Nitrogen - Carbon-linked (azolylphenyl) oxazolidinones with potent antibacterial activity against the fastidious gram-negative organisms Haemophilus influenzae and Moraxella catarrhalis [1]

Full text of DOI:10.1021/jm980546k

5144
J . Med. Chem. 1998, 41, 5144-5147
Communications to the Editor
pounds are potent antibacterials in vitro and in vivo
against numerous important Gram-positive human
pathogens including resistant strains of S. aureus
(MRSA), S. epidermidis (MRSE), and the enterococci
(VRE).12 Currently, linezolid is in phase III clinical
development for the treatment of Gram-positive infec-
tions. Recently, we have been interested in increasing
the Gram-positive activity as well as expanding the
spectrum of activity of the oxazolidinones to include the
fastidious Gram-negative organisms Haemophilus in-
fluenzae and Moraxella catarrhalis. In this Communica-
tion, we describe (pyrrolylphenyl)- and (pyrazolylphenyl)-
oxazolidinones which were discovered during the course
of an extensive SAR program which included the
replacement of the morpholine moiety of linezolid with
various aromatic five-membered nitrogen-containing
heterocycles (azoles). The 3-cyanopyrrole and 4-cyan-
opyrazole moieties were found to impart excellent broad-
spectrum antibacterial activity. Both PNU-171933 and
PNU-172576 were discovered to have potent antibacte-
rial activity in vitro versus Gram-positive and Gram-
negative pathogens. Also, both compounds are very
effective in vivo versus S. aureus and S. pneumoniae,
and these compounds are the first class of oxazolidino-
nes reported to demonstrate potent activity versus both
Gram-positive and Gram-negative bacteria.
Nitr ogen -Ca r bon -Lin k ed
(Azolylp h en yl)oxa zolid in on es w ith
P oten t An tiba cter ia l Activity Aga in st th e
F a stid iou s Gr a m -Nega tive Or ga n ism s
Ha em oph ilu s in flu en za e a n d Mor a xella
ca ta r r h a lis
Michael J . Genin,*,† Douglas K. Hutchinson,†
Debra A. Allwine,† J ackson B. Hester,†
D. Edward Emmert,† Stuart A. Garmon,†
Charles W. Ford,‡ Gary E. Zurenko,‡
J udith C. Hamel,‡ Ronda D. Schaadt,‡
Douglas Stapert,‡ Betty H. Yagi,‡ J anice M. Friis,§
Eric M. Shobe,§ and Wade J . Adams§
Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49001
Received September 28, 1998
The emergence of multidrug-resistant strains of Gram-
positive bacterial pathogens is a problem of ever in-
creasing significance.1-3 Organisms including methicillin-
resistant Staphylococcus aureus (MRSA)4 and Staphylo-
coccus epidermidis (MRSE),4 vancomycin-resistant en-
terococci (VRE),5,6 and penicillin- and cephalosporin-
resistant streptococci7 are continually challenging sci-
entists, physicians, and patients. In the case of VRE,
most of these isolates are also resistant to other
antibiotics as well, and mortality rates of >35% have
been reported with VRE infection.8 In addition, many
MRSA isolates are only susceptible to vancomycin which
is considered a drug of last resort.4,9 Of particular
concern is the recent emergence of staphylococcal strains
with reduced susceptibility to vancomycin, the so-called
vancomycin-intermediate strains, in the United States10
and J apan.11 Fortunately, vancomycin therapy still
effected a cure in these patients. However, the prob-
ability that more highly vancomycin-resistant S. aureus
isolates will be seen in a clinical setting is growing.
Since vancomycin is considered a last resort therapy for
serious infections, the spread of such an organism could
be catastrophic. If such infections are to be controlled,
new and powerful antibiotics will be required that act
via novel mechanisms.
The oxazolidinones are one such class of totally
synthetic antibacterial agents with potent activity
against Gram-positive organisms.12 They have been
shown to selectively bind to the 50S ribosomal subunit
and to inhibit bacterial translation at the initiation
phase of protein synthesis.13,14 Because of this unique
mechanism of action, the oxazolidinones are not cross-
resistant with other antibiotics. An extensive structure-
activity relationship (SAR) study within Pharmacia &
Upjohn led to the discovery of eperezolid (1, PNU-
100592) and linezolid (2, PNU-100766).15 These com-
Ch em istr y. The (azolylphenyl)oxazolidinones of in-
terest were prepared as outlined below. Various syn-
thetic routes were employed depending on the nature
of the azole heterocycle. The pyrrole derivative 3, PNU-
171933, was prepared as shown in Scheme 1. Reaction
of benzylamine with 3,4-difluoronitrobenzene (5) yielded
6 which was hydrogenated to give 7. Bis-protection of 7
with benzyl chloroformate yielded the fully protected
p-dianiline 8. Conversion of 8 to the oxazolidinone 9 was
accomplished via the use of standard methods using
n-butyllithium and (R)-(-)-glycidyl butyrate.15 The ox-
azolidinone side-chain alcohol of 9 was then converted
to the acetamide 10 via an intermediate phthalimide.15
Hydrogenation of 10 resulted in complete deprotection
† Medicinal Chemistry Research.
‡ Infectious Diseases Research.
§ Drug Metabolism Research.
10.1021/jm980546k CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/01/1998

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5145
Sch em e 1a
a
Reagents: (a) BnNH2, DIEA, CH3CN, 90 °C, 84%; (b) H2, 5% Pt/C, THF; (c) N,N-dimethylaniline, benzyl chloroformate, THF, 0 °C-
rt, 72% for steps b and c; (d) (i) n-BuLi, THF -78 °C, (ii) (R)-(-)-glycidyl butyrate, -78 °C-rt, 77%; (e) MsCl, NEt3, CH2Cl2; (f) potassium
phthalimide, CH3CN, 95 °C, 93% for steps e and f; (g) hydrazine hydrate, MeOH, 80 °C, 99%; (h) Ac2O, pyridine, 0 °C-rt, 89%; (i) H2, 10%
Pd/C, EtOH, 78%; (j) HOAc, reflux, 85%; (k) NH2OH, MeOH/CH2Cl2, K2CO3, 72% E/Z mixture; (l) PPh3, CH3CN/CCl4, 72%.
Sch em e 2a
a
Reagents: (a) hydrazine hydrate, K2CO3, CH3CN, 94%; (b) NaOAc, EtOH, reflux, 65%; (c) H2, 10% Pd/C, MeOH, 96%; (d) benzyl
chloroformate, NaHCO3, THF, 98%; (e) (i) n-BuLi, THF, -78 °C, (ii) (R)-(-)-glycidyl butyrate, -78 °C-rt, 79%; (f) MsCl, NEt3, CH2Cl2,
98%; (g) NaN3, DMF, 65 °C, 93%; (h) (i) PPh3, THF, (ii) H2O, 65 °C, 77%; (i) Ac2O, pyridine, CH2Cl2, 99%; (j) NH3/MeOH, KCN, 70 °C,
34%; (k) SOCl2, DMF, 0 °C, 71%.
to give the key intermediate (aminophenyl)oxazolidi-
none 11. Condensation of 11 with 2,4-dimethoxy-2,5-
dihydrofurancarboxaldehyde (12) yielded the 3-formylpyr-
rolyl intermediate 13.16 This material was then converted
to the oxime 14 with hydroxylamine in good yields.
Dehydration of 14 with triphenylphosphine17 resulted
in the formation of the [(3-cyanopyrrolyl)phenyl]oxazo-
lidinone 3, PNU-171933.
The regioselective synthesis of the pyrazole analogues
was performed as outlined in Scheme 2. Treatment of
5 with hydrazine hydrate in acetonitrile gave an excel-
lent yield of the arylhydrazine 15. This material was
then condensed with (ethoxycarbonyl)malondialde-
hyde18 (16) in refluxing ethanol to give the 4-carb-
ethoxypyrazole intermediate 17.19 Reduction of the nitro
group of 17 followed by protection with benzyl chloro-

5146 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Communications to the Editor
Ta ble 1. In Vitro Antibacterial Activity, Minimum Inhibitory
Concentration (MIC, µg/mL)
171933) demonstrated excellent broad-spectrum activity
versus several Gram-positive and the fastidious Gram-
negative pathogens. The 4-cyanopyrazole congener 4
(PNU-172576) also has excellent broad-spectrum anti-
bacterial activity. Interestingly, the ester analogue 21,
although equivalent to linezolid versus Gram-positive
organisms, was not active against the fastidious Gram-
negative bacteria. Finally, the carboxamide congener 22
was a poor antibacterial agent. Thus, PNU-171933 and
PNU-172576 are very active antibacterial agents with
MICs ) <0.125-0.5 µg/mL versus Gram-positive bac-
teria and MICs ) 2-4 µg/mL against the fastidious
Gram-negative organisms H. influenzae and M. ca-
tarrhalis. These compounds are several times more
potent versus all organisms tested than the benchmark
vancomycin.
compound
S.a.a S.a.b S.e.c
S.p.d
E.f.e H.inf.f M.cat.g
PNU-171933
PNU-172576
13
14
21
0.5
0.5
0.25 e0.125 e0.125
0.5 e0.125 e0.125
0.25
0.5
0.25
0.5
4
4
4
2
1
2
1
0.25 0.25 e0.125 e0.125
1
4
>16
4
0.5
4
0.25 e0.125
4
1
2
1
>16
>16
16
16
>32
>16
>16
8
8
>32
22
16
4
1
0.5
2
0.5
1
0.5
0.5
>16
linezolid
eperezolid
vancomycin
2
1
1
4
2
4
4
1
a
b
Methicillin-susceptible S. aureus UC 9213. Methicillin-
resistant S. aureusUC 6685. c Methicillin-resistant S. epidermidis
UC 12084. S. pneumoniae UC 9912. e E. faecalis UC 9217. f H.
d
influenzae UC 30063. g M. catarrhalis UC 30610. Minimum inhibi-
tory concentration, lowest concentration of drug (µg/mL) that
inhibits visible growth of the organism.
Because of their outstanding in vitro activity, 13, 14,
PNU-171933, and PNU-172576 were tested in vivo
versus S. aureus and S. pneumoniae in mouse models
of human infection.15 The analogues were dosed orally
and compared to either eperezolid, linezolid, or vanco-
mycin as controls (Table 2). When a lethal systemic
infection of S. aureus was employed, PNU-171933 and
PNU-172576 were as effective as the eperezolid control.
Against S. pneumoniae, PNU-171933 was also as effec-
tive as eperezolid. The cyanopyrazole analogue, PNU-
172576, was tested against the same infection with
linezolid as a control, and it was found to be 1 order of
magnitude more effective in this model than linezolid.
The intermediate oxime 14 also retained in vivo activity
although it was somewhat less potent than eperezolid.
The aldehyde 13, however, was devoid of in vivo activity.
This is probably due to problems with bioavailability
and/or metabolic stability.
formate yielded 18 which was subjected to the asym-
metric oxazolidinone ring formation15 to give the key
intermediate alcohol 19. The alcohol side chain was
converted to the acetamide 21 by previously described
methods15 via the intermediate azide 20. This material
was subjected to ammonolysis to give the carboxamide
22 which was converted to the nitrile 4, PNU-172576,
via dehydration with thionyl chloride.
Biologica l Resu lts. The oxazolidinones prepared
above were tested in vitro versus a panel of Gram-
positive and Gram-negative bacterial isolates. Minimum
inhibitory concentration (MIC) values were determined
using standard agar dilution methods.15 Also, in vivo
efficacy after oral administration was determined via a
lethal systemic S. aureus and S. pneumoniae infection
in mice.15 All of the pyrroles tested had excellent in vitro
antibacterial activity (Table 1). The intermediate alde-
hyde 13 and oxime 14 were very active; however, due
to the potential metabolic instability and/or reactivity
of the aldehyde and the oxime moieties, they were not
evaluated further. The 3-cyanopyrrole analogue 3 (PNU-
In light of the outstanding in vitro and in vivo activity
of PNU-171933 and PNU-172576, the pharmacokinetics
of both analogues were evaluated in male Sprague-
a
Ta ble 2. In Vivo Antibacterial Activity (ED50
,
mg/kg/day)
organism
S. aureus
strain
compound
ED50
control ED50
UC 9213
PNU-171933
PNU-172576
1.9 (1.1-2.7)
1.2 (0.8-2.0)
4.0 (0.1-6.6)
3.3 (1.8-7.0)
eperezolid
13
>20
1.7 (0.9-2.6)
vancomycin
2.6 (2.2-2.9)
eperezolid
1.4 (0.8-2.0)
eperezolid
14
5.5 (3.5-9.1)
0.6 (0.1-1.0)
0.35 (0.2-0.6)
S. pneumoniae
UC 15087
PNU-171933
PNU-172576
3.2 (2.0-4.9)
linezolid
a
Effective dose50 is the amount of drug required after oral administration (mg/kg/day) to cure 50% of infected mice subjected to a
lethal systemic infection. Numbers in parentheses are 95% confidence ranges. Data shown are from one experiment (n ) 36 mice/drug).
Ta ble 3. Single-Dose Pharmacokinetics of PNU-171933, PNU-172576, and Linezolid in Male Sprague-Dawley Rats
b
compound
route
dosea (mg/kg)
Cmax (µM)
tmaxc (h)
t1/2âd (h)
Vsse (L/kg)
CL/Ff (mL/min/kg)
Fg (%)
PNU-171933
iv
po
iv
po
iv
po
9.9
42.9
9.0
46.8
10
26.1 ( 2.8
31 ( 1.7
29.2 ( 3.9
29.4 ( 4.0
na
na
10 ( 1.2
na
4.0 ( 0.0
na
5.15 ( 0.22
5.14h
0.52 ( 0.07
1.18 ( 0.21
1.43 ( 0.49
1.04 ( 0.29
1.25 ( 0.15
10.5
na
92
na
80
na
nc
PNU-172576
linezolid
5.35 ( 0.81
0.43 ( 0.11
nc
0.72
nc
5.35h
0.95
1.05
25
15.8
0.3
na
109
a
b
d
n ) 3. Maximum plasma concentration. c Time at which Cmax is achieved. Harmonic mean apparent terminal disposition half-life.
e Steady-state volume of distribution ) dose[(AUMC/(AUC)2], where AUMC is the area under the first moment curve [AUMC ) ∫ C(t)t
∞
o
dt] and AUC is the area under the concentration-time curve [AUC ) o∫ C(t)t dt]. f Clearance ) dose/AUC. Absolute oral bioavailability
∞
g
h
assuming linear pharmacokinetics ) [(AUC/dose)po/(AUC/dose)iv] × 100. Harmonic mean apparent terminal disposition half-life for iv
dose; half-life for po dose could not be calculated because of prolonged absorption or slow clearance.

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5147
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Dawley rats following single-dose intravenous and oral
administration (Table 3). Both analogues demonstrated
excellent pharmacokinetic profiles. Absolute oral bio-
availability (F) was very good at 92% and 80%, respec-
tively. In addition, blood levels (Cmax) after oral dosing
were high, and the harmonic mean apparent terminal
disposition half-lives (t1/2â) were greater than 5 h for
each drug. Also, the volume of distribution (Vss) was
moderate, and the mean systemic clearance (CL/F) was
low for each analogue (1.18 ( 0.21 mL/min/kg for PNU-
171933 and 1.04 ( 0.29 mL/min/kg for PNU-172576).
In conclusion, in an effort to expand the spectrum of
antibacterial activity of the oxazolidinones to include
Gram-negative organisms, the (azolylphenyl)oxazolidi-
none subclass has been developed. Certain members of
this class have very potent activity versus both Gram-
positive and Gram-negative organisms. In particular,
the 3-cyanopyrrole and 4-cyanopyrazole congeners 3 and
4 (PNU-171933 and PNU-172576) had S. aureus MICs
e 0.5 µg/mL and H. influenzae and M. catarrhalis MICs
) 2-4 µg/mL. In addition, both analogues have out-
standing pharmacokinetic profiles. Furthermore, these
compounds are more effective than linezolid and eper-
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models of human infection. A detailed account of this
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