Exploration of the activity of 7-pyrrolidino-8-methoxyisothiazoloquinolones against methicillin-resistant staphylococcus aureus (MRSA)

Article abstract of DOI:10.1021/jm101604v

Figure Presented. A series of 7-(3′-substituted)pyrrolidino-8- methoxyisothiazoloquinolone (ITQ) analogues were prepared, and their antibacterial potency against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aure

Full text of DOI:10.1021/jm101604v

ARTICLE
pubs.acs.org/jmc
Exploration of the Activity of 7-Pyrrolidino-8-
methoxyisothiazoloquinolones against Methicillin-Resistant
Staphylococcus aureus (MRSA)
Ha Young Kim, Jason A. Wiles, Qiuping Wang, Godwin C. G. Pais, Edlaine Lucien, Akihiro Hashimoto,
David M. Nelson, Jane A. Thanassi, Steven D. Podos, Milind Deshpande, Michael J. Pucci, and
Barton J. Bradbury*
Achillion Pharmaceuticals, Inc., 300 George Street, New Haven, Connecticut 06511, United States
ABSTRACT: A series of 7-(30-substituted)pyrrolidino-8-methoxyisothiazoloquinolone (ITQ) analogues were
prepared, and their antibacterial potency against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-
resistant Staphylococcus aureus (MRSA), and Escherichia coli were compared. Many of these analogues had MIC
e 0.25 μg/mL against quinolone-resistant MRSA strains. The stereochemical preference was explored for a
series of 100-methyl-30-aminomethylpyrrolidine analogues. Antibacterial activity was generally more favorable
with 30-R, 100-S configuration. Substitution on the 30-aminomethyl nitrogen tended to decrease activity, while
potency was maintained with disubstitution or aryl substitution at the 100-carbon. The 7-[(R)-3-((S)-1-aminoethyl)pyrrolidin-1-yl]
analogue (6a(R,S)) and the (R)-7-[3-(2-aminopropan-2-yl)pyrrolidin-1-yl] analogue (7a(R)) were found to be the ITQs with the
most promising antibacterial profiles. The MICs of these select ITQs versus a panel of clinical MRSA strains were determined, and the
ITQs were found to have 8- to 16-fold greater potency than linezolid. These analogues were also evaluated for inhibition of the target
enzymes, topoisomerase IV and DNA gyrase, from both wild-type and multidrug resistant strains. The ITQs were up to >30 times
more inhibitory against these targets than the fluoroquinolone moxifloxacin.
’ INTRODUCTION
several possible mechanisms still under investigation.13 The
ITQs, best represented by the direct ciprofloxacin analogue
A-628249 (1, Figure 1), were previously shown to be more
potent against S. aureus than their quinolone counterparts.
However, these initial compounds possessed selectivity and
toxicity issues14 and none were advanced into clinical develop-
ment. Recently, we reported new variations of ITQs and related
compounds containing functionalized aryls and heteroaryls
attached via a CꢀC bond at the 7-position15,16 and structural
modifications at positions 4a, 6, 8, and 9.17,18 Many of these
compounds demonstrated (i) excellent in vitro antibacterial
activities against MRSA, (ii) strong inhibitory activities against
DNA gyrase and topoisomerase IV, (iii) weak activity against
human topoisomerase II, and (iv) decreased cytotoxicity against
human cell lines relative to previously reported ITQs. These data
indicate that addition of an 8-methoxy group affected an increase
in activity against MRSA and a decrease in cytotoxicity against
human Hep2 laryngeal carcinoma cells. In our continuing efforts
to develop ITQs with potent anti-MRSA and improved pharma-
cological properties, we tested the extension of this finding to the
more traditional saturated aminocyclic type of 7-position sub-
stituents, particularly pyrrolidines that are known to enhance
Gram-positive activity.11,19,20 Here we report the synthesis, anti-
MRSA activity, cytotoxicity, and target enzyme activity of
8-methoxy ITQs with 7-aminocyclic substituents, focusing on a
Methicillin-resistant Staphylococcus aureus (MRSA) continues to
be a leading cause of nosocomial infections.1 Over the past several
years, community-associated MRSA strains have been involved in
an increasing number of serious infections not originating in the
hospital setting.2 Despite the availability of several classes of
antibiotics including the relatively recently introduced oxazolidi-
nones (linezolid) and lipopeptides (daptomycin), these infections
cause significant morbidity and mortality. Resistance to a number
of antibacterial agents has slowly, but steadily, increased over time
including reports of clinical isolates resistant to vancomycin, a
widely used antistaphylococcal drug.3,4 The current clinical land-
scape suggests that both the prudent use of existing drugs coupled
with new antibacterial discovery are required to combat these
serious medical issues.5 A valuable new addition to our antibac-
terial arsenal for MRSA infections would be a new bactericidal,
oral/iv agent effective against resistant isolates. The quinolone
class of antibacterials could fill this need; however, most MRSA
clinical isolates are sufficiently resistant to quinolones, rendering
them ineffective in successfully curing many infections.
Isothiazoloquinolones (ITQs, Figure 1), originally reported
by Chu and co-workers,6ꢀ9 are a class of potent antibacterial
agents related to the quinolones10 that also inhibit the type II
bacterial topoisomerases DNA gyrase (GyrA/GyrB) and topoi-
somerase IV (GrlA/GrlB).11,12 These compounds probably
resemble quinolones in their mechanism of action in that they
form stable ternary complexes with the topoisomerases and
cleaved DNA, thus acting as a “poison” and inhibiting DNA
replication.13 This leads to relatively rapid bacterial cell death by
Received: December 17, 2010
Published: March 22, 2011
r
2011 American Chemical Society
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potent series of 7-(30-aminomethylpyrrolidine) ITQs and the
effects of various substitutions and stereochemistry.
designation for the appended 30-N-substitution pattern in the
included table. The stereochemical configurations of compounds
with chiral 30- and 100-positions are designated in parentheses
(see, for example, compound 6b(R,S) shown in Figure 2). In
some cases the appended 3-position amine or alkylamine was
Boc-protected prior to the substitution reaction to improve
workup and purification of the 7-substituted ITQ and was then
subsequently deprotected with standard conditions to give the
target ITQ analogue. In no case of using an unprotected
appended amine did alkylation result in the unwanted regioi-
somer under these conditions. Also, the substitution reaction
resulted in varying degrees (up to 20%) of demethylation of the
starting ITQ core 2. This side product was removed during final
purification via preparative reverse-phase HPLC.
’ CHEMISTRY
Overview. The target ITQs were prepared according to
general Scheme 1 by nucleophilic aromatic substitution com-
monly used in the preparation of quinolones.11 The key ITQ
intermediate 2 was prepared according to published meth-
ods.18,21 The 7-position substituents of the corresponding target
compound sets 3ꢀ12 are listed in Figure 2 with a letter
Preparation of N-Alkyl-Substituted Pyrrolidines. Pyrroli-
dines used to make target compound sets 4ꢀ12 and their
isomers were obtained from commercial sources, prepared
following literature procedures22ꢀ30 or synthesized in manners
similar to the representative examples outlined below.
Reductive amination of 1-protected 3-aminomethylpyrroli-
dines with formic acid and formaldehyde24 led to dimethylation
and was used to prepare the pyrrolidines needed for 5k(S), 6k(R,
rac), 6k(R,S), and 7k(R). The monomethyl amines (b series
compounds) were prepared by simple reduction of the Boc-
protected amine.25 The monoethylamines (c) were prepared by
alkylation in the presence of cesium hydroxide according to the
procedure of Salvatore et al.31 The N-fluoroethyl intermediates
needed for preparation of 5d(S), 6d(R,R), 6d(R,S), and 7d(R)
were made by treatment of the primary amines with 2-fluoroethyl
4-methylbenzenesulfonate.32
Figure 1. Numbering scheme of isothiazoloquinolone 1 and quinolone
ciprofloxacin.
Scheme 1a
N-Protected pyrrolidine 13 was prepared according to a
published procedure22 and then used to synthesize pyrrolidine
intermediates 14ꢀ17 (Scheme 2). Compound 13 was alkylated
by reductive amination using commercially available cyclopro-
panecarboxaldehyde to afford N-(cyclopropylmethyl)-1-[(R)-
1-((S)-1-phenylethyl)pyrrolidin-3-yl]ethanamine 14 in 93% yield.
a Reagents and conditions: (a) piperazine or various pyrrolidines (1.1ꢀ
1.2 equiv), N,N-diisopropylethylamine (5 equiv), DMSO, 120 °C, 15 h;
(b) deprotection if needed; (c) purification by preparative HPLC,
10ꢀ44% yield overall.
Figure 2. Structures and numbering of the R7 substituents of ITQ series 3ꢀ12.
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Scheme 2a
Scheme 4a
a Reagents and conditions: (a) 2-nitropropane (excess), K2CO3 (0.1
equiv), EtOH, reflux; (b) cyclopropylamine (excess, neat), room temp;
(c) BH3 Me2S, room temp.
3
a Reagents and conditions. (a) For 14: c-PrCHO. For 15: (CH2)4CO.
For 16: CH3COCH3 (1.05 equiv). NaBH(OAc)3 (1.4 equiv), AcOH
(1 equiv), DCE, room temp, 6ꢀ24 h, 90ꢀ93%. (b) (CF3CO)2O (1.3
equiv), CHCl3, room temp, 15 h, quant; (c) LAH (2 equiv), THF, room
temp, 70 °C, 3 days, 87%.
Scheme 5a
Scheme 3a
a Reagents and conditions: (a) EDCI (1.5 equiv), HOBt (1.3 equiv),
2-methylpropan-1-amine (1.1 equiv) in DMF, room temp, 18 h, 84%;
(b) LAH (4 equiv) in THF reflux 18 h, quant; (c) EDCI (1.5 equiv),
HOBt (1.3 equiv), isobutyric acid (1 equiv) in DMF, room temp,
18 h, 76%.
a Reagents and conditions: (a) c-PrNH2 (1 equiv), NaBH(OAc)3 (1.4
equiv), AcOH (1 equiv), DCE, room temp, 24 h, 97% for 19a and 96%
for 19b; (b) BH3 THF (5 equiv), THF, room temp, 24 h, 94% for 20a
3
and 88% for 20b.
The N-cyclopropyl substituted pyrrolidine used for analogue
7h(rac) was prepared from maleimide (21) according to
Scheme 4. Reaction with 2-nitropropane gave the exocyclic
R,β-unsaturated maleimeide (22), which reacted slowly with
cyclopropylamine to produce adduct 23. This was reacted with-
out isolation with borane dimethyl sulfide complex to give the
desired intermediate 24, which was later debenzylated and
treated with ITQ core 2.
The isobutyl substituted pyrrolidine intermediates for analo-
gues 5g(rac) and 7g(R) were prepared from the commercially
available carboxy-substituted pyrrolidone 25 and intermediate
28, respectively, according to Scheme 5 below.
The same conditions were used to prepare cyclopentylamine 15
and isopropylamine 16 from cyclopentanone and acetone in 93%
and 90% yield, respectively. The trifluoroethylamine compound
17 was obtained by treatment of 13 with trifluoroacetic anhy-
dride in chloroform at room temperature to provide the trifluor-
oacetamide intermediate in quantitative yield. This intermediate
was subsequently reduced with lithium aluminum hydride
in refluxing tetrahydrofuran to give 17 in 87% yield. The
intermediates 14, 15, 16, and 17 were debenzylated under
standard reductive conditions (hydrogenolysis over Pearlman’s
catalyst) and were then used to prepare compounds 6i(R,rac),
6j(R,rac), 6f(R,rac), and 6e(R,rac), respectively, shown in
Scheme 1.
Preparation of the 3-(N-cyclopropyl-1-aminoethyl)pyrrolidine
side chain for compound 6h(R,rac) and 6h(S,rac) could not
be accomplished with this same reductive amination strategy, nor
could it be prepared from alkylation of the primary amine by
nucleophilic substitution with a halocyclopropane. Rather,
S-phenylethylpyrrolidinones 18a and 18b, prepared according
to previously published methods,22 were separately treated with
cyclopropylamine, sodium triacetoxyborohydride,33 and acetic
acid in 1,2-dicholoroethane to yield the N-cyclopropylpyrrolidi-
nones 19a and 19b (Scheme 3). Further reduction with a
solution of boraneꢀtetrahydrofuran complex provided the sec-
ondary amines 20a and 20b in high yield (91% and 88%,
respectively, over two steps). Debenzylation and subsequent
treatment with 2 gave compounds 6h(R,rac) and 6h(S,rac),
respectively. The corresponding pyrrolidine for preparation of N-
cyclopropyl analogue 5h(S) was prepared from R-methylbenzyl
protected (R)-3-carboxy-2-pyrrolidone according to the proce-
dure of Takemura.34
The 3-10-aminopropylpyrroldine diastereomers 9b(R,S) and
9b(R,R) were prepared from the common intermediate 32
(Scheme 6). Reduction of the cyclopropyl intermediate 31,
prepared according to Miyauchi35 and used for the synthesis of
8a(R), did not produce the expected N-methylcyclopropyl
intermediate but instead also effected ring-opening to give 32
as a mixture of diastereomers. This mixture was used directly to
prepare 9b(R,S) and 9b(R,R), which were separated/purified by
preparative HPLC.
Preparation of Heterocycle-Substituted Pyrrolidine Ana-
logues. Reports36 of bulkier and/or more lipophilic 7-substitu-
tents leading to increased activity against Gram-positive strains
prompted exploration of a series of heterocycles, such as
thialzolyl, oxazolyl, and pyridyl moieties, in the 100-position
of the 30-aminomethylpyrrolidines. We employed literature
procedures37ꢀ40 to provide the heterocycle-containing pyrroli-
dines necessary for ITQs 10a(R,rac), 11a(R,rac), 12a(R,rac)
shown in Figure 2.
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Scheme 6a
a Reagents and conditions: (a) LAH (4 equiv), THF, 0 °C f reflux, 18 h; (b) H2 (1 atm), Pd(OH)2 (cat.), EtOH, 45 °C; (c) 2 (0.9 equiv), N,N-
diisopropylethylamine (5 equiv), DMSO, 120 °C, 3 h; (d) preparative HPLC to separate isomers, 20% for 9b(R,S) and 7% for 9b(R,R).
’ BIOLOGICAL EVALUATION AND DISCUSSION
results in toxicity to 50% of the cells), demonstrating that
although cytotoxicity tracks with MIC, it can be separated.
Alkyl substitution on the 100-nitrogen of the 30-(100-methyl)
aminomethylpyrrolidine series (compound series 6) offered
improvement in cytotoxicity but did not improve Gram-positive
potency as seen for the 30-aminomethylpyrrolidine series 5.
Potency generally decreased with increased bulk of the substitu-
ent, and disubstitution of the nitrogen also decreased MQRSA
potency as demonstrated by comparing 6k(R,S) to its mono-
substituted counterpart 6a(R,S). Compound 6h(R,rac) with a
cyclopropyl substituent was equipotent to its 100-desmethyl
counterpart 5h(S) and its nonsubstituted counterpart 6a(R,rac)
with a greater than 2-fold advantage in cytotoxicity.
Each compound was screened for antibacterial activity using
standard techniques41 against methicillin-sensitive S. aureus (MSSA)
strain and a methicillin-resistant and quinolone-resistant S. aureus
(MQRSA) strain and compared to the marketed quinolones
moxifloxacin (MXF) and ciprofloxacin (CIP) as well as the
previously reported ITQ, 1 (Table 1). Breadth of spectrum was
addressed through testing against E. coli. Microbial specificity was
determined with a 72 h cytotoxicity assay using human Hep2 cells
(laryngeal carcinoma).42
To understand if the addition of an 8-methoxy group would
provide the same benefit previously reported16 of reducing
cytotoxicity while maintaining or increasing Gram-positive po-
tency, we first prepared compound 3, the 8-methoxy version of 1.
There was a 4-fold improvement in cytotoxicity, while the Gram-
positive potency remained the same; however, there was a loss of
potency against E. coli. Exploring other aminocyclics at the
7-position, we found that the 3-aminopyrrolidines 4a and 4b
were slightly more potent against MQRSA, consistent with
reported SAR of standard quinolones showing enhanced Gram-
positive activity of 7-pyrrolidines versus other aminocyclics.11,19,20,28
There was a slight stereochemical preference for the 30-S-isomers.
Alkyl substitution on the nitrogen (4b) both decreased potency and
improved cyctotoxicity.
The 100-dimethyl series 7 analogues were also highly potent
anti-MQRSA agents. Comparison with corresponding com-
pounds in the monomethyl series 6 showed a mixed trend for
MICs, with MQRSA moving slightly up or down but Gram-
negative MICs generally increasing. Cytotoxicity was diminished
(higher CC50) for all except 7d(R). The 100-cyclopropyl analogue
8a(R) had the strongest MSSA activity of all the compounds with
equally impressive MQRSA and E. coli MIC. The 100-ethyl
isomers 9b(R,S) and 9b(R,R) had slightly less antibacterial
activity than the corresponding 100-methyl series (6) compounds.
Interestingly, substitution with aryl groups on the 100-methy-
lene did not significantly impact MQRSA potency as exemplified
by compound 10a(R,rac) with an MIC of 0.06 μg/mL compared
to 0.06 μg/mL of the corresponding 100-methyl analogue 6a(R,
rac). One drawback to the series of 100-aryl susbstituted analo-
gues was the greater degree of cytotoxicity.
Further Evaluation of Select ITQs. Compounds 6a(R,S) and
7a(R) were chosen for further in vitro profiling because they
exhibited strong anti-MQRSA MICs of 0.06 μg/mL. We eval-
uated their in vitro antibacterial activities by performing suscept-
ibility testing against two panels of recent clinical isolates of
S. aureus (one panel of 10 strains and one panel of 70 strains).
Table 2 lists (i) the MIC ranges of the selected ITQs and
reference compounds against these panels of S. aureus strains,
(ii) the MICs at which 50% of the isolates are inhibited (MIC50),
and (iii) the MICs at which 90% of the isolates are inhibited
(MIC90). From a comparison of activity in the 10-strain panel,
the MIC50 of ITQ 6a(R,S) was 2-fold better than 7a(R), though
their MIC90 values were the same. Additional susceptibility
testing against a collection of 70 recent S. aureus clinical isolates
showed that 7a(R) was 256 times more potent than levofloxacin
(LFX) and 4- to 16-fold better than the clinical standard of care
against MRSA, vancomycin (VAN).
Insertion of a methylene between the 30-nitrogen and the
pyrrolidine ring, compounds 5a(S) and 5a(R), improved
MQRSA activity. Monoalkylation of the terminal amine of the
30-aminomethylpyrrolidine reduced cytotoxicity as seen with
5b(S), 5f(S), and 5h(S) while maintaining or improving
MQRSA activity. ITQ 5h(S) was exceptionally potent versus
MSSA and MQRSA with MICs of 0.004 and 0.06 μg/mL,
respectively. The increasing potency seen with increased
bulk prompted the synthesis of the isobutyl analogue 5g(rac);
however, MQRSA potency was greatly decreased to 4 μg/mL
(at best 2 μg/mL considering 2ꢁ dilution as the racemic
mixture). Dimethylation (5k(S)) also decreased anti-MQRSA
activity.
Substitution on the 100-methylene of the 30-aminomethylpyr-
rolidines to give the diastereomer series of compounds, 6a,
resulted in a differential in stereochemical preference. We ob-
served a strong preference for the 30-R-isomer, showing a 4- to 16-
fold improvement in MQRSA MIC over the 30-(S) isomers. Of all
the diastereomers, the 6a(R,S) isomer was the most potent with
an MIC of 0.06 μg/mL against the highly resistant S. aureus strain.
The stereochemical preference of the 100-carbon was pronounced
for the more potent 30-R isomer, with the 100-S isomer showing a
4-fold improvement against MQRSA (6a(R,R) vs 6a(R,S)).
However, no preference was seen when comparing the less active
100-S isomers 6a(S,R) and 6a(S,S). The stereochemistry did have
an impact on cytotoxicity for both diaseteromers, with the 100-S
isomers showing lower CC50 (the concentration of drug that
We further evaluated dual activities of 7a(R) and 6a(R,S)
along with the N-alkylated ITQs 6b(R,S) and 6d(R,S) against
their target enzymes topoisomerase IV and DNA gyrase. Anti-
bacterial agents having dual activities against these biochemical
targets are desirable, as they should reduce the selection of
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Table 1. Antibacterial and Cytotoxic Activities of 6-Fluoro-8-methoxy-ITQsa
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Table 1. Continued
a Abbreviations: MIC, minimum inhibitory concentration; hep2, Hep2 laryngeal carcinoma cells; MSSA, methicillin-sensitive Staphylococcus aureus
ATCC 29213; MQRSA, methicillin- and quinolone-resistant Staphylococcus aureus ATCC 700699; Ec, Escherichia coli ATCC 25922; CIP, ciprofloxacin;
MXF, moxifloxacin; n.e., not evaluated. b Minimum inhibitory concentrations (MICs) are expressed in μg/mL. c 72 h cytotoxic activities (CC50) are
expressed in μM. d Reference for the method of preparation of the 7-position pyrrolidine. e Not applicable. f Commercially available.
resistant organisms and should remain active against preexisting
strains with mutations in one of the two targets. Their activities
were compared with those of the fluoroquinolones CIP, gemi-
floxacin (GEM), and moxifloxacin (MFX). The in vitro results
reported in Table 3 are (i) MICs against S. aureus and E. coli and
(ii) inhibitory activities against their target enzymes from
S. aureus, wild-type topoisomerase IV and DNA gyrase, as well
as the corresponding enzymes from staphylococcal mutants
expressing fluoroquinolone resistance. The mutant enzymes
possess previously reported mutations in their quinolone-
resistance-determining regions (QRDRs).11 The select analo-
gues displayed stronger inhibition activities than the comparator
quinolones against the target enzymes especially against enzymes
from resistant bacteria. Notably, when compared with ciproflox-
acin, 7a(R) and 6a(R,S) with MRSA MIC of 0.09 and 0.06 μg/mL,
respectively, showed a 91-fold increase in inhibition of wild-type
DNA gyrase activity for 7a(R), and 6a(R,S) showed a 75-fold
increase in inhibition of wild-type topoisomerase IV activity for
6a(R,S). Similar improvements in inhibition of enzymes from
resistant bacteria were seen with >48-fold and >62-fold increases
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against mutant DNA gyrase for 7a(R) and 6a(R,S), respectively,
and 95-fold and 121-fold increases in inhibition observed for
mutant topoisomerase IV for 7a(R) and 6a(R,S), respectively.
Alkylated analogues 6b(R,S) and 6d(R,S) were similarly active
against target enzymes compared with the selected analogues
7a(R) and 6a(R,S). We note that this result demonstrated a
significant improvement in inhibition of the S. aureus enzymes and
provided a further basis for additional in vitro profiling. This
potent inhibition of both the gyrase and topoisomerase IV target
enzymes probably accounts for the excellent antibacterial activity
even against quinolone-resistant strains and suggests that resis-
tance development may be slow to emerge among clinical isolates.
’ SUMMARY OF RESULTS
Table 2. Activities of Select ITQs against Staphylococcal
Clinical Isolatesa
In conclusion, we described the syntheses and antibacterial
activities of 8-methoxy ITQs having structural modifications at
the 7-position. The structureꢀactivity and structureꢀtoxicity
relationships are summarized in Figure 3. We found that 7-(30-
aminomethylpyrrolidino) ITQs were generally more potent than
7-(30-aminopyrrolidine) analogues and that the R-isomer of the
30-methylaminopyrrolidines were more potent (up to 16-fold)
than the corresponding S-isomer. Substitution at the 100-carbon
of the 30-methylaminopyrrolidines improved potency, and the
S-100-methyl analogues were slightly more potent than the
corresponding R-isomers. The dimethyl substitution series (7)
and the cyclopropyl series (8) were also highly potent com-
pounds. Bulky substitution with aryl groups at the 100-position (9,
10, and 11) did not significantly diminish MRSA potency but
reduced bacterial selectivity as measured by toxicity against hep2
laryngeal cells. While bulk seemed tolerable at the 100-position,
N-substitution was generally not favorable except for N-cyclopropyl
MIC (μg/mL)
compd
organism (no. of strains)
range
50%
90%
6a(R,S)
7a(R)
7a(R)
LFX
MRSA (10)
MRSA (10)
MRSA (70)
MRSA (70)
MRSA (70)
MRSA (70)
MRSA (70)
MRSA (70)
0.004ꢀ0.5
0.008ꢀ1
0.008ꢀ0.5
0.12 to >16
1ꢀ2
0.06
0.13
0.06
16
0.5
0.5
0.25
>16
2
LZD
1
OXA
4 to >16
0.25ꢀ1
0.5ꢀ2
>16
0.5
1
>16
0.5
1
QPS/DPS
VAN
a Abbreviations: LFX, levofloxacin; LZD, linezolid; MIC, minimum
inhibitory concentration; MRSA, methicillin-resistant Staphylococcus
aureus; OXA, oxacillin; QPS, quinupristin; DPS, dalfopristin; VAN,
vancomycin; 50% and 90%, MIC50 and MIC90, respectively.
Table 3. In Vitro Activities of Select ITQsa
Sa enzyme Inhibitionb
MIC c
compd
TopoIV WT
TopoIV Ser80Phed
DNA gyrase WT
DNA gyrase Ser84Leud
MSSA
MRSA
Ec
64
CIP
3
57
62
>200
>200
>200
3.2
0.25
32
GEM
0.4
1
9.8
5.6
0.03
2
4
MXF
11.9
0.47
1.16
0.66
0.6
7.7
0.06
2
4
6a(R,S)
6b(R,S)
6d(R,S)
7a(R)
0.04
0.11
0.06
0.12
0.68
1.1
0.002
0.008
0.002
0.004
0.06
0.25
0.13
0.06
0.02
0.03
0.06
0.06
18.7
12
1.32
0.68
4.2
a Abbreviations: CIP, ciprofloxacin; Ec, Escherichia coli ATCC 25922; GEM, gemifloxacin; MRSA, methicillin-resistant Staphylococcus aureus ATCC
700699; MSSA, methicillin-sensitive Staphylococcus aureus ATCC 29213; MXF, moxifloxacin. b Inhibitions of wild-type and mutant S. aureus
topoisomerase IV (TopoIV) decatenation (IC50) and DNA gyrase supercoiling (IC50) are expressed in μM. The values listed are the averages of
multiple experiments. c Minimum inhibitory concentration (MIC) is expressed in μg/mL. The values listed are the modal values of multiple experiments.
d Indicates position of substituted amino acid due to mutation in gene that encodes enzyme product.
Figure 3. Summary of structureꢀactivity and structureꢀtoxicity relationships of 7-(30-aminomethylpyrrolidino) ITQs (substitution effects relative to
unsubstituted).
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and N-2-fluoroethyl analogues, particularly for more bulky
substituents and N-dimethylation.
temperature. The resulting mixture was filtered through a B€uchner
funnel on a layer of Celite, washing with methanol. The filtrate was
evaporated under reduced pressure to afford debenzylated pyrrolidiny-
lamine product in quantitative yields. The residue obtained was used in
the next coupling reaction without further purification.
Exceptionally potent compounds 6a(R,S) (MIC = 0.06 μg/mL)
and 7a(R) (MIC = 0.09 μg/mL) against MQRSA emerged from
this study. The MIC50 and MIC90 against a panel of MRSA strains
proved their superior efficacy against commercially established
Gram-positive antibacterial agents. This activity tracks with their
inhibition of target enzymes topoisomerase IV and DNA gyrase
from MRSA strains. These compounds seem to be much less
affected by mutations in the QRDRs of the target enzymes than
conventional quinolones, and this translates to improved anti-
bacterial activity against quinolone-resistant isolates.
Generally, isothiazoloquinolones are a viable class of antibacterial
agents with exceptional potency against MRSA strains, even those
that are quinolone resistant. Further in vitro and in vivo profiling
studies for 6a(R,S) and 7a(R) are underway in our laboratories to
evaluate their antibacterial activities and other pharmaceutical
properties. From preliminary single-dose studies (unpublished
results), data for 7a(R) indicated efficacy in both mouse sepsis
and thigh infection models with reductions in bacterial numbers
equivalent to or greater than those of positive control compound.
General Coupling Procedure for Compounds 3ꢀ12. The
general procedures for preparation and purification (preparative
RP-HPLC using mass-based fraction collection) of ITQ analogues are
outlined as follows. tert-Butyl (S)-1-((R)-pyrrolidin-3-yl)ethylcarbamate
(0.95 g, 4.44 mmol) and diisopropylethylamine (3.52 mL, 20.20 mmol)
were added to a suspension of 9-cyclopropyl-6,7-difluoro-8-methoxy-
isothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione (1.31 g, 4.04 mmol) in
dimethylsulfoxide (12.0 mL). The reaction mixture was heated on an oil
bath for 15 h at 120 °C and cooled to room temperature. The resulting
zsolution was purified by preparative RP-HPLC using mass-based
fraction collection to provide the desired mass only fractions. The
fractions of product were combined and lyophilized. The resulting
solid was suspended in ethyl acetate, treated with concentrated HCl
or a solution of 1.25 M HCl in methanol. The suspension was soni-
cated, centrifuged, and decanted to remove solvents. This consecu-
tive step of acidification and solidification was repeated three times.
The solid product obtained was dried under vacuum, dissolved in
water, and lyophilized to afford the desired product (596 mg, 30%,
HCl salt) as a brown solid. Yields and analytical data for the analogues
described in this report are listed below.
’ EXPERIMENTAL SECTION
General. All nonaqueous reactions were performed under an atmo-
sphere of dry Ar (99.99%) using oven-dried glassware and anhydrous
solvents. The purity of all target compounds (>95%) was verified via
HPLCꢀMS using the following two methods: (i) 20 min gradient
elution of increasing concentrations of CH3CN in water (5ꢀ95%)
containing 0.1% TFA with a flow rate of 1.0 mL/min and UV detection
at 254 nm on a Waters X-bridge C18 150 mm ꢁ 4.6 mm, 3.5 μm column
(method 1); (ii) 20 min gradient elution of increasing concentrations of
MeOH in water (5ꢀ95%) containing 0.1% TFA with a flow rate of
1.0 mL/min and UV detection at 254 nm on a Waters X-bridge C8 150
mm ꢁ 4.6 mm, 3.5 μm column (method 2). Low-resolution mass
spectra were recorded on a Thermo Finnigan Surveyor MSQ instrument
(operating in APCI mode) equipped with a Gilson liquid chromato-
graph. Unless noted otherwise, the quasi-molecular ions, [M þ H]þ,
observed in the low-resolution mass spectra were the base peaks. NMR
spectra were recorded using a Bruker Avance 300 spectrometer (1H at
9-Cyclopropyl-6-fluoro-8-methoxy-7-(piperazin-1-yl)isothia-
zolo[5,4-b]quinoline-3,4(2H,9H)-dione Hydrochloride (3). Yield:
32%. 1H NMR (DMSO-d6) δ 9.32 (br, 1H), 7.59 (d, JH,F = 12.2 Hz, 1H),
3.74 (m, 1H), 3.70 (s, 3H), 3.48 (br, 4H), 3.18 (br, 4H), 1.08 (m, 2H), 0.90
(m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.3 (s). LCMS: m/z calcd for
C18H19FN4O3S ([M]þ) 390; found 391 ([M þ H]þ).
(R)-7-(3-Aminopyrrolidin-1-yl)-9-cyclopropyl-6-fluoro-8-
methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione Hy-
drochloride (4a(R)). Yield: 26%. 1H NMR (DMSO-d6) δ 8.25 (br s,
2H), 7.58 (d, JH,F = 13.8 Hz, 1H), 3.89ꢀ3.60 (m, 6H), 3.56 (s, 3H),
2.32ꢀ2.26 (m, 1H), 2.07ꢀ2.01 (m, 1H), 1.15ꢀ1.12 (m, 2H), 0.95 (m,
2H). 19F NMR (DMSO-d6) δ ꢀ125.5 (s). LCMS: m/z calcd for
C18H19FN4O3S ([M]þ) 390; found 391 ([M þ H]þ).
(S)-7-(3-Aminopyrrolidin-1-yl)-9-cyclopropyl-6-fluoro-8-
methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione Hy-
drochloride (4a(S)). Yield: 30%. 1H NMR (TFA-d) δ 7.92 (d, JH,F
=
300.1 MHz, 13C at 75.5 MHz, and 19F at 282.4 MHz). All 13C and 19
F
13.2 Hz, 1H), 4.41ꢀ4.27 (m, 3H), 4.16 (m, 3H), 3.65 (s, 3H),
2.68ꢀ2.54 (m, 1H), 2.52ꢀ2.39 (m, 1H), 1.54ꢀ1.46 (m, 2H),
1.28ꢀ1.21 (m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.5 (s). LCMS:
m/z calcd for C18H19FN4O3S ([M]þ) 390; found 391 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-8-methoxy-7-[3-(methylamino)
pyrrolidin-1-yl]isothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione
Hydrochloride (4b(R)). Yield:20%. 1HNMR(DMSO-d6) δ9.06 (br s,
1H), 7.59 (d, JH,F = 13.5 Hz, 1H), 3.87ꢀ3.66 (m, 6H), 3.67 (s, 3H), 2.65
(br s, 3H), 2.39ꢀ2.24 (m, 1H), 2.18ꢀ2.06 (m, 1H), 1.17ꢀ1.11 (m, 2H),
0.98ꢀ0.93 (m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.3 (s). LCMS: m/z
calcd for C19H21FN4O3S ([M]þ) 404; found 405 ([M þ H]þ).
NMR spectra were broadband 1H decoupled. The chemical shifts for 1H
and 13C are reported in parts per million (δ) relative to external TMS and
were referenced to signals of residual protons in the deuterated solvent.
The chemical shifts for 19F are reported in parts per million (δ) relative to
1
13
13
external CFCl3. 1Hꢀ H COSY, 1Hꢀ C HMQC, 1Hꢀ C HMBC, and
13C APT spectra were used routinely for assignment of signals.
Boc Protection of 100-Position Amines. The general procedure
to protect primary or secondary 100-position amines of the pyrrolidines
was done as follows. The 100-position amine (1.0 equiv) was taken in
dichloromethane, and a solution of potassium carbonate (2.10 equiv) in
water (same volume to dichloromethane) was added. Di-tert-butyl
dicarbonate (1.05 equiv) was added to the solution, and the resulting
solution was stirred 18 h at room temperature. After completion of the
reaction, the layers were separated and the aqueous layer was extracted
with dichloromethane (three times). The combined organic layer was
dried over sodium sulfate and concentrated.
(S)-9-Cyclopropyl-6-fluoro-8-methoxy-7-[3-(methylamino)
pyrrolidin-1-yl]isothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione
1
Hydrochloride (4b(S)). Yield: 18%. H NMR (DMSO-d6) δ 9.28
(br s, 2H), 7.58 (d, JH,F = 14.1 Hz, 1H), 3.85ꢀ3.68 (m, 6H), 3.57 (s, 3H),
2.63 (t, J = 5.1 Hz, 3H), 2.39ꢀ2.28 (m, 1H), 2.20ꢀ2.11 (m, 1H),
1.18ꢀ1.12 (m, 2H), 0.98ꢀ0.92 (m, 2H). 19F NMR (DMSO-d6) δ
ꢀ125.4 (s). LCMS: m/z calcd for C19H21FN4O3S ([M]þ) 404; found
405 ([M þ H]þ).
Debenzylation of N-Benzylpyrrolidine. The general proce-
dure to remove the protective benzyl group of pyrrolidine analogues was
done as follows. A solution of each same amount (wt to wt ratio) of
N-benzylpyrrolidine and palladium hydroxide (20 wt % of Pd on carbon,
60% moisture) in ethanol (0.05 M solution) was heated on an oil bath at
40 °C for 12 h under hydrogen atmosphere and cooled to room
(S)-7-[3-(Aminomethyl)pyrrolidin-1-yl]-9-cyclopropyl-6-
fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (5a(S)). Yield: 18%. 1H NMR (D2O) δ 7.29
(d, JH,F = 15 Hz, 1H), 3.87ꢀ3.47 (m, 8H), 3.18ꢀ3.06 (m, 2H), 2.67ꢀ2.57
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Journal of Medicinal Chemistry
ARTICLE
(m, 1H), 2.27ꢀ2.14 (m, 1H), 1.82ꢀ1.67 (m, 1H), 1.25ꢀ1.16 (m, 2H),
1.05ꢀ0.95 (m, 2H). 19F NMR (D2O) δ ꢀ123.9 (s). LCMS: m/z calcd for
C19H21FN4O3S ([M]þ) 404; found 405 ([M þ H]þ).
1.29 (d, J = 6.5 Hz, 3H, methyl), 1.26 (d, J = 6.5 Hz, 3H, methyl
epimer), 1.14 (m, 2H, c-Pr), 0.95 (m, 2H, c-Pr). 19F NMR (DMSO-
d6) δ ꢀ125.4 (s, 1F), ꢀ125.5 (s, 1F). LCMS: m/z calcd for
C20H23FN4O3S ([M]þ) 418; found 419 ([M þ H]þ).
(R)-7-[3-(Aminomethyl)pyrrolidin-1-yl]-9-cyclopropyl-6-
fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (5a(R)). Yield: 22%. 1H NMR (D2O) δ 7.19
(d, JH,F = 14.1 Hz, 1H), 3.71ꢀ3.31 (m, 5H), 3.39 (s, 3H), 3.13ꢀ2.99 (m,
2H), 2.61ꢀ2.44 (m, 1H), 2.20ꢀ2.06 (m, 1H), 1.75ꢀ1.58 (m, 1H), 1.23ꢀ
1.06 (m, 2H), 1.01ꢀ0.84 (m, 2H). 19F NMR (D2O) δ ꢀ124.1 (s). LCMS:
m/z calcd for C19H21FN4O3S ([M]þ) 404; found 405 ([M þ H]þ).
(S)-9-Cyclopropyl-6-fluoro-8-methoxy-7-(3-((methylamino)-
methyl)pyrrolidin-1-yl)isothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (5b(S)). Yield: 14%. 1H NMR (DMSO-d6)
δ 8.98 (br s, 1H), 7.55 (d, J = 13.2 Hz, 1H), 3.58 (m, 3H), 3.46 (s, 3H),
2.74 (m, 1H), 2.51 (t, J = 5.2 Hz, 2H), 2.50 (s, 3H), 2.09 (m, 1H), 1.73
(m, 1H), 0.98 (m, 4H). 19F NMR (DMSO-d6) δ ꢀ125.6 (s). LCMS: m/z
calcd for C20H23FN4O3S ([M]þ) 418; found 419 ([M þ H]þ).
(S)-9-Cyclopropyl-6-fluoro-7-(3-((2-fluoroethylamino)-
methyl)pyrrolidin-1-yl)-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (5d(S)).Yield: 13%. 1HNMR(DMSO-
d6) δ9.37(br, 1H), 9.31(br, 1H), 7.55(d, JH,F = 14.0 Hz, 1H), 4.87 (t, J=4.6
Hz, 1H, -CH2F), 4.72 (t, J = 4.6 Hz, 1H, -CH2F), 3.81ꢀ3.68 (m, 2H), 3.62
(m, 2H), 3.52 (s, 3H, methoxy), 3.48 (m, 1H), 3.38 (m, 1H), 3.29 (m, 1H),
3.11 (m, 2H), 2.68 (m, 1H), 2.17 (m, 1H), 1.80 (m, 1H), 1.12 (m, 2H, c-Pr),
0.95 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.6 (s). LCMS: m/z calcd
for C21H24F2N4O3S ([M]þ) 450; found 451 ([M þ H]þ).
7-[(R)-3-((R)-1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-6-
fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione
Hydrochloride (6a(R,R)). Yield: 22%. 1H NMR (DMSO-d6) δ 8.28 (br,
3H), 7.54 (d, JH,F = 14.0 Hz, 1H), 3.82ꢀ3.50 (m, 2H), 3.51 (s, 3H,
methoxy), 3.50ꢀ3.43 (m, 3H), 3.27 (m, 1H), 2.41 (m, 1H), 2.19 (m, 1H),
1.76 (m, 1H), 1.25 (d, J = 6.5 Hz, 3H, methyl), 1.23 (m, 2H, c-Pr), 0.93 (m,
2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.5 (s). LCMS: m/z calcd for
C20H23FN4O3S ([M]þ) 418; found 419 ([M þ H]þ).
7-[(R)-3-((S)-1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (6a(R,S)). Yield: 30%. 1H NMR (DMSO-d6)
δ 8.15 (br, 3H), 7.55 (d, JH,F = 13.9 Hz, 1H), 3.86ꢀ3.51 (m, 3H), 3.52
(s, 3H, methoxy), 3.51ꢀ3.45 (m, 2H), 3.29 (m, 1H), 2.42 (m, 1H), 2.09
(m, 1H), 1.75 (m, 1H), 1.29 (d, J = 6.5 Hz, 3H, methyl), 1.14 (m, 2H,
c-Pr), 0.95 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS:
m/z calcd for C20H23FN4O3S ([M]þ) 418; found 419 ([M þ H]þ).
(S)-7-[3-(1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-6-
fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (6a(S,rac)). Yield: 22%. 1H NMR (DMSO-
d6) δ 8.13 (br, 3H), 7.56 (d, JH,F = 14.0 Hz, 1H), 7.55 (d, JH,F = 14.0
Hz, 1H, ArH epimer), 3.83ꢀ3.34 (m, 5H), 3.52 (s, 3H, methoxy),
3.27 (m, 1H), 2.48ꢀ2.03 (m, 2H), 1.76 (m, 1H), 1.29 (d, J = 6.5 Hz,
3H, methyl), 1.24 (d, J = 6.5 Hz, 3H, methyl epimer), 1.24 (m,
2H, c-Pr), 0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s,
1F), ꢀ125.5 (s, 1F). LCMS: m/z calcd for C20H23FN4O3S ([M]þ)
418; found 419 ([M þ H]þ).
(S)-9-Cyclopropyl-6-fluoro-7-(3-((isopropylamino)methyl)-
pyrrolidin-1-yl)-8-methoxyisothiazolo[5,4-b]quinoline-3,4
(2H,9H)-dione Hydrochloride (5f(S)). Yield: 25%. 1H NMR
(CD3OD) δ 7.53 (d, J = 13.8 Hz, 1H), 3.79ꢀ3.29 (m, 6H), 3.48
(s, 3H), 3.14ꢀ3.04 (m, 2H), 2.63ꢀ2.48 (m, 1H), 2.27ꢀ2.14 (m, 1H),
1.83ꢀ1.65 (m, 1H), 1.29 (d, J = 4.5 Hz, 6H), 1.21ꢀ1.08 (m, 2H),
0.98ꢀ0.87 (m, 2H). 19F NMR (CD3OD) δ ꢀ125.9 (s). LCMS: m/z
calcd for C22H27FN4O3S ([M]þ) 446; found 447 ([M þ H]þ).
9-Cyclopropyl-6-fluoro-7-(3-((isobutylamino)methyl)-
pyrrolidin-1-yl)-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (5g(rac)). Yield: 15%. 1H NMR
(CD3OD) δ 7.49 (d, J = 14.1 Hz, 1H), 3.72ꢀ3.51 (m, 5H), 3.46 (s, 3H),
3.17ꢀ3.08 (m, 2H), 2.87 (d, J = 6.3 Hz, 2H), 2.67ꢀ2.57 (m, 1H), 2.24ꢀ
2.17 (m, 1H), 2.04ꢀ1.98 (m, 1H), 1.81ꢀ1.75 (m, 1H), 1.15 (d, J = 6.3 Hz,
2H), 0.99ꢀ0.88 (m, 8H). 19F NMR (CD3OD) δ ꢀ125.6 (s). LCMS: m/z
calcd for C23H29FN4O3S ([M]þ).460; found 461 ([M þ H]þ).
7-[(S)-3-((R)-1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (6a(S,R)). Yield: 10%. 1H NMR (DMSO-d6)
δ 8.20 (br, 2H), 7.55 (d, JH,F = 13.9 Hz, 1H), 3.86ꢀ3.51 (m, 3H),
3.52 (s, 3H, methoxy), 3.51ꢀ3.45 (m, 2H), 3.29 (m, 1H), 2.42 (m, 1H),
2.09 (m, 1H), 1.75 (m, 1H), 1.29 (d, J = 6.5 Hz, 3H, methyl),
1.14 (m, 2H, c-Pr), 0.95 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ
ꢀ121.0 (s). LCMS: m/z calcd for C20H23FN4O3S ([M]þ) 418; found
419 ([M þ H]þ).
7-[(S)-3-((S)-1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (6a(S,S)). Yield: 35%. 1H NMR (DMSO-d6)
δ 7.42 (d, JH,F = 14.1 Hz, 1H), 3.63 (m, 2H), 3.45ꢀ3.33 (m, 3H), 3.36
(s, 3H, methoxy), 2.97 (m, 1H), 2.23ꢀ1.99 (m, 2H), 1.64 (m, 1H), 1.08
(d, J = 5.7 Hz, 3H, methyl), 1.04 (m, 2H, c-Pr), 0.82 (m, 2H, c-Pr). 19F
NMR (DMSO-d6) δ ꢀ126.0 (s). LCMS: m/z calcd for C20H23FN4O3S
([M]þ) 418; found 419 ([M þ H]þ).
(S)-9-Cyclopropyl-7-(3-((cyclopropylamino)methyl)py-
rrolidin-1-yl)-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (5h(S)). Yield: 15%. 1H NMR
(DMSO-d6) δ 9.46 (br, 2H), 7.53 (d, JH,F = 14.0 Hz, 1H), 7.16 (br, 1H),
3.81ꢀ3.66 (m, 2H), 3.61 (m, 2H), 3.55 (s, 3H, methoxy), 3.52ꢀ3.42 (m,
1H), 3.13 (m, 2H), 2.70 (m, 2H), 2.16 (m, 1H), 1.80 (m, 1H), 1.13 (m, 2H,
c-Pr), 0.96 (m, 4H, c-Pr), 0.73 (m, 2H, c-Pr). 19F NMR (DMSO-d6)
δ ꢀ120.8 (s). LCMS (APCI): m/z calcd for C22H25FN4O3S ([M]þ) 444;
found 445 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[1-(methyl-
amino)ethyl]pyrrolidin-1-yl}isothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6b(R,rac)). Yield: 39%. 1H
NMR (DMSO-d6) δ 9.08 (br, 1H), 8.89 (br, 1H), 7.55 (d, JH,F
=
14.0 Hz, 1H), 6.30 (br, 1H), 3.81ꢀ3.64 (m, 3H), 3.55 (m, 2H), 3.52 (s,
3H, methoxy), 3.29 (m, 1H), 2.56 (s, 3H, -NMe), 2.55 (s, 3H, -NMe
epimer), 2.28ꢀ2.03 (m, 2H), 1.78 (m, 1H), 1.29 (d, J = 6.6 Hz, 3H,
methyl), 1.25 (d, J = 6.6 Hz, 3H, methyl epimer), 1.13 (m, 2H, c-Pr),
0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z
calcd for C21H25FN4O3S ([M]þ) 432; found 433 ([M þ H]þ).
9-Cyclopropyl-6-fluoro-8-methoxy-7-{(R)-3-[(R)-1-(methyl-
amino)ethyl]pyrrolidin-1-yl}isothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6b(R,R)). Yield: 18%. 1H NMR
(DMSO-d6) δ 8.84 (br, 1H), 8.69 (br, 1H), 7.56 (d, JH,F = 14.0 Hz,
1H), 3.81ꢀ3.64 (m, 3H), 3.59ꢀ3.47 (m, 2H), 3.53 (s, 3H, methoxy),
3.29 (m, 1H), 2.58 (s, 3H, -NMe), 2.28ꢀ2.03 (m, 2H), 1.77 (m, 1H),
1.24 (d, J = 6.6 Hz, 3H, methyl), 1.14 (m, 2H, c-Pr), 0.96 (m, 2H, c-Pr).
(S)-9-Cyclopropyl-7-(3-((dimethylamino)methyl)pyrrolidin-1-
yl)-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
1
dione Hydrochloride (5k(S)). Yield: 27%. H NMR (DMSO-d6)
δ 7.50 (d, J = 14.2 Hz, 1H), 3.71 (m, 5H), 3.48 (m, 7H), 3.17 (t, J =
6.3 Hz, 1H), 2.74 (m, 4H), 2.18 (m, 1H), 1.71 (m, 1H), 1.07 (m, 2H),
0.89 (m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.7 (s). LCMS: m/z calcd
for C21H25FN4O3S ([M]þ) 432; found 433 ([M þ H]þ).
(R)-7-[3-(1-Aminoethyl)pyrrolidin-1-yl]-9-cyclopropyl-6-
fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
dione Hydrochloride (6a(R,rac)). Yield: 38%. 1H NMR (DMSO-
d6) δ 8.23 (br, 1H), 8.20 (br, 1H), 7.56 (d, JH,F = 14.0 Hz, 1H), 6.20
(br, 2H), 3.82ꢀ3.50 (m, 3H), 3.60ꢀ3.45 (m, 2H), 3.52 (s, 3H,
methoxy), 3.28 (m, 1H), 2.41 (m, 1H), 2.14 (m, 1H), 1.76 (m, 1H),
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Journal of Medicinal Chemistry
ARTICLE
19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd for
C21H25FN4O3S ([M]þ) 432; found 433 ([M þ H]þ).
δ ꢀ125.5 (s). LCMS: m/z calcd for C22H24F4N4O3S ([M]þ) 500; found
501 ([M þ H]þ).
9-Cyclopropyl-6-fluoro-8-methoxy-7-{(R)-3-[(S)-1-(methyl-
amino)ethyl]pyrrolidin-1-yl}isothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6b(R,S)). Yield: 23%. 1H NMR
(DMSO-d6) δ 9.07 (br, 1H), 8.86 (br, 1H), 7.55 (d, JH,F = 13.9 Hz,
1H), 3.82ꢀ3.64 (m, 3H), 3.61ꢀ3.45 (m, 2H), 3.55 (s, 3H, methoxy),
3.29 (m, 1H), 2.78ꢀ2.66 (m, 1H), 2.55 (s, 3H, -NMe), 2.07 (m, 1H),
1.75 (m, 1H), 1.29 (d, J = 6.6 Hz, 3H, methyl), 1.14 (m, 2H, c-Pr), 0.95
(m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd
for C21H25FN4O3S ([M]þ) 432; found 433 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-7-{3-[1-(isopropylamino)ethyl]-
pyrrolidin-1-yl}-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,
9H)-dione Hydrochloride (6f(R,rac)). Yield: 17%. 1H NMR (DMSO-
d6) δ 9.01 (br, 1H), 8.44 (br, 1H), 7.55 (d, JH,F = 14.0 Hz, 1H), 3.82ꢀ3.62
(m, 3H), 3.60ꢀ3.32 (m, 3H), 3.53 (s, 3H, methoxy), 2.52 (m, 1H),
2.36ꢀ2.04 (m, 2H), 1.79 (m, 1H), 1.37ꢀ1.19 (m, 9H), 1.13 (m, 2H, c-Pr),
0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd
for C23H29FN4O3S ([M]þ) 460; found 461 ([M þ H]þ).
(R)-9-Cyclopropyl-7-{3-[1-(cyclopropylamino)ethyl]pyrroli-
din-1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4-
(S)-9-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[1-(methyl-
amino)ethyl]pyrrolidin-1-yl}isothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6b(S,rac)). Yield: 19%. 1H
1
(2H,9H)-dione Hydrochloride (6h(R,rac)). Yield: 37%. H NMR
(DMSO-d6) δ 9.40 (br, 1H), 9.18 (br, 1H), 7.55 (d, JH,F = 14.0 Hz, 1H),
3.82ꢀ3.64 (m, 3H), 3.62ꢀ3.39 (m, 3H), 3.52 (s, 3H, methoxy), 2.70 (m,
2H), 2.31ꢀ2.05 (m, 1H), 1.81 (m, 1H), 1.38 (d, J = 6.6 Hz, 3H, methyl),
1.35 (d, J = 6.7 Hz, 3H, methyl epimer), 1.17ꢀ0.75 (m, 8H, c-Pr). 19F
NMR (DMSO-d6) δ ꢀ125.2 (s, 1F), ꢀ125.4 (s, 1F). LCMS: m/z calcd
for C23H27FN4O3S ([M]þ) 458; found 459 ([M þ H]þ).
NMR (DMSO-d6) δ 8.99 (br, 1H), 8.70 (br, 1H), 7.55 (d, JH,F
=
14.0 Hz, 1H), 3.82ꢀ3.61 (m, 3H), 3.60ꢀ3.37 (m, 2H), 3.52 (s, 3H,
methoxy), 3.29 (m, 1H), 2.79ꢀ2.67 (m, 1H), 2.56 (s, 3H, -NMe), 2.55
(s, 3H, -NMe epimer), 2.29ꢀ2.04 (m, 1H), 1.76 (m, 1H), 1.29 (d,
J = 6.6 Hz, 3H, methyl), 1.25 (d, J = 6.6 Hz, 3H, methyl epimer), 1.14
(m, 2H, c-Pr), 0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4
(s). LCMS: m/z calcd for C21H25FN4O3S ([M]þ) 432; found 433
([M þ H]þ).
(S)-9-Cyclopropyl-7-{3-[1-(cyclopropylamino)ethyl]pyrroli-
din-1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4-
1
(2H,9H)-dione Hydrochloride (6h(S,rac)). Yield: 26%. H NMR
(R)-9-Cyclopropyl-7-{3-[1-(ethylamino)ethyl]pyrrolidin-1-yl}-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-
(DMSO-d6) δ 9.32 (br, 1H), 9.12 (br, 1H), 7.55 (d, JH,F = 14.0 Hz, 1H),
7.20 (br, 1H), 3.81ꢀ3.64 (m, 3H), 3.64ꢀ3.38 (m, 3H), 3.52 (s, 3H,
methoxy), 2.73 (m, 2H), 2.30ꢀ2.05 (m, 1H), 1.79(m, 1H), 1.38(d, J=6.7
Hz, 3H, methyl), 1.35 (d, J = 6.7 Hz, 3H, methyl epimer), 1.17ꢀ0.75 (m,
8H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd for
C23H27FN4O3S ([M]þ) 458; found 459 ([M þ H]þ).
1
dione Hydrochloride (6c(R,rac)). Yield: 25%. H NMR (DMSO-
d6) δ 9.04 (br, 1H), 8.73 (br, 1H), 7.56 (d, JH,F = 14.0 Hz, 1H), 6.52 (br,
1H), 3.81ꢀ3.68 (m, 3H), 3.56 (m, 2H), 3.52 (s, 3H, methoxy), 3.34 (m,
1H), 3.03ꢀ2.96 (m, 2H), 2.59 (m, 1H), 2.26ꢀ2.04 (m, 1H), 1.79 (m,
1H), 1.33ꢀ1.19 (m, 6H), 1.13 (m, 2H, c-Pr), 0.94 (m, 2H, c-Pr). 19F
NMR (DMSO-d6) δ ꢀ125.3 (s, 1F), ꢀ125.4 (s, 1F). LCMS: m/z calcd
for C22H27FN4O3S ([M]þ) 446; found 447 ([M þ H]þ).
(R)-9-Cyclopropyl-7-{3-[1-(cyclopropylmethylamino)ethyl]-
pyrrolidin-1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoli-
1
ne-3,4(2H,9H)-dione Hydrochloride (6i(R,rac)). Yield: 12%. H
9-Cyclopropyl-7-{(R)-3-[(S)-1-(ethylamino)ethyl]pyrrolidin-
1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4
(2H,9H)-dione Hydrochloride (6c(R,S)). Yield: 27%. 1H NMR
(DMSO-d6) δ 9.15 (br, 1H), 8.81 (br, 1H), 7.49 (d, JH,F = 14.0 Hz, 1H),
7.20 (br, 1H), 3.79ꢀ3.59 (m, 3H), 3.49 (m, 2H), 3.48 (s, 3H, methoxy),
3.30 (m, 1H), 2.95 (m, 2H), 2.61 (m, 1H), 2.06 (m, 1H), 1.78 (m, 1H),
1.27 (d, J = 6.6 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H), 1.09 (m, 2H, c-Pr), 0.90
(m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd
for C22H27FN4O3S ([M]þ) 446; found 447 ([M þ H]þ).
NMR (DMSO-d6) δ 9.08 (br, 1H), 8.82 (br, 1H), 7.55 (d, JH,F = 14.0 Hz,
1H), 3.81ꢀ3.62 (m, 2H), 3.61ꢀ3.46 (m, 2H), 3.52 (s, 3H, methoxy), 3.38
(m, 1H), 2.99ꢀ2.51 (m, 3H), 2.49 (m, 2H), 2.30ꢀ2.03 (m, 1H), 1.79
(m, 1H), 1.31 (d, J = 6.6 Hz, 3H, methyl), 1.27 (d, J = 6.7 Hz, 3H, methyl
epimer), 1.13 (m, 2H, c-Pr), 0.94 (m, 2H, c-Pr), 0.58 (m, 2H, c-Pr), 0.39
(m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd for
C24H29FN4O3S ([M]þ) 472; found 473 ([M þ H]þ).
(R)-7-{3-[1-(Cyclopentylamino)ethyl]pyrrolidin-1-yl}-9-cyclo-
propyl-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,
9H)-dione Hydrochloride (6j(R,rac)). Yield: 44%. 1H NMR (DMSO-
d6) δ9.27 (br, 1H), 8.78 (br, 1H), 7.55 (d, JH,F = 14.0 Hz, 1H), 6.53 (br, 1H),
3.92ꢀ3.46 (m, 5H), 3.52 (s, 3H, methoxy), 3.37 (m, 1H), 2.68 (m, 1H),
2.50ꢀ1.91 (m, 4H), 1.89ꢀ1.61 (m, 5H), 1.54 (m, 2H), 1.33 (d, J = 6.6 Hz,
3H, methyl), 1.30 (d, J = 6.6 Hz, 3H, methyl epimer), 1.16 (m, 2H, c-Pr),
0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.3 (s, 1F), ꢀ125.4
(s, 1F). LCMS: m/z calcd for C25H31FN4O3S ([M]þ) 486; found 487
([M þ H]þ).
9-Cyclopropyl-6-fluoro-7-{(R)-3-[(R)-1-(2-fluoroethylamino)-
ethyl]pyrrolidin-1-yl}-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6d(R,R)). Yield: 29%. 1H NMR
(DMSO-d6) δ 9.41 (br, 1H), 9.19 (br, 1H), 7.55 (d, JH,F = 13.9 Hz, 1H),
6.20 (br, 1H), 4.91 (m, 1H, -CH2F), 4.75 (m, 1H, -CH2F), 3.82ꢀ3.63 (m,
2H), 3.60ꢀ3.25 (m, 6H), 3.53 (s, 3H, methoxy), 2.63 (m, 1H), 2.26 (m,
1H), 1.81 (m, 1H), 1.30 (d, J= 6.6 Hz, 3H), 1.13 (m, 2H, c-Pr), 0.94 (m, 2H,
c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s). LCMS: m/z calcd for
C22H26F2N4O3S ([M]þ) 464; found 465 ([M þ H]þ).
(R)-9-Cyclopropyl-7-{3-[1-(dimethylamino)ethyl]pyrrolidin-
1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,
9H)-dione Hydrochloride (6k(R,rac)). Yield: 36%. 1H NMR (DMSO-
d6) δ 10.17 (br, 1H), 7.55 (d, JH,F = 14.0 Hz, 1H), 3.82ꢀ3.70 (m, 3H),
3.70ꢀ3.44 (m, 3H), 3.52 (s, 3H, methoxy), 2.75 (m, 3H), 2.69 (m, 3H),
2.56 (m, 1H), 2.39ꢀ2.03 (m, 1H), 1.76 (m, 1H), 1.30 (d, J = 6.7 Hz,
3H, methyl), 1.25 (d, J = 6.7 Hz, 3H, methyl epimer), 1.15 (m, 2H, c-Pr),
0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6) δ ꢀ125.4 (s, 1F), ꢀ125.5
(s, 1F). LCMS: m/z calcd for C22H27FN4O3S ([M]þ) 446; found 447
([M þ H]þ).
9-Cyclopropyl-6-fluoro-7-{(R)-3-[(S)-1-(2-fluoroethylamino)-
ethyl]pyrrolidin-1-yl}-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (6d(R,S)). Yield: 14%. 1H NMR
(DMSO-d6) δ 9.32 (br, 1H), 9.09 (br, 1H), 7.55 (d, JH,F = 13.9 Hz, 1H),
4.88 (m, 1H, -CH2F), 4.72 (m, 1H, -CH2F), 3.82ꢀ3.64(m, 3H),3.61ꢀ3.25
(m, 5H), 3.53 (s, 3H, methoxy), 2.62 (m, 1H), 2.09 (m, 1H), 1.80 (m, 1H),
1.34 (d, J = 6.6 Hz, 3H), 1.16 (m, 2H, c-Pr), 0.94 (m, 2H, c-Pr). 19F NMR
(DMSO-d6) δ ꢀ125.3 (s). LCMS: m/z calcd for C22H26F2N4O3S ([M]þ)
464; found 465 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-8-methoxy-7-{3-[1-(2,2,2-trifluor-
oethylamino)ethyl]pyrrolidin-1-yl}isothiazolo[5,4-b]quinoline-
3,4(2H,9H)-dione Hydrochloride (6e(R,rac)). Yield: 15%. 1H NMR
(DMSO-d6) δ7.56(d, JH,F = 14.0 Hz, 1H), 4.09ꢀ3.16 (m, 9H), 3.52 (s, 3H,
methoxy), 2.26ꢀ1.99 (m, 2H), 1.76 (m, 1H), 1.28 (d, J = 6.5 Hz, 3H,
methyl), 1.13 (m, 2H, c-Pr), 0.94 (m, 2H, c-Pr). 19F NMR (DMSO-d6)
9-Cyclopropyl-7-{(R)-3-[(S)-1-(dimethylamino)ethyl]pyrroli-
din-1-yl}-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,
1
4(2H,9H)-dione Hydrochloride (6k(R,S)). Yield: 31%. H NMR
(DMSO-d6) δ 10.07 (br, 1H), 7.52 (d, JH,F = 14.4 Hz, 1H), 3.73
(m, 2H), 3.57ꢀ3.41 (m, 2H), 3.49 (s, 3H, methoxy), 3.19ꢀ2.96 (m,
1H), 2.31ꢀ1.84 (m, 3H), 2.16 (s, 6H, N,N-dimethyl), 1.61ꢀ1.35 (m, 1H),
3277
dx.doi.org/10.1021/jm101604v |J. Med. Chem. 2011, 54, 3268–3282

Journal of Medicinal Chemistry
ARTICLE
1
1.11 (m, 2H), 0.98ꢀ0.82 (m, 5H). 19F NMR (DMSO-d6) δ ꢀ126.1
(s). LCMS: m/z calcd for C22H27FN4O3S ([M]þ) 446; found 447
([M þ H]þ).
ylidene)pyrrolidine-2,5-dione (22). H NMR (CDCl3) δ 7.42ꢀ7.25
(m, 5H), 4.69 (s, 2H), 3.20 (t, J = 1.5 Hz, 2H), 2.34 (t, J = 1.2 Hz, 3H),
1.86 (s, 3H). 13C NMR (CDCl3) δ 173.5, 169.4, 149.7, 136.3, 128.8,
128.6, 127.7, 119.0, 41.9, 34.1, 24.1, 20.7. A mixture of 22 (2.0 g, 8.7
mmol) and cyclopropylamine (10 mL) was stirred for 50 days at room
temperature. Excess cyclopropylamine was then removed in vacuo. To
(R)-7-[3-(2-Aminopropan-2-yl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dio-
ne Hydrochloride (7a(R)). Yield: 21%. 1H NMR (TFA-d) δ 8.00 (d,
JH,F = 13.0 Hz, 1H, aromatic H-5), 4.20 (m, 1H, pyrrolidine H-50),
4.13ꢀ3.94 (m, 4H, c-Pr CH, pyrrolidine H-2, H-20, and H-5), 3.75 (s, 3H,
methoxy), 3.00 (m, 1H, pyrrolidine H-3), 2.42 (m, 1H, pyrrolidine H-40),
2.16 (m, 1H, pyrrolidine H-4), 1.62 (s, 3H, pyrrolidine CH3), 1.60 (s, 3H,
pyrrolidine CH3), 1.48 (m, 2H, c-Pr), 1.22 (m, 2H, c-Pr). 19F NMR (D2O):
δ ꢀ124.4 (s). LCMS: m/z calcd for C21H25FN4O3S ([M]þ) 432; found
433 ([M þ H]þ).
the remaining residue (crude 23) was added BH3 Me2S (10 mL, neat),
3
and the mixture was stirred for 30 days at room temperature. To the
reaction mixture was added H2O and aqueous NaOH. The mixture was
then refluxed for 2 h and extracted with CH2Cl2 (3 ꢁ 20 mL). The
organic layer was separated, dried (Na2SO4), and evaporated under
reduced pressure. The remaining residue (crude 24) was dissolved in
CH2Cl2 (20 mL) and treated with Boc2O (2.09 g, 9.6 mmol) and DMAP
(0.05 g). The reaction mixture was stirred for 15 h at room temperature
and then treated with H2O. The mixture was extracted with CH2Cl2
(3 ꢁ 20 mL), and the combined organic extracts were dried (Na2SO4)
and evaporated under reduced pressure. The residue was treated with
EtOH (20 mL) and 20% Pd(OH)2/C (1.0 g). Then the mixture was
stirred for 15 h at 60 °C under an atmosphere of H2 (∼1 atm). The sup-
ported catalyst was removed by filtration and the solvent removed under
reduced pressure. The remaining residue was treated with 2 (0.45 g,
1.4 mmol), i-Pr2NEt (1 mL), and DMSO (10 mL), and the mixture was
heated for 7 h at 120 °C. The solvent was removed in vacuo and the
remaining residue was purified by HPLC to give the TFA salt. This
material was treated with AcOEt (10 mL) and concentrated HCl
(0.3 mL), then dried in vacuo to afford 180 mg of the title compound.
1H NMR (DMSO-d6) δ 9.22 (bs, NH, 1H), 9.04 (bs, NH, 1H), 7.57 (d,
JH,F = 13.2 Hz, H-5, 1H), 3.83ꢀ3.70 (m, c-Pr CH, 2H), 3.59ꢀ3.48 (m,
CH2, 2H), 3.17 (s, OCH3, 3H), 2.85ꢀ2.71 (m, CH2, 2H), 2.14ꢀ2.02
(m, CH2, 1H), 1.91ꢀ1.74 (m, CH2, 1H), 1.42 (s, CH3, 3H), 1.40 (s,
CH3, 3H), 1.36ꢀ0.70 (m, c-Pr CH2, 8H). 19F NMR (DMSO-d6)
δ ꢀ125.1 (s).
(S)-7-[3-(2-Aminopropan-2-yl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dio-
ne Hydrochloride (7a(S)). Yield: 28%. 1HNMR(CD3CO2D) δ7.80(d,
JH,F = 13.9 Hz, 1H), 4.00ꢀ3.78 (m, 3H), 3.69 (m, 1H), 3.65 (s, 3H,
methoxy), 2.77 (m, 1H), 2.24 (m, 1H), 2.15ꢀ1.81 (m, 4H), 1.55 (s, 6H,
dimethyl), 1.31 (m, 2H, c-Pr), 1.14 (m, 2H, c-Pr). 19F NMR (CD3CO2D)
δ ꢀ124.6 (s). LCMS: m/z calcd for C21H25FN4O3S ([M]þ) 432; found
433 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-8-methoxy-7-(3-(2-(methylamino)-
propan-2-yl)pyrrolidin-1-yl)isothiazolo[5,4-b]quinoline-3,4
(2H,9H)-dione Hydrochloride (7b(R)). Yield: 45%. 1H NMR
(DMSO-d6) δ 8.93 (br s, 1H), 7.55 (d, J = 13.8 Hz, 1H), 3.77 (m,
3H), 3.61 (m, 1H), 3.54 (s, 3H), 3.54 (m, 2H), 2.63 (m, 1H), 2.47 (s,
3H), 2.04 (m, 1H), 1.81 (m, 1H), 1.32 (s, 3H), 1.29 (s, 3H), 1.34 (m,
2H), 0.93 (m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.2 (s). LCMS: m/z
calcd for C22H27FN4O3S ([M]þ) 446; found 447 ([M þ H]þ).
(R)-9-Cyclopropyl-7-(3-(2-(ethylamino)propan-2-yl)pyrroli-
din-1-yl)-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4-
(2H,9H)-dione Hydrochloride (7c(R)). Yield: 34%. 1H NMR
(DMSO-d6) δ 8.75 (br s, 1H), 7.55 (d, J = 14.9 Hz, 1H), 3.61 (m, 5H),
3.53 (s, 3H), 2.99 (m, 2H), 2.65 (m, 1H), 2.05 (m, 1H), 1.81 (m, 1H),
1.36 (s, 3H), 1.33 (s, 3H), 1.27 (t, J = 7.3 Hz, 3H), 1.14 (m, 2H), 0.94
(m, 2H). 19F NMR (DMSO-d6) δ ꢀ125.1 (s). LCMS: m/z calcd for
C23H29FN4O3S ([M]þ) 460; found 461 ([M þ H]þ).
(R)-7-[3-(1-Aminocyclopropyl)pyrrolidin-1-yl]-9-cyclopropyl-
6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dio-
ne Hydrochloride (8a(R)). Yield: 10%. 1H NMR (CD3OD) δ 7.69 (d,
JH,F = 14.1 Hz, 1H), 3.89ꢀ3.59 (m, 5H), 3.60 (s, 3H), 2.83ꢀ2.56 (m, 1H),
2.20ꢀ2.13 (m, 1H), 1.81ꢀ1.67 (m, 1H), 1.27ꢀ1.25 (m, 2H), 1.04ꢀ1.00
(m, 6H). 19F NMR (CD3OD) δ ꢀ126.2 (s). LCMS: m/z calcd for
C21H23FN4O3S ([M]þ) 430; found 431 ([M þ H]þ).
(R)-9-Cyclopropyl-6-fluoro-7-(3-(2-(2-fluoroethylamino)pro-
pan-2-yl)pyrrolidin-1-yl)-8-methoxyisothiazolo[5,4-b]quinoline-
3,4(2H,9H)-dione Hydrochloride (7d(R)). Yield: 35%. 1H NMR
(DMSO-d6) δ 7.49 (d, J = 13.8 Hz, 1H), 4.85 (t, J = 4.2 Hz, 1H), 4.69
(t, J = 6.0 Hz, 1H), 3.48 (s, 3H), 3.74ꢀ3.27 (m, 7H), 2.69ꢀ2.58 (m, 1H),
2.06ꢀ1.96 (m, 1H), 1.84ꢀ1.69 (m, 1H), 1.33 (d, J= 7.5 Hz, 6H), 1.12ꢀ1.05
(m, 2H), 0.92ꢀ0.85 (m, 2H). 19FNMR(DMSO-d6) δ18.3 (s, 1F), ꢀ125.2
(s, 1F). LCMS: m/z calcd for C23H28F2N4O3S ([M]þ) 478; found 479
([M þ H]þ).
9-Cyclopropyl-6-fluoro-8-methoxy-7-((R)-3-((S)-1-(methyla-
mino)propyl)pyrrolidin-1-yl)isothiazolo[5,4-b]quinoline-3,4-
(2H,9H)-dione Hydrochloride (9b(R,S)). Amide 31 (1.2g, 3.5mmol)
in THF (10 mL) was added dropwise to a suspension of LAH (0.53 g,
14.0 mmol) in THF (10 mL) at 0 °C. After the addition was complete, the
reaction mixture was refluxed for 18 h, cooled to 0 °C, and quenched
carefully by the addition of 10% aqueous NaOH. The resulting white
precipitate was removed by filtration, and the filtrate was transferred to a
separatory funnel. The layers were separated, and the aqueous layer was
extracted with EtOAc. The organic extract and layer were combined, dried
(Na2SO4), and evaporated under reduced pressure to give 0.8 g of amine 32
as a light yellow oil. This crude material (mixture of diastereomers) was
used without purification for the synthesis of 9b(R,S) and 9b(R,R) via
subsequent debenzylation, treatment with 2, and HPLC purification. Yield:
20%. 1H NMR (CD3OD) δ 7.54 (d, J = 14.4 Hz, 1H), 3.85ꢀ3.45 (m, 6H),
3.50 (s, 3H), 2.68 (s, 3H), 2.62ꢀ2.48 (m, 1H), 2.15ꢀ2.10 (m, 1H),
1.94ꢀ1.71 (m, 3H), 1.15ꢀ1.14 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H),
0.98ꢀ0.84 (m, 2H). 19F NMR (CD3OD) δ ꢀ126.0. LCMS: m/z calcd
for C22H27FN4O3S 446 ([Mþ]); found 447 ([M þ H]þ,).
(R)-9-Cyclopropyl-6-fluoro-7-(3-(2-(isobutylamino)propan-2-
yl)pyrrolidin-1-yl)-8-methoxyisothiazolo[5,4-b]quinoline-3,4-
(2H,9H)-dione Hydrochloride (7g(R)).Yield: 5%. 1HNMR(CD3OD)
δ 7.65 (d, J = 9.3 Hz, 1H), 3.97ꢀ3.57 (m, 5H), 3.57 (s, 3H), 2.97 (d, J = 6.3
Hz, 2H), 2.84ꢀ2.75 (m, 1H), 2.17ꢀ1.88 (m, 3H), 1.51 (s, 3H), 1.48
(s, 3H), 1.33ꢀ1.24 (m, 2H), 1.10 (d, J = 6.3 Hz, 8H). 19F NMR (CD3OD)
δ ꢀ124.6 (s). LCMS: m/z calcd for C25H33FN4O3S ([M]þ) 488; found
489 ([M þ H]þ).
9-Cyclopropyl-7-(3-(2-(cyclopropylamino)propan-2-yl)pyrr-
olidin-1-yl)-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,
4(2H,9H)-dione Hydrochloride (7h(rac)). A mixture of 1-benzyl-
1H-pyrrole-2,5-dione 21 (5.0 g, 26.7 mmol), 2-nitropropane (50 mL),
K2CO3 (0.5 g, 3.6 mmol), and EtOH (25 mL) was refluxed for 15 h. The
volatiles were then removed under reduced pressure, and the remaining
residue was treated with CHCl3 (30 mL) and H2O (30 mL). The
organic layer was separated, dried (Na2SO4), and evaporated under
reduced pressure. The crude product was purified by flash column
chromatography (SiO2) to afford 2.5 g (41%) of 1-benzyl-3-(propan-2-
9-Cyclopropyl-6-fluoro-8-methoxy-7-((R)-3-((R)-1-(methyla-
mino)propyl)pyrrolidin-1-yl)isothiazolo[5,4-b]quinoline-3,4-
(2H,9H)-dione Hyrdochloride (9b(R,R)). Yield: 7%. 1H NMR
(CD3OD) δ 7.57 (d, J = 14.1 Hz, 1H), 3.90ꢀ3.49 (m, 6H), 3.50 (s,
3H), 2.69 (m, 3H), 2.59ꢀ2.51 (m, 1H), 2.24ꢀ2.15 (m, 1H), 1.86ꢀ1.64
(m, 3H), 1.21ꢀ1.12 (m, 2H), 1.00 (t, J = 7.5 Hz, 3H), 0.98ꢀ0.88 (m, 2H).
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19F NMR (CD3OD) δ ꢀ126.2 (s). LCMS: m/z calcd for C22H27FN4O3S
446 ([Mþ]); found 447 ([M þ H]þ).
3H, -NCCH3Ph), 1.04ꢀ0.79 (m, 9H). 13C NMR (CDCl3) δ 145.6,
128.1, 127.0, 126.5, 65.7, 56.4, 54.1, 52.7, 45.9, 44.1 (43.8, epimer), 27.5,
24.4, 22.7, 19.3 (19.1, epimer). LCMS: m/z calcd for C17H28N2 ([M]þ)
260; found 261 ([M þ H]þ).
(R)-7-{3-[Amino(thiazol-2-yl)methyl]pyrrolidin-1-yl}-9-cycl-
opropyl-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4-
1
2,2,2-Trifluoro-N-{1-[(R)-1-((S)-1-phenylethyl)pyrrolidin-
3-yl]ethyl}ethanamine (17). A solution of amine 11 (0.13 g,
0.60 mmol) and trifluoroacetic anhydride (0.11 mL, 0.77 mmol) in chloro-
form (5 mL) was stirred for 15 h under an argon atmosphere at room
temperature. To the resulting solution was added water (10 mL), and the
appropriate layer was extracted with dichloromethane (3 ꢁ 10 mL). The
organic layer was washed with a 5% aqueous solution of NaHCO3
(15 mL) and dried over sodium sulfate. The solvent was evaporated to
afford the trifluoroacetate (0.19 g, quantitative) as a light yellow oil. 1H
NMR (CDCl3) δ 9.02 (br, 1H), 7.35 (m, 5H, ArH), 4.02ꢀ3.75 (m, 1H, -
NCHPh, epimers), 3.35ꢀ3.02 (m, 2H), 2.87ꢀ2.62 (m, 1H), 2.42ꢀ1.59
(m, 5H), 1.46 (d, J = 6.6 Hz, 3H, -COCF3NCCH3), 1.40 (d, J = 6.6 Hz,
3H, -COCF3NCCH3, epimer), 1.32 (d, J = 6.5 Hz, 3H, -NCCH3Ph),
1.32 (d, J = 6.5 Hz, 3H, -NCCH3Ph, epimer). 13C NMR (CDCl3)
δ 157.1, 144.2, 128.3, 127.5, 127.2, 126.9, 65.1, 56.8, 53.1 (52.5, epimer),
51.7 (51.2, epimer), 40.5, 27.9, 24.4 (22.4, epimer), 18.7 (17.5, epimer).
19F NMR(CDCl3) δ ꢀ75.6(s, 1F). LCMS: m/z calcd forC16H21F3N2O
([M]þ) 314; found 315 ([M þ H]þ). A 1.0 M solution of lithium
aluminum hydride (1.14 mL, 1.14 mmol) in tetrahydrofuran was added
to a solution of the trifluoroacetate (0.18 g, 0.57 mmol) in dry
tetrahydrofuran (5 mL) at room temperature. The mixed solution was
heated for 3 days at 70 °C and then cooled to room temperature. The
reaction mixture was quenched by cautious addition of water (1 mL), and
to themixturewas added a 15% aqueous solution of NaOH (10 mL). The
solution was filtered through a B€uchner funnel to remove immersions
and extracted with ethyl acetate (3 ꢁ 10 mL). The combined extracts
were dried over sodium sulfate and concentrated to give the desired
product 15 (0.15 g, 87%) as an oil. 1H NMR (CDCl3) δ 7.30 (m, 5H,
ArH), 3.28ꢀ3.08(m, 2H), 2.72ꢀ2.52 (m, 2H), 2.47ꢀ2.28 (m, 3H), 2.11
(m, 1H), 1.95 (m, 1H), 1.69 (m, 1H), 1.64 (s, 2H, -NCH2CF3), 1.46 (s,
2H, -NCH2CF3, epimer), 1.40 (d, J = 6.6 Hz, 3H, -NCCH3Ph), 1.39 (d, J
= 6.3 Hz, 3H, - NCCH3Ph, epimer), 1.05 (d, J = 6.3 Hz, 3H, -NHCCH3),
0.98(d, J = 6.7Hz, 3H, -NHCCH3, epimer). 13C NMR(CDCl3) δ 145.2,
128.2, 127.7, 126.8, 115.0, 65.7, 56.6, 52.7, 43.7 (43.3, epimer), 31.1
(30.2, epimer), 27.6, 26.2, 22.8 (22.7, epimer), 18.4. 19F NMR (CDCl3)
δ ꢀ71.6 (s, 3F), ꢀ71.9 (s, 3F). LCMS: m/z calcd for C16H23F3N2
([M]þ) 300; found 301 ([M þ H]þ).
(2H,9H)-dione Hydrochloride (10a(R,rac)). Yield: 33%. H NMR
(DMSO-d6) δ 8.90 (m, 1H), 7.97 (m, 1H), 7.88 (m, 1H), 7.55 (d, JH,F
=
14.3 Hz, 1H), 5.18 (br, 1H), 4.97 (m, 1H), 3.73 (m, 2H), 3.61ꢀ3.44
(m, 2H), 3.50 (s, 3H, methoxy), 3.32 (m, 1H), 2.88 (m, 1H), 2.26 (m, 1H),
2.25 (m, 1H), 1.90 (m, 1H), 1.11 (m, 2H, c-Pr), 0.95 (m, 2H, c-Pr). 19F
NMR (DMSO-d6) δ ꢀ125.4 (s, 1F), ꢀ125.7 (s, 1F). LCMS: m/z calcd for
C22H22FN5O3S2 ([M]þ) 487; found 488 ([M þ H]þ).
(R)-7-{3-[Amino(oxazol-2-yl)methyl]pyrrolidin-1-yl}-9-cyclo-
propyl-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,
9H)-dione Hydrochloride (11a(R,rac)). Yield: 25%. 1H NMR
(DMSO-d6) δ 8.96 (m, 1H), 8.03 (m, 1H), 7.55 (m, 1H), 7.39 (d, JH,F
=
14.2 Hz, 1H), 4.80 (m, 1H), 3.80 (br, 1H), 3.75 (m, 2H), 3.52 (s, 3H,
methoxy), 3.47 (m, 1H), 2.88 (m, 1H), 2.54 (m, 1H), 2.26 (m, 1H), 1.95
(m, 1H), 1.70 (m, 1H), 1.14 (m, 2H, c-Pr), 0.95 (m, 2H, c-Pr). 19F NMR
(DMSO-d6) δ ꢀ125.4 (s, 1F), ꢀ125.6 (s, 1F). LCMS: m/z calcd for
C22H22FN5O4S ([M]þ) 471; found 472 ([M þ H]þ).
(R)-7-{3-[Amino(pyridin-2-yl)methyl]pyrrolidin-1-yl}-9-cyclo-
propyl-6-fluoro-8-methoxyisothiazolo[5,4-b]quinoline-3,4(2H,
9H)-dione Hydrochloride (12a(R,rac)). Yield: 21%. 1H NMR
(DMSO-d6) δ 8.75ꢀ8.58 (m, 3H), 7.91ꢀ7.83 (m, 1H), 7.60ꢀ7.37 (m,
3H), 5.00 (br, 1H), 4.45 (m, 1H), 3.69 (m, 2H), 3.51 (m, 1H), 3.40 (s, 3H,
methoxy), 3.06 (m, 1H), 2.74 (m, 1H), 2.21 (m, 1H), 1.90 (m, 1H), 1.60 (m,
1H), 1.05 (m, 2H, c-Pr), 1.05 (m, 2H, c-Pr). 19F NMR (DMSO-d6)
δ ꢀ125.4 (s, 1F), ꢀ125.8 (s, 1F). LCMS: m/z calcd for C24H24FN5O3S
([M]þ) 481; found 482 ([M þ H]þ).
N-(Cyclopropylmethyl)-1-[(R)-1-((S)-1-phenylethyl)pyrro-
lidin-3-yl]ethanamine (14). The amine 11 (0.11 g, 0.52 mmol),
sodium triacetoxyborohydride (0.16 g, 0.73 mmol), and acetic acid (29.7
μL, 0.52 mmol) were successively added to a solution of cyclopropane-
carboxaldehyde (41.4 μL, 0.55 mmol) in 1,2-dichloroethane (5 mL).
The reaction mixture was allowed to stir for 6 h under an argon
atmosphere at room temperature. The resulting solution was quenched
by adding 1 N NaOH (10 mL). After being stirred for 5 min, the aqueous
solution was extracted with dichloromethane (3 ꢁ 10 mL). The organic
layer was washed with brine (15 mL) and dried over sodium sulfate. The
solvent was evaporated to provide the desired product 12 (0.13 g, 93%)
1
as an oil. H NMR (CDCl3) δ 7.19 (m, 5H, ArH), 3.10 (m, 1H, -
(R)-4-[1-(Cyclopropylamino)ethyl]-1-((S)-1-phenylethyl)-
pyrrolidin-2-one (19a). Cyclopropylamine (44.9 μL,0.65 mmol),
sodium triacetoxyborohydride (0.19 g, 0.91 mmol), and acetic acid
(36.7 μL, 0.65 mmol) were successively added to a solution of 8a
(0.15 g, 0.65 mmol) in 1,2-dichloroethane (5 mL). The reaction
mixture was stirred for 24 h under an argon atmosphere at room
temperature and then quenched by adding 1 N NaOH (10 mL). After
being stirred for 5 min, the resulting aqueous solution was extracted with
dichloromethane (3 ꢁ 10 mL). The organic layer was washed with brine
(15 mL) and dried over sodium sulfate. The solvent was evapo-
rated to give the desired product 9a (0.17 g, 97%) as an oil. 1H NMR
(CDCl3) δ 7.28 (m, 5H, ArH), 5.47 (m, 1H, -NCHPh), 3.40ꢀ2.89
(m, 2H, -pyrrolidine CH2, C-5), 2.75ꢀ1.94 (m, 6H), 1.50 (d, J = 7.2 Hz,
3H, -NCCH3Ph), 0.96 (d, J = 6.3 Hz, 3H, -c-PrNCCH3), 0.86 (d, J =
6.3 Hz, 3H, -c-PrNCCH3, epimer), 0.49ꢀ0.20 (m, 4H, c-Pr). 13C NMR
(CDCl3) δ 173.6, 140.0, 128.4, 127.4, 127.3, 56.5, 48.7, 44.4, 37.5, 34.9,
28.1, 18.0, 16.0 (15.9, epimer), 7.1. LCMS: m/z calcd for C17H24N2O
([M]þ) 272; found 273 ([M þ H]þ).
NCHPh), 2.79ꢀ1.35 (m, 12H), 1.29 (d, J = 6.6 Hz, 3H, -NCCH3Ph),
0.93 (d, J = 6.3 Hz, 3H, -c-PrCNCCH3), 0.87 (d, J = 6.3 Hz, 3H,
-c-PrCNCCH3, epimer), 0.48ꢀ0.25 (m, 2H, c-Pr), 0.05 (m, 2H, c-Pr).
13C NMR (CDCl3) δ 145.5, 128.1, 127.0, 126.6, 65.7, 56.5, 52.7, 43.7,
27.5, 27.0, 23.1 (23.0, epimer), 18.4 (18.3, epimer), 11.4, 10.3, 3.5, 3.1.
LCMS: m/z calcd for C18H28N2 ([M]þ) 272; found 273 ([M þ H]þ).
N-{1-[(R)-1-((S)-1-Phenylethyl)pyrrolidin-3-yl]ethyl}cyclope-
ntanamine (15). The procedure for the preparation of 12 was used for this
compound from cyclopentanone. The product 13 was obtained as an oil.
1
Yield: 93%. H NMR (CDCl3) δ 7.24 (m, 5H, ArH), 3.05 (m, 1H, -
NCHPh), 2.90ꢀ2.52 (m, 3H), 2.38ꢀ2.07 (m, 3H), 2.06ꢀ1.63 (m, 4H),
1.62ꢀ1.32 (m, 6H), 1.29 (d, J = 6.6 Hz, 3H, -NCCH3Ph), 1.24ꢀ1.02 (m,
2H), 0.94 (d, J = 6.2 Hz, 3H, -c-pentyl-NCCH3), 0.88 (d, J = 6.3 Hz, 3H, -c-
pentyl-NCCH3, epimer). 13C NMR (CDCl3) δ 145.6, 128.1, 126.9, 126.5,
65.6, 56.9, 55.5, 52.6, 44.0 (43.7, epimer), 34.0, 32.8, 27.9, 23.6, 23.0, 18.9
(18.7, epimer). LCMS: m/z calcd for C19H30N2 ([M]þ) 286; found 287
([M þ H]þ).
N-{1-[(R)-1-((S)-1-Phenylethyl)pyrrolidin-3-yl]ethyl}prop-
an-2-amine (16). The procedure for the preparation of 12 was used
for this compound from acetone. The product 14 was obtained as a light
yellow oil. Yield: 90%. 1H NMR (CDCl3) δ 7.19 (m, 5H, ArH), 3.08 (m,
1H, -NCHPh), 2.77 (m, 1H), 2.63 (m, 1H), 2.48 (m, 1H), 2.41ꢀ2.13
(m, 2H), 2.12ꢀ1.62 (m, 3H), 1.54ꢀ1.32 (m, 2H), 1.29 (d, J = 6.6 Hz,
(S)-4-[1-(Cyclopropylamino)ethyl]-1-((S)-1-phenylethyl)-
pyrrolidin-2-one (19b). The procedure for the preparation of 9a
was used for this compound from starting material 8b. The product 9b
was obtained as an oil. Yield: 96%. 1H NMR (CDCl3) δ 7.25 (m, 5H,
ArH), 5.49 (m, 1H, -NCHPh), 3.40ꢀ2.87 (m, 2H, -pyrrolidine CH2,
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C-5), 2.75ꢀ1.95 (m, 6H), 1.48 (d, J = 7.2 Hz, 3H, -NCCH3Ph), 1.08
(d, J = 6.4 Hz, 3H, -c-PrNCCH3), 1.02 (d, J = 6.4 Hz, 3H, -c-PrNCCH3,
epimer), 0.49ꢀ0.20 (m, 4H, c-Pr). 13C NMR (CDCl3) δ 173.4, 140.1,
128.2, 127.1, 126.7, 56.6, 48.6, 45.7, 37.3, 34.8, 27.9, 18.1, 15.8 (15.7,
epimer), 6.9. LCMS: m/z calcd for C17H24N2O ([M]þ) 272; found
273 ([M þ H]þ).
separated, and the aqueous layer was extracted with EtOAc. The organic
extract and layer were combined, dried (Na2SO4), and evaporated under
reduced pressure to give 2 g (quant) of amine 27 as a light yellow oil.
This crude material was used without purification in the next synthetic
step. An analytical sample was obtained by preparative TLC (solvent
system CHCl3/MeOH/28% aq NH4OH 100:10:1). 1H NMR (CDCl3)
δ 7.32ꢀ7.21 (m, 5H), 3.59 (d, J = 17.4, 12.6 Hz, 2H), 2.77 (dd, J = 9.0,
7.8 Hz, 1H), 2.66ꢀ2.58 (m, 1H), 2.57 (d, J = 6.9 Hz, 2H), 2.52ꢀ2.46
(m, 1H), 2.39 (d, J = 6.9 Hz, 2H), 2.40ꢀ2.26 (m, 1H), 2.47 (dd, J = 9.1,
6.4 Hz, 1H), 2.05ꢀ1.92 (m, 1H), 1.77ꢀ1.68 (m, 1H), 1.51ꢀ1.42 (m,
2H), 0.88 (d, J = 6.6 Hz, 6H).
N-{1-[(R)-1-((S)-1-Phenylethyl)pyrrolidin-3-yl]ethyl}cyclopr-
opanamine (20a). A 1.0 M solution of boraneꢀtetrahydrofuran complex
(3.30 mL, 3.30 mmol) in tetrahydrofuran was added to a solution of 9a
(0.18 g, 0.66 mmol) in tetrahydrofuran (10 mL), and the mixture was
allowed to stir for 24 h at room temperature. The solvent was evaporated
under reduced pressure, and dichloromethane (20 mL) was added to the
residue. The mixture was washed with a saturated sodium chloride aqueous
solution (15 mL) and dried over sodium sulfate. The solvent was
evaporated, and a 5 N sodium hydroxide aqueous solution (5 mL) was
added to the residue. The solution was heated for 3 h on an oil bath at
100 °C, cooled to room temperature, and extracted with dichloromethane
(3 ꢁ 15 mL). The organic layer was dried over sodium sulfate and
evaporated to provide the desired product 10a (0.16 g, 94%) as an oil.
1H NMR (CDCl3) δ 7.32ꢀ7.18 (m, 5H, ArH), 3.14 (m, 1H, -NCHPh),
2.74 (m, 1H, -c-PrNCH), 2.65ꢀ2.24 (m, 3H, -pyrrolidine CH2, C-2,
and -NH), 2.19ꢀ1.96 (m, 3H, -pyrrolidine CH2, C-5, and -pyrrolidine
CH, C-3), 1.89 (m, 1H, -c-Pr CH), 1.61ꢀ1.35 (m, 2H, -pyrrolidine CH2,
C-4), 1.34 (d, J = 6.6 Hz, 3H, -NCCH3Ph), 1.05 (d, J = 6.3 Hz, 3H, -c-
PrNCCH3), 0.99 (d, J = 6.3 Hz, 3H, -c-PrNCCH3, epimer), 0.49ꢀ0.15
(m, 4H, c-Pr). 13C NMR (CDCl3) δ 145.8, 128.3, 127.1, 126.5, 65.9, 58.0,
56.8, 52.8, 43.8, 28.5, 27.6, 23.3, 19.0 (18.7, epimer), 7.5, 6.0. LCMS: m/z
calcd for C17H26N2 ([M]þ) 258; found 259 ([M þ H]þ).
(R)-N-(2-(1-Benzylpyrrolidin-3-yl)propan-2-yl)-2-methyl-
propan-1-amine (30). 30 was prepared using procedures analo-
gous to those described above for 27. Amide 29 was isolated as a pale
yellow oil (1.0 g, 76% yield). 1H NMR (CDCl3) δ 7.32ꢀ7.24 (m, 5H),
6.87 (br s, 1H), 3.63 (d, J = 12.6 Hz, 1H), 3.51 (d, J = 12.9 Hz, 1H),
2.98ꢀ2.92 (m, 1H), 2.89 (d, J = 9.0 Hz, 1H), 2.29ꢀ2.10 (m, 4H),
1.91ꢀ1.73 (2H, m), 1.34 (d, J = 9.6 Hz, 6H), 1.10 (t, J = 6.9 Hz, 6H).
13C NMR (CDCl3) δ 177.1, 138.8, 128.7, 128.3, 127.1, 60.2, 55.7, 55.3,
54.6, 47.6, 36.6, 25.7, 25.6, 24.4, 19.8. LCꢀMS m/z calcd for
C18H28N2O 288 ([Mþ]); found 289 ([M þ H]). As described above
for compound 26, amide 29 (1 g, 3.47 mmol) was reduced using LAH
(0.53 g, 13.9 mmol) to give 0.7 g of amine 30 as a light yellow oil. The
isolated crude material was used without purification in the next synthetic
step. An analytical sample was obtained by preparative TLC (solvent system
CHCl3/MeOH/28% aq NH4OH 100:10:1). 1H NMR (CDCl3): δ
7.32ꢀ7.23 (m, 5H), 3.66 (d, J = 12.9 Hz, 1H), 3.53 (d, J = 12.9 Hz, 1H),
2.69ꢀ2.60 (m, 2H), 2.45ꢀ2.26 (m, 5H), 1.83ꢀ1.76 (m, 1H), 1.68ꢀ1.51
(m, 2H), 0.99 (d, J = 2.5 Hz, 6H), 0.88 (dd, J = 6.6, 0.6 Hz, 6H). LCꢀMS
m/z calcd for C18H30N2 274 ([Mþ]); found 275 ([M þ H]).
N-{1-[(S)-1-((S)-1-Phenylethyl)pyrrolidin-3-yl]ethyl}cyclopr-
opanamine (20b). The procedure for the preparation of 10a was used
for this compound from starting material 9b. The product 10b was obtained
as an oil. Yield: 88%. 1H NMR (CDCl3) δ 7.33ꢀ7.18 (m, 5H, ArH), 3.15
(m, 1H, -NCHPh), 2.73 (m, 1H, -c-PrNCH), 2.63ꢀ2.24 (m, 3H, -
pyrrolidine CH2, C-2 and -NH), 2.19ꢀ1.97 (m, 3H, -pyrrolidine CH2,
C-5, and -pyrrolidine CH, C-3), 1.85 (m, 1H, -c-Pr CH), 1.59ꢀ1.38 (m,
2H, -pyrrolidine CH2, C-4), 1.35 (d, J = 6.6 Hz, 3H, -NCCH3Ph), 1.06
(d, J = 6.3 Hz, 3H, -c-PrNCCH3), 0.99 (d, J = 6.3 Hz, 3H, -c-PrNCCH3,
epimer), 0.49ꢀ0.16 (m, 4H, c-Pr). 13C NMR (CDCl3) δ 145.6, 128.0, 126.9,
126.5, 65.7, 57.6, 56.6, 52.7, 43.4, 28.3, 27.4, 23.1, 18.8 (18.5, epimer), 7.1,
6.0. LCMS: m/z calcd for C17H26N2 ([M]þ) 258; found 259 ([M þ H]þ).
N-((1-Benzylpyrrolidin-3-yl)methyl)-2-methylpropan-1-
amine (27). To a stirred solution of acid 25 (2.2 g, 10.0 mmol) in DMF
(20 mL) were added EDCI (2.89 g, 15.1 mmol) and HOBt (1.77 g,
13.1 mmol). 2-Methylpropan-1-amine (1.11 mL, 11.1 mmol) was
added, and the reaction mixture was stirred for 18 h at room tempera-
ture. The reaction mixture was then diluted with H2O (200 mL) and
extracted with CHCl3. The organic extracts were combined, dried
(Na2SO4), and evaporated under reduced pressure. The remaining
crude material was purified by silica gel flash column chromatography
(eluent 0ꢀ2% MeOH in CHCl3) to give 2.3 g (84%) of 26 as a white
solid. 1H NMR (CDCl3) δ 7.35ꢀ7.22 (m, 5H), 5.87 (br s, 1H), 4.54 (d,
J = 14.7 Hz, 1H), 4.34 (d, J = 14.7 Hz, 1H), 3.51 (dd, J = 9.6, 7.2 Hz, 1H),
3.39 (t, J = 8.7 Hz, 1H), 3.08ꢀ3.01 (m, 3H), 2.78 (dd, J = 16.8, 9.6 Hz,
1H), 2.62 (dd, J = 16.8, 9.6 Hz, 1H), 1.78ꢀ1.66 (m, 1H), 0.88 (d, J = 6.3
Hz, 6H). 13C NMR (CDCl3) δ 172.6, 172.0, 136.0, 128.8, 128.1, 127.7,
49.2, 47.1, 46.6, 37.8, 35.0, 28.4, 20.0. LCꢀMS m/z calcd for
C16H22N2O2 274 ([Mþ]); found 275 ([M þ H]). Anal. Calcd for
C16H22N2O2 C 70.04, H 8.08, N 10.21; found C 70.01, H 8.20, N 10.25.
Amide 26 (2.3 g, 8.4 mmol) in THF (20 mL) was added dropwise to a
suspension of LAH (1.27 g, 33.5 mmol) in THF (20 mL) at 0 °C. After
the addition was complete, the reaction mixture was refluxed for 18 h,
cooled to 0 °C, and quenched carefully by the addition of 10% aqueous
NaOH. The resulting white precipitate was removed by filtration, and
the filtrate was transferred to a separatory funnel. The layers were
Biological Assays. Antimicrobial susceptibility, cytotoxicity, topoi-
somerase IV assays, and DNA gyrase assays were conducted using
methods described previously.18
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: 203-624-7000. Fax: 203-752-5454. E-mail:
bbradbury@achillion.com.
’ ACKNOWLEDGMENT
We thank Christy Thoma and Jijun Cheng for biology
technical support of this work.
’ ABBREVIATIONS USED
ITQ, isothiazoloquinolone; CIP, ciprofloxacin; GEM, gemiflox-
acin; LVX, levofloxacin; LFX, levofloxacin; MXF, moxifloxacin;
VAN, vancomycin; LZD, linezolid; OXA, oxacillin; QPS/DPS,
quinupristin/dalfopristin; MIC, minimum inhibitory concentra-
tion; MRSA, methicillin-resistant Staphylococcus aureus; MSSA,
methicillin-sensitive Staphylococcus aureus; MQRSA, methicillin-
and quinolone-resistant Staphylococcus aureus; Ec, Escherichia coli
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reduction. The amount of fluorescence or absorbance is proportional to
the number of living cells and corresponds to the cells metabolic activity.
Damaged and nonviable cells have lower innate metabolic activity and
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human laryngeal carcinoma cell line was incubated with drug concen-
trations at 37 °C for 72 h to generate eight-point CC50 data.
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