The synthesis and antibacterial activity of 1,3,4-thiadiazole phenyl oxazolidinone analogues

Article abstract of DOI:10.1016/j.bmcl.2003.07.018

Replacement of the morpholine C-ring of linezolid 1 with a 1,3,4-thiadiazolyl ring leads to oxazolidinone analogues 5 having potent antibacterial activity against both gram-positive and gram-negative organisms. Conversion of the C5 acetamide group to a thioacetamide further increases the potency of these compounds.

Full text of DOI:10.1016/j.bmcl.2003.07.018

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4193–4196
The Synthesis and Antibacterial Activity of 1,3,4-Thiadiazole
Phenyl Oxazolidinone Analogues
Lisa M. Thomasco,* Robert C. Gadwood, Elizabeth A. Weaver, Jason M. Ochoada,
Charles W. Ford, Gary E. Zurenko, Judith C. Hamel, Douglas Stapert,
Judy K. Moerman, Ronda D. Schaadt and Betty H. Yagi
Discovery-Chemistry and Discovery-Infectious Diseases, Pharmacia Corporation, 7000 Portage Road, Kalamazoo, MI 49001, USA
Received 5 May 2003; accepted 30 July 2003
Abstract—Replacement of the morpholine C-ring of linezolid 1 with a 1,3,4-thiadiazolyl ring leads to oxazolidinone analogues 5
having potent antibacterial activity against both gram-positive and gram-negative organisms. Conversion of the C5 acetamide
group to a thioacetamide further increases the potency of these compounds.
# 2003 Elsevier Ltd. All rights reserved.
Decades of antibiotic use have resulted in the develop-
ment of widespread resistance to commonly prescribed
antibacterial agents. Nosocomial infections by methi-
cillin-resistant Staphylococus aureus (MRSA)1 and van-
comycin-resistant Enterococcus (VRE)2 are particularly
serious problems. Recently, a glycopeptide-intermediate
S. aureus (GISA) isolate with reduced susceptibility to
vancomycin3 was reported. The spread of resistance of
MRSA to vancomycin would be especially devastating.
Within the community, penicillin- and cephalosporin-
resistant Staphylococus pneumoniae (PRSP)4 has
become an increasing problem. In view of the increased
resistance of gram-positive bacteria to currently avail-
able antibacterial agents, there is an urgent need for new
antibacterial agents.
with improved antibacterial potency.6,7 More recently,
Genin and co-workers have reported that replacement
of the morpholine C-ring of linezolid with a carbon–
carbon linked pyrazole ring system afforded oxazolidi-
none analogues (4) having improved antibacterial
potency against fastidious gram-negative organisms.8
With this in mind, we were interested in preparing oxa-
zolidinone analogues (5) having other carbon-linked
heteroaromatic C-rings. In this letter, we describe the
synthesis and antibacterial activity of oxazolidinone
analogues having 1,3,4-thiadiazole C-rings.
The oxazolidinones are a novel class of totally synthetic
antibacterial agents. Linezolid (1) is the first oxazolidi-
none approved for the treatment of gram-positive bac-
terial infections in humans.5 Linezolid is active against
multidrug-resistant strains of staphylococci, strepto-
cocci and enterococci, but is only weakly active (or
inactive) against gram-negative organisms. Early SAR
studies, conducted by workers at DuPont, revealed that
oxazolidinone analogues having a phenyl or 4-pyridyl
C-ring resulted in oxazolidinone analogues (2 and 3)
In order to facilitate the rapid preparation of multiple
1,3,4-thiadiazolyl phenyl oxazolidinone analogues, an
*Corresponding author. Tel.: +1-860-715-0521; fax: +1-860-686-
0543; e-mail: lisa.m.thomasco@pfizer.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.018

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L. M. Thomasco et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4193–4196
advanced intermediate was sought that could readily be
converted to the target compounds. The thiobenzhy-
drazide 6 was selected as the key intermediate, since the
1,3,4-thiadiazoles would be readily available from 6 by
reaction with carboxylic acid chlorides. Synthesis of 6
was carried out as shown in Scheme 1. Thus, diazotiza-
tion of the known9 amine 7 followed by reaction with
potassium cyanide gave the nitrile 8. Addition of
hydrogen sulfide to 8 in hot DMFafforded the thio-
amide 9. Alkylation of 9 with methyl triflate and treat-
ment of the intermediate isothioamide 10 with H2S led
to the dithioester 11, which was immediately treated
with hydrazine to afford the desired thiobenzhydrazide
6.10,11
Oxazolidinone analogues bearing a C-5 thioacetamide
group are known to have dramatically increased
potency against both gram-positive and gram-negative
organisms.12,13 In order to evaluate this modification in
1,3,4-thiadiazolyl phenyl oxazolidinones, several exam-
ples were converted to a C-5 thioacetamide. Reaction of
5a and 5q with Lawesson’s Reagent in refluxing dioxane
afforded the thioamides 12a and 12b. Thioamides 12c
and 12d were prepared by reaction of the Fmoc-pro-
tected acetamide oxazolidinones 5r and 5s with Law-
esson’s Reagent followed by deprotection with
piperidine (Scheme 3).
All of the analogues were tested in vitro against a panel
of gram-positive and fastidious gram-negative bac-
teria.14 Selected compounds were also evaluated for in
vivo efficacy against S. aureus in a mouse bacteremia
model.15 The in vitro and in vivo activity of the 1,3,4-
thiadiazole analogues 5a–u and 12a–d are shown in
Table 1. All of these analogues exhibited good to excel-
lent antibacterial activity, including good activity
against fastidious gram-negative organisms. In many
cases, the compounds had superior activity to linezolid.
As seen in Table 1, the in vitro activity of the 1,3,4-
thiadiazolyl phenyl oxazolidinones is relatively insensi-
tive to the nature of the substituent at the 2-position. As
expected, the thioacetamide analogues (12a–d) are
extremely potent against both gram-positive and fastid-
ious gram-negative organisms. Several of the 1,3,4-thia-
diazole analogues had in vivo activity comparable to
linezolid, but the thioamides lacked oral activity pre-
sumably due to metabolism of the thioamide. For
example, in rat pharmacokinetic studies, 12c was found
to have a clearance of 34.3 mL/min/kg and its bioavail-
able fraction was only 14%.
Once in hand, reaction of the thiobenzhydrazide 6 with
readily available acid chlorides in refluxing THFaffor-
ded a variety of 1,3,4-thiadiazolyl phenyl oxazolidinone
analogues in good yields (Scheme 2).
Simple functional group manipulations provided addi-
tional analogues. For example, hydrolysis of the acetate
5g (MeOH/K2CO3) yielded 5l. Reaction of the ester 5h
with methanolic ammonia gave 5m. Compound 5n was
prepared by acidic hydrolysis (H2SO4/H2O) of 5j.
Reaction of ketone 5i with hydroxylamine afforded the
oxime 5o. Oxidation of 5k with NaIO4 gave the sulfox-
ide 5p, while reaction of 5k with OXONE1 yielded the
sulfone 5q.
Reaction of 6 with Fmoc-glycyl chloride or Fmoc-sar-
cosine acid chloride (prepared from Fmoc-sarcosine and
thionyl chloride using a catalytic amount of DMF) in
refluxing THFafforded the Fmoc-protected 2-amino-
methyl-1,3,4-thiadiazole analogues 5r and 5s, respec-
tively. Removal of the Fmoc-protecting group with
piperidine afforded 5t and 5u (Scheme 3).
Oxazolidinone analogues that contain a 1,3,4-thiadi-
azole C-ring represent a new class of oxazolidinone
antibacterial agents having excellent activity against
both gram-positive and fastidious gram-negative
organisms. These compounds could be prepared in a
straightforward manner from the key intermediate
thiobenzhydrazide 6. Reaction of 6 with a variety of
acid chlorides leads to 1,3,4-thiadiazoles in good yield.
1,3,4-Thiadiazolyl phenyl oxazolidinones that incorpo-
rate a C-5 thioacetamide moiety are extraordinarily
potent antibacterial agents. The 1,3,4-thiadiazolyl
phenyl oxazolidinones are useful extensions of the SAR
of the oxazolidinone class of antibacterial agents.
Scheme 1. (a) NaNO2, 2 N HCl; (b) CuCN, KCN, EtOAc; (c) H2S,
Et3N, DMF, 100 ꢀC; (d) CF3SO3CH3, THF, CH2Cl2; (e) H2S, pyri-
.
dine, THF, CH2Cl2; (f) NH2NH2 H2O, THF, CH2Cl2.
Scheme 3. (a) FmocNH(R)CH2COCl, THF, reflux; (b) piperidine, rt,
30 min; (c) Lawesson’s, dioxane, reflux; (d) piperidine, rt, 30 min.
Scheme 2. (a) RCOCl, THF, reflux, 60–95%.

L. M. Thomasco et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4193–4196
4195
Table 1. In vitro and in vivo activity of 1,3,4-thiadiazole oxazolidinone analogues against selected organisms
Compd
R
X
MIC (mg/mL)a
ED50
(mg/kg)
SAi
SAb
SAc
SEd
SPe
EFf
HIg
MCh
5ak
5bl
5c
5d
5e
H
OH
CH3
CH3CH2
FCH2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
1
1
1
1
1
2
1
8
1
2
1
1
0.5
4
2
8
4
8
4
1
1
0.5
0.5
1
1
1
8
1
1
1
1
0.5
4
2
8
4
4
4
0.25
0.5
<0.125
0.25
0.25
4
0.25
0.125
<0.125
0.25
0.25
0.25
<0.125
<0.125
<0.125
0.25
0.25
<0.125
0.25
<0.125
<0.125
0.25
<0.5
<0.125
<0.5
<0.125
0.06
<0.5
1
1
0.5
0.5
1
1
1
8
0.5
1
1
1
1
2
1
4
2
1
2
4
4
2
4
4
8
4
4
2
2
2
4
2
16
4
4
4
2
4
8
4
8
4
2
2.2 (1.8)
13 (3.8)
6.2 (3.5)
9.2 (3.8)
8.6 (4.0)
5.8 (2.2)
18.5 (3.7)
NTj
5f
CH3OCH2
AcOCH2
EtO2C
CH3CO(CH2)2
NCCH2
CH3SCH2
HOCH2
H2NCO
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5t
5u
12a
12b
12c
12d
1
0.25
2
4
>16
4
4
>16
4
16
4
4
8
0.25
0.25
0.25
0.25
0.25
0.5
0.5
0.5
1
7.6 (2.2)
>20 (2.9)
NTj
8.7 (3.1)
12.4 (2.0)
>20 (3.5)
>20 (5)
NTj
NTj
21.3 (11.5)
NTj
>20 (5.6)
>20 (5.0)
>20 (8)
>20 (4.3)
H2NCOCH2
HON=C(CH3)(CH2)2
CH3S(O)CH2
CH3SO2CH2
H2NCH2
MeHNCH2
H
4
2
4
1
2
1
1
4
0.25
0.25
<0.5
<0.5
2–4
<0.125
0.5
<0.5
<0.5
2–4
<0.125
0.25
<0.5
<0.5
1–2
0.125
0.25
<0.5
<0.5
1–2
0.25
0.5
<0.5
<0.5
8–16
CH3SO2CH2
H2NCH2
CH3NHCH2
—
S
S
S
<0.5
<0.5
16
<0.5
0.5–1
aMIC, minimum inhibitory concentration.
bSA, Staphylococcus aureus UC19213 (methicillin-susceptible S. aureus).
cSA, S. aureus UC112673 (methicillin-resistant S. aureus, Ciprofloxicin1 resistant, Rifampin1 resistant, Imipenem1 resistant).
dSE, Staphylococcus epidermidis UC112084 (methicillin-resistant S. epidermidis).
eSP, Streptococcus pneumoniae UC19912 (penicillin-sensitive S. pneumoniae).
fEF, Enterococcus faecalis UC19217 (vancomycin-resistant E. faecalis).
gHI, Haemophilus influenzae 30063 (Ampicillin1 resistant, b-lactamase +).
hMC, Moraxella catarrhalis 30607 (b-lactamase +).
iAgainst S. aureus UC19213 in mice. Oral administration. Value in parentheses is the ED50 for linezolid, which is used as a control.
jNot tested.
kPrepared using formic acid as the solvent.
lPrepared using triphosgene in refluxing THF.
Preparation of (S)-4-[5-[(acetylamino)methyl]-2-oxo-3-
oxazolidinyl]-2-fluorobenzenecarbothioic acid hydrazide
(6)16. To a stirred solution of the aniline 7 (10.0g, 37.4
mmol) in 2 N HCl (44 mL), cooled to 0 ꢀC, was added a
solution of NaNO2 (3.9 g, 56.1 mmol) in H2O (24 mL).
The reaction mixture was stirred at 0 ꢀC for 90 min,
then neutralized with solid NaHCO3 (pH 7). In a sepa-
rate flask, a mixture of copper cyanide (4.4 g, 48.6
mmol) and potassium cyanide (3.6 g, 56.1 mmol) was
suspended in H2O (36 mL) and ethyl acetate (73 mL).
The resulting mixture was cooled to 0 ꢀC and the neu-
tralized diazonium salt was added via cannula over 20
min. The dark reaction mixture was stirred at 0 ꢀC for
45 min and then filtered through a pad of Celite. The
filter cake was washed with ethyl acetate (5Â100 mL).
The filtrate phases were separated and the aqueous
phase was extracted with ethyl acetate (300 mL). The
combined organic phases were dried (MgSO4), filtered
and concentrated to a small volume (75 mL). The
orange solution was purified by filtration through a plug
of silica gel using 5% CH3OH in ethyl acetate as the
eluent to afford 7.42 g (26.8 mmol, 72%) of the nitrile 8
as a yellow solid. Mp 164–167 ꢀC. 1H NMR (DMSO-d6)
d 8.23 (m, 1H), 7.92 (t, J=9 Hz, 1H), 7.74 (dd, J=14, 2
Hz, 1H), 7.52 (dd, J=9, 2 Hz, 1H), 4.76 (m, 1H), 4.14
(t, J=9 Hz, 1H), 3.75 (dd, J=9, 6 Hz, 1H), 3.40 (t, J=5
Hz, 2H), 1.80 (2, 3H); 13C NMR (DMSO-d6) d 170.4,
161.0 (d, J=251 Hz), 154.2, 145.0 (d, J=11 Hz), 134.8,
114.5 (d, J=14 Hz), 105.3 (d, J=25 Hz), 94.0 (d, J=15
Hz), 72.5, 47.6, 41.7, 22.8; IR (mull) 3361, 2417, 2363,
2232, 2180, 2114, 1757, 1745, 1664, 1625, 1513, 1442,
1410, 1219, 1207 cmÀ1. [a]2D5=À35.5ꢀ (c 1.06, DMSO)
Anal. calcd for C13H12FN3O3: C, 56.32; H, 4.36; N,
15.16. Found: C, 56.16; H, 4.34; N, 14.83.
To a stirred solution of the nitrile 8 (8.84, 31.9 mmol) in
DMF(35 mL) was added triethylamine (11.2 mL, 79.8
mmol). The reaction was heated to 100 ꢀC and H2S was
bubbled into the flask for 1 h. The excess H2S was swept
out of the reaction mixture with a stream of N2 over 30
min, while the mixture cooled to 60 ꢀC. The reaction
mixture was then poured onto ice (50 mL) and stirred

4196
L. M. Thomasco et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4193–4196
until the ice melted. The resulting yellow precipitate was
filtered and dried in a vacuum oven (30 ꢀC, 12 h) to give
7.9 g (25.4 mmol, 80%) of the thioamide 9. Mp 116–
119 ꢀC. 1H NMR (DMSO-d6) d 10.1 (s, 1H), 9.5 (s, 1H),
8.24 (t, J=6 Hz, 1H), 7.74 (t, J=9 Hz, 1H), 7.50 (dd,
J=13, 2 Hz, 1H), 7.31 (dd, J=9, 2 Hz, 1H), 4.76 (m,
1H), 4.14 (t, J=9 Hz, 1H), 3.76 (dd, J=9, 7 Hz, 1H),
3.43 (t, J=5 Hz, 2H), 1.83 (s, 3H); 13C NMR (CDCl3) d
195.8, 170.5, 157.3 (d, J=246.8 Hz), 154.4, 141.7 (d,
J=7.9 Hz), 132.3, 124.8 (d, J=13.6 Hz), 113.4, 105.2
(d, J=28.4 Hz), 72.4, 47.7; 41.8, 22.9; IR (mull) 3344,
3153, 1755, 1734, 1669, 1644, 1616, 1550, 1506, 1409,
1395, 1342, 1219, 1206, 1197 cmÀ1. HRMS (EI) calcd
for C13H14FN3O3S 311.0740, found 311.0744.
[a]2D5=À32 (c 0.99, DMSO). Anal. calcd for
C13H14FN3O3S: C, 50.15; H, 4.53; N, 13.50; S, 10.30.
Found: C, 50.54; H, 4.70; N, 13.04; S, 9.60.
General procedure for preparation of 1,3,4-thiadiazole
oxazolidinone analogues from acid chlorides. To a stirred
suspension of the thiobenzhydrazide 6 (1.0 mmol) in dry
THF(10 mL) was added the acid chloride (1.2–2.0
mmol). The reaction mixture was heated at reflux for 1–
6 h or until complete reaction was evident by TLC. The
cooled reaction mixture was concentrated. The resulting
residue was dissolved in CH3OH/CH2Cl2, adsorbed
onto silica gel and purified by silica gel chromatography
using 2.5–5% CH3OH in CH2Cl2 as the eluent to afford
the desired thiadiazole.
References and Notes
1. Rybak, M. J. J. Hosp. Infect. 2001, 49, S25.
2. Linden, P. K. Drugs 2002, 62, 425.
3. Khurshid, M. A.; Chou, T.; Carey, R.; Larsen, R.;
Conover, C.; Bornstein, S. L. MMWR Morb. Mortal. Wkly.
Rep. 2000, 48, 1165.
To a stirred suspension of the thioamide 9 (5.0 g, 16.1
mmol) in dry THF(80 mL) and CH Cl2 (80 mL) was
2
4. Thornsberry, C.; Sahm, D. F.; Kelly, L. J.; Critchley, I. A.;
Jones, M. E.; Evangelista, A. T.; Kalrowsky, J. A. Clin. Infect.
Dis. 2002, 34, S4.
5. Brickner, S. J. Curr. Pharm. Des. 1996, 2, 175.
6. Park, C. H.; Brittelli, C. L.; Wang, J.; Marsh, F. D.;
Gregory, W. A.; Wuonola, M. A.; McRipley, R. J.; Eberly,
V.S; Slee, A. M.; Forbes, M. J. Med. Chem. 1992, 1156.
7. Carlson, R. K.; Park, C.-H.; Gregory, W. A. US Patent
5,130,316, 1992. CAN 113:172003.
8. Lee, C. S.; Allwine, D. A.; Barbachyn, M. R.; Grega, K. C.;
Dolak, L. A.; Ford, C. W.; Jensen, R. J.; Seest, E. P.; Hamel,
J. C.; Schaadt, R. D.; Stapert, D.; Yagi, B. H.; Zurenko, G. E.;
Genin, M. J. Bioorg. Med. Chem. 2001, 9, 3243.
9. Hutchinson, D. K. US Patent 5,910,504, 1999. CAN
131:18998.
10. Kohrt, A.; Hartke, K. Liebigs Ann. Chem. 1992, 595.
11. Stillings, M. R.; Welbourne, A. P.; Walter, D. S. J. Med.
Chem. 1986, 29, 2280.
added methyl trifluoromethanesulfonate (2.0 mL, 17.7
mmol). The reaction was stirred at rt for 30 min, during
which time the reaction mixture became homogeneous.
Pyridine (3.9 mL, 48.3 mmol) was added and then H2S
gas was bubbled through the reaction mixture for 1 h
during which time the reaction mixture turned red-
orange in color. Excess H2S was swept out of the reac-
tion with a stream of nitrogen over 30 min. Hydrazine
monohydrate (2.6 mL, 53.1 mmol) was added and the
reaction mixture was stirred at room temperature for an
additional h during which time the red-orange color
disappeared. The reaction mixture was concentrated.
The resulting residue was dissolved in THF/CH3OH,
adsorbed onto silica gel and purified by silica gel chro-
matography using 2.5–5% CH3OH in CH2Cl2 as the
eluent to afford 2.51 g (7.7 mmol, 48%) of 6 as a yellow
solid. Mp 207–208 ꢀC. H NMR (DMSO-d6) d 12.1 (s,
1
12. Tokuyama, R.; Takahashi, Y.; Tomita, Y.; Suzuki, T.;
Yoshida, T.; Iwasaki, N.; Kado, N.; Okezaki, E.; Nagata, O.
Chem. Pharm. Bull. 2001, 49, 347.
1H), 8.23 (t, J=6, Hz, 1H), 7.54 (t, J=9, Hz, 1H), 7.45
(dd, J=14, 2 Hz, 1H), 7.29 (dd, J=9, 2, Hz, 1H), 6.2 (s,
1H), 4.73 (m, 1H), 4.11 (t, J=9, Hz, 1H), 3.77 (dd,
J=9, 6, Hz, 1H), 3.40 (t, J=5, Hz, 2H), 1.81 (s, 3H);
13C NMR (DMSO-d6) d 176.3, 170.5, 157.1 (d, J=245
Hz), 154.4, 140.0 (d, J=11 Hz), 131.9 (d, J=4 Hz), 123.4
(d, J=14 Hz), 113.5 (d, J=3 Hz), 105.2 (d, J=28 Hz),
72.3, 47.6, 41.8, 22.9; IR (mull) 3369, 3353, 3297, 1752,
1666, 1620, 1556, 1507, 1419, 1401, 1218, 1190, 1153,
1109, 865 cmÀ1; [a]D25=À25ꢀ (c 1.05, DMSO). Anal.
calcd for C13H15FN4O3S: C, 47.85; H, 4.63; N, 17.17; S,
9.82. Found: C, 48.03; H, 4.72; N, 16.86; S, 9.39.
13. Hester, J. B. US Patent 6,362,189, 2000.
14. National Committee for Clinical Laboratory Standards.
Methods for Dilution Antimicrobial Susceptibility Test for
Bacteria that Grow Aerobically, 6th ed.; Approved Standard;
NCCLS Document M7-A6; NCCLS; 940 West Valley Road,
Suite 1400, Wayne, PA, 2003; 19087 (broth micro dilution
method).
15. Ford, C. W.; Hamel, J. C.; Wilson, D. M. Antimicrob.
Agents Chemother. 1996, 40, 1508.
16. Gadwood, R. C.; Thomasco, L. M.; Anderson, D. J. US
Patent 5,977,373, 1999.