5.61 mmol) was dissolved in anhy-
drous DCM (20 mL) and the solution was cooled to ꢀ15 °C. After
10 min, dimethyl sulfide (0.5 ml, 6.32 mmol) was added drop wise.
The white suspension was stirred at ꢀ15 °C for 20 min, then a DCM
solution (5 mL) of compound 30 (2.78 g, 3.51 mmol) was added
over 30 min. The resulting suspension was stirred at ꢀ15 °C for
30 min, and triethylamine (0.8 ml, 5.6 mmol) was added. The solu-
tion became clear in a minute and stirring was continued at ꢀ10 °C
for 2 h. The reaction was quenched by adding saturated aqueous
NaHCO3 solution and the organic layer was separated. The aqueous
layer was extracted twice with DCM (2 ꢂ 50 mL). The combined
organic layer was washed with water 50 mL), brine (50 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo. The
crude was purified by column chromatography (Silica gel,
12:1:0.5 DCM/MeOH/NH4OH) to yield the product (2.49 g 90%) as
white solid. 1H NMR (400 MHz, CDCl3) d 1H NMR (400 MHz, CDCl3),
6.21 (d, J = 41.6 Hz, 2H), 5.27 (t, J = 6.6 Hz, 1H), 5.19 (s, 1H), 5.05 (t,
J = 7.0 Hz, 1H), 5.00–4.93 (m, 1H), 4.93–4.87 (m, 1H), 4.72–4.54 (m,
1H), 4.39 (q, J = 6.6 Hz, 1H), 4.36–4.27 (m, 1H), 4.11–4.06 (m, 1H),
4.05–3.99 (m, 5H), 3.50–3.41 (m, 1H), 3.36 (d, J = 6.1 Hz, 1H), 3.29
(s, 1H), 3.26 (s, 3H), 3.24–3.20 (m, 5H), 3.18–3.12 (m, 1H), 2.70–
2.51 (m, 4H), 2.41 (d, J = 12.0 Hz, 1H), 2.20 (dt, J = 16.6, 9.6 Hz,
4H), 2.15–2.08 (m, 2H), 2.01 (d, J = 4.6 Hz, 1H), 1.97 (s, 3H), 1.93
(s, 1H), 1.86 (s, 3H), 1.72–1.65 (m, 1H), 1.65–1.57 (m, 1H), 1.53
(d, J = 14.9 Hz, 1H), 1.47–1.38 (m, 1H), 1.34–1.28 (m, 7H), 1.22 (t,
J = 6.2 Hz, 1H), 1.18 (d, J = 7.3 Hz, 1H), 1.09 (ddd, J = 23.5, 15.0,
8.7 Hz, 3H), 0.97 (d, J = 8.6 Hz, 3H), 0.84 (dd, J = 6.6, 3.3 Hz, 3H),
0.82–0.72 (m, 3H). 13C NMR (101 MHz, CDCl3) d 211.4, 210.7,
177.7, 176.6, 173.3, 170.0, 169.9, 101.8, 100.4, 97.7, 95.4, 85.1,
82.8, 81.2, 73.8, 72.6, 70.7, 69.2, 68.4, 63.2, 62.3, 60.7, 50.9, 49.8,
5.1.55. (400-(Methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin (37)
Compound 35 (1.057 g, 7.3 mmol) was added to a solution of
compound 34 (1.95 g, 2.4 mmol) in MeOH (10 mL). The mixture
was stirred under argon at 60 °C for 6 h, after that the solution
was cooled to room temperature and diluted with excess ethyl
acetate (100 mL). The organic layer was washed with saturated
aqueous NaHCO3 solution (30 mL), water (20 mL), brine (20 mL),
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The crude was purified by column chromatography column (Silica
gel, 4:3:2:0.1 Hexane/EtOAc/MeOH/NH4OH) to give compound 37
as light yellow solid (2.0 g, 92% yield). 1H NMR (400 MHz, CDCl3)
d (ppm) 7.43 (d, J = 8.2 Hz, 2H), 7.21(d, 2H), 5.04 (d, J = 4.6 Hz,
1H), 4.62 (d, J = 8.6 Hz, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.24–4.15
(m, 3H), 3.67 (d, J = 6.4 Hz, 2H), 3.63 (s, 3H), 3.46 (s, 3H), 3.26
(d, J = 21.0 Hz, 6H), 3.05 (s, 1H), 2.95 (d, J = 14.7 Hz, 3H), 2.81
(d, J = 7.1 Hz, 3H), 2.66 (s, 3H), 2.44 (d, J = 10.1 Hz, 2H), 2.31
(d, J = 10.5 Hz, 9H), 2.18 (s, 3H), 2.15 (d, J = 0.5 Hz, 1H), 2.02
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
17
(t, J = 11.7 Hz, 3H), 1.92 (dd, J = 15.0, 5.0 Hz, 2H), 1.29 (s, 3H), 1.21
(t, J = 6.9 Hz, 10H), 1.14 (s, 3H), 1.12–1.02 (m, 10H), 0.88 (t, J =
7.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) d (ppm) 178.6, 139.4,
132.2, 128.8, 121.2, 102.8, 95.0, 94.8, 83.8, 83.3, 76.8, 74.2, 73.5,
70.9, 70.1, 68.1, 67.2, 67.2, 65.7, 63.2, 62.6, 62.5, 56.9, 49.6, 49.5,
45.4, 45.2, 43.2, 42.3, 42.2, 41.1, 40.9, 40.5, 40.4, 36.2, 31.3, 29.0,
27.5, 26.7, 22.7, 21.9, 21.3, 18.8, 16.2, 15.0, 14.6, 11.2, 9.2, 7.2.
HRMS (ESI) m + 2/2z Calcd for C49H85N3O12 [M+2H+]: 453.8061,
found 453.8055.
22.8, 21.2, 20.1, 18.2, 16.8, 15.2, 12.3, 10.8, 8.8. HRMS (ESI) m/z
Calcd for C52H90N7O14 [M+H+]: 1036.6540, found 1036.6550.
5.1.58. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxybutanamide (39c)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin 37 (0.20 g, 0.18 mmol) and 4-azido-N-((tert-
butyldimethylsilyl)oxy)butanamide 51c (0 .08 g, 0.31 mmol) fol-
lowed by TBS deprotection with caesium fluoride as described for
the synthesis of 39a, gave 39c as light yellow solid (0.173 g, 80%).
1H NMR (400 MHz, CD3OD) d (ppm) 8.30 (s, 1H), 7.76 (d, J = 7.2 Hz,
2H), 7.34 (d, J = 7.7 Hz, 2H), 5.44 (s, 1H), 5.06 (d, J = 4.7 Hz, 1H),
4.51–4.35 (m, 3H), 4.30–4.19 (m, 2H), 3.60 (dd, J = 23.4, 16.5 Hz,
3H), 3.45 (d, J = 25.7 Hz, 1H), 3.32 (s, 3H), 3.27–3.16 (m, 3H), 2.99
(d, J = 14.7 Hz, 1H), 2.84–2.69 (m, 2H), 2.63–2.43 (m, 2H), 2.30 (d,
J = 14.5 Hz, 10H), 2.24–2.04 (m, 15H), 1.95 (dd, J = 13.7, 6.5 Hz,
3H), 1.91–1.77 (m,3H), 1.77–1.64 (m, 3H), 1.49–1.30 (m, 3H),
1.27 (s, 3H), 1.21 (d, J = 6.0 Hz, 6H), 1.16 (d, J = 6.1 Hz, 11H), 1.03
(dd, J = 19.7, 6.7 Hz, 7H), 0.85 (dd, J = 14.1, 7.0 Hz, 3H). 13C NMR
(126 MHz, CD3OD) d (ppm) 180.7, 177.2, 149.4, 141.0, 131.9,
131.6, 127.7, 123.7, 104.9, 97.6, 85.5, 80.4, 78.8, 78.4, 76.4, 76.2,
74.5, 73.6, 73.1, 70.9, 69.9, 69.4, 66.9, 65.7, 64.9, 57.2, 55.6, 51.1,
48.2, 47.5, 44.9, 44.3, 43.9, 41.5, 37.7, 34.0, 33.6, 33.4, 32.8, 31.7,
31.4, 28.5, 28.2, 24.6, 23.2, 22.7, 20.3, 18.2, 16.7, 15.3, 12.5, 10.8,
5.1.56. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxyacetamide (39a)
(400-(Methylamino)-N(methyl)(4-ethynylbenzyl))azithromycin
37 (0.14 g, 0.16 mmol) and 2-Azido-N-((tert-butyldimethyl silyl)
oxy)ethaneamide 51a (0 .06 g, 0.23 mmol) were dissolved in anhy-
drous THF (5 mL) and purged with Ar for 15 min. Copper(I) iodide
(0.01 g, 0.08 mmol) and Hunig’s base (0.06 mL, 0.31 mmol) were
then added to the reaction mixture. The reaction mixture was
purged with Ar for additional 15 min and stirring continued for
12 h. Caesium fluoride (0.04 g, 0.24 mmol) and MeOH (5 mL) were
added to the mixture to remove TBS protecting group and the reac-
tion continued for an additional 2 h. The reaction was quenched by
adding a solution of 4:1 saturated NH4Cl/NH4OH (30 mL) and
extracted with 20% MeOH/CH2Cl2 (3 ꢂ 30 mL). The organic layer
was dried over Na2SO4 and concentrated in vacuo. The crude pro-
duct was purified by preparative TLC (Silica gel, 5:1:1 EtOAc/
MeOH/NH4OH) to give the product (0.131 g, 80% yield) as light yel-
low solid. 1H NMR (400 MHz, CD3OD) d (ppm) 8.33 (s, 1H), 7.79 (d,
J = 6.5 Hz, 2H), 7.37 (d, J = 7.1 Hz, 2H), 5.46 (s, 1H), 5.07 (d,
J = 4.6 Hz, 3H), 4.44 (d, J = 6.9 Hz, 1H), 4.32–4.18 (m, 3H), 3.69 (s,
2H), 3.64–3.56 (m, 3H), 3.48 (d, J = 25.5 Hz, 2H), 3.34 (s, 3H),
3.32–3.21 (m, 3H), 3.09–2.93 (m, 3H), 2.85–2.64 (m, 3H), 2.41 (t,
J = 20.9 Hz, 12H), 2.33–2.20 (m, 3H), 2.19–2.09 (m, 3H), 2.00 (dd,
J = 25.3, 18.4 Hz, 3H), 1.95–1.83 (m, 3H), 1.82–1.70 (m, 3H), 1.52–
1.39 (m, 3H), 1.32 (s, 3H), 1.24 (d, J = 6.3 Hz, 3H), 1.22–1.12 (m,
13H), 1.08 (s, 3H), 1.03 (d, J = 7.2 Hz, 3H), 0.96–0.82 (m, 3H). 13C
NMR (126 MHz, CD3OD) d (ppm) 180.5, 177.3, 149.4, 140.9,
131.9, 131.6, 127.7, 124.6, 105.0, 102.0, 97.7, 85.6, 80.3, 78.9,
78.3, 76.3, 76.0, 73.6, 73.2, 73.0, 71.0, 69.8, 69.5, 67.0, 65.5, 65.0,
59.0, 51.0, 47.6, 45.0, 44.5, 44.1, 41.4, 37.7, 33.9, 33.2, 32.6, 31.6,
31.4, 28.6, 28.2, 24.6, 23.2, 23.0, 20.2, 18.2, 16.5, 15.3, 12.3, 10.7,
8.7. HRMS (ESI) m+2/2z Calcd for
C
53H93N7O14 [M+2H+]:
525.8385, found 525.8385.
5.1.59. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxypentanamide (39d)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin 37 (0.14 g, 0.15 mmol) and 5-azido-N-((tert-
butyldimethylsilyl)oxy)pentanamide 51d (0.06 g, 0.23 mmol) fol-
lowed by TBS deprotection with caesium fluoride as described for
the synthesis of 39a, gave 39d as light yellow solid (0.125 g, 75%).
1H NMR (400 MHz, CD3OD) d (ppm) 7.72 (s, 1H), 7.19 (d, J = 7.9 Hz,
2H), 6.77 (d, J = 8.0 Hz, 2H), 4.47 (d, J = 5.0 Hz, 1H), 3.85 (d,
J = 6.7 Hz, 3H), 3.63 (dd, J = 17.6, 4.5 Hz, 2H), 3.09 (s, 2H), 3.04–
2.97 (m, 3H), 2.75 (s, 3H), 2.68 (dt, J = 3.3, 1.6 Hz, 2H), 2.43 (d,
J = 14.3 Hz, 2H), 2.19 (dd, J = 7.4, 4.6 Hz, 2H), 2.12 (d, J = 11.6 Hz,
1H), 1.89 (d, J = 18.4 Hz, 15H), 1.64 (d, J = 7.1 Hz, 5H), 1.59–1.46
(m, 6H), 1.37 (dd, J = 16.4, 7.9 Hz, 7H), 1.15 (t, J = 7.3 Hz, 1H),
1.03 (s, 3H), 0.85 (td, J = 14.5, 7.5 Hz, 3H), 0.73 (s, 3H), 0.60 (ddd,
J = 18.8, 13.2, 6.4 Hz, 15H), 0.49 (s, 3H), 0.43 (d, J = 7.3 Hz, 3H),
0.32 (d, J = 6.8 Hz, 3H), 0.27 (t, J = 7.4 Hz, 3H) .13C NMR (126 MHz,
CD3OD) d (ppm) 180.2, 176.9, 172.9, 149.1, 140.6, 131.6, 127.3,
122.8, 104.7, 97.1, 85.3, 80.1, 78.6, 78.0, 76.1, 75.8, 74.3, 73.1,
71.1, 69.7, 66.6, 64.6, 64.5, 62.8, 58.7, 56.9, 55.5, 52.7, 51.6, 50.8,
47.3, 44.5, 44.1, 43.9, 41.4, 37.3, 33.6, 33.0, 32.7, 31.3, 30.0, 28.6,
26.7, 24.2, 23.1, 22.7, 20.0, 17.9, 16.4, 16.1, 15.0, 12.2, 10.6, 8.2.
HRMS (ESI) m/z Calcd for C54H94N7O14 [M+H+]: 1064.6853, found
1064.6854.
8.8. HRMS (ESI) m + 2/2z Calcd for
511.8228, found 511.8230.
C
51H89N7O14 [M+2H+]:
5.1.57. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxypropanamide (39b)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin 37 (0.20 g, 0.218 mmol) and 3-azido-N-((tert-
butyldimethylsilyl)oxy)propanamide 51b (0.08 g, 0.32 mmol) fol-
lowed by TBS deprotection with caesium fluoride as described for
the synthesis of 39a, gave 39b as light yellow solid (0.178 g, 79%).
1H NMR (500 MHz, CD3OD) d (ppm) 8.26 (d, J = 10.7 Hz, 1H), 7.78
(d, J = 7.6 Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H), 5.48 (s, 1H), 5.13–5.07
(m, 2H), 4.74 (s, 3H), 4.46 (d, J = 7.2 Hz, 2H), 4.32–4.22 (m, 1H),
3.69 (d, J = 18.3 Hz, 3H), 3.67–3.57 (m, 1H), 3.57–3.48 (m, 2H),
3.38 (d, J = 13.2 Hz, 3H), 3.33–3.25 (m, 1H), 3.02 (dd, J = 23.0,
16.2 Hz, 3H), 2.86–2.75 (m, 3H), 2.74–2.65 (m, 3H), 2.44 (s, 3H),
2.31–2.19 (m, 3H), 2.19–2.10 (m, 2H), 2.09–1.97 (m, 3H), 1.94–
1.87 (m, 9H), 1.87–1.82 (m, 2H), 1.81–1.72 (m, 3H), 1.53–1.40
(m, 3H), 1.35 (d, J = 11.2 Hz, 3H), 1.27 (t, J = 11.9 Hz, 13H), 1.24–
1.19 (m, 3H), 1.16 (d, J = 6.7 Hz, 3H), 1.10 (s, 3H), 1.05 (d,
J = 7.5 Hz, 3H), 0.96–0.86 (s, 3H). 13C NMR (126 MHz, CD3OD) d
(ppm) 180.4, 177.1, 169.8, 149.2, 140.8, 131.8, 131.3, 127.5,
123.5, 104.8, 97.3, 85.4, 80.3, 78.9, 78.3, 76.2, 76.0, 74.5, 73.4,
72.9, 70.9, 69.9, 69.2, 66.8, 65.6, 65.0, 63.0, 58.8, 57.0, 55.8, 50.9,
48.1, 47.5, 44.8, 43.9, 41.3, 37.7, 33.9, 32.7, 31.6, 28.3, 24.5, 23.3,
5.1.60. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxyhexanamide (39e)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin 37 (0.09 g, 0.10 mmol) and 6-azido-N-((tert-
butyldimethylsilyl)oxy)hexanamide 51e (0.04 g, 0.16 mmol) fol-
lowed by TBS deprotection with caesium fluoride as described for
the synthesis of 39a, gave 39e as light yellow solid (0.090 g, 80%).
1H NMR (400 MHz, CD3OD) d (ppm) 8.27 (s, 1H), 7.74 (d, J = 8.1 Hz,
2H), 7.32 (d, J = 8.1 Hz, 2H), 5.05 (d, J = 5.0 Hz, 1H), 4.78 (dd,
J = 10.0, 2.3 Hz, 3H), 4.37 (dd, J = 15.3, 7.2 Hz, 3H), 4.23 (dd,
J = 12.4, 5.5 Hz, 2H), 4.00 (dq, J = 13.6, 6.9 Hz, 1H), 3.66 (d,
J = 17.8 Hz, 1H), 3.60–3.52 (m, 2H), 3.46 (dd, J = 9.9, 5.4 Hz, 1H),
3.29 (d, J = 12.7 Hz, 3H), 3.26–3.16 (m, 3H), 3.02–2.93 (m, 1H),
2.78–2.68 (m, 2H), 2.59–2.46 (m, 1H), 2.27 (d, J = 16.0 Hz, 3H),
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

18
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
2.22–2.15 (m, 3H), 2.10 (t, J = 8.1 Hz, 2H), 2.03 (d, J = 16.0 Hz, 3H),
1.97–1.91 (m, 5H), 1.91–1.86 (m, 3H), 1.83 (d, J = 3.9 Hz, 2H), 1.73–
1.63 (m, 2H), 1.60–1.47 (m, 3H), 1.32 (dd, J = 21.9, 6.6 Hz, 6H), 1.26
(d, J = 5.2 Hz, 3H), 1.23–1.17 (m, 6H), 1.17–1.08 (m, 11H), 1.01 (dd,
J = 17.4, 6.0 Hz, 11H), 0.89–0.79 (m, 3H). 13C NMR (126 MHz, CD3-
OD) d (ppm) 180.2, 177.0, 173.3, 149.2, 140.7, 131.8, 131.3, 127.4,
122.9, 104.8, 97.2, 85.4, 80.1, 78.8, 78.3, 76.4, 75.9, 73.3, 73.2, 71.3,
69.9, 69.1, 66.6, 64.9, 58.7, 55.6, 53.0, 52.0, 50.9, 47.5, 44.7, 44.2,
43.9, 41.4, 37.4, 34.2, 33.7, 33.1, 32.6, 31.6, 30.3, 28.5, 28.2, 27.5,
26.6, 24.4, 22.9, 22.7, 20.0, 18.0, 16.4, 16.1, 12.4, 10.6, 8.4. HRMS
(ESI) m+2/2z Calcd for C55H97N7O14 [M+2H+]: 539.8541, found
539.8539.
(0.13 mL, 1.41 mmol). Then the mixture was heated to 40 °C in a
pressure tube and stirring continued for 48 h. The reaction mixture
was cooled and diluted with DCM (100 mL) and washed with sat-
urated NaHCO3 (50 mL), water (50 mL), and brine (50 mL). The
organic layer was dried over anhydrous Na2SO4, filtered and con-
centrated in vacuo. The solid crude product 41 (1.1 g, 95%) was suf-
ficiently pure to be used for the next reaction without any further
purification. 1H NMR (400 MHz, CDCl3) d (ppm) 7.38 (d, J = 8.1 Hz,
2H), 7.17 (d, J = 8.1 Hz, 2H), 5.06–5.00 (m, 1H), 4.94 (dd, J = 10.6,
2.3 Hz, 1H), 4.86 (dd, J = 10.5, 7.6 Hz, 1H), 4.79 (d, J = 4.4 Hz, 1H),
4.45 (d, J = 7.6 Hz, 1H), 4.13 (t, J = 5.6 Hz, 1H), 3.97 (dq, J = 12.5,
6.2 Hz, 1H), 3.64 (d, J = 13.8 Hz, 1H), 3.57–3.44 (m, 2H), 3.40 (d,
J = 4.3 Hz, 1H), 3.29 (dd, J = 12.3, 4.8 Hz, 1H), 3.20 (s, 3H), 3.02–
2.94 (m, 2H), 2.74 (td, J = 12.2, 4.2 Hz, 1H), 2.67–2.58 (m, 1H),
2.53 (q, J = 7.2 Hz, 2H), 2.28 (d, J = 14.9 Hz, 2H), 2.19 (s, 3H), 2.14
(d, J = 6.8 Hz, 3H), 2.10 (d, J = 7.4 Hz, 3H), 2.04 (d, J = 5.4 Hz, 3H),
1.99–1.89 (m, 1H), 1.82–1.70 (m, 3H), 1.68–1.34 (m, 3H), 1.28–
1.17 (m, 13H), 1.13 (d, J = 7.1 Hz, 6H), 1.04–0.91 (m, 10H), 0.89–
0.80 (m, 3H). 13C NMR (101 MHz, CDCl3) d (ppm) 176.5, 175.5,
171.0, 170.0, 140.8, 132.0, 128.4, 128.1, 128.0, 120.7, 101.4, 96.5,
84.3, 83.7, 80.6, 79.8, 77.9, 76.4, 75.3, 74.1, 72.9, 71.2, 69.0, 65.6,
61.9, 61.1, 58.2, 49.4, 48.9, 45.5, 42.9, 41.4, 36.9, 35.1, 34.4, 31.0,
29.6, 27.1, 25.2, 23.6, 21.9, 21.6, 20.6, 17.9, 14.1, 11.7, 11.1, 8.3.
HRMS (ESI) m+2/2z Calcd for C48H80N2O13 [M+2H+]: 446.2825,
found 446.2810.
5.1.61. (Azithromycin-400-(methylamino)-N(methyl)(4-benzyl-
triazolyl))-N-hydroxyheptanamide (39f)
Reaction of (400-(methylamino)-N(methyl)(4-ethynylbenzyl))
azithromycin 37 (0.16 g, 0.18 mmol) and 7-azido-N-((tert-
butyldimethylsilyl)oxy)heptanamide 51f (0.08 g, 0.27 mmol) fol-
lowed by TBS deprotection with caesium fluoride as described for
the synthesis of 39a, gave 39f as light yellow solid (0.154 g, 80%).
1H NMR (400 MHz, CD3OD)
d (ppm) 7.71 (s, 1H), 7.18 (d,
J = 8.1 Hz, 2H), 6.77 (d, J = 8.1 Hz, 2H), 4.49 (d, J = 5.0 Hz, 1H),
3.83 (dd, J = 10.2, 7.1 Hz, 3H), 3.72–3.63 (m, 2H), 3.50–3.40 (m,
1H), 3.11 (d, J = 16.2 Hz, 1H), 3.02 (d, J = 7.0 Hz, 1H), 2.99–2.95
(m, 1H), 2.90 (dd, J = 17.7, 14.2 Hz, 1H), 2.75 (s, 3H), 2.71–2.62
(m, 3H), 2.43 (d, J = 14.7 Hz, 1H), 2.24 (d, J = 7.0 Hz, 1H), 2.18 (dd,
J = 7.5, 4.6 Hz, 1H), 2.00 (t, J = 14.8 Hz, 1H), 1.75 (d, J = 10.6 Hz,
9H), 1.70–1.58 (m, 6H), 1.55–1.44 (m, 3H), 1.44–1.34 (m, 3H),
1.33–1.30 (m, 3H), 1.31–1.25 (m, 3H), 1.19–1.10 (m, 2H), 1.04 (d,
J = 7.4 Hz, 3H), 0.76 (dd, J = 20.3, 12.8 Hz, 3H), 0.70 (s, 3H), 0.68–
0.61 (m, 6H), 0.60 (dt, J = 12.7, 4.4 Hz, 12H), 0.53–0.46 (m, 10H),
0.43 (d, J = 7.5 Hz, 5H), 0.34–0.24 (m, 3H). 13C NMR (126 MHz, CD3-
OD) d (ppm) 180.5, 177.4, 173.6, 149.4, 149.0, 140.7, 132.3, 131.9,
131.5, 127.6, 123.2, 122.9, 105.1, 97.3, 85.6, 80.2, 78.9, 78.2, 76.5,
75.8, 73.5, 71.6, 70.1, 69.3, 66.7, 65.1, 64.7, 58.7, 55.8, 53.3, 52.2,
50.9, 47.7, 44.9, 44.5, 44.3, 41.8, 37.7, 34.4, 33.4, 32.9, 32.0, 31.6,
30.7, 30.3, 28.9, 28.0, 27.4, 23.3, 20.2, 18.4, 16.7, 16.5, 12.6, 10.9,
5.1.64. ((400-Oxo)-30-O-acetyl)-40-N-(4-ethynylbenzyl))
clarithromycin (42)
A solution of N-chlorosuccinimide (0.034 g, 0.23 mmol) in DCM
(5 mL) was stirred at ꢀ15 °C for 10 min then dimethyl sulfide
(0.020 mL, 0.26 mmol) was added drop wise to form a white turbid
solution. After stirring for 20 min, a DCM (5 mL) solution of com-
pound 40 (0.128 g, 0.144 mmol) was added over a period of
°
30 min and the resulting suspension was stirred at ꢀ15⁄⁄⁄ C for
30 min. Subsequently, TEA (0.030 mL, 0.23 mmol) was added and
the suspension cleared up within a min. Stirring continued at
ꢀ10 °C for 2 h and the reaction was quenched with saturated
NaHCO3 (10 mL). The organic layer extracted with DCM (50 mL),
dried with anhydrous Na2SO4, filtered, and concentrated in vacuo.
Residue was purified by preparative chromatography (Silica gel,
Hexane/EtOAc/MeOH (3:2:0.05) to give compound 31 as a yellow
solid (0.055 g, 45%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.32 (s,
1H), 7.77 (d, J = 7.7 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 5.09 (d,
J = 9.2 Hz, 1H), 4.96–4.91 (m, 1H), 4.46 (s, 2H), 4.39 (d, J = 7.2 Hz,
1H), 4.19 (d, J = 6.4 Hz, 1H), 3.81–3.60 (m, 5H), 3.48 (s, 1H), 3.28–
3.19 (m, 4H), 3.10–2.95 (m, 5H), 2.92–2.82 (m, 1H), 2.67 (s, 1H),
2.54 (s, 1H), 2.41 (s, 6H), 2.30 (s, 1H), 2.27–2.16 (m, 7H), 2.13
(dd, J = 20.0, 10.6 Hz, 4H), 1.94–1.69 (m, 6H), 1.62 (d, J = 14.6 Hz,
1H), 1.50–1.41 (m, 1H), 1.42–1.31 (m, 4H), 1.26–1.13 (m, 16H),
1.13–1.01 (m, 12H), 0.86–0.76 (m, 3H). 13C NMR (126 MHz, MeOD)
d (ppm) 212.0, 177.2, 172.2, 142.0, 133.0, 129.9, 122.5, 101.8, 97.9,
84.5, 81.5, 80.5, 79.8, 78.5, 78.2, 76.0, 74.2, 73.9, 73.1, 70.7, 69.9,
62.6, 59.1, 51.8, 51.1, 50.9, 46.6, 46.0, 39.8, 39.6, 39.2, 39.1, 39.1,
38.6, 37.4, 32.1, 22.3, 21.8, 21.7, 21.1, 20.9, 20.4, 19.4, 18.5, 17.3,
16.7, 16.3, 15.1, 13.7, 12.8, 11.1, 10.1, 9.9. HRMS (ESI) m/z Calcd
for C48H74NO14 [M+H+]: 888.5104, found 888.5113.
8.6. HRMS (ESI) m+2/2z Calcd for
C
56H99N7O14 [M+2H+]:
546.8620, found 546.8621.
5.1.62. ((30-O-Acetyl)-40-N-(4-ethynylbenzyl))clarithromycin (40)
(40-Ethynylbenzyl)clarithromycin (0.15 g, 0.177 mmol) was dis-
solved in anhydrous DCM (5 mL) and acetic anhydride (0.04 mL,
0.442 mmol) added to the solution. The mixture was left to stir at
room temperature under Ar for 3 days after which TLC shows full
consumption of the starting material. The reaction was washed
with NaHCO3, brine and H2O, the organic layer was dried with
anhydrous Na2SO4 and concentrated in vacuo to give compound
40 (128 mg, 81%). 1H NMR (400 MHz, CDCl3) d 7.38 (d, J = 8.2 Hz,
2H), 7.19 (d, J = 8.1 Hz, 2H), 5.01 (dd, J = 11.1, 2.2 Hz, 1H), 4.87 (d,
J = 4.8 Hz, 1H), 4.74 (dd, J = 10.6, 7.4 Hz, 1H), 4.48 (d, J = 7.4 Hz,
1H), 3.94 (s, 1H), 3.86 (dt, J = 12.1, 6.1 Hz, 1H), 3.66 (t, J = 11.1 Hz,
2H), 3.63 (s, 1H), 3.52 (d, J = 6.5 Hz, 1H), 3.46 (s, 1H), 3.40 (dd,
J = 11.3, 6.7 Hz, 1H), 3.18 (s, 1H), 3.07 (s, 3H), 3.02 (d, J = 6.2 Hz,
1H), 2.99–2.91 (m, 5H), 2.80 (dt, J = 14.2, 7.2 Hz, 1H), 2.73 (d,
J = 4.5 Hz, 1H), 2.59–2.47 (m, 2H), 2.29–2.24 (m, 1H), 2.20–2.15
(m, 9H), 2.06–2.01 (m, 4H), 1.93–1.76 (m, 3H), 1.71 (t, J = 12.8 Hz,
1H), 1.62 (d, J = 11.6 Hz, 1H), 1.57–1.47 (m, 3H), 1.42 (ddd,
J = 13.2, 8.3, 3.1 Hz, 1H), 1.37 (t, J = 5.2 Hz, 1H), 1.36–1.28 (m, 4H),
1.25–1.17 (m, 11H), 1.14 (t, J = 7.6 Hz, 3H), 1.08 (t, J = 6.0 Hz, 8H),
0.85 (t, J = 7.5 Hz, 3H), 0.79 (t, J = 7.4 Hz, 2H). HRMS (ESI) m + z Calcd
for C48H76NO14 [M+H+]: 890.5260, found 890.5256.
5.1.65. ((400-Oxo)-30-O-acetyl)-40-N-(4-ethynylbenzyl))
azithromycin (43)
A solution of N-chlorosuccinimide (0.46 g, 3.47 mmol) in DCM
(5 mL) was stirred at ꢀ15 °C for 10 min. Then DMS (0.30 mL,
3.90 mmol) was added drop wise to form a white solution. After
stirring for 20 min a DCM (5 mL) solution of compound 41
(1.93 g, 2.17 mmol) was added over 30 min and the resulting sus-
pension was stirred at ꢀ15 °C for 30 min. Subsequently, TEA
(0.5 ml, 3.47 mmol) was added and the reaction cleared up within
5.1.63. ((30-O-Acetyl)-40-N-(4-ethynylbenzyl))azithromycin (41)
To the solution of (40-ethynylbenzyl)azithromycin 14 (1.00 g,
1.18 mmol) in DCM (10 mL) was added acetic anhydride
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S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
19
a min. Stirring continued at ꢀ10 °C for 2 h and the reaction was
quenched with saturated aqueous NaHCO3 solution (20 mL), extra
DCM (20 mL) was added and the organic layer was separated. The
aqueous layer was again extracted with DCM (2 ꢂ 10 mL) and the
combined organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The crude was purified by column chro-
matography (Silica gel, 12:1:0.5 DCM/MeOH/NH4OH) to give the
title compound 43 (1.79 g, 93% yield) as white solid. 1H NMR
water several times to remove DMSO. The organic layer was dried
over Na2SO4 and concentrated to give light yellow solid product
(1.63 g, 85% yield). 1H NMR (400 MHz, CDCl3) d (ppm) 7.37 (d,
J = 8.0 Hz, 2H), 7.18 (d, J = 6.7 Hz, 2H), 5.26 (s, 1H), 5.06 (d,
J = 10.0 Hz, 1H), 4.98 (d, J = 8.5 Hz, 1H), 4.95–4.76 (m, 2H), 4.72
(dd, J = 12.6, 6.2 Hz, 1H), 4.62 (t, J = 7.4 Hz, 1H), 4.50 (s, 1H), 4.44
(d, J = 7.6 Hz, 1H), 4.09 (t, J = 7.3 Hz, 1H), 4.03 (dd, J = 12.6, 5.9 Hz,
1H), 3.70–3.60 (m, 1H), 3.60–3.52 (m, 1H), 3.51–3.42 (m, 1H),
3.34 (d, J = 19.2 Hz, 1H), 3.28–3.14 (m, 2H), 3.09 (d, J = 7.6 Hz,
1H), 3.01 (d, J = 4.1 Hz, 1H), 2.91 (d, J = 4.3 Hz, 1H), 2.82–2.61 (m,
3H), 2.61–2.52 (m, 1H), 2.35–2.25 (m, 1H), 2.24–2.11 (m, 6H),
2.10–2.00 (m, 4H), 1.95–1.84 (m, 2H), 1.83–1.64 (m, 4H), 1.56
(ddd, J = 20.9, 13.1, 6.4 Hz, 2H), 1.49–1.32 (m, 3H), 1.30–1.14 (m,
10H), 1.12 (d, J = 8.3 Hz, 2H), 1.06 (s, 3H), 1.03 (d, J = 4.9 Hz, 3H),
1.03–0.97 (m, 4H), 0.94 (dd, J = 15.2, 8.7 Hz, 3H), 0.91–0.75 (m,
3H). 13C NMR (126 MHz, CDCl3) d (ppm) 182.2, 175.8, 173.7,
169.7, 145.2, 142.9, 140.2, 132.2, 131.9, 128.6, 128.1, 120.9,
106.6, 105.0, 103.7, 97.4, 83.7, 78.3, 75.5, 74.3, 73.7, 72.5, 71.0,
70.6, 69.9, 69.3, 65.5, 65.3, 61.7, 60.9, 59.3, 49.9, 49.4, 42.7, 40.4,
39.7, 37.0, 31.9, 29.2, 22.9, 21.5, 19.3, 17.9, 15.2, 14.1, 11.7, 11.0,
(400 MHz, CDCl3)
d (ppm) 7.37 (d, J = 8.1 Hz, 2H), 7.17 (d,
J = 7.3 Hz, 2H), 5.18–5.04 (m, 1H), 5.05–4.73 (m, 2H), 4.65 (d,
J = 9.7 Hz, 1H), 4.48–4.34 (m, 1H), 4.20 (d, J = 4.3 Hz, 1H), 4.16–
4.06 (m, 1H), 3.95 (dd, J = 15.2, 8.5 Hz, 1H), 3.72–3.57 (m, 2H),
3.56–3.37 (m, 3H), 3.31 (dd, J = 16.4, 9.8 Hz, 1H), 3.23 (t,
J = 5.2 Hz, 1H), 3.19 (s, 1H), 3.07 (d, J = 6.2 Hz, 1H), 3.05–2.91 (m,
2H), 2.86–2.55 (m, 2H), 2.49 (d, J = 10.1 Hz, 1H), 2.27 (dd, J = 11.5,
6.2 Hz, 3H), 2.20 (d, J = 5.6 Hz, 3H), 2.17–2.13 (m, 2H), 2.11 (d,
J = 5.3 Hz, 2H), 2.08–1.95 (m, 5H), 1.83–1.70 (m, 2H), 1.66–1.46
(m, 2H), 1.44–1.34 (m, 3H), 1.34–1.16 (m, 14H), 1.13 (dd, J = 6.8,
4.2 Hz, 4H), 1.10–1.01 (m, 4H), 1.00–0.91 (m, 3H), 0.90–0.73 (m,
3H). 13C NMR (126 MHz, CDCl3) d (ppm) 211.6, 177.2, 176.5,
170.0, 169.7, 140.6, 132.0, 128.3, 120.6, 100.5, 94.6, 83.6, 75.8,
75.3, 74.3, 73.9, 73.0, 72.4, 72.2, 71.2, 69.8, 69.0, 68.0, 65.6, 63.1,
61.8, 58.3, 51.2, 49.5, 48.9, 45.1, 37.1, 36.9, 35.2, 34.4, 29.5, 23.0,
22.1, 21.5, 21.1, 20.6, 18.1, 15.5, 14.3, 12.3, 11.3, 9.0, 7.7. HRMS
10.3, 8.8. HRMS (ESI) m/z Calcd for
C49H79N2O13 [M+H+]:
903.5577, found 903.5568.
5.1.68. ((400-(N,N-Dimethylaminomethyl)-40-N-(4-ethynylbenzyl))-
clarithromycin (47)
(ESI) m/z Calcd for
C
48H77N2O13 [M+H+]: 889.5420, found
889.5412.
N,N-Dimethylmethylamine 46 (4.22 mL, 8.46 mmol, 2 M in
THF) was added to a solution of compound 44 (0.10 g, 0.11 mmol)
in MeOH (20 mL). The solution was heated at 60 °C for 6 h. MeOH
and residual methylamine was evaporated off to give light yellow
solid which was again dissolved in MeOH (10 mL) and heated at
90 °C for three days after which TLC showed complete conversion.
Excess MeOH was evaporated off to give compound 47 as yellow
solid (0.08 g, 83%). 1H NMR (400 MHz, CDCl3) d (ppm) 7.43 (d,
J = 8.2 Hz, 3H), 7.26–7.20 (m, 2H), 5.04 (dd, J = 11.0, 2.2 Hz, 1H),
4.98 (d, J = 5.0 Hz, 1H), 4.37 (d, J = 7.2 Hz, 1H), 4.07–4.00 (m, 1H),
3.94 (s, 1H), 3.79 (s, 1H), 3.75 (s, 2H), 3.73–3.69 (m, 2H), 3.66
(dd, J = 8.5, 5.8 Hz, 1H), 3.62 (dd, J = 6.6, 3.6 Hz, 1H), 3.61–3.57
(m, 1H), 3.50–3.42 (m, 2H), 3.39 (dd, J = 12.3, 7.7 Hz, 3H), 3.29
(dd, J = 10.2, 7.2 Hz, 1H), 3.21 (d, J = 3.9 Hz, 1H), 3.16 (d,
J = 3.7 Hz, 2H), 3.09 (s, 3H), 3.06 (t, J = 4.5 Hz, 4H), 3.05–3.01 (m,
4H), 2.99 (d, J = 8.0 Hz, 1H), 2.91–2.80 (m, 1H), 2.66 (s, 1H), 2.62
(s, 1H), 2.52 (dd, J = 15.6, 7.6 Hz, 2H), 2.36 (s, 9H), 2.23 (d,
J = 4.3 Hz, 4H), 2.18–2.15 (m, 1H), 2.07 (s, 1H), 2.03 (s, 2H), 1.99
(d, J = 6.8 Hz, 1H), 1.90 (dt, J = 30.0, 10.5 Hz, 6H), 1.64–1.52 (m,
2H), 1.49–1.42 (m, 1H), 1.41–1.33 (m, 6H), 1.31–1.22 (m, 10H),
1.22–1.17 (m, 5H), 1.15–1.01 (m, 25H), 0.91 (dt, J = 14.0, 5.6 Hz,
3H), 0.87–0.78 (m, 5H). 13C NMR (126 MHz, CDCl3) d (ppm)
175.7, 169.9, 167.8, 132.3, 132.0, 130.9, 128.8, 128.5, 121.1,
102.8, 96.4, 83.4, 81.3, 78.3, 78.0, 76.5, 76.0, 74.3, 70.9, 70.3, 69.0,
68.3, 68.2, 67.6, 63.6, 58.0, 57.8, 50.8, 50.6, 49.4, 47.3, 45.3, 44.9,
39.3, 38.8, 38.7, 37.2, 36.9, 31.3, 30.4, 29.7, 28.9, 23.7, 23.0, 21.6,
21.0, 19.8, 18.5, 18.1, 16.0, 16.0, 15.1, 14.1, 12.4, 11.0, 10.6, 9.3.
HRMS (ESI) m + 2/2z Calcd for C49H82N2O13 [M+2H+]: 453.2903
found 453.2892.
5.1.66. ((400-Epoxy)-30-O-acetyl)-40-N-(4-ethynylbenzyl))
clarithromycin (44)
Compound 42 (0.40 g, 0.45 mmol) was reacted with NaH (60%
dispersion in mineral oil) (0.040 g, 0.99 mmol) and trimethyloxo-
sulfonium iodide (0.218 g, 0.99 mmol) as described for the synthe-
sis of compound 32 to give compound 44 after purification by
preparative chromatography (Silica gel, Hexane/EtOAc/EtOH
(4:1:0.5), as yellow solid (0.35 g, 77%). 1H NMR (400 MHz, CDCl3)
d (ppm) 7.40 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 4.88 (d,
J = 3.5 Hz, 1H), 4.84–4.73 (m, 2H), 4.56 (d, J = 7.5 Hz, 1H), 3.98–
3.91 (m, 1H), 3.69 (s, 1H), 3.67–3.61 (m, 2H), 3.49 (d, J = 14.0 Hz,
2H), 3.34 (s, 1H), 3.06 (s, 3H), 3.04 (s, 2H), 2.90 (d, J = 4.1 Hz, 1H),
2.81–2.67 (m, 3H), 2.64 (d, J = 4.1 Hz, 1H), 2.49 (dt, J = 21.9,
7.4 Hz, 1H), 2.38 (dt, J = 9.9, 4.9 Hz, 1H), 2.35–2.25 (m, 2H), 2.21
(s, 3H), 2.18 (s, 1H), 2.15 (s, 1H), 2.08 (s, 3H), 1.83 (d, J = 4.8 Hz,
1H), 1.81–1.71 (m, 2H), 1.43–1.32 (m, 3H), 1.30–1.22 (m, 9H),
1.19 (t, J = 5.8 Hz, 7H), 1.12 (t, J = 9.0 Hz, 4H), 1.04–0.97 (m, 10H),
0.92 (d, J = 6.5 Hz, 3H), 0.88 (dd, J = 10.4, 4.7 Hz, 2H). 13C NMR
(101 MHz, CDCl3) d (ppm) 216.1, 178.8, 171.3, 169.8, 140.7,
131.9, 128.4, 120.6, 99.7, 96.0, 83.7, 79.7, 78.7, 73.8, 71.5, 68.2,
63.1, 61.5, 60.4, 58.2, 50.3, 49.4, 46.7, 41.6, 41.1, 38.3, 37.0, 36.4,
35.7, 33.4, 31.3, 29.7, 24.6, 21.3, 21.1, 20.2, 19.2, 17.9, 14.3, 14.1,
10.9, 10.7, 7.8. HRMS (ESI) m/z Calcd for C49H76NO14 [M+H+]:
902.5260, found 902.5260.
5.1.67. ((400-Epoxy)-30-O-acetyl)-40-N-(4-ethynylbenzyl))
azithromycin (45)
NaH (0.2 g, 4.67 mmol, 60%w/w) was added to an oven dried
three necked flask and was washed with petroleum ether (ꢂ3).
The flask was immediately flushed with Ar and dry DMSO (6 mL)
was introduced through a septum. The mixture was stirred at room
temperature under Ar. Then trimethyloxosulfonium iodide (1.05 g,
4.67 mmol) was added over a period of 5 min. After hydrogen gas
ceased to evolve, the resulting yellow clear solution was treated
with a solution of compound 43 (1.88 g, 2.12 mmol) in anhydrous
THF (5 mL) over 10 min and stirring continued for 2 h after which
TLC (4:1:0.5 Hex/EtOAc/EtOH) showed a near quantitative conver-
sion to a new product. THF was evaporated off, EtOAc (20 mL) was
added to the remaining residue and the mixture was washed with
5.1.69. ((400-(N,N-Dimethylaminomethyl)-40-N-(4-ethynylbenzyl))-
azithromycin (48)
Reaction of compound 45 (2.00 g, 2.2 mmol) and N,N-dimethyl-
methyl amine 46 (8.92 mL, 168.3, 2 M in THF) mmol) in anhydrous
methanol (20 mL) as described for the synthesis of 47, gave 48 as
white solid (2.0 g, 90%) after purification by column chromatogra-
phy (Silica gel, 20:1:0.1 DCM, MeOH, and NH4OH). 1H NMR
(400 MHz, CDCl3)
d (ppm) 7.38 (t, J = 5.6 Hz, 2H), 7.19 (d,
J = 8.0 Hz, 2H), 4.98 (d, J = 4.7 Hz, 1H), 4.80 (dd, J = 10.7, 7.5 Hz,
1H), 4.61 (dd, J = 16.8, 8.0 Hz, 1H), 4.53–4.47 (m, 1H), 4.32 (dd,
J = 11.6, 6.2 Hz, 1H), 4.08 (t, J = 7.2 Hz, 1H), 4.02 (q, J = 6.4 Hz,
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

20
S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
1H), 3.70 (dt, J = 9.6, 5.5 Hz, 1H), 3.67–3.59 (m, 2H), 3.54 (d,
J = 6.2 Hz, 1H), 3.49 (s, 1H), 3.41 (ddd, J = 22.7, 11.0, 6.6 Hz, 2H),
3.31 (d, J = 7.3 Hz, 1H), 3.20–3.12 (m, 1H), 3.11–2.98 (m, 3H),
2.92 (d, J = 3.3 Hz, 1H), 2.85 (d, J = 2.4 Hz, 1H), 2.78 (dd, J = 17.2,
10.1 Hz, 1H), 2.73 (s, 1H), 2.65 (dd, J = 14.3, 7.6 Hz, 2H), 2.57 (d,
J = 9.5 Hz, 1H), 2.45 (dt, J = 23.1, 10.2 Hz, 1H), 2.31 (d, J = 11.6 Hz,
7H), 2.24–2.16 (m, 3H), 2.13 (dd, J = 11.4, 4.5 Hz, 1H), 2.10–2.01
(m, 4H), 2.00–1.78 (m, 6H), 1.68 (dd, J = 23.3, 12.9 Hz, 2H), 1.23
(qd, J = 15.5, 7.1 Hz, 17H), 1.13 (dd, J = 9.7, 4.5 Hz, 3H), 1.09–0.98
(m, 8H), 0.86 (ddd, J = 11.8, 10.2, 6.2 Hz, 3H). 13C NMR (101 MHz,
CDCl3) d (ppm) 178.0, 169.7, 140.7, 131.8, 128.3, 120.5, 100.4,
95.6, 84.2, 83.5, 78.8, 76.8, 76.0, 74.9, 74.2, 73.3, 71.4, 70.2, 70.1,
67.5, 67.4, 61.9, 61.3, 58.4, 57.9, 53.6, 49.0, 47.3, 47.2, 45.1, 44.7,
41.9, 41.4, 40.1, 36.8, 36.5, 31.5, 30.6, 30.0, 27.0, 26.5, 21.7, 21.3,
20.9, 18.5, 16.1, 15.7, 14.7, 11.1, 9.38, 7.5. ESI MS m/z Calcd for
5.1.72. (Azithromycin-(40-N-(4-benzyltriazolyl))-400-(N,N-dime-
thylaminomethyl))-N-hydroxyhexanamide (50a)
Compound 48 (0.17 g, 0.18 mmol) and 6-Azido-N-((tert-butyl-
dimethyl silyl)oxy)hexanamide 51e (0.08 g, 0.30 mmol) were
dissolved in anhydrous THF (5 mL) and stirred under Ar at room
temperature. Copper(I) iodide (0.02 g, 0.09 mmol) and Hunig’s base
(0.07 mL, 0.37 mmol) were added to the mixture and stirring con-
tinued for 12 h. Caesium fluoride (0.04 g, 0.28 mmol) and MeOH
(4 mL) were added to the mixture to remove TBS protecting group
and stirring continued for an additional 2 h. A solution of 4:1 sat-
urated NH4Cl/NH4OH (30 mL) was added to the reaction mixture
and extracted with 20% MeOH/CH2Cl2 (3 ꢂ 30 mL). The organic
layer was dried over Na2SO4 and concentrated in vacuo. The crude
product was purified by preparative chromatography (Silica gel,
5:1:1 EtOAc/MeOH/NH4OH) to give the product (0.16 g, 80%) as
light yellow solid. 1H NMR (400 MHz, CD3OD) d (ppm) 7.74 (s,
1H), 7.20 (d, J = 7.8 Hz, 2H), 6.83 (d, J = 7.9 Hz, 2H), 3.83 (s, 2H),
3.74 (d, J = 7.3 Hz, 1H), 3.60–3.46 (m, 2H), 3.26 (d, J = 13.0 Hz,
1H), 2.99–2.89 (m, 3H), 2.78 (s, 1H), 2.75–2.67 (m, 3H), 2.37 (d,
J = 3.6 Hz, 3H), 2.23–2.10 (m, 2H), 1.96 (t, J = 14.2 Hz, 3H), 1.80–
1.64 (m, 11H), 1.60–1.45 (m, 3H), 1.43–1.30 (m, 11H), 1.28 (s,
1H), 1.22 (d, J = 6.6 Hz, 1H), 1.13 (dd, J = 24.4, 9.4 Hz, 3H), 1.02 (d,
J = 15.3 Hz, 2H), 0.93–0.81 (m, 1H), 0.74 (dd, J = 14.2, 7.2 Hz, 3H),
0.68 (dd, J = 10.0, 4.1 Hz, 5H), 0.67–0.58 (m, 3H), 0.55 (d,
J = 6.7 Hz, 6H), 0.48 (d, J = 4.3 Hz, 6H), 0.38 (d, J = 7.5 Hz, 3H),
0.33–0.21 (m, 10H). 13C NMR (126 MHz, CD3OD) d (ppm) 180.5,
178.8, 177.3, 173.7, 172.8, 149.4, 141.7, 131.8, 127.5, 122.8,
105.1, 102.9, 97.3, 86.5, 80.7, 78.8, 77.9, 76.3, 75.9, 73.7, 72.8,
71.4, 70.0, 69.2, 64.7, 63.2, 61.6, 60.1, 55.7, 53.1, 52.1, 50.9, 48.5,
47.3, 44.5, 44.1, 43.9, 38.3, 37.6, 32.9, 32.4, 32.3, 32.2, 31.9, 31.7,
30.2, 28.4, 27.9, 23.1, 22.7, 19.7, 18.1, 16.4, 12.4, 10.7, 8.4. HRMS
(ESI) m+2/2z Calcd for C55H97N7O14 [M+2H+]: 539.8541, found
539.8549.
C
51H86N3O13 [M+H+]: 948.61.
5.1.70. (Clarithromycin-(40-N-(4-benzyltriazolyl))-400-(N,N-dime-
thylaminomethyl))-N-hydroxyhexanamide (49a)
Reaction of compound 47 (0.05 g, 0.06 mmol) with 6-Azido-N-
((tert-butyldimethylsilyl)oxy)hexanamide
51e
(0.075 g,
0.249 mmol) followed by TBS removal with caesium fluoride as
described for the synthesis of compound 5a, gave 49a as light yel-
low solid (0.02 g, 44%). 1H NMR (400 MHz, CD3OD) d (ppm) 8.36 (s,
1H), 7.81 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 5.12 (d,
J = 10.8 Hz, 1H), 4.45 (t, J = 6.9 Hz, 3H), 4.31 (d, J = 7.2 Hz, 1H),
4.09 (d, J = 6.5 Hz, 1H), 3.90 (d, J = 13.4 Hz, 1H), 3.72 (d,
J = 13.5 Hz, 2H), 3.59 (d, J = 12.8 Hz, 2H), 3.20 (dd, J = 19.5, 9.0 Hz,
3H), 3.11 (d, J = 15.2 Hz, 2H), 3.00 (d, J = 8.4 Hz, 6H), 2.89 (s, 1H),
2.65 (s, 1H), 2.59 (s, 3H), 2.42 (s, 6H), 2.34 (s, 4H), 2.28 (s, 1H),
2.21 (s, 1H), 2.10 (t, J = 7.4 Hz, 4H), 1.96 (dd, J = 15.3, 7.4 Hz, 5H),
1.82 (s, 7H), 1.73–1.60 (m, 5H), 1.44–1.33 (m, 9H), 1.27 (d,
J = 10.6 Hz, 7H), 1.22 (dd, J = 12.4, 6.3 Hz, 7H), 1.21–1.16 (m, 6H),
1.17–1.09 (m, 17H), 1.06 (d, J = 7.5 Hz, 4H), 0.91 (d, J = 10.9 Hz,
5H), 0.84 (dd, J = 9.5, 5.1 Hz, 5H). 13C NMR (101 MHz, CDCl3) d
(ppm) 175.7, 147.5, 129.7, 125.8, 102.9, 96.8, 94.9, 78.3, 76.1,
74.3, 71.3, 70.9, 69.0, 68.3, 67.5, 51.3, 50.7, 50.7, 50.1, 50.5, 49.4,
47.2, 45.3, 45.3, 44.6, 39.4, 37.5, 36.8, 33.7, 31.9, 29.7, 28.6, 23.2,
22.7, 21.6, 21.3, 19.7, 18.1, 16.0, 15.9, 15.1, 14.1, 12.3, 10.6, 9.2,
7.8. HRMS (ESI) m/z Calcd for C55H93N6O15 [M+H+] 1077.6693,
found 1077.6692.
5.1.73. (Azithromycin-(40-N-(4-benzyltriazolyl))-400-(N,N-dime-
thylaminomethyl))-N-hydroxyheptanamide (50b)
Reaction of compound 48 (0.08 g, 0.09 mmol) with 7-Azido-N-
((tert-butyldimethylsilyl)oxy)heptanamide 51f (0.04 g, 0.14 mmol)
followed by TBS removal with caesium fluoride as described for the
synthesis of compound 50a, gave 50b as light yellow solid (0.069 g,
70%). 1H NMR (400 MHz, CD3OD) d (ppm) 7.46 (s, 1H), 6.92 (d,
J = 8.0 Hz, 2H), 6.56 (d, J = 8.0 Hz, 2H), 3.55 (d, J = 6.2 Hz, 2H),
3.47 (d, J = 7.2 Hz, 1H), 3.33–3.17 (m, 2H), 2.99 (d, J = 12.9 Hz,
1H), 2.76 (d, J = 13.2 Hz, 1H), 2.67 (dd, J = 14.5, 9.8 Hz, 3H), 2.51
(s, 1H), 2.43 (ddd, J = 6.2, 4.9, 2.3 Hz, 2H), 2.26 (t, J = 15.8 Hz, 1H),
2.09 (d, J = 5.7 Hz, 3H), 1.98–1.83 (m, 2H), 1.69 (dd, J = 14.5,
9.6 Hz, 2H), 1.55–1.37 (m, 13H), 1.35–1.23 (m, 2H), 1.22 (d,
J = 13.4 Hz, 3H), 1.16–1.01 (m, 11H), 0.87 (t, J = 11.8 Hz, 3H), 0.75
(d, J = 15.1 Hz, 3H), 0.46 (d, J = 19.0 Hz, 6H), 0.44–0.40 (m, 3H),
0.35 (dt, J = 15.7, 9.2 Hz, 6H), 0.32–0.25 (m, 6H), 0.20 (t,
J = 5.5 Hz, 6H), 0.11 (d, J = 7.5 Hz, 3H), 0.07–0.06 (m, 6H). 13C
NMR (126 MHz, CD3OD) d (ppm) 180.4, 180.1, 177.3, 173.5,
172.7, 149.5, 141.7, 141.2, 131.8, 131.4, 127.4, 122.8, 105.1,
102.8, 97.3, 86.1, 80.3, 78.9, 76.5, 75.7, 73.7, 72.8, 71.5, 70.1,
69.2, 64.6, 63.5, 61.7, 60.1, 55.7, 53.2, 52.1, 51.0, 48.5, 47.6, 47.4,
44.1, 43.8, 38.5, 38.0, 37.6, 32.1, 31.5, 29.0, 28.8, 27.7, 26.8, 23.1,
22.4, 20.0, 18.2, 16.4, 16.0, 12.6, 10.8, 8.5. HRMS (ESI) m+2/2z Calcd
for C56H99N7O14 [M+2H+]: 546.8620, found 546.8611.
5.1.71. (Clarithromycin-(40-N-(4-benzyltriazolyl))-400-(N,N-dime-
thylaminomethyl))-N-hydroxyheptanamide (49b)
Reaction of compound 47 (0.024 g, 0.022 mmol) with 7-Azido-
N-((tert-butyldimethylsilyl)oxy)heptanamide
51f
(0.075 g,
0.249 mmol) followed by TBS removal with caesium fluoride as
described for the synthesis of compound 5a, gave 49b as a light
yellow solid (0.021 g, 83%). 1H NMR (400 MHz, CD3OD) d 8.37 (s,
1H), 7.82 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 5.14–5.07 (m,
1H), 4.45 (t, J = 7.0 Hz, 2H), 4.30 (d, J = 7.2 Hz, 1H), 4.09 (d,
J = 6.5 Hz, 1H), 3.92 (d, J = 13.0 Hz, 1H), 3.72 (d, J = 12.3 Hz, 2H),
3.64–3.57 (m, 2H), 3.34 (d, J = 2.4 Hz, 2H), 3.09 (s, 1H), 3.04–2.97
(m, 7H), 2.89 (s, 2H), 2.59 (s, 3H), 2.49 (s, 7H), 2.36 (s, 3H), 2.28
(s, 1H), 2.15 (s, 1H), 2.11–2.03 (m, 5H), 1.92 (dd, J = 13.5, 6.7 Hz,
6H), 1.83 (d, J = 10.2 Hz, 4H), 1.61 (d, J = 7.5 Hz, 6H), 1.42–1.33
(m, 12H), 1.28 (s, 6H), 1.23 (d, J = 6.0 Hz, 5H), 1.19 (d, J = 7.2 Hz,
4H), 1.17–1.08 (m, 16H), 1.05 (d, J = 7.5 Hz, 4H), 0.97 (s, 1H), 0.91
(d, J = 11.7 Hz, 4H), 0.84 (t, J = 7.4 Hz, 4H). 13C NMR (101 MHz,
cd3od) d 223.5, 179.1, 149.8, 132.2, 127.7, 123.6, 105.6, 99.1,
83.2, 80.8, 79.1, 77.0, 73.5, 71.6, 70.5, 69.7, 63.3, 60.6, 53.5, 52.2,
51.4, 48.7, 47.8, 47.4, 41.5, 38.1, 33.1, 32.2, 31.5, 30.9, 30.6,
28.2, 27.6, 23.1, 20.0, 19.8, 18.1, 17.7, 16.9, 13.7, 12.2, 11.3 HRMS
(ESI) m+2/2z Calcd for C56H97N6O15 [M+2H+]: 546.3461, found
546.3469.
5.2. In vitro HDAC inhibition: SAMDI assay
The maleimide-presenting SAMs and expression of HDAC8
enzyme were prepared as previously reported.21 To obtain IC50 val-
ues, we incubated isoform-optimized substrates (50
lM, detailed
below) with enzyme (250 nM, detailed below) and inhibitor (at
Please cite this article in press as: Tapadar, S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.045

S. Tapadar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
21
concentrations ranging from 1 nM to 1.0 mM) in HDAC buffer
5.5. Real-time quantitative RT-PCR analysis
(25.0 mM Tris–HCl, pH 8.0, 140 mM NaCl, 3.0 mM KCl, 1.0 mM
MgCl2, and 0.1 mg/mL BSA) in 96-well microtiter plates (60 min,
37 °C). Solution-phase deacetylation reactions were quenched with
trichostatin A (TSA) and transferred to SAMDI plates to immobilize
the substrate components. SAMDI plates were composed of an
array of self-assembled monolayers (SAMs) presenting maleimide
in standard 384-well format for high-throughput handling capabil-
ity. Following immobilization, plates were washed to remove buf-
fer constituents, enzyme, inhibitor, and any unbound substrate and
analyzed by MALDI mass spectrometry using automated protocols.
Deacetylation yields in each triplicate sample were determined
from the integrated peak intensities of the molecular ions for the
substrate and the deacetylated product ion by taking the ratio of
the former over the sum of both. Yields were plotted with respect
to inhibitor concentration and fitted to obtain IC50 values for each
isoformꢀinhibitor pair.
Total RNA was isolated with TRIzol reagent (Life Technologies)
by following the manufacturer’s instruction. For the reverse tran-
scription reaction, TaqMan reverse transcription reagents (Life
Technologies) were used as described previously. For quantitative
RT-PCR analysis, PCR amplifications were performed by using SYBR
Green Universal Master Mix (Life Technologies). In brief, reactions
were performed in triplicate containing 2ꢂ ꢃUniversal Master
Mix, 1
lL of template cDNA, 500 nM primers in a final volume of
12.5 L, and they were analyzed in a 96-well optical reaction plate
l
(USA Scientific). Reactions were amplified and quantified by using a
StepOnePlus Real-Time PCR System and the manufacturer’s corre-
sponding software (StepOnePlus Software v2.3; Life Technologies).
The relative quantities of mRNAs were obtained by using the com-
parative Ct method and were normalized by using human cyclophi-
lin as an endogenous control. For semiquantitative RT-PCR analysis,
PCR amplifications were performed with PrimeSTAR Max poly-
merase (Takara) by following the manufacturer’s instruction. The
Isoform-optimized substrates were prepared by traditional
FMOC solid-phase peptide synthesis (reagents supplied by Ana-
spec) and purified by semi-preparative HPLC on a reverse-phase
C18 column (Waters). The peptide GRKacFGC was prepared for
HDAC1 and HDAC8 experiments, whereas the peptide GRKacYGC
was prepared for HDAC6 experiments.
primer for TNF-a a
, IL-1b were described previousery.40,41 IL-1 : 50-
CGAGCCAATGATCAGTACCTC-30 and 30-CACCCATATATTTCACTG -50.
Acknowledgements
HDAC1, HDAC6, and HDAC2 were purchased from BPS Bio-
sciences. The catalytic domain of HDAC8 was expressed as previ-
ously reported.21e Briefly, an amplicon was prepared by PCR with
the following primers: forward (50ꢀ30) TATTCTCGAGGA-CCACA
TGCTTCA and reverse (50ꢀ30) ATAAGCTAGCATG-GAGGAGCCGGA.
A pET21a construct bearing the genetic insert between the NheI
and XhoI restriction sites was transformed into Escherichia coli
BL21(DE3) (Lucigen) and expressed by standard protocols.
Following purification by affinity chromatography, the His-tagged
enzyme-containing fractions were purified by FPLC (AKTA) on a
superdex size-exclusion column (GE), spin-concentrated, and
stored at ꢀ80 °C in HDAC buffer with 10% glycerol.
This work was financially supported by NIH RO1 Grant
R01CA131217 (A.K.O.), by Georgia Institute of Technology through
Cullen-Peck fellowship (A.K.O.), and by NIH RO1 Grant
RO1DC005843 (J.D.L.). We thank ACS division of Medicinal
Chemistry for a travel award to Idris Raji.
Supplementary data
Supplementary data (1H NMR, 13C NMR spectral and NF-
activity in NTHi infected BEAS-2B cells treated with 1 M of tested
jB
l
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2015.10.045.
5.3. Cell viability assay
References and notes
All cell lines used in this study (A549, MCF-7 and VERO) were
maintained in DMEM (Lonza, GA) supplemented with 10% fetal
bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA) and 1% Peni-
cillin–Streptomycin. Prior to treatment with various drug concen-
trations and subsequent incubation for 72 h, cells were incubated
in a 96 well plate for 24 h. Cell viability was measured using the
MTS assay (CellTiter 96 Aqueous One Solution and CellTiter 96
Non-Radioactive Cell Proliferation Assays, Promega, Madison, WI)
protocol as described by the manufacturer. For all drugs tested,
DMSO concentration was maintained at 0.1%.
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