The effect of remote chirality on the antibacterial activity of indolinyl, tetrahydroquinolyl and dihydrobenzoxazinyl oxazolidinones

Article abstract of DOI:10.1016/j.bmcl.2003.07.029

The oxazolidinones are promising agents for the treatment of infections caused by gram-positive bacteria, including multidrug-resistant strains. In ongoing studies we have discovered that a strategically placed chiral center of appropriate absolute configuration improves the antibacterial activity of indolinyl oxazolidinone analogues (gram-positive MIC's<0.5 μg/mL for the most potent congeners). The design, synthesis, antibacterial activity and pharmacokinetic profile of a selected series of α-methylated indoline derivatives and a related set of tetrahydroquinolyl and dihydrobenzoxazinyl analogues are discussed.

Full text of DOI:10.1016/j.bmcl.2003.07.029

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4235–4239
The Effect of Remote Chirality on the Antibacterial Activity of
Indolinyl, Tetrahydroquinolyl and Dihydrobenzoxazinyl
Oxazolidinones
Fred L. Ciske, Michael R. Barbachyn,* Michael J. Genin, Kevin C. Grega, Chi Sing Lee,
Lester A. Dolak, Eric P. Seest, William Watt, Wade J. Adams, Janice M. Friis,
Charles W. Ford and Gary E. Zurenko
Pfizer, 7000 Portage Road, Kalamazoo, MI 49001, USA
Received 5 May 2003; accepted 14 July 2003
Abstract—The oxazolidinones are promising agents for the treatment of infections caused by gram-positive bacteria, including
multidrug-resistant strains. In ongoing studies we have discovered that a strategically placed chiral center of appropriate absolute
configuration improves the antibacterial activity of indolinyl oxazolidinone analogues (gram-positive MIC’s<0.5 mg/mL for the
most potent congeners). The design, synthesis, antibacterial activity and pharmacokinetic profile of a selected series of a-methylated
indoline derivatives and a related set of tetrahydroquinolyl and dihydrobenzoxazinyl analogues are discussed.
# 2003 Published by Elsevier Ltd.
The oxazolidinones, exemplified by linezolid (1), are a
promising group of antibacterials that have recently
emerged as important therapeutic agents for the treat-
ment of infections caused by gram-positive bacteria.1
Importantly, linezolid’s spectrum of activity includes
multidrug-resistant strains of the staphylococci, strep-
tococci and enterococci, including recently described
Contemporaneous synthetic studies led to the prepara-
tion of closely related indolinyl oxazolidinones such as
(Æ)-4.6 Compound (Æ)-4 exhibits interesting levels of
gram-positive antibacterial activity (S.aureus MIC=4–8
mg/mL). Moreover, it was found to be very well toler-
ated in extended rat safety studies, showing no sig-
nificant adverse events after oral doses of 100 mg/kg bid
for 30 days.7 Based on these results, we decided to
glycopeptide-intermediate
Staphylococcus
aureus
(GISA) isolates with reduced susceptibility to vanco-
mycin.2 Because of their significance, extensive synthetic
investigations into the structure–activity relationships
(SARs) of the oxazolidinones have been conducted at
Pharmacia and elsewhere. Early SAR studies revealed
that the incorporation of a strategically placed methyl
group in indanone derivatives significantly improved
antibacterial activity. For example the MICs of the
unsubstituted indanone 2 range from 8 to 16 mg/mL for
S.aureus whereas the 2-methyl indanone congener 3
(mixture of diastereomers) has MICs=2–4 mg/mL.
Thus, alkyl substitution adjacent to the carbonyl moiety
confers enhanced antibacterial activity to the
pharmacophore.3À5
*Corresponding author. Tel.: +1-269-833-7726; fax: +1-269-833-
9629; e-mail: michael.r.barbachyn@pfizer.com
0960-894X/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.07.029

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F.L.Ciske et al./ Bioorg.Med.Chem.Lett.13 (2003) 4235–4239
investigate the effects of methylation at the 2-position of
indolinyl oxazolidinones and at the analogous position
of the related tetrahydroquinoline and dihydrobenzox-
azines (see generic structure 5). Herein we describe the
synthesis, antibacterial activity and pharmacokinetic
profile of selected examples in this series. Like the inda-
none series, these compounds exhibit increased levels of
antibacterial activity against gram-positive bacteria
when compared to their unsubstituted counterparts. In
addition, the configuration of the newly introduced ste-
reocenter plays an important role in the antibacterial
activity. Furthermore, some analogues have notable
activity against the fastidious gram-negative organisms
Haemophilus influenzae and Moraxella catarrhalis.8
Preparative scale separation of the diastereomeric mix-
tures on a Chiralcel1 OJ column provided the pure
diastereomers
(R,R)-18–20a
and
(S,R)-18–20b
(X=CH2, CH2CH2, CH2O). The a and b descriptors
refer to the absolute configuration of the stereocenter in
the indoline, tetrahydroquinoline or dihydrobenzox-
azine ring, with b corresponding to the configuration
depicted in Figure 1 (vide infra). Several X-ray structure
determinations enabled an unequivocal assignment of
the absolute configurations of the indoline and tetra-
hydroquinoline C-2 chiral centers and the dihy-
drobenzoxazine C-3 chiral center. A representative
example, diastereomer 18b, is shown (Fig. 1).
The individual indoline diastereomers 18a and b were
converted to the acetamide derivatives (R,S)-21a and
(S,S)-21b, respectively (Scheme 3). Cbz-deprotection
gave 22a and 22b which were converted to the formyl
and hydroxymethylacetyl analogues 23a,b and 24a,b as
shown. The preparation of analogues bearing C-5
thioamide side chains was also examined and in this
regard, two approaches gave desired analogues. Initial
reaction of 21a and 21b with Lawesson’s Reagent fol-
lowed by Cbz-deprotection with HBr/HOAc and for-
mylation gave thioamides 25a and 25b. Alternatively,
22a was treated with Lawesson’s Reagent followed by
acylation and deprotection to afford the hydroxy-
methylacetyl analogue 26a. The desired analogues in the
tetrahydroquinoline and benzoxazine series were pre-
pared in a similar fashion yielding compounds 27–30a
and 27–29b.
Chemistry
The synthesis of the desired analogues is outlined in
Schemes 1–3. Requisite bis-Cbz protected intermediates
were prepared as shown in Scheme 1. The known indole
69 was reduced toindoline 7 with sodium cyanoboro-
hydride. Reduction of the nitro group via catalytic
hydrogenation afforded 8, which was bis-protected with
Cbz–Cl togive 9. The tetrahydroquinoline 13 was pre-
pared from the commercially available quinoline 10.
.
Reduction of the nitro group with SnCl2 2H2O gave 11,
which was converted to the tetrahydro intermediate 12
via hydrogenation using PtO2/MeOH. This material
was bis-protected as above to give 13. The benzoxazine
bis-carbamate 17 was constructed from the known pre-
cursor 14.10 Alkylation of the phenol with chloro-
acetone gave 15. Phthalimide deprotection using excess
hydrazine is accompanied by ring-formation to give the
intermediate imine 16. The imine was then reduced with
NaBH4 and protected to afford 17.
Biology
The analogues prepared above were tested in vitro versus
a panel of gram-positive and fastidious gram-negative
bacteria (Table 1). Minimum inhibitory concentration
(MIC) values were determined using standard agar dilu-
tion methods.12 Also shown are comparator compounds
Compounds 9, 13 and 17 were converted to the corre-
sponding (5R)-(hydroxymethyl)oxazolidinones 18–20
via chemistry previously described (Scheme 2).11
.
Scheme 1. (a) NaBH3CN; (b) H2, Pd/C, MeOH; (c) CbzCl, NaHCO3, acetone; (d) SnCl2 H2O/EtOH, reflux; (e) H2, PtO2, MeOH/H2SO4; (f) KI,
.
K2CO3, ClCH2COCH3, reflux; (g) NH2NH2 H2O, THF, reflux; (h) NaBH4, MeOH.

F.L.Ciske et al./ Bioorg.Med.Chem.Lett.13 (2003) 4235–4239
4237
linezolid and 4. Considering the indoline series first, it
can be seen that methylation at the 2-position imparts a
2- to4-fold increase in activity ( 24a vs 4). This is in
agreement with what we predicted based on historical
observations for the indanones 2 and 3 (vide supra).
Moreover, the stereochemistry of the new chiral center
is critical. Only the appropriate absolute configuration
at the 2-position of the indolinyl ring leads to an
enhancement in MICs. Thus, in all instances the (R)-
isomer is approximately 4- to 8-fold more active than
the corresponding (S)-isomer (e.g., 23a vs 23b). Explor-
ing substitution on the indoline nitrogen it was found
that the formyl group was preferred over the hydroxy-
methyl acetyl moiety, and analogue 23a exhibits potent
antibacterial activity with gram-positive MICs in the
range of <0.5–1 mg/mL. Furthermore, the thioamide
side-chain analogue of this congener, 25a, is even more
effective against gram-positive organisms and also has
very interesting activity against the fastidious gram-
negative organisms. The MICs of 25a against H.influen-
zae and M.catarrhalis are 2 and 1 mg/mL, respectively.
Methylation of the corresponding tetrahydroquinolinyl
(27–28) and dihydrobenzoxazinyl (29–30) oxazolidi-
nones also confers enhanced antibacterial activity to
these pharmacophores and in all cases the (R)-isomer
is more active. As was seen with the indolines, formyl
and hydroxyacetyl appendages at the ring nitrogen
increase potency, with formyl substitution being
favored. Formyl substituted tetrahydroquinolines (e.g.,
28a) were comparable to the indolines while the ben-
zoxazine analogues were slightly less active. Con-
sistent with previously studied oxazolidinones,
thioacetamide derivatives are significantly more active
than acetamides.13
Selected compounds were also evaluated for in vivo
efficacy against S.aureus in a lethal systemic infection
model in the mouse (Table 2).14 With ED50s of 1.8 and
7.5 mg/kg, respectively, the formyl and hydroxymethyl
acetyl indoline analogues, 24a and 25a, are comparable
in activity to linezolid. These compounds were also
subjected to a single-dose pharmacokinetic perfor-
mance assessment in the male Sprague–Dawley rat
(Table 2). All of the selected analogues have reasonable
oral bioavailabilty (F=30.8–56.0%). However, the
maximum serum concentrations (Cmax) are generally
Scheme 2. (a) n-BuLi, (R)-glycidyl butyrate, THF; (b) chiral HPLC.
Scheme 3. (a) NosCl, Et3N, CH2Cl2; (b) NH4OH, CH3CN/IPA; (c) Ac2O; (d) H2, Pd/C; (e) HCO2H, (i-Pr)2NEt, (EtO)2P(O)CN; (f) (1) ClCO-
CH2OAc; (2) K2CO3/MeOH; (g) chiral HPLC; (h) Lawesson’s reagent; (i) (1) HBr/HOAc; (2) HCO2H, (i-Pr)2NEt, Ac2O.
Figure 1. X-ray structure of intermediate 18b.

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F.L.Ciske et al./ Bioorg.Med.Chem.Lett.13 (2003) 4235–4239
Table 1. In vitro activity of indolinyl, tetrahydroquinolinyl and dihydrobenzoxazinyl oxazolidinones
Compd
*
R
X
Y
MIC (mg/mL)
SA1
SA2
SE
SP
EF
HI
MC
22a
22b
23a
23b
24a
24b
25a
25b
26a
27a
27b
28a
28b
(R)–
(S)–
(R)–
(S)–
(R)–
(S)–
(R)–
(S)–
(R)–
(R)–
(S)–
(R)–
(S)–
H
H
–CH2–
O
O
O
O
O
O
S
S
S
O
O
S
8
32
1
8
4
8
<0.5
2
<0.5
2
8
32
1
8
2
8
<0.5
2
<0.5
1
4
16
<0.5
4
0.5
4
—
<0.5
4
0.5
16
64
1
16
>64
>64
8
>64
8
>16
2
32
8
64
>64
4
64
8
16
1
16
2
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
CHO
CHO
C(O)CH2OH
C(O)CH2OH
CHO
2
2
1
8
<0.5
2
<0.5
1
<0.5
1
<0.5
<0.5
2
<0.5
<0.5
<0.5
<0.5
2
CHO
C(O)CH2OH
CHO
CHO
32
64
4
4
16
1
8
4
8
CHO
CHO
<0.5
1
<0.5
1
<0.5
<0.5
<0.5
<0.5
<0.5
2
S
16
2
29a
29b
30a
4
Linezolid (1)
(R)–
(S)–
(R)–
—
CHO
CHO
CHO
–CH2O–
–CH2O–
–CH2O–
–CH2–
—
O
O
S
O
—
4
8
2
8
1
4
1
4
0.5
1
4
32
1
4
1–2
64
64
32
16
16
8
32
2
8
8–16
<0.5
8
2–4
<0.5
4
2–4
<0.5
2
1–2
C(O)CH2OH
—
—
0.5–1
MIC, minimum inhibitory concentration; SA1, Staphylococcus aureus UC19213 (MSSA); SA2, S.aureus UC112673 (MRSA, CIPROR, RIFR,
IMIPENR); SE, Staphylococcus epidermidis UC112084 (MRSE); SP, Streptococcus pneumoniae UC19912 (PSSP); EF, Enterococcus faecalis
UC19217 (VSEF); HI, Haemophilus influenzae 30063 (AMPR, b-lactamase+); MC, Moraxella catarrhalis 30607 (b-lactamase+).
Table 2. Efficacy in a murine model and single-dose pharmacokinetics in rats
e
f
h
Compd
ED50 (mg/kg)a
Route
Dosed
(mg/kg)
Cmax
(mg/mL)
tmax
(h)
t1/2bg
(h)
Vss
CLi
(mL/min/kg)
Fj
(%)
(L/kg)
1.28
24a
23a
25a
1.8 (2.5)b
Not Tested
7.5 (4.0)b
iv
po20.73
iv
po19.76
iv
po19.2
9.92
15.1
nak
1.8
na
1.7
na
0.1
na
24
l
na
55.4
na
3.8
0.5
2.8
na
0.8
na
0.61
nc
nc
nc
9.84
9.83
16.1
13.6
1.27
0.64
18.4
56
28.9
7.2
0.5
0.5
na
na
1.13
30.8
109
Linezolid
5.6 (3.9)c
iv
po25
10
nc
na
1.05
0.95
na
0.72
10.5
15.8
0.3
nc
aS.aureus UC19213 systemic infection mouse model, po administration.
bOrally administered linezolid control in parentheses.
cSubcutaneously administered vancomycin control in parentheses.
dN=3.
eMaximum plasma concentration.
fTime at which Cmax achieved.
gHarmonic mean apparent terminal disposition half-life.
hSteady state volume of distribution.
iClearance.
jAbsolute oral bioavailability assuming linear pharmacokinetics.
kna, not applicable.
lnc, not calculated.
lower and the clearance (CL) typically higher than the
comparator.
might be inferred that the methyl of the (R)-diaste-
reomer is either occupying a hydrophobic pocket and/or
promoting a more energetically stable conformation for
adjacent nitrogen substituents that is preferred by the
binding site. In addition, the indoline derivatives (23–25a)
have reasonable oral bioavailability as well as in vivo
efficacy comparable to that of linezolid. With these results
in hand, further evaluation of related oxazolidinones
appears tobe warranted.
In summary, we have prepared a series of a-methylated
indolinyl, tetrahydroquinolyl and dihydrobenzoxazinyl
oxazolidinone derivatives which demonstrate a unique
stereochemical relationship with antibacterial activity.
The (R)-configurational isomers are always more active
and represent an interesting structure–activity trend. It

F.L.Ciske et al./ Bioorg.Med.Chem.Lett.13 (2003) 4235–4239
4239
References and Notes
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