Synthesis and antibacterial activity of novel lincomycin derivatives. I. Enhancement of antibacterial activities by introduction of substituted azetidines

Article abstract of DOI:10.1038/ja.2015.134

The synthesis and antibacterial activity of (7S)-7-sulfur-Azetidin-3-yl lincomycin derivatives are described. Modification was achieved by a simple reaction of (7R)-7-O-methanesulfonyllincomycin and the corresponding substituted azetidine-2-Thiol. Several

Full text of DOI:10.1038/ja.2015.134

The Journal of Antibiotics (2016), 1–6
2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16
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&
ORIGINAL ARTICLE
Synthesis and antibacterial activity of novel lincomycin
derivatives. I. Enhancement of antibacterial activities
by introduction of substituted azetidines
Ko Kumura, Yoshinari Wakiyama, Kazutaka Ueda, Eijiro Umemura, Takashi Watanabe, Eiki Shitara,
Hideki Fushimi, Takuji Yoshida and Keiichi Ajito
The synthesis and antibacterial activity of (7S)-7-sulfur-azetidin-3-yl lincomycin derivatives are described. Modification was
achieved by a simple reaction of (7R)-7-O-methanesulfonyllincomycin and the corresponding substituted azetidine-2-thiol.
Several compounds first showed moderate antibacterial activity against Streptococcus pneumoniae and Streptococcus pyogenes
with erm gene as lincomycin derivatives.
The Journal of Antibiotics advance online publication, 13 January 2016; doi:10.1038/ja.2015.134
INTRODUCTION
derivative10 (Figure 2). Although some of these compounds have
different mode of actions from lincosamide, we attempted to
incorporate the azetidine ring to lincosamide at the C-7 position via
sulfur atom with (7R) configuration to generate novel lincomycin
antibiotics that are active against S. pneumoniae and S. pyogenes with
erm gene.
Lincosamide antibiotics are prokaryotic translation inhibitors that act
on the 50S ribosome in a similar manner to macrolide antibiotics.
Clindamycin (CLDM) derived from lincomycin (LCM) is a useful
semisynthetic antibiotic (Figure 1) that is effective against staphylo-
coccal and streptococcal infections. However, those lincosamides show
almost no antibacterial activity against resistant pathogens such as
Streptococcus pneumoniae and Streptococcus pyogenes with erm gene.
Erm methyltransferases methylate A2058Ec of rRNA and diminish the
affinity of clinically important macrolides, lincosamides and strepto-
gramin B.1 Emergence of resistant bacteria is a serious concern in
clinical sites.2 The crystal structures of bacterial 23S ribosomal rRNA
complexed with CLDM showed several hydrogen bonds between a
sugar moiety of CLDM and the peptidyl transferase cavity composed
of such as A2058Ec, G2520Ec and A2059Ec.3 This finding explains the
reason why chemical modifications of the sugar moiety of lincosamide
result in drastic loss of antibacterial activity.4 On the other hand, there
is a hydrophobic space around the C-7 position of CLDM in the
crystal structure and it is consistent with the fact that modifications at
the C-7 position of LCM tend to give comparable antibacterial activity
to that of LCM.5,6 Sztaricskai et al.5 reported compounds 1 and 2,
which have a heteroaryl group via sulfur atom with (7R) configura-
tion. We supposed that (7S) configuration is more suitable for target
interaction from reported structure-activity relationships (SAR)6 and
the three-dimensional structural information. So far, we synthesized
many (7S)-7-sulfur-substituted LCM analogues,7 and we describe the
first-generation derivatives among them in this report. Thus, we
intended to incorporate an azetidine ring instead of the aromatic
heterocycle. The azetidine ring is sometimes used in antibiotics
chemistry, for example, β-lactam antiinfective, new quinolone and
Chemistry
Although synthesis of lincosamide derivatives that have a sulfide group
at the C-7 position with (7R) configuration was reported by a simple
SN2 reaction using CLDM,5,11 preparation of lincosamide derivatives
that have a sulfide group with (7S) configuration is limited because a
starting material is a natural compound with a single stereochemistry
at the C-7. Bannister12 reported introduction of an alkyl thiol to the
C-7 position with (7S) configuration utilizing epimine prepared form
LCM. This method is unique but requires many steps, and a source of
sulfide is limited to dithioacetals or monothioacetals. To establish
practical synthetic route, we utilized 2,3,4-tris-O-(trimethylsilyl)linco-
mycin (5),13 which was prepared in two steps from LCM as shown in
Scheme 1. After methanesulfonylation of the 7-OH of LCM, nucleo-
philic substitution of 6 with 4 afforded 7 in 48% yield. Removal of
trimethylsilyl groups followed by the deprotection of the amine
afforded 8 in 73% yield. For the synthesis of N-substituted derivatives,
8 was converted to 9 by reprotecting hydroxyl groups of 8. N-Aryl
analogues 10a, 10b and 10d were prepared by an SNAr reaction
between 9 and aryl fluoride or chloride. N-Alkyl analogues (11a–c)
were synthesized via addition of 9 to acrylic acid derivatives and
reaction of 9 with corresponding acid chloride furnished urea
aminoglycoside: tebipenem,8 delafloxacin9 and
a
tobramycin derivatives and amides (12a–e).
Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., Yokohama, Japan
Correspondence: Dr K Ajito, Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan.
E-mail: keiichi.ajito@meiji.com
Received 18 August 2015; revised 7 November 2015; accepted 19 November 2015

Antibacterial activity of lincomycin derivatives
K Kumura et al
2
Compound 10e that has the side chain of tebipenem was prepared antibacterial activity against those resistant strains as lincomycin
from 6 with 3-(4,5-dihydrothiazol-2-yl)azetidine-1-thiol8 in a similar
derivatives. Thus, we confirmed that the (7S)-7-sulfur-azetidin-3-yl-
manner as shown in Scheme 2.
LCM analogue was an attractive framework against the target pathogens.
RESULTS AND DISCUSSION
EXPERIMENTAL PROCEDURE
The antibacterial activities of novel lincomycin derivatives against
S. pneumoniae, S. pyogenes and Haemophilus influenzae are
summarized in Table 1. CLDM showed potent activity against
susceptible pathogens, but MICs against the resistant strains were
General
1H NMR spectra were measured with a JEOL JNM-GSX 400 (Tokyo, Japan,
400 MHz) spectrometer in CDCl3 or CD3OD with 0.03% tetramethylsilane as
an internal standard. Mass spectra were obtained on a JEOL JMS-FABmate
spectrometer or JEOL JMS-700 mass spectrometer or Agilent Technologies
6530-Q-TOF LC/MS mass spectrometer (Agilent Technologies, Santa Clara,
CA, USA). The optical rotations were recorded with Jasco P-2300 digital
polarimeter (Jasco, Tokyo, Japan). Column chromatography was performed
with silica gel (Kanto Chemical, Tokyo, Japan: 60N; spherical, neutral).
larger than 128 μg ml− 1
. Compound 8 exhibited comparable
antibacterial activities to LCM against susceptible Gram-positive
strains. Although the antibacterial activities of 8 against resistant
bacteria were not detected, the result prompted us to explore the SAR
of (7S)-7-sulfur-azetidin-3-yl-LCM derivatives. As for the compounds
10a–d that have an aryl group directly to azetidine, all the compounds
except 10c showed more potent antibacterial activities against
susceptible Gram-positive strains and resistant strains with mef gene
than CLDM. It is notable that 10b, 10d and 10e slightly exhibit
antibacterial activity against resistant strains with erm gene. As for
N-alkyl derivatives (11a–c), antibacterial activities were weak and
tert-Butyl 3-mercaptoazetidine-1-carboxylate (4)
To a solution of 3 (2.48 g) in tetrahydrofuran (35 ml) were added triphenyl-
phosphine (5.64 g), diethyl azodicarboxylate (3.92 ml) and the mixture was
stirred at room temperature for 20 min. To the mixture was added thiobenzoic
acid (2.53 ml) and the mixture was stirred at room temperature for 1 h. The
mixture was concentrated in vacuo and the residue was purified by silica gel
almost the same as those of N-unsubstituted compound 8, and their column chromatography (hexane–AcOEt) to give a colorless amorphous
(6.0 g). To a stirred solution of the compound obtained above (6.0 g) in
MeOH (50 ml) was added NaOMe (772 mg) and the reaction mixture was
stirred for 1 h. Saturated aqueous NaHCO3 was added to the mixture and
extracted with AcOEt and the organic phase was dried over Na2SO4, filtered
and concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane–AcOEt) to afford 4 (1.81 g, 67%) as a colorless solid.
1H NMR (400 MHz, CDCl3) δ 4.32–4.37 (m, 2H), 3.77–3.82 (m, 2H),
3.61–3.71 (m, 1H), 2.00 (d, J = 8.5 Hz, 1H), 1.39–1.50 (m, 9H).
weak activities were probably due to their high hydrophilicity. On the
other hand, amides 12d and 12e derived from acid chlorides showed
similar antibacterial profile to 10b, 10d and 10e. Furthermore, urea
derivatives 12b and 12c showed stronger activities against resistant
S. pneumoniae and S. pyogenes with erm gene than 10b, 10d and 10e.
The antibacterial activities of 12b against susceptible Gram-positive
strains are the strongest in this series, and 12c showed stronger
antibacterial activities against H. influenzae than CLDM.
7-O-Methanesulfonyl-2,3,4-tris-O-(trimethylsilyl)lincomycin (6)
To a cold (0 °C) solution of 5 (4.0 g) in CHCl3 (20 ml) were added
triethylamine (2.45 ml) and methanesulfonyl chloride (990 μl) and the mixture
was stirred at room temperature for 3 h. The mixture was diluted with CHCl3
and washed with 10% aqueous NaHCO3. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane–AcOEt) to afford 6 (4.2 g, 93%) as a
colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 11.0 Hz, 1H),
5.16 (d, J = 5.6 Hz, 1H), 5.10–5.15 (m, 1H), 4.75 (dt, J = 3.3, 10.3 Hz, 1H),
4.15 (dd, J = 5.6, 9.5 Hz, 1H), 3.90 (d, J = 9.7 Hz, 1H), 3.76 (d, J = 2.3 Hz, 1H),
3.52 (dd, J = 2.3, 9.4 Hz, 1H), 3.15–3.20 (m, 1H), 3.09 (s, 3H), 2.99 (dd, J = 3.7,
10.2 Hz, 1H), 2.40 (s, 3H), 2.11 (s, 3H), 1.92–2.09 (m, 3H), 1.79–1.89 (m, 1H),
1.40 (d, J = 6.8 Hz, 3H), 1.23–1.35 (m, 4H), 0.89 (t, J = 6.7 Hz, 3H),
0.17 (s, 9H), 0.14 (s, 9H), 0.13 (s, 9H); MS (ESI) m/z 701 (M+H)+.
CONCLUSIONS
To generate novel lincomycin derivatives, which are effective against
resistant S. pneumoniae and S. pyogenes with erm gene, we synthesized
a series of (7S)-7-sulfur-azetidin-3-yl-LCM derivatives by utilizing the
simple substitution reaction. N-Unsubstituted compound 8 showed
comparable antibacterial activities to LCM against susceptible Gram-
positive strains. N-Aryl, amide and urea derivatives showed weak
antibacterial activities against S. pneumoniae and S. pyogenes with erm
gene. Especially, urea derivatives 12b and 12c first exhibited moderate
X
X
Me
N
S
O
HN
HO
Me
7
Me
NR
O
HN
HO
Me
7
R
O
N
6
Me
N
6
Me
O
7(S)-7-Deoxy-7-[1-(tert-butoxycarbonyl)azetidin-3-ylthio]-2,3,4-
tris-O-(trimethylsilyl)lincomycin (7)
To a solution of 6 (10.5 g) and K2CO3 (4.15 g) in N,N-dimethylformamide
(50 ml) was added 4 (1.9 g) and the mixture was stirred at 80 °C for 15 h. After
cooled to room temperature, the mixture was diluted with AcOEt and washed
with brine. The organic phase was dried over Na2SO4, filtered and concentrated
in vacuo. The residue was purified by silica gel column chromatography
SMe
SMe
HO
OH
HO
OH
Lincomycin (LCM): R =
Clindamycin (CLDM): R =
OH
Cl
1: R = H; X = CH
2: R = Me; X = N
Figure 1 Structures of lincosamides.
HO
O
N
O
OH
HO
H2N
OH
N
O
F
OH
S
OH
Me
N
H2N
O
O
O
H
H
N
Me
OH
NH2
S
Cl
F
HO
N
HN
NH2
N
O
OH
HN
O
H2N
O
OH
F
Tebipenem
Tobramycin derivative
Delafloxacin
Figure 2 Structures of antibiotics that have an azetidine ring.
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Antibacterial activity of lincomycin derivatives
K Kumura et al
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a, b
HO
NBoc
HS
NBoc
4
3
NR2
Me
O
Me
7
Me
O
HN
Me
7
R
S
N
HN
c, d
Me
N
O
LCM
Me
O
TMSO
SMe
R1O
SMe
TMSO
OTMS
R1O
OR1
5: R = OH
6: R = OMs
f
e
7: R1 = TMS, R2 = Boc
8: R1 = H, R2 = H
c, d
g
Ar
N
NH
Me
Me
O
Me
7
O
Me
7
S
S
h, i
N
N
HN
HO
HN
Me
Me
TMSO
O
O
SMe
SMe
TMSO
OTMS
HO
OH
10a: Ar = 2-NO2Ph
10b: Ar = 4-NO2Ph
10c: Ar = 4-NH2Ph
9
j
l, i
10d: Ar = 1,3-benzoxazol-2-yl
k, i
O
O
R3
R4
N
N
Me
Me
O
HN
HO
Me
7
O
HN
HO
Me
7
S
S
N
N
Me
Me
O
O
SMe
SMe
HO
OH
HO
OH
12a: R4 = NMe2
11a: R3 = OMe
11b: R3 = NMe2
11c: R3 = morpholin-4-yl
12b: R4 = piperidin-1-yl
12c: R4 = morpholin-4-yl
12d: R4 = phenyl
12e: R4 = 2-NO2-phenyl
Scheme 1 Synthesis of 7(S)-sulfur-substituted lincomycin derivatives. Reagents: (a) thiobenzoic acid, DEAD, PPh3, THF; (b) NaOMe MeOH; (c) TMSCl,
HMDS, pyridine; (d) 6 N AcOH, MeOH; (e) MsCl, TEA, CHCl3; (f) 4, K2CO3, DMF; (g) 1N HCl, MeOH then trifluoroacetic acid; (h) XAr, TEA, DMF; (i) 1 N
HCl, MeOH; (j) H2, Pd/C, MeOH; (k) CH2CHCOR3, EtOH; (l) R4COCl, DMAP, TEA, CHCl3.
Synthesis of 7(S)-7-(azetidin-3-ylthio)-7-deoxylincomycin (8)
N
N
To a solution of 7 (280 mg) in MeOH (2 ml) was added 1 N hydrochloric acid
(1 ml) and the mixture was stirred at room temperature for 20 min. The
mixture was diluted with AcOEt and washed with 10% aqueous NaHCO3. The
organic phase was dried over Na2SO4, filtered and concentrated in vacuo to give
white solid (205 mg). Trifluoroacetic acid (1 ml) was added to the solid and
stirred at room temperature for 20 min and concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (AcOEt–
S
Me
Me
O
HN
HO
Me
O
Me
S
OMs
O
N
N
a, b
HN
Me
Me
O
SMe
TMSO
SMe
29
HO
OH
TMSO
OTMS
MeOH) to afford 8 (99 mg, 73%) as a colorless solid. [α]D +118° (c 1.0,
10e
CHCl3); 1H NMR (400 MHz, CD3OD) δ 7.91 (s, 1H), 5.26 (d, J = 5.6 Hz, 1H),
4.36–4.50 (m, 3H), 4.21 (d, J = 9.7 Hz, 1H), 4.12–4.17 (m, 1H), 4.09 (dd,
J = 5.6, 10.3 Hz, 1H), 3.98 (ddd, J = 3.5, 7.4, 10.8 Hz, 2H), 3.78 (d, J = 3.4 Hz,
1H), 3.59–3.66 (m, 1H), 3.56 (dd, J = 3.4, 10.4 Hz, 1H), 3.36–3.51 (m, 3H),
2.61 (s, 3H), 2.34–2.45 (m, 1H), 2.34–2.44 (m, 1H), 2.19–2.30 (m, 1H) 2.18 (s,
3H), 1.96–2.14 (m, 2H), 1.33–1.44 (m, 4H), 1.31 (d, J = 6.8 Hz, 3H), 0.93
(t, J =7.1 Hz, 3H); MS (FAB (fast atom bombardment)) m/z 477 (M+H)+; high
resolution mass spectrometry (HRMS) (FAB) m/z calcd for C21H40N3O5S2
478.2409, found 478.2408 (M+H)+.
6
Scheme 2 Synthesis of 10e. Reagents: (a) 3-(4,5-dihydrothiazol-2-yl)
azetidine-1-thiol hydrochloride, K2CO3, DMF; (b) 1 N HCl/AcOEt, MeOH.
30
(hexane–AcOEt) to afford 7 (5.7 g, 48%) as a colorless solid. [α]D +76°
(c 0.46, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 10.7 Hz, 1H), 5.21
(d, J = 5.6 Hz, 1H), 4.50 (dt, J = 1.7, 10.3 Hz, 1H), 4.29–4.16 (m, 2H), 4.15
(dd, J = 5.6, 9.5 Hz, 1H), 3.98 (d, J =9.9 Hz, 1H), 3.75–3.80 (m, 2H), 3.64–3.71
(m, 2H), 3.59 (dd, J = 2.4, 9.5 Hz, 1H), 3.35 (dq, J = 1.7, 7.1 Hz, 1H), 3.14–3.19
(m, 1H), 2.97 (dd, J = 3.7, 10.7 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 1.89–2.05
(m, 2H), 1.77–1.87 (m, 1H), 1.41 (s, 9H), 1.24–1.30 (m, 4H), 1.22
7(S)-7-(Azetidin-3-ylthio)-7-deoxy-2,3,4-tris-O-(trimethylsilyl)
lincomycin (9)
(d, J =7.1 Hz, 3H), 0.84–0.89 (m, 3H), 0.16 (m, 9H), 0.12 (m, 9H), 0.11 To a cold (0 °C) solution of 8 (3.48 g) in pyridine were added trimethylsilyl
(m, 9H); MS (ESI) m/z 794 (M+H)+.
chloride (4.59 ml) and hexamethyldisilazane (7.5 ml) and the mixture was
The Journal of Antibiotics

Antibacterial activity of lincomycin derivatives
K Kumura et al
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stirred at room temperature for 1 h. The reaction mixture was poured into 10%
aqueous NaHCO3 and extracted with AcOEt. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The resulting residue was dissolved
in MeOH (30 ml) and 6 N AcOH (3.59 ml) was added to the solution. After the
mixture was stirred at room temperature for 3.5 h, the mixture was poured into
10% aqueous NaHCO3 and extracted with AcOEt. The organic phase was dried
over Na2SO4, filtered and concentrated in vacuo. The resulting residue was
purified by NH silica gel column chromatography (hexane–AcOEt) to afford 9
(3.0 g, 60%) as a colorless solid. [α]D +78° (c 1.2, CHCl3); 1H NMR
28
(400 MHz, CDCl3) δ 7.48 (d, J = 11.0 Hz, 1H), 5.23 (d, J = 5.6 Hz, 1H), 4.47
(dt, J = 1.8, 10.5 Hz, 1H), 4.17 (dd, J = 5.6, 9.5 Hz, 1H), 4.00 (d, J =10.2 Hz,
1H), 3.83–3.90 (m, 2H), 3.73–3.81 (m, 1H), 3.70 (d, J = 2.2 Hz, 1H), 3.57–3.65
(m, 3H), 3.36 (dq, J = 1.8, 7.1 Hz, 1H), 3.14–3.20 (m, 1H), 2.98 (dd, J = 3.7,
11 Hz, 1H), 2.43 (s, 3H), 2.41 (d, J = 6.1 Hz, 1H), 2.18 (s, 3H), 1.92–2.09
(m, 3H), 1.73–1.90 (m, 1H), 1.68 (br s, 2H), 1.25–1.32 (m, 4H), 1.23
(d, J = 7.1 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 0.17 (s, 9H), 0.14 (s, 9H), 0.12
(s, 9H); MS (ESI) m/z 694 (M+H)+.
7(S)-7-[1-(Benzo[d]oxazol-2-yl)azetidin-3-ylthio]-7-deoxylincomycin
(10d)
To a solution of 9 (60 mg) in N,N-dimethylformamide (500 μl) were added 2-
chlorobenzoxazole (9.9 μl), triethylamine (12.1 μl) and the mixture was stirred
at room temperature for 3 h The mixture was diluted with AcOEt and washed
with 10% aqueous NaHCO3. The organic phase was dried over Na2SO4, filtered
and concentrated in vacuo. To the resulting residue were added MeOH (1 ml)
and 1 M hydrochloric acid (1 ml) and the reaction mixture was stirred at room
temperature for 10 min. The mixture was diluted with AcOEt and washed with
10% aqueous NaHCO3. The organic phase was dried over Na2SO4, filtered and
concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (AcOEt–MeOH) to afford 10d (51 mg, 99%) as a colorless
28
solid. [α]D +103° (c 0.99, CHCl3); H NMR (400 MHz, CD3OD) δ 7.29–7.35
(m, 2H), 7.19 (dt, J = 1.1, 7.5 Hz, 1H), 7.09 (dt, J = 1.1, 7.5 Hz, 1H), 5.27
(d, J = 5.6 Hz, 1H), 4.62–4.72 (m, 2H), 4.33 (dd, J = 2.9, 9.7 Hz, 1H), 4.07–4.21
(m, 5H), 3.71–3.75 (m, 1H), 3.57 (dd, J = 3.4, 10.2 Hz, 1H), 3.52 (dq, J = 2.9,
6.8 Hz, 1H), 3.23 (dd, J = 5.6, 8.0 Hz, 1H), 2.99 (dd, J = 4.4, 10.5 Hz, 1H), 2.43
(s, 3H), 2.17 (s, 3H), 1.95–2.15 (m, 3H), 1.80–1.90 (m, 1H), 1.27–1.38
(m, 7H), 0.88–0.94 (m, 3H); MS (FAB) m/z 595 (M+H)+; HRMS (FAB) m/z
calcd for C28H43N4O6S2 595.2624, found 595.2633 (M+H)+.
7(S)-7-Deoxy-7-[1-(2-nitrophenyl)azetidin-3-ylthio]lincomycin (10a)
Reaction of 9 with 2-fluoronitrobenzene gave 10a as a yellow solid in 57%
yield by a similar procedure to 10d. [α]D +53° (c 0.75, CHCl3); 1H NMR
29
(400 MHz, CD3OD) δ 7.78 (dd, J = 1.6, 8.3 Hz, 1H), 7.45 (ddd, J = 1.6, 7.1,
8.5 Hz, 1H), 6.80 (ddd, J = 1.2, 7.1, 8.3 Hz, 1H), 6.72 (dd, J = 1.2, 8.5 Hz, 1H),
5.26 (d, J = 5.6 Hz, 1H), 4.39 (t, J = 8.2 Hz, 1H), 4.27–4.36 (m, 2H), 4.16
(d, J =9.7 Hz, 1H), 4.09 (dd, J = 5.6, 10.2 Hz, 1H), 3.91–4.01 (m, 1H),
3.72–3.78 (m, 2H), 3.70–3.72 (m, 1H), 3.56 (dd, J = 3.2, 10.2 Hz, 1H), 3.51
(dq, J = 2.7, 7.0 Hz, 1H), 3.23 (dd, J = 5.6, 8.0 Hz, 1H), 2.99 (dd, J = 4.6,
10.7 Hz, 1H), 2.41 (s, 3H), 2.15 (s, 3H), 2.03–2.14 (m, 2H), 1.93–2.03 (m, 1H),
1.84 (td, J = 10.2, 12.8 Hz, 1H), 1.29–1.35 (m, 7H), 0.87–0.94 (m, 3H);
MS (FAB) m/z 599 (M+H)+; HRMS (FAB) m/z calcd for C27H43N4O7S2
599.2573, found 599.2572 (M+H)+.
7(S)-7-Deoxy-7-[1-(4-nitrophenyl)azetidin-3-ylthio]lincomycin (10b)
Reaction of 9 with 4-fluoronitrobenzene gave 10b as a yellow solid in 56% yield
by a similar procedure to 10d. [α]D +94° (c 0.87, CHCl3); 1H NMR
30
(400 MHz, CD3OD)
δ 8.06–8.12 (m, 2H), 6.40–6.46 (m, 2H), 5.27
(d, J = 5.4 Hz, 1H), 4.50 (t, J = 8.0 Hz, 1H), 4.45 (t, J = 8.0 Hz, 1H), 4.33
(dd, J = 2.7, 9.7 Hz, 1H), 4.19 (d, J = 9.7 Hz, 1H), 4.05–4.13 (m, 2H), 3.84–3.91
(m, 2H), 3.71–3.74 (m, 1H), 3.57 (dd, J = 3.2, 10.2 Hz, 1H), 3.52 (dq, J = 2.7,
7.3 Hz, 1H), 3.23 (dd, J = 5.5, 7.9 Hz, 1H), 3.00 (dd, J =4.5, 10.6 Hz, 1H),
2.41 (s, 3H), 2.16 (s, 3H), 2.04–2.14 (m, 2H), 1.94–2.04 (m, 1H), 1.85
(td, J = 10.2, 13.0 Hz, 1H), 1.27–1.37 (m, 7H), 0.87–0.94 (m, 3H); MS (FAB)
m/z 599 (M+H)+; HRMS (FAB) m/z calcd for C27H43N4O7S2 599.2573, found
599.2567 (M+H)+.
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K Kumura et al
5
7(S)-7-[1-(4-Aminophenyl)azetidin-3-ylthio]-7-deoxylincomycin (10c) CD3OD) δ 5.25 (d, J = 5.6 Hz, 1H), 4.24 (dd, J = 2.5, 9.7 Hz, 1H), 4.14
(d, J = 9.7 Hz, 1H), 4.08 (dd, J = 5.6, 10.4 Hz, 1H), 3.61–3.82 (m, 7H), 3.55
(dd, J = 3.2, 10.4 Hz, 1H), 3.41 (dq, J = 2.5, 7.0 Hz, 1H), 3.24 (dd, J = 5.2,
8.0 Hz, 1H), 2.96–3.05 (m, 3H), 2.75 (t, J = 7.2 Hz, 2H), 2.42 (s, 3H), 2.36
(t, J = 7.2 Hz, 2H), 2.17 (s, 3H), 2.04–2.16 (m, 2H), 1.93–2.03 (m, 1H),
1.80–1.90 (m, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J = 7.0 Hz, 3H), 0.87–0.95
(m, 3H); MS (FAB) m/z 564 (M+H)+; HRMS (FAB) m/z calcd for
C25H46N3O7S2 564.2777, found 564.2770 (M+H)+.
To a solution of 10b (127 mg) in MeOH (4 ml) was added PtO2 (77.2 mg) and
the mixture was stirred in hydrogen atmosphere at room temperature for 1 h.
The mixture was filtered through Celite and the filtrate was concentrated in
vacuo. The resulting residue was purified by silica gel column chromatography
30
(AcOEt–MeOH) to afford a colorless solid (77 mg, 64%). [α]D +104° (c 1.2,
CHCl3); 1H NMR (400 MHz, CD3OD) δ 6.64–6.75 (m, 2H), 6.33–6.44
(m, 2H), 5.27 (d, J =5.6 Hz, 1H), 4.28 (dd, J = 2.4, 9.7 Hz, 1H), 4.18–4.25
(m, 1H), 4.16 (d, J = 9.7 Hz, 1H), 4.09 (dd, J = 5.6, 10.2 Hz, 2H), 3.89–3.98
(m, 1H), 3.70 (d, J = 3.2 Hz, 1H), 3.56 (dd, J = 3.2, 10.2 Hz, 2H), 3.51–3.54
(m, 1H), 3.49 (dq, J = 2.4, 7.1 Hz, 1H), 3.23 (dd, J = 5.5, 7.7 Hz, 1H), 3.00
(dd, J = 4.5, 10.5 Hz, 1H), 2.42 (s, 3H), 2.17 (s, 3H), 2.03–2.14 (m, 2H),
1.94–2.03 (m, 1H), 1.85 (td, J = 10.2, 12.8 Hz, 1H), 1.29–1.36 (m, 7H),
0.87–0.95 (m, 3H); MS (FAB) m/z 569 (M+H)+; HRMS (FAB) m/z calcd for
C27H45N4O5S2 569.2831, found 569.2838 (M+H)+.
7(S)-7-Deoxy-7-{1-[2-(morpholin-4-yl)ethyl]azetidin-3-ylthio}
lincomycin (11c)
Reaction of 9 with 4-acryloylmorpholine gave 11c as a colorless solid in 70%
29
yield by a similar procedure to 11b. [α]D +91° (c 1.1, CHCl3); 1H NMR
(400 MHz, CD3OD) δ 5.26 (d, J = 5.6 Hz, 1H), 4.24 (dd, J =2.4, 10.1 Hz, 1H),
4.14 (d, J = 10.1 Hz, 1H), 4.08 (dd, J = 5.6, 10.7 Hz, 1H), 3.71–3.81 (m, 2H),
3.61–3.71 (m, 6H), 3.49–3.58 (m, 5H), 3.42 (dq, J = 2.4, 6.9 Hz, 1H), 3.20–3.26
(m, 1H), 3.01–3.09 (m, 2H), 2.99 (dd, J = 4.6, 10.5 Hz, 1H), 2.75 (t, J = 7.6 Hz,
2H), 2.42 (s, 3H), 2.41 (t, J = 7.6 Hz, 2H), 2.18 (s, 3H), 2.05–2.16 (m, 2H),
1.94–1.99 (m, 1H), 1.80–1.89 (m, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J = 6.9 Hz,
3H), 0.88–0.95 (m, 3H); MS (FAB) m/z 619 (M+H)+; HRMS (FAB) m/z calcd
for C28H51N4O7S2 619.3199, found 619.3192 (M+H)+.
7(S)-7-Deoxy-7-[1-(4,5-dihydrothiazol-2-yl)azetidin-3-ylthio]
lincomycin (10e)
To a solution of 6 (150 mg) and K2CO3 (88.7 mg) in N,N-dimethylformamide
(1.5 ml) was added 3-(4,5-dihydrothiazol-2-yl)azetidine-1-thiol hydrochloride
(90.2 mg) and the mixture was stirred at 80 °C for 5 h. After cooled to room
temperature, the mixture was diluted with AcOEt and washed with brine. The
organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The
residue was purified by silica gel column chromatography (hexane–AcOEt) to
give a colorless solid (95 mg). To a solution of the compound obtained above
(50 mg) in MeOH was added 1 M hydrochloric acid (1 ml) and the reaction
mixture was stirred at room temperature for 5 min. The mixture was diluted
with AcOEt and extracted with H2O. The aqueous phase was neutralized with
10% aqueous NaHCO3 and extracted with AcOEt. The organic phase was dried
over Na2SO4, filtered and concentrated in vacuo to afford 10e (30 mg, 47%) as
a colorless solid. [α]D28 +103° (c 0.99, CHCl3); 1H NMR (400 MHz, CD3OD) δ
5.26 (d, J = 5.6 Hz, 1H), 4.40 (t, J = 8.0 Hz, 1H), 4.35 (t, J = 8.2 Hz, 1H), 4.30
(dd, J = 2.6, 9.6 Hz, 1H), 4.15 (d, J = 9.6 Hz, 1H), 4.09 (dd, J = 5.6, 10.2 Hz,
1H), 3.98 (tt, J =5.5, 7.8 Hz, 1H), 3.94 (t, J = 7.5 Hz, 2H), 3.79–3.85 (m, 2H),
3.69–3.72 (m, 1H), 3.56 (dd, J = 3.2, 10.2 Hz, 1H), 3.47 (dq, J = 2.6, 7.1 Hz,
1H), 3.38 (t, J = 7.5 Hz, 2H), 3.24 (dd, J = 5.5, 7.9 Hz, 1H), 2.99 (dd, J = 4.6,
10.7 Hz, 1H), 2.42 (s, 3H), 2.19 (s, 3H), 2.05–2.18 (m, 2H), 1.95–2.03 (m, 1H),
1.85 (td, J = 10.2, 12.8 Hz, 1H), 1.31–1.37 (m, 4H), 1.29 (d, J = 7.1 Hz, 3H),
0.88–0.95 (m, 3H); MS (FAB) m/z 563 (M+H)+; HRMS (FAB) m/z calcd for
C24H43N4O5S2 563.2396, found 563.2388 (M+H)+.
7(S)-7-Deoxy-7-[1-(dimethylaminocarbonyl)azetidin-3-ylthio]
lincomycin (12a)
To a solution of 9 (100 mg) in CHCl3 (700 μl) were added triethylamine
(20.2 μl), dimethylcarbamoyl chloride (13.2 μl) and N,N-dimethylaminopyr-
idine (17.6 mg) and the mixture was stirred at room temperature for 3.5 h. The
mixture was diluted with AcOEt and washed with 10% aqueous NaHCO3.
The organic phase was dried over Na2SO4, filtered and concentrated in vacuo.
The residue was purified by silica gel column chromatography (hexane–AcOEt)
to give a colorless solid (95 mg). To a stirred solution of the compound
obtained above (95 mg) in MeOH (1 ml) was added 1 M hydrochloric acid
(1 ml) and the reaction mixture was stirred at room temperature for 10 min.
The mixture was diluted with AcOEt and extracted with H2O. The aqueous
phase was neutralized with 10% aqueous NaHCO3 and extracted with AcOEt.
The organic phase was dried over Na2SO4, filtered and concentrated in vacuo to
29
afford 12a (55 mg, 70%) as a colorless solid. [α]D +66° (c 1.1, CHCl3);
1H NMR (400 MHz, CD3OD) δ 5.26 (d, J = 5.6 Hz, 1H), 4.32–4.42 (m, 2H),
4.29 (dd, J = 2.6, 9.7 Hz, 1H), 4.15 (dd, J = 0.73, 9.7 Hz, 1H), 4.09 (dd, J = 5.6,
10.4 Hz, 1H), 3.80–3.88 (m, 3H), 3.69–3.71 (m, 1H), 3.56 (dd, J = 3.2, 10.4 Hz,
1H), 3.46 (dq, J = 2.6, 7.1 Hz, 1H), 3.24 (dd, J =5.5, 7.9 Hz, 1H), 2.99
(dd, J = 4.6, 10.5 Hz, 1H), 2.84 (s, 6H), 2.42 (s, 3H), 2.19 (s, 3H), 2.04–2.16
(m, 2H), 1.94–2.02 (m, 1H), 1.84 (td, J = 10.1, 12.9 Hz, 1H), 1.31–1.37
(m, 4H), 1.29 (d, J = 7.1 Hz, 3H), 0.89–0.94 (m, 3H); MS (FAB) m/z 549
(M+H)+; HRMS (FAB) m/z calcd for C26H44N5O6S3 549.2781, found
549.2782 (M+H)+.
7(S)-7-Deoxy-7-{1-[2-(dimethylaminocarbonyl)ethyl]azetidin-3-
ylthio}lincomycin (11b)
To a solution of 9 (60 mg) in EtOH (1 ml) was added N,N-dimethylacrylamide
(8.9 μl) and the mixture was stirred at room temperature for 15 min and at
50 °C for 3 h. The mixture was concentrated in vacuo and the residue was
dissolved in MeOH (1 ml) and 1 M hydrochloric acid (1 ml) was added to the
solution. The mixture was stirred at room temperature for 10 min and diluted
with AcOEt and extracted with H2O. The aqueous phase was neutralized with
10% aqueous NaHCO3 and extracted with AcOEt. The organic phase was dried
over Na2SO4, filtered and concentrated in vacuo to afford 11b (37 mg, 74%) as
7(S)-7-Deoxy-7-{1-[(morpholin-4-yl)carbonyl]azetidin-3-ylthio}
lincomycin (12c)
Reaction of 9 with 4-morpholinylcarbonyl chloride gave 12c as a colorless solid
29
in 70% yield by a similar procedure to 12a. [α]D +69° (c 0.86, CHCl3); 1H
a colorless solid. [α]D +90° (c 1.1, CHCl3); 1H NMR (400 MHz, CD3OD)
29
NMR (400 MHz, CD3OD) δ 5.26 (d, J = 5.6 Hz, 1H), 4.33–4.45 (m, 2H), 4.29
(dd, J = 2.7, 9.5 Hz, 1H), 4.15 (d, J = 9.5 Hz, 1H), 4.09 (dd, J = 5.6, 10.4 Hz,
1H), 3.83–3.91 (m, 3H), 3.70 (d, J = 3.3 Hz, 1H), 3.59–3.64 (m, 4H), 3.55
(dd, J = 3.3, 10.4 Hz, 1H), 3.46 (dq, J = 2.7, 7.0 Hz, 1H), 3.27–3.33 (m, 4H),
3.23 (dd, J = 5.2, 7.9 Hz, 1H), 2.99 (dd, J = 4.6, 10.7 Hz, 1H), 2.42 (s, 3H),
2.18 (s, 3H), 2.04–2.17 (m, 2H), 1.94–2.02 (m, 1H), 1.79–1.89 (m, 1H),
δ 5.26 (d, J = 5.6 Hz, 1H), 4.23 (dd, J =2.4, 9.7 Hz, 1H), 4.14 (d, J = 9.7 Hz, H),
4.09 (dd, J = 5.6, 10.2 Hz, 1H), 3.62–3.82 (m, 5H), 3.55 (dd, J =3.2, 10.2 Hz,
1H), 3.42 (dq, J = 2.4, 6.9 Hz, 1H), 3.19–3.26 (m, 1H), 3.02–3.09 (m, 4H),
2.93–3.02 (m, 1H), 2.91 (s, 3H), 2.74 (t, J = 7.3 Hz, 2H), 2.42 (s, 3H), 2.39
(t, J = 7.3 Hz, 2H), 2.18 (s, 3H), 2.03–2.16 (m, 2H), 1.94–2.02 (m, 1H), 1.84
(td, J = 10.0, 12.9 Hz, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J = 6.9 Hz, 3H), 1.31–1.37 (m, 4H), 1.29 (d, J = 7.0 Hz, 3H), 0.88–0.95 (m, 3H); MS (FAB) m/z
0.87–0.95 (m, 3H); MS (FAB) m/z 577 (M+H)+; HRMS (FAB) m/z calcd for 591 (M+H)+; HRMS (FAB) m/z calcd for C26H47N4O7S2 591.2886, found
C26H49N4O6S2 577.3094, found 577.3089 (M+H)+.
591.2891 (M+H)+.
7(S)-7-Deoxy-7-{1-[2-(methoxycarbonyl)ethyl]azetidin-3-ylthio}
7(S)-7-Deoxy-7-[1-(phenylcarbonyl)azetidin-3-ylthio]lincomycin
lincomycin (11a)
(12d)
Reaction of 9 with methyl 3-butenoate gave 11a as a colorless solid in 74% yield Reaction of 9 with benzoyl chloride gave 12d as a colorless solid in 67% yield
by a similar procedure to 11b. [α]D30 +86° (c 1.1, CHCl3); 1H NMR (400 MHz, by a similar procedure to 12a. [α]D +90° (c 0.74, CHCl3); 1H NMR
30
The Journal of Antibiotics

Antibacterial activity of lincomycin derivatives
K Kumura et al
6
(400 MHz, CD3OD) δ 7.84–7.90 (m, 2H), 7.72–7.79 (m, 1H), 7.65–7.72 In vitro antibacterial activity
(m, 2H), 5.21 (d, J = 5.6 Hz, 1H), 4.22 (dd, J =2.7, 9.3 Hz, 1H), 4.19–4.11
(m, 2H), 4.06 (dd, J = 5.6, 10.3 Hz, 1H), 4.05–4.02 (m, 1H), 3.68–3.77 (m, 1H),
3.66 (d, J = 3.2 Hz, 1H), 3.54–3.58 (m, 2H), 3.52 (dd, J = 3.2, 10.3 Hz, 1H),
3.33 (dq, J = 2.7, 6.8 Hz, 1H), 3.19 (dd, J = 5.2, 7.9 Hz, 1H), 2.94 (dd, J = 4.6,
10.7 Hz, 1H), 2.34 (s, 3H), 2.10–2.16 (m, 1H), 2.08 (s, 3H), 2.02–2.07 (m, 1H),
1.89–2.00 (m, 1H), 1.76–1.87 (m, 1H), 1.26–1.35 (m, 4H), 1.16–1.21 (m, 3H),
0.87–0.94 (m, 3H); MS (FAB) m/z 618 (M+H)+; HRMS (FAB) m/z calcd for
C27H44N3O7S3 618.2341, found 618.2340 (M+H)+.
MIC was determined by the agar dilution method. Test strains were subjected
to seed culture using sensitivity test broth (Nissui Pharmaceutical, Tokyo,
Japan) cultured on blood agar plate for S. pneumoniae, S. pyogenes and
H. influenzae. A 5-μl portion of cell suspension of the test strains having
about 106 CFU per ml was inoculated into sensitivity disk agar (Nissui
Pharmaceutical) supplemented with 5% horse blood and incubated at 37 °C for
20 h. Then, MIC was defined as the lowest drug concentration that prevented
visible growth.
7(S)-7-Deoxy-7-[1-(2-nitrophenylcarbonyl)azetidin-3-ylthio]
lincomycin (12e)
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Reaction of 9 with 2-nitrobenzoyl chloride gave 12e as a yellow solid in 72%
yield by a similar procedure to 12a. [α]D +88° (c 1.0, CHCl3); 1H NMR
30
ACKNOWLEDGEMENTS
(400 MHz, CD3OD) δ 8.17–8.23 (m, 1H), 7.79–7.85 (m, 1H), 7.69–7.74
(m, 1H), 7.52–7.57 (m, 1H), 5.19–5.28 (m, 1H), 4.55–4.66 (m, 1H), 4.25–4.41
(m, 2H), 3.98–4.20 (m, 3H), 3.78–3.86 (m, 1H), 3.68–3.73 (m, 1H), 3.41–3.63
(m, 2H), 3.17–3.27 (m, 1H), 3.17–3.27 (m, 1H), 2.93–3.02 (m, 1H), 2.36–2.44
(m, 3H), 2.03–2.24 (m, 5H), 1.92–2.03 (m, 1H), 1.78–1.90 (m, 1H), 1.22–1.37
(m, 7H), 0.87–0.95 (m, 3H); MS (FAB) m/z 627 (M+H)+; HRMS (FAB) m/z
calcd for C28H43N4O8S2 627.2522, found 627.2516 (M+H)+.
We thank Mr A Tamura and Dr T Okutomi for valuable scientific discussion.
We are grateful to Professor Emeritus Dr M Konno for supervision through our
in-house drug discovery program in lincomycin field. We are also grateful to
Ms T Miyara, Ms S Miki, Ms K Kaneda, Dr T Murata and Mr S Sato for
contribution toward analytical chemistry, Mr T Matsuhira for biological
studies, and Ms M Takagi for manuscript.
7(S)-7-Deoxy-7-[1-(piperidin-1-yl)azetidin-3-ylthio]lincomycin
(12b)
1
2
3
4
5
6
7
8
9
Weisblum, B. Erythromycin resistance by ribosome modification. Antimicrob. Agents
Chemother. 39, 577–585 (1995).
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modifications and future applications. Heterocycles 89, 281–352 (2014).
Schlünzen, F. et al. Structural basis for the interaction of antibiotics with the peptidyl
transferase centre in eubacteria. Nature 413, 814–821 (2001)
Bannister, B. Modifications of lincomycin involving the carbohydrate portion. Part I. The
2-O-methyl and 2-deoxy-analogues. J. Chem. Soc. Perkin I 23, 3025–3030 (1972).
Sztaricskai, F. et al. Semisynthetic modification of antibiotic lincomycin. J. Antibiot.
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To a cold (0 °C ) solution of 9 (80 mg) in CH2Cl2 (0.5 ml) were added
piperidine (12.5 μl), pyridine (20.5 μl) and triphosgene (12.6 mg), the mixture
was stirred at room temperature for 1 h. The mixture was diluted with AcOEt
and washed with 10% aqueous NaHCO3. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane–AcOEt) to give a colorless amorphous
(22 mg). To a stirred solution of the compound obtained above (22 mg) in
MeOH (1 ml) was added 1 M hydrochloric acid (1 ml) and the reaction mixture
was stirred at room temperature for 10 min. The mixture was diluted with
AcOEt and washed with 10% aqueous NaHCO3. The organic phase was dried
over Na2SO4, filtered and concentrated in vacuo to afford 12b (16 mg, 23%) as
29
1
Isoda, T. et al. Syntheses and pharmacokinetic studies of prodrug esters for the
development of oral carbapenem, L-084. J. Antibiot. 4, 241–247 (2006)
Remy, M. J., Tow-Keoght, A. C., MacConnell, S. T., Dalton, M. J. & DeVito, A. J. Activity
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and characterization. J. Antimicrob. Chemother. 67, 2814–2820 (2012).
a colorless solid. [α]D +100° (c 0.72, CHCl3); H NMR (400 MHz, CD3OD)
δ 5.26 (d, J =5.6 Hz, 1H), 4.39 (t, J = 7.8 Hz, 1H), 4.34 (t, J = 7.8 Hz, 1H), 4.29
(dd, J = 2.6, 9.6 Hz, 1H), 4.16 (d, J = 9.6 Hz, 1H), 4.09 (dd, J = 5.6, 10.4 Hz,
1H), 3.80–3.90 (m, 3H), 3.70 (d, J = 3.2 Hz, 1H), 3.56 (dd, J = 3.2, 10.4 Hz,
1H), 3.46 (dq, J = 2.6, 7.1 Hz, 1H), 3.22–3.30 (m, 5H), 2.99 (dd, J = 4.6,
10.7 Hz, 1H), 2.42 (s, 3H), 2.19 (s, 3H), 2.05–2.17 (m, 2H), 1.95–2.03 (m, 1H),
1.85 (td, J = 10.2, 12.8 Hz, 1H), 1.59–1.66 (m, 2H), 1.49–1.57 (m, 4H),
1.31–1.38 (m, 4H), 1.29 (d, J = 7.1 Hz, 3H), 0.90–0.94 (m, 3H); 13C NMR
(125 MHz, CD3OD) δ 178.0, 164.0, 90.5, 72.0, 71.3, 70.4, 69.9, 69.5, 63.9, 61.3,
60.1, 54.3, 46.9, 42.9, 42.3, 39.2, 38.7, 37.0, 33.2, 27.0, 25.5, 22.6, 21.0, 14.6,
14.5; MS (EI) m/z 588 M+; HRMS (FAB) m/z calcd for C27H49N4O6S2
589.3094, found 589.3096 (M+H)+.
10 Igarashi, K. Chemical modification of tobramycin. Jpn J. Antibiot. 17 (Suppl),
S187–S194 (1979).
11 Berkov-Zrihen, Y. et al. Synthesis and evaluation of hetero- and homodimers of
ribosome-targeting antibiotics: antimicrobial activity, in vitro inhibition of translation,
and drug resistance. J. Med. Chem. 56, 5613–5625 (2013)
12 Bannister, B. The S-Alkylation of sulphides by an activated carbohydrate epimine under
acidic catalysis: the formation of α-acetamido-sulphides. Part 4. Reactions with
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13 Houtman, R. L. & Mich, P. (The Upjohn Company), Trimethylsilyl ethers of lincomycin
and its compounds. US3418414 (1966).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics