Synthesis and antibacterial activity of novel lincomycin derivatives. III. Optimization of a phenyl thiadiazole moiety

Article abstract of DOI:10.1038/ja.2017.59

Lincomycin derivatives that have a 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl thio moiety at the 7-position were synthesized. 5-Substituted 2-nitrophenyl derivatives showed potent antibacterial activities against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene. Antibacterial activities of the 4,5-di-substituted 2-nitrophenyl derivatives were generally comparable to those of telithromycin (TEL) against S. pneumoniae with erm gene and clearly superior to those of TEL against S. pyogenes with erm gene. Compounds 6 and 10c that have a methoxy group at the 5-position of the benzene ring exhibited activities comparable to TEL against Haemophilus influenzae. These results suggest that lincomycin derivatives modified at the 7-position would be promising compounds as a clinical candidate. We would like to dedicate this article to the special issue for late Professor Dr. Hamao Umezawa in The Journal of Antibiotics.

Full text of DOI:10.1038/ja.2017.59

The Journal of Antibiotics (2017), 1–9
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
www.nature.com/ja
&
ORIGINAL ARTICLE
Synthesis and antibacterial activity of novel lincomycin
derivatives. III. Optimization of a phenyl
thiadiazole moiety
Ko Kumura, Yoshinari Wakiyama, Kazutaka Ueda, Eijiro Umemura, Takashi Watanabe, Mikio Yamamoto,
Takuji Yoshida and Keiichi Ajito
Lincomycin derivatives that have a 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl thio moiety at the 7-position were synthesized.
5-Substituted 2-nitrophenyl derivatives showed potent antibacterial activities against Streptococcus pneumoniae and
Streptococcus pyogenes with erm gene. Antibacterial activities of the 4,5-di-substituted 2-nitrophenyl derivatives were generally
comparable to those of telithromycin (TEL) against S. pneumoniae with erm gene and clearly superior to those of TEL against
S. pyogenes with erm gene. Compounds 6 and 10c that have a methoxy group at the 5-position of the benzene ring exhibited
activities comparable to TEL against Haemophilus influenzae. These results suggest that lincomycin derivatives modified at the
7-position would be promising compounds as a clinical candidate. We would like to dedicate this article to the special issue for
late Professor Dr. Hamao Umezawa in The Journal of Antibiotics.
The Journal of Antibiotics advance online publication, 5 July 2017; doi:10.1038/ja.2017.59
INTRODUCTION
S. pneumoniae and an improved pharmacokinetic profile (Figure 2).
Macrolide antibiotics have been widely used for respiratory infections Lincosamide antibiotics inhibit protein synthesis acting on 50S
over many years. Especially, clarithromycin1 and azithromycin2 ribosome in a similar way to macrolide antibiotics. This similar
(Figure 1) are clinically important macrolides derived from a natural mode of action led to cross-resistance against clinically problematic
product, erythromycin (EM). Clarithromycin is distinct from EM in S. pneumoniae and S. pyogenes with erm gene.13 Thus, CLDM shows
improved pharmacokinetics because of its stability in acidic condition. almost no antibacterial activity against these resistant pathogens as
Azithromycin has a long plasma half-life and good tissue distribution, shown in Table 1. CLDM, however, is attractive because of its safety
and shows stronger antibacterial activity than EM against and effectiveness against resistant pathogens with efflux pump. On the
Gram-negative bacteria such as Haemophilus influenzae. However, an other hand, macrolide antibiotics, including ketolides, are influenced
emergence of macrolide-resistant bacteria, such as Streptococcus by efflux pumps of resistant S. pneumoniae and S. pyogenes with mef
pneumonia, has been causing serious clinical problems.3,4 Ketolides, gene. In addition, CLDM has no gastrointestinal side effect based on
such as TEL,5 cethromycin,6 solithromycin7 and nafithromycin8 the mode of action of a motilin receptor known in EM.14 As a rare
(Figure 1), newly developed/derived from natural EM, show case, furthermore, CLDM has been reported to be effective for invasive
antibacterial activities against resistant pathogens. Much effort has group A streptococcal infections caused by S. pyogenes.15 A simple
been made to launch ketolides, which were effective against macrolide- chemical structure of CLDM compared with that of macrolide is a
resistant bacteria, but only TEL has been marketed so far. Its safety positive characteristic, too. On the basis of these reasons, we initiated
problems,9 however, make it difficult to be used in the clinical site. chemical modifications of lincosamide in order to generate a novel
Another promising class is 16-membered azalides10 that are effective antibacterial agent that is effective against resistant S. pneumoniae and
against resistant S. pneumoniae and S. pyogenes with erm gene. These S. pyogenes with erm and mef genes.
compounds, however, are still under research process and have not
entered clinical phase. Thus, currently there is no clinically available with CLDM showed that the sugar moiety of CLDM had
drug that is effective against resistant S. pneumoniae with erm and mef several hydrogen bonding and played
pivotal role in its
genes and free of safety or taste problem.
binding mode.16 On the other hand, a hydrophobic space was
The crystal structures of bacterial 23S ribosomal RNA complexed
a
Lincomycin (LCM)11 was produced by Streptomyces lincolnensis and observed around the C-7 position of CLDM in X-ray crystallographic
has been used as antibacterial agent mainly against Gram-positive analysis. It is known that modifications at the C-7 position of
bacteria. Chemical modification of LCM led to clindamycin LCM tend to give comparable antibacterial activity to that of
(CLDM)12 that has an enhanced antibacterial activity against LCM.17,18 We reported chemical modifications of LCM and clarified
Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd, Yokohama, Japan
Correspondence: Dr K Ajito, Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd, Yokohama, Japan.
E-mail: keiichi.ajito@meiji.com
Received 19 February 2017; revised 9 April 2017; accepted 21 April 2017

Synthesis of novel lincomycin derivatives
K Kumura et al
2
Me
N
O
N
Me
Me
Me
Me
O
HO
Me
OMe
Me
HO
HO
Me
OH
Me
Me
Me
Me
OH
OH
Me
NMe2
Me
NMe2
Me
Me
HO
NH
O
O
O
O
O
O
O
O
NMe2
Me
O
Me
HO
Me
Me
Me
O
O
O
O
OMe
Me
OMe
Me
O
O
O
Me
Me
Me
Me
O
O
OH
OH
O
O
Me
Me
Clarithromycin (CAM)
Azithromycin (AZM)
Cethromycin (ABT-773)
N
H2N
N
N
N
N
N
N
N
S
N
N
NH2
Me
O
O
O
O
O
H
Me
Me
Me
Me
Me
Me
O
Me
Me
Me
Me
O
N
OMe
Me
HO
OMe
Me
N
OMe
Me
NMe2
Me
O
NMe2
Me
NMe2
Me
O
O
Me
Me
HO
HO
O
O
O
O
O
O
O
O
O
Me
Me
Me
O
O
O
O
O
O
Me
F
Me
Me
Telithromycin (TEL)
Solithromycin
Nafithromycin
Figure 1 Clinically important macrolides, clarithromycin and azithromycin, and representative novel ketolides.
O2N
O
Me
N
O
O Me
N
N
7
R
O
N
S
Me
Me
N
6
S
HN
HO
O Me
O Me
Me
7
7
S
SMe
N
N
HN
HN
Me
Me
O
O
HO
OH
HO
HO
SMe
HO
HO
SMe
Lincomycin (LCM): R =
Clindamycin (CLDM): R =
OH
Cl
OH
OH
1
2
Figure 2 Structures of LCM, CLDM, compounds 1 and 2.
that (7S)-7-arylthio-7-deoxylincomycin derivatives19–22 and (7S)-7- Chemistry
(azetidin-3-yl-thio)-7-deoxylincomycin derivatives23 exhibited moder- Introductions of a substituent at the 4- and 5-positions of the
ate to strong antibacterial activities against S. pneumoniae and 2-nitrophenyl group using a nucleophilic aromatic substitution
S. pyogenes with erm gene. Compound 1 that has a substituted (SNAr) reaction of fluoro-benzene and
a nucleophile were
azetidin-3-yl-thio moiety at the 7-position exhibited moderate planned. Mono-fluoro compounds 4a and 4b, and difluoro
antibacterial activities against S. pneumoniae and S. pyogenes with compound 4c were prepared by our method reported previously,
erm gene. Compound 2 that tethered a phenyl thiadiazol-2-yl thio which was the Mitsunobu reaction of 2, 3, 4-tris-O-(trimethylsilyl)
moiety to the 7-position showed antibacterial activities against those lincomycin (3)24 and the corresponding thiols followed by acidic
resistant pathogens. The ortho nitro group of 2 played a key role for treatment (Scheme 1). A major reason of relatively low yield of
the enhancement of antibacterial activity of S. pneumoniae with erm these Mitsunobu reactions was explained by generation of an
gene.22 In this article, we report further optimization of the phenyl N-connected byproduct in the thiadiazole moiety instead of the
moiety of 2 in order to achieve potent antibacterial activity against desired S-connected derivative. Compound 6 was synthesized by
S. pneumoniae and S. pyogenes with erm gene.
an SNAr reaction of (7S)-7-deoxy-7-mercaptolincomyicn (5)21 and
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Synthesis of novel lincomycin derivatives
K Kumura et al
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Table 1 Antibacterial activities of LCM, CLDM and our reported LCM derivatives (MIC; μg ml− 1 a
)
No.
Test organismb
Characteristics
LCM
CLDM
1
2
1
Streptococcus pneumoniae DP1 TypeI
S. pneumoniae #2
Susceptible
1
1
0.13
0.13
0.13
4128
4128
4128
4128
4128
0.13
0.13
0.13
4128
0.13
8
0.13
0.13
0.13
16
0.06
0.06
0.13
4
2
Susceptible
3
S. pneumoniae #3
Susceptible
0.25
4128
4128
4128
128
128
1
4
S. pneumoniae #4
ermB methylase (c)
ermB methylase (c)
ermB methylase (c) + mefE
ermB methylase (i)
ermB methylase (i)
mefE efflux
5
S. pneumoniae #5
8
8
6
S. pneumoniae #6
16
64
7
S. pneumoniae #7
8
4
8
S. pneumoniae #8
ND
0.13
0.13
0.13
4
4
9
S. pneumoniae #9
0.06
0.06
0.06
4
10
11
12
13
14
15
16
S. pneumoniae #10
Streptococcus pyogenes Cook
S. pyogenes #2
mefE efflux
1
Susceptible
0.13
4128
0.25
8
ermB methylase (c)
mefE efflux
S. pyogenes #3
0.13
4
0.13
16
Haemophilus influenzae #1
H. influenzae #2
Susceptible
Susceptible
16
8
8
8
H. influenzae #3
Susceptible
16
32
16
64
Abbreviations: c, constitutive; i, inducible; CLDM, clindamycin; LCM, lincomycin.
aGray shading strains are target strains.
bAll strains except standard organisms were clinically isolated.
O2N
R1
R2
N
N
S
Me
Me
S
O Me
O Me
7
7
OH
N
N
HN
HN
a, b
Me
Me
O
O
HO
HO
SMe
TMSO
TMSO
SMe
OTMS
OH
3
4a: R1 = F; R2 = H
4b: R1 = H; R2 = F
4c: R1 = F; R2 = F
O2N
OMe
OMe
N
N
S
Me
Me
S
O Me
O Me
7
SH
7
N
N
HN
HO
HN
c
Me
Me
O
O
SMe
HO
HO
SMe
HO
OH
OH
5
6
Scheme 1 Synthesis of (7S)-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) ArSH, DEAD, PPh3, toluene, 0 °C, 30 min,
then rt, 3 h; (b) 1 N HCl, MeOH, rt, 30 min, two steps 34% from 3 to 4a, two steps 19% from 3 to 4b, two steps 33% from 3 to 4c; (c) 2-(4,5-dimethoxy-
2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thiadiazole, NaHMDS, DMF, rt, 20 min, 67%.
2-(4,5-dimethoxy-2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thiadiazole. of 5-fluoro compound 4b with amines, such as dimethylamine
The derivatization of 4a, 4b and 4c is shown in Scheme 2. and methylamine, gave 7b and 8b in good yields. Compounds 7c,
Compound 7a that has a 4-dimethylamino group was obtained 8c and 9c were obtained from 4,5-difluoro compound 4c
from 4-fluoro compound 4a. Synthesis of 2-nitro-4-methoxy in the similar way to 4b. Methoxy derivatives 10b and 10c were
derivative using sodium methoxide and 4a was unsuccessful prepared by reaction of sodium methoxide with 4b and 4c,
probably because of poor reactivity of 4-fluoro atom in 4a. Treatment respectively.
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Synthesis of novel lincomycin derivatives
K Kumura et al
4
O2N
O2N
R
NMe2
N
N
N
N
S
NMe2
Me
Me
S
S
O Me
7
O Me
a
4a
7
S
N
N
HN
HN
HO
Me
Me
O
O
HO
HO
SMe
SMe
OH
HO
OH
b
c
7a
4b
4c
7b: R = H
7c: R = F
O2N
O2N
O2N
R
R
F
N
N
N
N
S
N
S
N
S
NHMe
OMe
NH
Me
Me
Me
S
S
S
O Me
7
O Me
7
O Me
7
N
N
HN
N
HN
HN
HO
Me
Me
Me
O
O
O
HO
HO
SMe
HO
HO
SMe
HO
HO
9c
SMe
OH
OH
OH
e
d
d
f
f
8b: R = H
8c: R = F
4c
10b: R = H
10c: R = F
4b
4c
4b
4c
Scheme 2 Synthesis of (7S)-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) 2M dimethylamine in methanol, 80 °C, 16 h,
81%; (b) 2M dimethylamine in methanol, rt, 1 h, 87%; (c) 2M dimethylamine in methanol, 50 °C, 3.5 h, 87%; (d) 40% methylamine in methanol, rt, 1 h,
75% from 4b to 8b, 98% from 4c to 8c; (e) 2-aminoethanol, 50 °C, 2 h, 86%; (f) 28% sodium methoxide methanol solution, rt, 64% from 4b to 10b, 70%
from 4c to 10c.
RESULTS AND DISCUSSION
4,5-dimethoxy derivative 6 showed an antibacterial profile similar to
Antibacterial activities of 2,4- or 2,5-di-substituted derivatives are 10c. It indicates that electronic property of the substituent at the
shown in Table 2. 4-Fluoro derivative 4a showed somehow enhanced 4-position is not important for antibacterial activity. Antibacterial
antibacterial activities against S. pneumoniae and S. pyogenes with activities of the 2,4,5-tri-substituted derivatives (7c, 8c, 10c and 6)
erm gene compared with 2, and its potency especially against against S. pneumoniae with erm gene were generally comparable to
S. pneumoniae with erm and mef gene (strain #6) was clearly enhanced. those of TEL and they were significantly superior to TEL against
In contrast, dimethylamino derivative 7a exhibited enhanced S. pyogenes with erm gene. As for antibacterial activity against
antibacterial activities against susceptible strains of S. pneumoniae
H. influenzae, 10c and 6 exhibited generally stronger activities
and S. pyogenes, but the effect of the dimethylamino group on than 7c, 8c and 9c and their activities have been getting closer to
antibacterial activity against resistant strains of S. pneumoniae (#4–8) those of TEL.
was unclear. In the case of 5-fluoro derivative 4b, the antibacterial
activity decreased against almost all of the test organisms. It should be CONCLUSIONS
A series of LCM derivatives that have the 5-(2-nitrophenyl)-1,3,4-
thiadiazol-2-yl thio moiety at the 7-position in S-configuration were
synthesized. Introductions of a substituent at the 4- and 5-positions of
the 2-nitrophenyl group were accomplished by the SNAr reaction of
fluoro-benzene 4a, 4b or 4c and a nucleophile. The other method was
the SNAr reaction of (7S)-7-deoxy-7-mercaptolincomyicn (5)21
and 5-(methylsulfonyl)-1,3,4-thiadiazole derivative (Scheme 1,
condition (c)). From the results of antibacterial activities of 2-nitro-
5-substituted phenyl derivatives, the modification led to significant
noted that the antibacterial activities of 5-dimethylamino derivative
7b, 5-methylamino derivative 8b and 5-methoxy derivative 10b
were significantly improved against all of the Gram-positive
pathogens compared with 2, and showed comparable antibacterial
activities against constitutive-resistant S. pneumoniae with erm gene
(#4 and 5) to those of TEL. The antibacterial activities of 7b, 8b and
10b against S. pyogenes with erm gene were further improved and 32
times stronger than that of TEL. The antibacterial activity of 10b
against H. influenzae was also improved compared with 2 but it is still
weaker than that of TEL. On the basis of the results obtained above, improvement in antibacterial activities against S. pneumoniae and
we performed further optimization focusing on the 4- and S. pyogenes with erm gene. Moreover, an additional substituent at the
4-position, such as a fluoro atom or a methoxy group, further
5-substituents on the benzene ring.
Antibacterial activities of 2,4,5-tri-substituted derivatives are shown improved the antibacterial activities of 2-nitro-5-substituted phenyl
in Table 3. As expected, compound 7c that has the 5-dimethylamino derivatives against S. pneumoniae and S. pyogenes with erm
group and the 4-fluoro group showed stronger antibacterial activities gene. Antibacterial activities of the 2-nitro-4,5-di-substituted phenyl
against S. pneumoniae and S. pyogenes with erm gene as well as derivatives (7c, 8c, 10c and 6) against S. pneumoniae with erm gene
S. pneumoniae with erm and mef gene (#6) than 5-dimethylamino were generally comparable to those of TEL, and they were significantly
derivative 7b did. Similar antibacterial profiles were confirmed superior to TEL against S. pyogenes with erm gene. Compounds 10c
not only in methylamine derivative 8c, but also 10c that has a and 6 that have a methoxy group at the 5-position of the benzene
methoxy group at the 5-position of the phenyl group. Furthermore, ring exhibited activities comparable to TEL against H. influenzae.
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Synthesis of novel lincomycin derivatives
K Kumura et al
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Synthesis of novel lincomycin derivatives
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Synthesis of novel lincomycin derivatives
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It is noteworthy that a single modification at the 7-position realized (dd, J = 10.2, 2.7 Hz, 1H), 3.28–3.38 (m, 1H), 3.10 (dd, J =10.2, 4.6 Hz, 1H),
2.40 (s, 3H), 2.17 (s, 3H), 2.03–2.15 (m, 2H), 1.86–2.02 (m, 2H), 1.57
(d, J = 7.1 Hz, 3H), 1.27–1.36 (m, 4H), 0.85–0.96 (m, 3H); HRMS (ESI) m/z
calcd for C26H36F2N5O7S3 664.1739, found 664.1735 (M+H)+.
comparable activity to that of TEL against target-resistant pathogens.
LCM analogs have a promising framework to overcome the resistant
S. pneumoniae and S. pyogenes with erm gene.
(7S)-7-Deoxy-7-[5-(4,5-dimethoxy-2-nitrophenyl)-1,3,4-thiadiazol-
2-ylthio]lincomycin (6)
EXPERIMENTAL PROCEDURE
General
To a solution of 521 (74 mg, 0.18 mmol) in N,N-dimethylformamide (0.5 ml)
were added 1 M sodium hexamethyldisilazane tetrahydrofuran solution
(0.33 ml) and 2-(4,5-dimethoxy-2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thia-
diazole (63 mg, 0.18 mmol) and the mixture was stirred at room temperature
for 20 min. The mixture was diluted with ethyl acetate and washed with water.
The organic phase was dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by preparative TLC
(CHCl3/CH3OH/28% aq NH4OH= 10/1/0.1) to afford 6 (75 mg, 67%) as a
colorless solid; MP 222–227 °C (dec.); [α]D23 +65° (c 0.20, CH3OH); IR (KBr)
3364.21, 2922.59, 2866.67, 2866.67, 2787.60, 2360.44, 1654.62, 1614.13,
1547.59, 1508.06 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.94 (br d, J = 9.0 Hz,
1H), 7.66 (s, 1H), 7.14 (s, 1H), 5.36 (d, J = 5.6 Hz, 1H), 5.27 (br s, 1H),
4.36–4.46 (m, 2H), 4.25 (d, J = 10.0 Hz, 1H), 4.15 (dd, J = 10.2, 5.6 Hz, 1H),
4.02 (s, 3H), 4.00 (s, 3H), 3.58 (dd, J = 10.0, 3.7 Hz, 1H), 3.33 (dd, J = 7.7,
5.2 Hz, 1H), 3.09 (dd, J =10.7, 4.4 Hz, 1H), 2.39 (s, 3H), 2.17 (s, 3H),
2.04–2.16 (m, 2H), 1.85–2.01 (m, 2H), 1.57 (d, J = 6.8 Hz, 3H), 1.26–1.40
(m, 4H), 0.85–0.94 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 178.94, 164.79,
164.40, 152.80, 150.67, 141.11, 118.40, 113.44, 108.11, 89.01, 71.70, 71.10,
69.37, 68.47, 68.19, 62.60, 56.73, 56.70, 53.15, 44.81, 41.81, 38.13, 38.04, 35.72,
21.59, 18.81, 14.75, 14.29; HRMS (ESI) m/z calcd for C28H42N5O9S3 688.2139,
found 688.2150 (M+H)+.
1H NMR spectra were measured with Varian Gemini-300 (Varian Inc.,
Palo Alto, CA, USA) for 300 MHz, JEOL JNM-GSX 400 (JEOL Ltd,
Tokyo, Japan) for 400 MHz or BRUKER Ascend 400 NMR spectrometer
(BRUKER Corporation, Coventry, UK) for 400 MHz in CDCl3 or CD3OD with
0.03% TMS as an internal standard. 13C NMR spectra were measured with
BRUKER Ascend 400 NMR spectrometer (BRUKER Corporation) for
100 MHz. 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). The melting points were measured with Yanaco MP-S
(Yanaco, Tokyo, Japan). The infrared spectra were measured with Jasco
FT/IR-410 (Jasco). Column chromatography was performed with silica gel
60N (Kanto Chemical, Tokyo, Japan, spherical, neutral).
(7S)-7-Deoxy-7-[5-(4-fluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-
ylthio]lincomycin (4a)
To a solution of 3 (240 mg, 0.39 mmol) in toluene (5 ml) at 0 °C were added
triphenylphosphine (150 mg, 0.57 mmol) and diethylazodicarboxylate (0.10 ml,
0.55 mmol) and stirred at 0 °C for 30 min and 5-(4-fluoro-2-nitrophenyl)-
1,3,4-thiadiazole-2-thiol (120 mg, 0.47 mmol) was added and stirred at room
temperature for 3 h. The mixture was concentrated under reduced pressure. To
the resulting mixture were added methanol (5 ml), 1N hydrochloric
acid (0.7 ml) and stirred at room temperature for 1 h. The mixture was
concentrated under reduced pressure and the resulting residue was dissolved by
water and washed with ethyl acetate. To the mixture was added NaHCO3, and
the mixture was extracted with ethyl acetate. The organic phase was
concentrated under reduced pressure and the resulting residue was purified
by preparative TLC (CHCl3/CH3OH/28% aq NH4OH= 20/1/0.1) to afford 4a
(132 mg, 34%) as colorless solid.
(7S)-7-Deoxy-7-[5-(4-dimethylamino-2-nitrophenyl)-1,3,4-
thiadiazol-2-ylthio]lincomycin (7a)
A solution of 4a (70 mg, 0.11 mmol) in 2M dimethylamine methanol solution
(1.0 ml) was stirred at 80 °C for 16 h in a sealed tube. The mixture was
concentrated under reduced pressure and the resulting residue was purified by
preparative TLC (CHCl3/CH3OH/28% aq NH4OH= 20/2/0.2) to afford 7a
(59 mg, 81%) as a colorless solid. [α]D +69° (c 1.0, CH3OH); 1H NMR
22
(400 MHz, CDCl3) δ 9.20 (d, J =9.0 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.09
(d, J = 2.7 Hz, 1H), 6.86 (dd, J = 8.8, 2.7 Hz, 1H), 5.39 (br s, 1H), 5.35
(d, J = 5.5 Hz, 1H), 4.37–4.45 (m, 1H), 4.23–4.31 (m, 2H), 4.14 (dd, J = 10.1,
5.5 Hz, 1H), 3.71 (d, J = 3.4 Hz, 1H), 3.57 (dd, J = 10.0, 3.4 Hz, 1H), 3.37
(dd, J = 8.2, 5.6 Hz, 1H), 3.11 (s, 6H), 3.06–3.11 (m, 1H), 2.38 (s, 3H), 2.19
(s, 3H), 2.04–2.18 (m, 2H), 1.85–2.00 (m, 2H), 1.54 (d, J = 7.1 Hz, 3H),
1.29–1.37 (m, 4H), 0.85–0.95 (m, 3H); HRMS (ESI) m/z calcd for
C28H43N6O7S3 671.2350, found 671.2346 (M+H)+.
[α]D +67° (c 0.73, CH3OH); 1H NMR (300 MHz, CDCl3) δ 9.09 (br d,
22
J = 9.1 Hz, 1H), 7.69–7.85 (m, 2H), 7.37–7.54 (m, 1H), 5.35 (d, J = 5.3 Hz,
1H), 5.21–5.33 (m, 1H), 4.31–4.54 (m, 2H), 4.24 (br d, J = 10.2 Hz, 1H), 4.16
(dd, J = 9.9, 5.3 Hz, 1H), 3.68–3.83 (m, 1H), 3.58 (dd, J = 10.2, 3.3 Hz, 1H),
3.35 (br s, 1H), 3.06–3.22 (m, 1H), 2.41 (s, 3H), 2.18 (s, 3H), 1.83–2.15
(m, 4H), 1.57 (d, J = 6.6 Hz, 3H), 1.26–1.42 (m, 4H), 0.81–1.01 (m, 3H);
HRMS (ESI) m/z calcd for C26H37FN5O7S3 646.1834, found 646.1841 (M+H)+.
(7S)-7-Deoxy-7-[5-(5-dimethylamino-2-nitrophenyl)-1,3,4-
thiadiazol-2-ylthio]lincomycin (7b)
(7S)-7-Deoxy-7-[5-(5-fluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-
ylthio]lincomycin (4b)
A solution of 4b (27 mg, 0.042 mmol) in 2M dimethylamine methanol solution
(1.0 ml) was stirred at room temperature for 1 h. The mixture was concen-
trated under reduced pressure and the resulting residue was purified by
preparative TLC (CHCl3/CH3OH/28% aq NH4OH= 20/2/0.2) to afford 7b
Reaction of 3 (690 mg, 1.01 mmol) with 5-(5-fluoro-2-nitrophenyl)-1,3,4-
thiadiazole-2-thiol (200 mg, 1.55 mmol) gave 4b as a colorless solid in 19%
yield by a similar procedure to 4a. [α]D +66° (c 0.18, CH3OH); 1H NMR
22
(300 MHz, CDCl3) δ 9.09 (d, J = 9.2 Hz, 1H), 8.12 (dd, J = 9.0, 4.9 Hz, 1H),
7.46 (dd, J = 8.1, 2.7 Hz, 1H), 7.36–7.43 (m, 1H), 5.36 (d, J = 5.6 Hz, 1H), 5.33
(br s, 1H), 4.33–4.48 (m, 2H), 4.23 (d, J = 10.0 Hz, 1H), 4.15 (dd, J = 10.0,
5.5 Hz, 1H), 3.71 (br s, 1H), 3.52–3.63 (m, 1H), 3.29–3.37 (m, 1H), 3.10
(dd, J = 10.5, 4.6 Hz, 1H), 2.40 (s, 3H), 2.19 (s, 3H), 2.05–2.16 (m, 2H),
1.85–2.02 (m, 2H), 1.58 (d, J = 7.0 Hz, 4H), 0.85–0.95 (m, 3H); HRMS (ESI)
m/z calcd for C26H37FN5O7S3 646.1834, found 646.1840 (M+H)+.
(24 mg, 87%) as a colorless solid. [α]D +73° (c 0.70, CH3OH); 1H NMR
22
(300 MHz, CDCl3) δ 8.94 (d, J = 8.5, 1H), 8.18 (d, J = 9.1 Hz, 1H), 6.69–6.79
(m, 2H), 5.36 (d, J = 5.4 Hz, 1H), 5.19–5.34 (m, 1H), 4.34–4.48 (m, 2H), 4.27
(br d, J = 10.2 Hz, 1H), 4.15 (dd, J = 9.9, 5.4 Hz, 1H), 3.72 (br d, J = 3.4 Hz,
1H), 3.59 (dd, J = 10.2, 3.4 Hz, 1H), 3.30–3.39 (m, 1H), 3.16 (s, 6H), 3.05–3.13
(m, 1H), 2.40 (s, 3H), 2.18 (s, 3H), 1.86–2.15 (m, 4H), 1.57 (d, J = 6.9 Hz, 3H),
1.21–1.42 (m, 4H), 0.85–0.95 (m, 3H); HRMS (ESI) m/z calcd for
C28H43N6O7S3 671.2350, found 671.2346 (M+H)+.
(7S)-7-Deoxy-7-[5-(4,5-difluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-
ylthio]lincomycin (4c)
(7S)-7-Deoxy-7-[5-(5-dimethylamino-4-fluoro-2-nitrophenyl)-1,3,4-
thiadiazol-2-ylthio]lincomycin (7c)
A solution of 4c (10 mg, 0.015 mmol) in 2M dimethylamine methanol solution
Reaction of 3 (240 mg, 0.39 mmol) with 5-(4,5-difluoro-2-nitrophenyl)-1,3,4-
thiadiazole-2-thiol gave (140 mg, 0.51 mmol) as a colorless solid in 33% yield
by a similar procedure to 4a. 1H NMR (300 MHz, CDCl3) δ 9.03 (d, J = 8.2 Hz,
1H), 7.95–8.04 (m, 1H), 7.59–7.68 (m, 1H), 5.35 (d, J =5.6 Hz, 1H), 5.29 (1.0 ml) was stirred at 50 °C for 3.5 h in a sealed tube. The mixture was
(br s, 1H), 4.29–4.51 (m, 2H), 4.09–4.27 (m, 2H), 3.67–3.78 (m, 1H), 3.57 concentrated under reduced pressure and the resulting residue was purified by
The Journal of Antibiotics

Synthesis of novel lincomycin derivatives
K Kumura et al
8
preparative TLC (CHCl3/CH3OH/28% aq NH4OH= 20/2/0.2) to afford 7c (CHCl3/CH3OH/28% aq NH4OH= 20/2/0.2) to afford 10b (11 mg, 64%) as a
23
(9.0 mg, 87%) as a colorless solid. [α]D +91° (c 0.35, CH3OH); 1H NMR
colorless solid. [α]D +56° (c 0.36, CH3OH); 1H NMR (300 MHz, CDCl3)
23
(400 MHz, CDCl3) δ 8.94 (br d, J = 9.0 Hz, 1H), 7.94 (d, J = 14.1 Hz, 1H), 6.86
(d, J = 8.8 Hz, 1H), 5.36 (d, J = 5.6 Hz, 1H), 5.28 (br d, 1H), 4.36–4.47
(m, 2H), 4.26 (d, J =10.0 Hz, 1H), 4.18–4.23 (m, 1H), 4.15 (dd, J = 10.0,
5.6 Hz, 1H), 3.68–3.75 (m, 1H), 3.58 (dd, J = 10.1, 3.5 Hz, 1H), 3.33
(dd, J = 7.9, 5.5 Hz, 1H), 3.16–3.19 (m, 1H), 3.15 (s, 3H), 3.15 (s, 3H),
3.07–3.13 (m, 1H), 2.40 (s, 3H), 2.03–2.16 (m, 3H), 1.85–2.02 (m, 3H),
1.57 (d, J = 7.1 Hz, 3H), 1.26–1.35 (m, 4H), 0.85–0.94 (m, 3H); HRMS (ESI)
m/z calcd for C28H42FN6O7S3 689.2256, found 689.2258 (M+H)+.
δ 8.91–9.14 (m, 1H), 8.16 (d, J =8.4 Hz, 1H), 7.05–7.20 (m, 2H), 5.36
(d, J =5.4 Hz, 1H), 5.09–5.34 (m, 1H), 4.56–4.73 (m, 1H), 4.34–4.50 (m, 1H),
4.08–4.29 (m, 1H), 3.94 (s, 3H), 3.58–3.83 (m, 2H), 2.69–3.00 (m, 2H),
2.46–2.64 (m, 1H), 1.99–2.40 (m, 7H), 1.87–1.95 (m, 1H), 1.51–1.65 (m, 3H),
1.17–1.48 (m, 3H), 0.82–0.99 (m, 1H); HRMS (ESI) m/z calcd for
C27H40N5O8S3 658.2034, found 658.2040 (M+H)+.
(7S)-7-Deoxy-7-[5-(4-fluoro-5-methoxy-2-nitrophenyl)-1,3,4-
thiadiazol-2-ylthio]lincomycin (10c)
(7S)-7-Deoxy-7-[5-(5-methylamino-2-nitrophenyl)-1,3,4-thiadiazol-
2-ylthio]lincomycin (8b)
Reaction of 4b (30 mg, 0.046 mmol) with 40% methylamine methanol solution
Reaction of 4c (14 mg, 0.021 mmol) gave 10c (10 mg, 70%) as a colorless solid
23
by a similar procedure to 10b. [α]D +84° (c 0.30, CH3OH); 1H NMR
(400 MHz, CDCl3) δ 8.94 (br d, J = 8.8 Hz, 1H), 7.96 (d, J = 10.2 Hz, 1H), 7.26
(d, J = 7.8 Hz, 1H), 5.36 (d, J = 5.6 Hz, 1H), 5.23–5.31 (m, 1H), 4.38–4.48
(m, 2H), 4.24 (d, J = 10.0 Hz, 1H), 4.14–4.17 (m, 1H), 4.03 (s, 3H), 3.71
(d, J =3.2 Hz, 1H), 3.58 (dd, J = 10.0, 3.4 Hz, 1H), 3.30–3.34 (m, 1H),
3.09 (dd, J = 10.5, 4.4 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 2.06–2.15
(m, 2H), 1.85–2.01 (m, 3H), 1.58 (d, J = 6.8 Hz, 3H), 1.28–1.37 (m, 4H),
0.87–0.93 (m, 3H); HRMS (ESI) m/z calcd for C27H39N5O8S3 676.1939, found
676.1947 (M+H)+.
(1.0 ml) gave 8b (23 mg, 75%) as a colorless solid by a similar procedure to 7b.
22
[α]D +73° (c 0.69, CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.89 (br d,
J = 8.5 Hz, 1H), 8.10–8.18 (m, 1H), 6.63–6.72 (m, 2H), 5.36 (d, J = 5.2 Hz,
1H), 5.17–5.34 (m, 1H), 4.92–5.04 (m, 1H), 4.34–4.48 (m, 2H), 4.27
(d, J = 9.9 Hz, 1H), 4.15 (dd, J =10.1, 5.2 Hz, 1H), 3.71–3.78 (m, 1H),
3.60 (dd, J = 9.9, 3.3 Hz, 1H), 3.29–3.38 (m, 1H), 3.11 (dd, J = 10.2,
4.7 Hz, 1H), 2.95 (d, J = 4.9 Hz, 3H), 2.40 (s, 3H), 2.16 (s, 3H), 1.81–2.14
(m, 4H), 1.56 (d, J = 6.6 Hz, 3H), 1.24–1.38 (m, 4H), 0.85–0.96 (m, 3H);
HRMS (FAB) m/z calcd for C27H41N6O7S3 657.2193, found 657.2192 (M+H)+.
In vitro antibacterial activity
Minimum inhibitory concentration (MIC) was determined by the agar dilution
method. Test strains were subjected to seed culture using sensitivity test broth
(STB, 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 (SDA, 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.
(7S)-7-Deoxy-7-[5-(4-fluoro-5-methylamino-2-nitrophenyl)-1,3,4-
thiadiazol-2-ylthio]lincomycin (8c)
Reaction of 4c (7.0 mg, 0.011 mmol) with 40% methylamine methanol solution
(1.0 ml) gave 8c (7.0 mg, 98%) as a colorless solid by a similar procedure to 7b.
22
MP 220–225 °C (dec.); [α]D +64° (c 0.47, CH3OH); IR (KBr) 3397.96,
2922.59, 2782.78, 2369.12, 1654.62, 1577.49 and 1527.35 cm− 1 1H NMR
;
(400 MHz, CD3OD) δ 7.98 (d, J = 11.9 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 5.28
(d, J = 5.6 Hz, 1H), 4.61 (dd, J =9.8, 3.0 Hz, 1H), 4.41–4.49 (m, 2H), 4.11
(dd, J =10.4, 5.6 Hz, 1H), 3.82 (d, J = 3.1 Hz, 1H), 3.55–3.63 (m, 2H),
3.00–3.05 (m, 1H), 2.92 (s, 3H), 2.40 (s, 3H), 2.04–2.10 (m, 1H), 2.03
(s, 3H), 1.82–1.89 (m, 1H), 1.58 (d, J = 7.1 Hz, 3H), 1.28–1.36 (m, 4H),
0.88–0.94 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 178.93, 165.07, 164.77,
151.06, 148.59, 142.50, 142.38, 135.50, 135.42, 123.96, 123.93, 112.79, 112.72,
112.67, 112.56, 88.99, 71.67, 71.11, 69.38, 68.47, 68.20, 62.61, 53.19, 44.76,
41.82, 38.11, 38.04, 35.72, 29.74, 21.59, 18.86, 14.72, 14.29; HRMS (ESI) m/z
calcd for C27H40FN6O7S3 675.2099, found 675.2103 (M+H)+.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank Dr E Shitara, Mr A Tamura, 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 LCM 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, Ms K Yamada for biological
studies, and Ms M Takagi for manuscript. We also thank Ms M Ishii for
direction in intellectual properties.
(7S)-7-Deoxy-7-{5-[4-fluoro-5-(2-hydroxyethylamino)-2-
nitrophenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (9c)
A solution of 4c (12 mg, 0.018 mmol) in 2-ethanolamine (0.5 ml) was stirred at
50 °C for 2 h. The mixture was diluted with ethyl acetate and washed with
water. The organic phase was dried over Na2SO4, filtered and concentrated
under reduced pressure. The resulting residue was purified by preparative TLC
(CHCl3/CH3OH/28% aq NH4OH= 20/2/0.2) to afford 9c (11 mg, 86%) as a
1
2
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colorless solid. [α]D +63° (c 0.42, CH3OH); 1H NMR (400 MHz, CD3OD) δ
23
8.00 (d, J = 11.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 5.28 (d, J =5.6 Hz, 1H), 4.62
(dd, J = 9.9, 3.0 Hz, 1H), 4.41–4.49 (m, 2H), 4.12 (dd, J =10.3, 5.6 Hz, 1H),
3.80–3.84 (m, 1H), 3.73 (t, J = 5.6 Hz, 2H), 3.58 (dd, J = 10.3, 3.2 Hz, 1H), 3.42
(t, J = 5.6 Hz, 2H), 3.25–3.29 (m, 1H), 3.03 (dd, J =10.5, 5.1 Hz, 1H), 2.40
(s, 3H), 2.16–2.27 (m, 1H), 2.04–2.10 (m, 1H), 2.04 (s, 3H), 1.99–2.03
(m, 1H), 1.81–1.91 (m, 1H), 1.58 (d, J = 7.1 Hz, 3H), 1.29–1.38 (m, 4H),
0.89–0.95 (m, 3H); HRMS (ESI) m/z calcd for C28H42FN6O8S3 705.2205, found
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8
(7S)-7-Deoxy-7-[5-(5-methoxy-2-nitrophenyl)-1,3,4-thiadiazol-2-
ylthio]lincomycin (10b)
A solution of 4b (17 mg, 0.026 mmol) in methanol (0.50 ml) was added 28%
methanol solution of sodium methoxide (0.10 ml) stirred at room temperature
for 1 h. The mixture was diluted with ethyl acetate and washed with water.
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reduced pressure. The resulting residue was purified by preparative TLC
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