Studies on 6-aminoquinolones: Synthesis and antibacterial evaluation of 6-amino-8-methylquinolones

Article abstract of DOI:10.1021/jm950558v

The 6-aminoquinolone had previously been identified as a new class of quinolone antibacterial agents. To continue our structure-activity relationship (SAR) study in this series, novel 6-amino-8-methylquinolone derivatives have now been synthesized and eva

Full text of DOI:10.1021/jm950558v

436
J . Med. Chem. 1996, 39, 436-445
Stu d ies on 6-Am in oqu in olon es: Syn th esis a n d An tiba cter ia l Eva lu a tion of
6-Am in o-8-m eth ylqu in olon es1
Violetta Cecchetti,† Arnaldo Fravolini,*,† Maria Cristina Lorenzini,† Oriana Tabarrini,† Patrizia Terni,‡ and
Tao Xin†
Istituto di Chimica Farmaceutica e Tecnica Farmaceutica, Universita` di Perugia, 06123 Perugia, Italy, and Mediolanum
Farmaceutici, via S. G. Cottolengo 31, 20143 Milano, Italy
Received J uly 27, 1995X
The 6-aminoquinolone had previously been identified as a new class of quinolone antibacterial
agents. To continue our structure-activity relationship (SAR) study in this series, novel
6-amino-8-methylquinolone derivatives have now been synthesized and evaluated for in vitro
antibacterial activity. We have shown that the coupled presence of a methyl group at the C-8
position with an amino group at C-6 is effective for enhancing antibacterial activity, particularly
against Gram-positive bacteria. The SARs associated with the N-1, C-6, and C-7 are discussed.
The 1,2,3,4-tetrahydroisoquinolinyl derivative 19v showed the highest antibacterial activity
with MIC values on Gram-positive bacteria superior to that of ciprofloxacin, especially against
Staphylococcus aureus strains, including those strains which are methicillin- and ciprofloxacin-
resistant.
In tr od u ction
Our previous chemometric study on quinolone anti-
bacterials2 suggested that an amino group might have
the appropriate steric and electronic requirements to
replace the standard fluorine atom at C-6, one of the
major factors indicated as responsible for the increased
activity of current quinolones (fluoroquinolones). On the
basis of this suggestion, a wide series of 6-aminoquino-
lones (1-5) was recently synthesized and evaluated for
in vitro antibacterial activity (Figure 1).3 The results
of this study showed that, while the C-6 fluorine atom
was still the best substituent, good activity could still
be obtained by replacing it with an amino group and
reoptimizing the other substituents. These findings
prompted us to extend our investigation in order to
outline a structure-activity relationship, increase po-
tency, and broaden the spectrum for this new class.
Indeed, although the new fluoroquinolones are generally
characterized by a broad antimicrobial spectrum, their
activities against Gram-positive organisms are limited,
with diminished potency especially against staphylo-
cocci, streptococci, and enterococci.4 In addition, the
majority of methicillin-resistant staphylococci (espe-
cially Staphylococcus aureus) are now resistant to all
of the currently available quinolones.4 Therefore, recent
efforts have been directed toward the synthesis of
compounds which have greater activities against these
organisms, such as tosufloxacin,5,6 clinafloxacin,6,7 DU
6859,6,8 Bay 3118,9 PD 138312,10 and PD 140248.10 On
the basis of these considerations, we hoped that with
the new series of 6-aminoquinolones, we could obtain
compounds with strong activity against all Gram-
positives and which would also be effective against
resistant strains such as methicillin-resistant staphy-
lococci.
F igu r e 1.
such as a methyl, as optimum to improve the activity
against Gram-positive bacteria, we synthesized a large
series of 6-amino-8-methyl-1-cyclopropylquinolones (19a-
v) variously substituted in the C-7 position (Figure 1).
This was also motivated by an observed favorable
antimicrobial effect due to the presence of the C-8
methyl group as reported by Miyamoto et al.11 In
addition, we have included a few data (compounds 20a -
22a ) employing 4-substituted phenyl groups as N-1
substituent instead of the usual cyclopropyl group.
Finally, in order to clarify the importance of the C-6
amino group, we also synthesized and assayed some
1-cyclopropyl-8-methylquinolones bearing
a
nitro
(24a ,i,j,v), a N-methylamino (26j), and a N,N-dimethy-
lamino (27j) group, as well as the usual fluorine atom
(30a ,i,v) at the C-6 position, with which a head to head
comparison with 6-amino-8-methylquinolones was made.
Ch em istr y
Our synthetic approach was focused on the prepara-
tion of the key intermediates ethyl 7-fluoro-8-methyl-
6-nitro-1-substituted-4-oxo-1,4-dihydroquinoline-3-car-
boxylates 10-12 in order to allow for easy diversification
of the substituent at the C-7 position.
The first attempt was achieved starting from 2,6-
difluoro-3-nitrotoluene12 which was reacted with the
appropriate amine (R1NH2) in order to obtain the
nucleophilic displacement of C-6 fluorine atom. The
successive reaction with (ethoxymethylene)malonate
Therefore, with these objectives in mind and consid-
ering the predictive indications of the previous chemo-
metric study2b that indicated a large substituent at C-8,
† Istituto di Chimica Farmaceutica e Tecnica Farmaceutica.
‡ Mediolanum Farmaceutici.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0436$12.00/0 © 1996 American Chemical Society

Studies on 6-Aminoquinolones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 437
Sch em e 1a
a
Reagents: (i) CH3COCl, AlCl3; (ii) Br2, NaOH, dioxane; (iii) 99.5% HNO3, 98% H2SO4; (iv) SOCl2; (v) (CH3)2NCHdCHCO2Et, Et3N,
toluene, 90 °C; (vi) NH2R1, EtOH/Et2O; (vii) K2CO3, DMF, 100 °C; (viii) R7H, methods A-D (see the Experimental Section); (ix) H2, Raney
Ni, CH3O(CH2)2OH; (x) 6 N HCl, EtOH, reflux; (xi) 1 N NaOH, reflux; (xii) 48% HBr, reflux.
Ch a r t 1. Heterocyclic Side Chains Employed as the R7
Substituent
(EMME) and ring closure should have provided the
desired key intermediates. Unfortunately, as previously
observed,3 the nucleophilic displacement occurs re-
giospecifically at the C-2 position without being the least
bit influenced by steric hindrance from the methyl group
at C-1.
Therefore, we planned an alternative pathway, shown
in Scheme 1, involving the Friedel-Crafts acetylation
of the 2-chloro-6-fluorotoluene. This reaction afforded
the desired acetyl derivative 6, as well as the other
meta-isomer, in 95:5 ratio, respectively, on the basis of
the 1H NMR spectrum. The failure to completely
remove this impurity forced us to use impure starting
material to carry out the following steps: oxidation,
nitration, and reaction with ethyl (dimethylamino)-
acrylate, after which the pure acrylate 9 was obtained
by column chromatography purification. The successive
substitution with appropriate amine (R1NH2) and ring
closure with potassium carbonate afforded the key
intermediates 10-12.
The desired acids 19-22 (see Table 1) were thus
synthesized by replacement of the C-7 fluorine atom of
10-12 with the selected heterocyclic side chain (shown
in Chart 1) followed by catalytic reduction and acid or
base hydrolysis. When 2,6-dimethylmorpholines (g,h )
or 1,2,3,4-tetrahydroisoquinoline (v) was used, it was
more convenient to carry out the hydrolysis step before
the nucleophilic reaction (Scheme 2). Thus, acid 23,
obtained from ester 10, was reacted with the above
heterocyclic bases to directly yield the desired 6-nitro-
quinolones 24g,h ,v, which were then reduced to the
target 6-aminoquinolones 19g,h ,v, respectively. The
other 6-nitroquinolones (24a ,i,j) assayed in this study
were, however, obtained from 6-nitro esters 13a ,i,j by
direct basic hydrolysis (Scheme 2).
was one-step N,N-dimethylated with trimethyl phos-
The ester 16j was chosen as a convenient intermedi-
ate to obtain an example of N-substituted 6-amino
derivatives. The ester 16j was elaborated, as in Scheme
3, in two ways: In the first, the C-6 amino group was
converted into its trifluoroacetamide derivative 25j
followed by methylation and hydrolysis to give the
6-(methylamino)quinolone 26j; in the second, the ester
phate and hydrolized to 6-(dimethylamino)quinolone
27j.
For the preparation of the the known 30a ,11 as well
as the unknown 6-fluoro-8-methylquinolones 30i,v (all
used for a comparative purpose), we did not use the
laborious synthetic procedure reported by Miyamoto et
al.,11 but we planned a shorter and more convenient

438 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Cecchetti et al.
Ta ble 1. Physical Properties for the
6-Amino-8-methyl-4-oxoquinolinecarboxylic Acids Tested in
This Study
Ta ble 2. Physical Properties for the
6-Substituted-1-cyclopropyl-8-methyl-4-oxoquinolinecarboxylic
Acids Tested in This Study
a
Yield is referred to a final step. b All compounds had elemental
analyses within (0.4% of theoretical values.
7-substituted acids 30 were directly obtained by cou-
pling reaction of borate complex 29 with the selected
heterocylic bases.
Biologica l Assa ys
All 6-amino-8-methylquinolone acids (19a -v, 20a ,
21a , and 22a ) as well as 6-substituted-8-methylqui-
nolone acids (24a ,i,j,v, 26j, 27j, and 30a ,i,v) prepared
for this study were tested in vitro against an assortment
of eight Gram-negative and five Gram-positive organ-
isms by conventional agar dilution procedure.13 The
minimum inhibitory concentrations (MICs, µg/mL) are
presented in Table 3. The geometric means of the MICs
for both Gram-positive and Gram-negative strains were
also calculated to facilitate a comparison of activity. In
addition, control drug ciprofloxacin and 6-aminoquino-
lones 1a -e,k ,q,v, 2a , 4a , and 5a , previously reported
by us,3 are included for comparative purposes.
A representative number of compounds was also
tested for their ability to inhibit the supercoiling activity
of DNA gyrase by using a previously described proto-
col.14 The IC50 values are reported in Table 4, together
with the clog P values.15
In addition, the in vitro antibacterial activity against
an assortment of Gram-positive clinical isolates, includ-
ing methicillin- and ciprofloxacin-resistant strains, was
evaluated for those 6-amino-8-methylquinolones which
showed highest Gram-positive antibacterial activity,
coupled with good Gram-negative antibacterial activity.
The MICs are presented in Table 5.
a
b
See the Experimental Section. All compounds had elemental
analyses within (0.4% of theoretical values. c Yield is that ob-
tained from Raney Ni reduction step as a final step.
alternative pathway, exploiting our key intermediate 10.
Thus, as depicted in Scheme 4, reduction of 10 gave the
6-amino derivative 28 which, by successive Balz-
Schiemann reaction, afforded, in only one step, the
6-fluoro derivative 29 as borate complex, indispensable
for the success of the following nucleophilic displace-
ment reaction. Finally, the desired 6-fluoro-8-methyl-
Finally, the in vivo potency of selected 19v, expressed
as the median effective dose (ED50, mg/kg), was deter-
mined in the experimental model of septicemia caused

Studies on 6-Aminoquinolones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 439
Sch em e 2a
a
Reagents: (i) 6 N HCl, EtOH, reflux; (ii) R7H, Et3N, CH3CN, reflux; (iii) 1 N NaOH, reflux; (iv) H2, Raney Ni, CH3O(CH2)2OH.
Sch em e 3a
a
Reagents: (i) (CF3CO)2O; (ii) CH3I, K2CO3, acetone, reflux; (iii) 20% NaOH, EtOH, reflux; (iv) (CH3O)3PO, 150 °C; (v) NaOH, reflux.
Sch em e 4a
a
Reagents: (i) H2, Raney Ni, CH3O(CH2)2OH; (ii) NaNO2, 6 N HCl, -5 °C; (iii) 50% HBF4; (iv) 180-200 °C; (v) R7H, Et3N, CH3CN,
reflux; (vi) 6 N HCl.
by S. aureus in mice after subcutaneous and oral
administration. Results are given in Table 6.
Among the 8-methyl series, a significant loss of
activity was observed when 4-substituted phenyl groups
were present at the N-1 position instead of the cyclo-
propyl group (compare 20a , 21a , and 22a vs 19a ). The
drop in activity was less noticeable for the 4-hydrox-
yphenyl group than for the 4-fluoro- and 4-methoxyphe-
nyl groups.
It should be noted that, relative to the 8-desmethyl
compounds 2a and 1q, derivatives 20a and 19q show
greatly diminished Gram-negative activity.
Substituent effects at the C-7 position were exten-
sively studied while maintaining the cyclopropyl group
at the N-1 position as well as the characteristic C-8
methyl group. The series of piperazinyl derivatives
19a -d was more active against Gram-negatives than
Gram-positives with the 4-methylpiperazinyl derivative
19a having the best balanced activity. The replacement
of the 4-piperazinyl nitrogen atom by a sulfur atom or
oxygen atom enhanced the activity against Gram-
positive bacteria without reducing their activity against
Gram-negatives (compare 19e,f vs 19c). This enhance-
Resu lts a n d Discu ssion
In a direct comparison (Table 3) between the 4-me-
thylpiperazinyl derivative of 6-amino-8-methyl series
19a and the previously reported counterparts having a
different C-8 substituent such as 1a (X ) CH), 4a (X )
CF), and 5a (X ) N) (see Figure 1), the 8-methyl
derivative 19a displays an increased potency against
both Gram-positives (4-8 times) and Gram-negatives
(2.5-3.5 times). In addition, matching the 8-methyl-
6-aminoquinolones 19a -e,k ,v, having various C-7 sub-
stituents, with the 8-unsubstituted counterparts 1a -
e,k ,v, it can be seen that the presence of the methyl
group at C-8 increased the potency against Gram-
positives, which rises to 37 times for the 4-hydroxypi-
peridinyl derivative 19k (vs 1k ) and to over 1000 times
for the 1,2,3,4-tetrahydroisoquinolinyl derivative 19v (vs
1v).

440 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Ta ble 3. In Vitro Antibacterial Activity (MICs, µg/mL)a
Cecchetti et al.
Gram-negative organisms
Gram-positive organisms
S. au. S. ep.
HCF
E. co.
E. cl.
P. vu.
P. st.
K. pn.
P. ae.
geometric means
ATCC
compd 8739
ISF
432
OMNFI CNUR CNUR ATCC
ATCC MPR ATCC
CPHL
A2
S. fe.
174
5
5
10031 S. en. 9027
5
6538 Berset C
LEP Br Gram-(-) Gram-(+)
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
19n
19o
19p
19q
19r
19s
19t
19u
19v
20a
21a
22a
24a
24i
24j
24v
26j
27j
30a
30i
30v
1a
0.03
0.125 0.03
0.03
0.125
0.125
0.06
0.5
0.25
0.125
1
4
4
4
8
32
8
16
8
8
8
16
32
>128
16
>128
64
>128
>128
32
64
64
2
>128
128
>128
64
64
64
64
>128
64
2
2
4
64
2
2
2
2
2
0.5
0.5
1
0.5
0.5
1
0.25
8
0.5
>128
4
64
64
0.5
1
0.03
0.03
0.03
0.03
0.02
0.03
0.03
0.08
0.03
0.03
0.03
0.03
0.5
0.06
1
0.016
0.5
0.25
0.03
0.03
0.125
0.06
1
0.06
0.06
0.03
0.25
0.125
0.125
1
1
0.25
0.5
2
1
1
2
1
1
4
4
1
1
2
4
64
4
0.5
1
4
1
0.5
1
2
1
0.06
1
1
8
2
1
1
4
1
4
4
16
16
0.5
1
0.205
0.225
0.158
0.348
0.362
0.189
0.451
0.336
0.225
0.268
0.495
0.414
6.168
0.641
20.749
0.504
22.627
11.313
0.763
2.141
4.000
0.105
9.513
76.109
2.378
13.454
41.498
38.054
45.254
11.313
64.000
0.145
0.591
0.451
0.642
0.451
0.836
0.451
0.451
0.984
6.727
107.635
1.834
0.699
0.416
0.033
1.000
1.319
5.278
2.000
0.123
0.218
0.122
0.107
0.123
0.122
0.106
0.162
1.132
0.079
128.000
0.287
12.120
18.379
0.275
0.246
0.186
0.024
111.430
84.448
18.370
4.595
9.189
8.000
3.482
3.031
24.251
0.162
0.046
0.046
4.000
10.556
42.224
6.964
1.320
4.000
128.000
27.857
10.556
8.000
6.063
0.092
0.03
0.06
0.25
0.03
0.06
0.03
0.03
0.03
0.03
0.03
0.06
0.125 0.125
0.06
0.03
0.06
0.06
0.125 0.06
0.06
0.5
0.125
0.125
0.125
0.125
0.125
0.125
0.06
0.25
1
0.06
>128
0.25
16
16
0.2
1
0.125
0.03
128
64
16
4
8
4
2
4
8
0.25
0.03
0.03
4
4
16
4
2
4
128
16
0.125
0.25
0.06
0.125
0.125
0.125
0.06
0.125
1
0.06
>128
0.25
16
0.06
0.03
0.06
0.06
1
1
0.008 0.008
1
0.03
0.03
1
0.03
0.5
0.125 0.03
1
0.016 0.016
32
16
2
4
4
0.008 0.008
1
>128
0.125 0.125
16
32
32
64
1
0.03
0.03
0.03
0.03
0.5
0.25
0.5
0.5
1
16
2
>128
2
64
16
1
4
4
0.25
16
>128
8
8
64
64
64
64
64
0.03
16
4
0.25
0.25
0.25
0.5
0.5
2
8
128
4
0.25
0.125
0.015
0.5
0.5
0.5
2
16
1
>128
2
64
>128
2
0.25
1
16
0.03
0.5
0.03
128
2
0.03
1
>128 >128 128
1
64
16
1
3
2
0.25
16
>128 >128 64
4
1
64
64
64
64
64
0.03
2
0.125 0.125
4
8
0.125 0.125
0.03
0.06
1
>128
>128
4
16
64
4
8
0.25
0.03
0.3
4
16
0.25
0.25
0.25
0.016
128
128
8
2
8
4
4
2
32
0.25
0.03
0.03
2
4
16
2
1
0.03
0.06
4
2
1
0.06
16
128
8
32
64
64
64
16
64
2
2
2
8
16
32
4
1
8
0.5
128
0.008 0.008
128
0.25
128
128
64
16
32
32
4
32
32
1
0.25
0.25
32
32
128
32
8
1
16
64
64
16
4
32
32
64
1
64
0.06
0.03
0.03
2
16
64
8
0.5
4
128
128
8
4
4
0.06
16
0. 25
16
64
64
64
0.5
64
0.03
8
16
4
1
2
0.25
1
16
0.03
0.03
0.03
2
16
64
8
0.5
4
128
4
4
4
2
4
0.5
64
0.03
64
64
64
64
>128
64
2
1
4
4
2
2
2
2
8
128
128
8
2
2
64
0.03
0.125 0.03
0.125 0.5
0.06
0.25
0.03
0.25
0.03
0.03
0.03
0.25
128
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.25
32
0.03
0.03
0.03
0.25
0.03
0.03
0.03
0.25
128
0.25
0.25
0.25
0.25
0.25
0.5
2
16
128
4
0.25
0.125
1b
1c
1d
1e
1k
1q
1v
2a
32
32
64
128
128
128
128
128
32
2
128
16
8
4
8
128
128
4
4
2
128
128
64
64
32
0.5
0.125 0.125
0.06
0.125
0.06
0.03
4
4
4
0.06
8
8
4
0.06
4a
5a
1
0.125 0.06
64
0.5
4
0.06
CPXb 0.015 0.015
0.06
0.015 0.015 0.06
a
Organisms selected are as follows: E. co., Escherichia coli; E. cl., Enterobacter cloacae; P. vu., Proteus vulgaris; P. st., Providencia
stuardii; K. pn., Klebsiella pneumonia; S. en., Shigella enteritidis; P. ae., Pseudomonas aeruginosa; S. au., Staphylococcus aureus; S. ep.,
b
Staphylococcus epidermidis; S. fe., Streptococcus faecalis. CPX ) ciprofloxacin.
Ta ble 4. Inhibitory Effect (IC50, µg/mL)a on Gyrase
Supercoiling Activity from E. coli, in Vitro Antibacterial
Activity (Geometric Mean MICs, µg/mL), and clog P by Selected
6-Amino-8-methylquinolones
Ta ble 5. In Vitro Antibacterial Activity (MIC, µg/mL)a of
Selected 6-Amino-8-methylquinolones on Clinical Gram-Positive
Isolates
S.
S.
E.
geometric mean MICs
compd S. aureus aureusb pneumoniae E. faecalis faecium
compd
IC50
Gram-negatives
Gram-positives
clog P
19e
19f
19h
19i
19j
19k
19v
0.06-0.13
0.06-0.13
0.06-0.13
2-8
4-8
2-4
1-2
1-2
2
1-2
1-2
0.5-2
0.13
0.5-2
0.5
0.25-0.5
0.05
0.13-0.5
0.13-0.25 0.13-2
0.25-0.5
0.06
0.5-1
2
0.5-2
1-32
1-2
19a
19e
19f
19h
19i
3.78
1.42
5.64
g50
3.22
5.86
5.74
3.58
3.14
0.68
0.205
0.362
0.189
0.336
0.225
0.268
0.495
0.105
0.450
0.033
1
0.695
1.672
0.951
1.989
2.595
3.114
0.508
3.305
4.549
-0.805
0.123
0.218
0.107
0.123
0.123
0.106
0.024
0.046
0.092
0.016-0.13 2-4
0.016-0.13 1-2
0.06-0.13
4-32
0.25-4
0.06-1
1-4
0.004-0.03 0.05-1
19j
CPXc 0.13-2
4-128
19k
19v
30v
CPXb
a
The range of MIC values for five isolates for each species.
Methicillin- and ciprofloxacin-resistant isolates (seven strains).
b
c CPX ) ciprofloxacin.
a
Calculated by the quantitative measurement of the supercoiled
8-unsubstituted-6-aminoquinolones 1e,3 and the mor-
pholinyl group, as in the case of its 6-fluoro-8-methyl
counterpart11 and Kanebo products.16 The 2,6-dialky-
lation of the morpholinyl moiety, as in 19g and cis-
derivative 19h , has a somewhat favorable effect on the
activity against Gram-positives (which reaches those of
b
DNA peak in an agarose gel by densitometric assay. CPX )
ciprofloxacin.
ment in Gram-positive antibacterial activity is in agree-
ment with what was already observed for both the
thiomorpholinyl group, as in the case of our previous

Studies on 6-Aminoquinolones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 441
Ta ble 6. Efficacy of 19v in the Experimental Model of
Septicemia Caused by S. aureusa in Mice
The DNA supercoiling assay (Table 4) shows that, in
general, the 6-amino-8-methylquinolones tested are 2-9
times less potent than ciprofloxacin as DNA gyrase
inhibitors, with the exception of 19h which is quite
inactive. Such a decrease in activity does not seem to
depend on the amino group at the C-6 position since
the 6-amino derivative 19v and its 6-fluoro counterpart
30v have the same inhibitory potency.
ED50, mg/kg
(95% confidence limits)
MIC,
compd
µg/mL
po
sc
19v
0.016
0.13
5 (3.8-6.6)
7 (5.1-9.7)
1.3 (1.1-1.5)
0.9 (0.8-1.1)
CPXb
a
b
See the Experimental Section. CPX ) ciprofloxacin.
Matching the Gram-positive MIC and DNA gyrase
values, we might think that 6-amino-8-methylquinolo-
nes have a better cell permeability against Gram-
positives than ciprofloxacin. Although it is now gener-
ally recognized that the more hydrophobic a compound,
the better its diffusion into Gram-positives,17 no close
correlation was found between MICs and clog P values
(Table 4); in fact, the last lipophilic agent in our study,
ciprofloxacin (clog P ) -0.805), is the third best agent
against Gram-positives.
ciprofloxacin) but an unfavorable effect against Gram-
negatives. The same good antibacterial activity was
also displayed by piperidinyl derivatives 19i-l,n ,p with
the 3,5-dimethyl derivative 19n showing a geometric
mean MIC of 0.079 µg/mL against Gram-positives on
the same order as those of ciprofloxacin (0.092 µg/mL).
On the contrary, the presence of gem-dimethyl group
in the C-3 position of piperidine moiety had a deleterious
effect on both Gram-positives and Gram-negatives
(compare 19m vs its monomethyl analogue 19l), as well
as the 3,4-dehydrogenation which gave completely inac-
tive 19o (compare vs 19i). Considering the piperidine
moiety, it was observed that the ring contraction to
pyrrolidinyl (q) or azetidinyl (r ) groups markedly de-
creased all antibacterial activity (compare 19q,r vs 19i)
with the pyrrolidinyl group again showing itself to be
an unsuitable C-7 substituent for the 6-aminoquinolo-
nes.3 On the contrary, a ring expansion to homopip-
eridine (s) permitted the activity to be maintained
(compare 19s vs 19i). Among the C-7 substituents, the
best overall antibacterial activity was displayed by
bicyclic 1,2,3,4-tetrahydroisoquinolinyl group. Indeed,
compound 19v was more potent against Gram-positives
than ciprofloxacin, with MIC values against S. aureus
of 0.008 and 0.06 µg/mL, respectively. While, its activity
against Gram-negatives was the best of all 6-aminoqui-
nolones, it was not as good as that of ciprofloxacin. The
saturation of 1,2,3,4-tetrahydroisoquinolinyl nucleus to
(()-trans-perhydroisoquinoline, as in 19u , caused a
marked decline in potency, particularly against Gram-
negatives.
We next turned our attention to the C-6 amino group,
and in an attempt to explore the importance of amino
group in the C-6 position, we prepared and assayed the
6-methylamino and 6-dimethylamino acids 26j and 27j,
some 6-nitro acids (24a ,i,j,v) which were intermediates
in the preparation of the 6-amino series, and 6-fluoro
analogues 30a ,i,v (Table 3). When compared with 19j,
the N-monomethylation of the C-6 amino group, as in
26j, lowered the antibacterial activity which disap-
peared completely against Gram-negatives with the
N,N-dimethylation, as in 27j. The reduced hydrophi-
licity caused by introducing a methyl group may account
for this diminished potency. The replacement of the
electron-donating C-6 amino group with the electron-
withdrawing nitro group resulted in a considerable loss
of activity against Gram-positives and the complete
disappearance of activity against Gram-negatives (com-
pare 24a ,i,j,v vs 19a ,i,j,v).
The interaction with topoisomerase II is central to the
activity of both quinolone and antitumor agents, so it
seemed of interest, from a safety standpoint,18 to also
determine the effects of 6-amino-8-methylquinolones,
tested on DNA-gyrase, on mammalian topoisomerase II.
All tested compounds resulted inactive in the decatena-
tion assay.
Since our main objective was to identify quinolones
with strong activity against Gram-positives, we broad-
ened our study on clinical isolates such as Streptococcus
pneumoniae, Enterococcus faecalis, Enterococcus faeci-
um, S. aureus, and methicillin- and ciprofloxacin-
resistant S. aureus that cause considerable clinical
problems. For this purpose, we selected the 6-amino-
8-methylquinolones, having excellent Gram-positive
antibacterial activity coupled with good Gram-negative
activity. The results reported in Table 5 show that all
of the tested compounds have extremely high activity
against staphylococci. The activity of 19v was also
excellent against methicillin- and ciprofloxacin-resistant
S. aureus. It must be pointed out that its MIC values
on S. pneumoniae isolates were 4-15 times lower than
those of ciprofloxacin and are probably better than any
other available agent.
In a preliminary in vivo study, a model of S. aureus
septicemia in mouse was used to evaluate the activity
of compound 19v (Table 6). Following oral administra-
tion, 19v protected mice to the same extent as ciprof-
loxacin. Considering that 19v had MIC 10 times lower
than ciprofloxacin on S. aureus in vitro, thought to have
been due to poor bioavailability, a subcutaneous study
was undertaken. Once again both compounds had a
similar effect. Therefore, further studies are needed in
order to clarify the in vivo efficacy of compound 19v.
In conclusion, in this study the amino group was
confirmed to be a good replacement for the C-6 fluorine
atom. In particular, as predicted by our previous
chemometric study,2b the introduction of a methyl group
at the C-8 position in the 6-aminoquinolones increased
the antibacterial activity, especially against Gram-
positives, and allowed some very potent antibacterial
agents to be identified. Among them, 6-amino-1-cyclo-
propyl-8-methyl-7-(1,2,3,4-tetrahydroisoquinolinyl)-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (19v) (MF
5137) is characterized by an extremely high activity
against staphylococci as well as those strains which are
methicillin- and ciprofloxacin-resistant. In addition, its
When the 6-amino-8-methyl derivatives 19a ,i,v were
matched with the 6-fluoro-8-methyl counterparts 30a,i,v,
contrasting indications were obtained. It can be seen
that the 6-amino-8-methyl-7-tetrahydroisoquinoline de-
rivative 19v was more active than its fluorurated
analogue 30v against both Gram-negative and Gram-
positive bacteria. This result was however reversed in
the case of the piperazinyl and piperidinyl derivatives.

442 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Cecchetti et al.
excess thionyl chloride was removed by distillation under
reduced pressure to give a mobile oil residue which was
dissolved in dry toulene (10 mL) and added to ethyl 3-(dim-
ethylamino)acrylate19 (3.06 g, 21.4 mmol) and dry Et3N (3.24
g, 32 mmol). The resulting solution was heated at 90 °C for 2
h. After cooling and filtering the insoluble material, the
solvent was evaporated to dryness and the residue was purified
by flash column chromatography eluting with the gradient
cyclohexane/EtOAc (90:10-30:70) to give 9 (4.60 g, 60%) as
activity against all streptococci is very high with MIC
values against isolated S. pneumoniae as much as 15
times lower than those of ciprofloxacin which makes it
probably better than any other agent around. An
expanded description of the microbiological, toxicologi-
cal, and pharmacokinetic profiles of 19v will be reported
elsewhere.
1
yellowish solid: mp 110-112 °C; H NMR (CDCl3) δ 0.95 (3
Exp er im en ta l Section
H, t, J ) 7 Hz, CH2CH3), 2.40 (3 H, d, J ) 3 Hz, CH3), 3.05
and 3.40 (each 3 H, bs, NCH3), 3.55 (2 H, q, J ) 7 Hz, CH2-
CH3), 7.90 (1 H, d, J ) 7.5 Hz, H-6), 8.00 (1 H, s, vinyl H).
Anal. (C15H16ClFN2O5) C, H, N.
Melting points were determined in capillary tubes (Bu¨chi
melting point apparatus) and are uncorrected. Elemental
analyses were performed on a Carlo Erba elemental analyzer,
Model 1106, and the data for C, H, and N are within 0.4% of
the theoretical values. 1H NMR spectra were recorded at 90
MHz (Varian EM 390 spectrometer) or 200 MHz (Bruker AC-
200 spectrometer) with Me4Si as internal standard. Chemical
shifts are given in ppm (δ), and the spectral data are consistent
with the assigned structures. Reagents and solvents were
purchased from common commercial suppliers and used as
received. Column chromatography separations were carried
out on Merck silica gel 40 (mesh 70-230) and flash chroma-
tography on Merck silica gel 60 (mesh 230-400). Organic
solutions were dried over anhydrous Na2SO4 and concentrated
with a Bu¨chi rotary evaporator at low pressure. Yields were
of purified product and were not optimized. All starting
materials were commercially available unless otherwise indi-
cated. The physical properties of the target acid derivatives
are summarized in Tables 1 and 2.
E t h yl 1-Cyclop r op yl-7-flu or o-8-m et h yl-6-n it r o-4-oxo-
1,4-d ih yd r oqu in olin e-3-ca r boxyla te (10). A stirred solu-
tion of 9 (1.5 g, 4 mmol) in EtOH (48 mL) and Et2O (18 mL)
was treated dropwise with cyclopropylamine (0.37 g, 6.5
mmol). After 15 min at room temperature, the mixture was
evaporated to dryness to give a residue which was solubilized
in dry DMF (5 mL). To this solution was added K2CO3 (1 g,
7.2 mmol), and the mixture was heated at 100 °C for 40 min.
After cooling, the reaction mixture was poured into ice-water.
The precipitated solid was filtered off, washed with water, and
dried to give 10 (1.3 g, 97%): mp 198-200 °C; 1H NMR (CDCl3)
δ 1.00-1.10 and 1.25-1.35 (each 2 H, m, cyclopropyl CH2),
1.45 (3 H, t, J ) 7.5 Hz, CH2CH3), 2.80 (3 H, d, J ) 3 Hz,
CH3), 3.90-4.10 (1 H, m, cyclopropyl CH), 4.40 (2 H, q, J )
7.5 Hz, CH2CH3), 8.65 (1 H, s, H-2), 8.95 (1 H, d, J ) 7.5 Hz,
H-5). Anal. (C16H15FN2O5) C, H, N.
1-(2-Ch lor o-4-flu or o-3-m et h ylp h en yl)et h a n on e (6).
Some drops of acetyl chloride were added to a mixture of
2-chloro-4-fluorotoluene (40 g, 0.28 mol) and AlCl3 (73.86 g,
0.55 mol) which was then heated to 40 °C to trigger the
reaction (generate HCl). After cooling to room temperature,
acetyl chloride (22 g, 0.28 mol) was added dropwise and the
reaction mixture was allowed to react for 2 h. The mixture
was then poured into ice-water and acidified with 2 N HCl.
The solution was extracted several time with CH2Cl2, and the
combined organic layers were washed with water, dried, and
evaporated to dryness to give a yellow oil (48.2 g, 93%)
containing 6, as well as the other meta-isomer, 1-(4-chloro-2-
fluoro-3-methylphenyl)ethanone, in 95:5 ratio, respectively, on
According to this procedure, compounds 11 and 12 were
prepared from 9, replacing cyclopropylamine with 4-fluoroa-
niline and 4-methoxyaniline, respectively. 11: mp 195-197
°C (95%). 12: mp 205-207 °C (90%).
Gen er a l P r oced u r es for Cou p lin g Rea ction . Meth od
A: P r ep a r a tion of Eth yl 1-Cyclop r op yl-8-m eth yl-6-n itr o-
7-(4-m eth yl-1-p ip er a zin yl)-4-oxo-1,4-d ih yd r oqu in olin e-3-
ca r boxyla te (13a ). The mixture of ester 10 (0.70 g, 2 mmol)
and N-methylpiperazine (0.80 g, 8 mmol) in dry pyridine (10
mL) was heated at 100 °C for 4 h. After removing the solvent,
the residue was triturated with EtOH and filtered, washing
with EtOH, to give 13a (0.62 g, 75%) which was reduced
without further purification: mp 210-213 °C; 1H NMR
(CDCl3) δ 0.75-1.50 (7 H, m, CH2CH3, cyclopropyl CH2), 2.40
(3 H, s, NCH3), 2.40-2.60 (5 H, m, piperazine CH2, cyclopropyl
CH), 2.70 (3 H, s, CH3), 2.95-3.25 (4 H, m, piperazine CH2),
4.40 (2 H, q, J ) 7 Hz, CH2CH3), 8.50 (1 H, s, H-2), 8.60 (1 H,
s, H-5).
1
the basis of H NMR spectrum. The material was used as is
in the next step: 1H NMR (CDCl3) δ 2.35 (3 H, d, J ) 2.3 Hz,
CH3), 2.60 (3 H, s, COCH3), 7.00 (0.95 H, t, J ) 8.5 Hz, H-5 of
the major isomer), 7.20 (0.05 H, d, J ) 8.5 Hz, H-5 of the minor
isomer), 7.35 (0.95 H, dd, J ) 6, 8.5 Hz, H-6 of the major
isomer), 7.65 (0.05 H, t, J ) 8.1, H-6 of the minor isomer).
2-Ch lor o-4-flu or o-3-m eth ylben zoic Acid (7). Bromine
(102.6 g, 0.64 mol) was added dropwise to a solution of NaOH
(85.6 g, 2.14 mol) in water (400 mL) mantained at 10 °C. The
mixture was then cooled to 0 °C, and a solution of 6 (40 g,
0.21 mol) in dioxane (400 mL) was added dropwise. After
stirring for 1 h at room temperature, the reaction mixture was
poured into water and extracted with CHCl3. The aqueous
solution was acidified with 37% HCl, and the white precipi-
tated solid was filtered off, washed with water and then with
Et2O, and dried to give 7 (39.5 g, 98%) (presumably slightly
impure, for the other isomer not detectable by 1H NMR
spectrum) which was used as is in the subsequent reaction:
1H NMR (CDCl3) δ 2.40 (3 H, d, J ) 2.3 Hz, CH3), 7.05 (1 H,
t, J ) 8.5 Hz, H-5), 7.90 (1 H, dd, J ) 6, 8.5 Hz, H-6).
In an analogous procedure, compounds 13i,j,l,r -t were
prepared from 10, while compounds 14a and 15a were
prepared from 11 and 12, respectively, by reaction with the
appropiate heterocylic amine.
Met h od B: P r ep a r a t ion of E t h yl 1-Cyclop r op yl-8-
m et h yl-6-n it r o-7-(3,3-d im et h yl-1-p ip er id in yl)-4-oxo-1,4-
d ih yd r oqu in olin e-3-ca r boxyla te (13m ). A mixture of ester
10 (0.8 g, 2.4 mmol), dry Et3N (0.96 g, 9.5 mmol), and 3,3-
dimethylpiperidine (1.08 g, 9.5 mmol) in dry CH3CN (15 mL)
was refluxed for 24 h. The solvent was then evaporated to
dryness, and the residue, treated with EtOH, gave a solid
which was purified by flash chromatography eluting with
CHCl3/MeOH (98:2) to give 13m (0.40 g, 39%): mp 192-193
1
°C; H NMR (CDCl3) δ 0.85-0.95 (2 H, m, cyclopropyl CH2),
1.00 (6 H, s, piperidine CH3), 1.15-1.30 (2 H, m, cyclopropyl
CH2), 1.45 (3 H, t, J ) 7 Hz, CH2CH3), 1.55-1.90 (4 H, m,
piperidine CH2), 2.55-2.70 (2 H, m, piperidine CH2), 2.75 (3
H, s, CH3), 3.10-3.25 (2 H, m, piperidine CH2), 3.90-4.05 (1
H, m, cyclopropyl CH), 4.40 (2 H, q, J ) 7 Hz, CH2CH3), 8.60
(1 H, s, H-2), 8.70 (1 H, s, H-5). Anal. (C23H29N3O5) C, H, N.
2-Ch lor o-4-flu or o-3-m eth yl-5-n itr oben zoic Acid (8). A
mixture of 99.5% HNO3 (4.7 mL), 98% H2SO4 (100 mL), and
acid 7 (20 g, 10.61 mol) was allowed to react at room
temperature for 1 h. The mixture was poured into ice-water,
and the resulting precipitate was filtered, dried, and recrystal-
lized from benzene to give a solid (22.8 g, 92%) containing 8,
as well as the isomer 4-chloro-2-fluoro-3-methyl-5-nitrobenzoic
acid, in 95:5 ratio, respectively, based on 1H NMR spectrum:
1H NMR (CDCl3) δ 2.45 (3 H, d, J ) 2.3 Hz, CH3), 8.50 (0.05
H, d, J ) 7.5 Hz, H-6 of the minor isomer), 8.60 (0.95 H, d, J
) 7.5 Hz, H-6 of the major isomer), 9.25 (1 H, bs, CO2H).
Eth yl 2-(2-Ch lor o-4-flu or o-3-m eth yl-5-n itr oben zoyl)-3-
(d im eth yla m in o)a cr yla te (9). A mixture of 8 (5 g, 21.4
mmol) and thionyl chloride (15 mL) was refluxed for 3 h. The
By a similar procedure, compounds 13c,d ,k ,n -p were
obtained from 10 by reaction with the appropiate heterocylic
amine. For piperazine derivative 13c, formylpiperazine was
employed as nucleophile and the protective formyl group was
removed in the successive hydrolysis step.
Meth od C: P r ep a r a tion of Eth yl 1-Cyclop r op yl-8-
m eth yl-6-n itr o-7-(3-m eth yl-1-p ip er a zin yl)-4-oxo-1,4-d ih y-
d r oqu in olin e-3-ca r boxyla te (13b). The mixture of ester 10

Studies on 6-Aminoquinolones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 443
(0.90 g, 2.7 mmol), 2-methylpiperazine (0.81 g, 8.1 mmol), and
K2CO3 (1.12 g, 8.1 mmol) in dry DMF (6 mL) was heated at
90 °C for 2 h. After cooling, the reaction mixture was poured
into water and extracted with CH2Cl2. The combined organic
layers were washed with water, dried, and evaporated to
dryness. The solid residue was purified by silica gel column
chromatography eluting with CHCl3/MeOH (97:3) to give 13b
(0.56 g, 50%): mp 126-128 °C; 1H NMR (CDCl3) δ 0.75-0.85
(2 H, m, cyclopropyl CH2), 1.00-1.30 (5 H, m, cyclopropyl CH2,
piperazine CH3), 1.40 (3 H, t, J ) 7 Hz, CH2CH3), 2.70 (3 H,
s, CH3), 2.80-3.20 (7 H, m, piperazine CH2, CH, NH), 3.85-
4.10 (1 H, m, cyclopropyl CH), 4.35 (2 H, q, J ) 7 Hz, CH2-
CH3), 8.45 (1 H, s, H-2), 8.60 (1 H, s, H-5). Anal. (C21H26N4O5)
C, H, N.
Pd/C (0.5 g) were added to a solution of 13o (0.5 g, 1.25 mmol)
in dry MeOH (15 mL), cooled to 0 °C. The stirred mixture
was maintained at this temperature for 30 min and then
filtered over charcoal. The solvent was evaporated to dryness,
and the resulting residue was purified by column chromatog-
raphy eluting with a gradient of CHCl3 to CHCl3/MeOH (95:
5) to give 16o (0.15 g, 33%): mp 273-275 °C; 1H NMR (CDCl3)
δ 0.80-1.25 (4 H, m, cyclopropyl CH2), 1.45 (3 H, t, J ) 7 Hz,
CH2CH3), 2.00-2.20 (4 H, m, pyridine CH2), 3.00 (3 H, s, CH3),
3.10-3.20 (2 H, m, pyridine CH2), 3.95-4.10 (1 H, m, cyclo-
propyl CH), 4.20 (2 H, bs, NH2), 4.45 (2 H, q, J ) 7 Hz, CH2-
CH3), 4.50-4.60 (2 H, t, J ) 5.5 Hz, pyridine CH), 8.50 (1 H,
s, H-5), 8.70 (1 H, s, H-2). Anal. (C21H25N3O3) C, H, N.
Gen er a l P r oced u r e for Hyd r olysis Rea ction . Meth od
F : P r ep a r a tion of 6-Am in o-1-cyclop r op yl-8-m eth yl-7-(4-
m eth yl-1-p ip er a zin yl)-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r -
boxylic Acid Dih yd r och lor id e (19a ). A solution of 16a
(0.20 g, 0.52 mmol) in EtOH (2 mL) and 6 N HCl (2 mL) was
refluxed for 2 h. After cooling, the crystalline-precipitated
solid was filtered off, washed with dry EtOH, and dried to give
19a (0.1 g, 45%) as a white solid: mp >300 °C; 1H NMR
(DMSO-d6) δ 0.70-1.30 (4 H, m, cyclopropyl CH2), 2.60 (3 H,
s, CH3), 2.80 (3 H, bd, NHCH3), 3.20-3.70 (9 H, m, piperazine
CH2, cyclopropyl CH), 7.50 (1 H, s, H-5), 8.65 (1 H, s, H-2),
10.80 (1 H, bs, CO2H). Anal. (C19H24N4O3‚2HCl) C, H, N.
By a similar procedure, compounds 13e,u were obtained
from 10 by reaction with the appropiate heterocylic amine.
Meth od D: P r ep a r a tion of Eth yl 1-Cyclop r op yl-8-
m eth yl-6-n itr o-7-(4-m or p h olin yl)-4-oxo-1,4-d ih yd r oqu in -
olin e-3-ca r boxyla te (13f). The mixture of ester 10 (0.9 g,
2.7 mmol) and morpholine (0.47 g, 5.4 mmol) in dry DMF (7
mL) was heated at 75 °C for 10 h. After cooling, the precipitate
was separated by filtration, washed with water and then with
1
EtOH, and dried to give 13f (0.35 g, 32%): mp >330 °C; H
NMR (CDCl3) δ 0.75-0.95 and 1.05-1.25 (each 2 H, m,
cyclopropyl CH2), 1.40 (3 H, t, J ) 7 Hz, CH2CH3), 2.70 (3 H,
s, CH3), 3.00-3.25 (4 H, m, morpholine CH2), 3.80-4.10 (5 H,
m, morpholine CH2, cyclopropyl CH), 4.40 (2 H, q, J ) 7 Hz,
CH2CH3), 8.55 (1 H, s, H-2), 8.65 (1 H, s, H-5). Anal.
(C20H23N3O6) C, H, N.
Using the above general procedure, target acids 19b-f,i-
q,s-u , 20a , and 21a were prepared from corresponding esters
16b-f,i-q,s-u , 17a , and 18a , as well as intermediate acid
1
23 from ester 10. Compound 23: mp 176-180 °C (90%); H
By a similar procedure, compound 13q was prepared from
10 by reaction with pyrrolidine.
NMR (DMSO-d6) δ 1.00-1.30 (4 H, m, cyclopropyl CH2), 2.85
(3 H, d, J ) 3 Hz, CH3), 4.30-4.50 (1 H, m, cyclopropyl CH),
8.80 (1 H, d, J ) 9 Hz, H-5), 8.85 (1 H, s, H-2), 13.00 (1 H, bs,
CO2H). Anal. (C14H11FN2O5) C, H, N.
Meth od E: P r ep a r a tion of 1-Cyclop r op yl-8-m eth yl-6-
n itr o-7-(1,2,3,4-tetr a h yd r o-2-isoqu in olin yl)-4-oxo-1,4-d i-
h yd r oqu in olin e-3-ca r boxylic Acid (24v). The mixture of
acid 23 (4 g, 13 mmol), 1,2,3,4-tetrahydroisoquinoline (7.5 g,
56 mmol), and Et3N (7.5 g, 74 mmol) in dry CH3CN (200 mL)
was heated at reflux for 40 h. After cooling, the resulting solid
was filtered and washed with CH3CN and then with EtOH to
give 24v (2.1 g, 38%): mp >300 °C; 1H NMR (DMSO-d6) δ
0.85-1.45 (4 H, m, cyclopropyl CH2), 2.75 (3 H, s, CH3), 2.85-
3.10 and 3.30-3.60 (each 2 H, m, isoquinoline CH2), 4.25-
4.50 (3 H, m, isoquinoline CH2, cyclopropyl CH), 7.20 (4 H,
bs, isoquinoline aromatic H), 8.50 (1 H, s, H-2), 8.90 (1 H, s,
H-5). Anal. (C23H21N3O5) C, H, N.
Meth od G: P r ep a r a tion of 6-Am in o-7-a zetid in yl-1-
cyclop r op yl-8-m eth yl-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r -
boxylic Acid (19r ). The suspension of 16r (0.1 g, 2.7 mmol)
in 1 N NaOH (2 mL) was refluxed for 3 h (until the suspension
became a solution). After cooling at room temperature, the
solution was filtered, diluted with water (4 mL), and brought
to pH 6 by adding a solution of 2 N HCl. The resulting
precipitate was filtered and washed with water to give 19r
(0.046 g, 50%): mp 230-231 °C; 1H NMR (DMSO-d6) δ 0.85-
1.00 and 1.15-1.30 (each 2 H, m, cyclopropyl CH2), 2.10-2.30
(2 H, m, azetidine CH2), 2.40 (3 H, s, CH3), 4.15-4.35 (5 H, m,
cyclopropyl CH, azetidine CH2), 4.95 (1 H, bs, NH2), 7.30 (1
H, s, H-5), 8.55 (1 H, s, H-2). Anal. (C17H19N3O3‚H2O) C, H,
N.
By a similar procedure, compounds 24g,h were prepared
from acid 23 by reaction with the appropiate heterocyclic
amine.
Gen er a l P r oced u r e for R ed u ct ion of 6-Nit r o Gr ou p
(NO2 w NH2): P r ep a r a tion of Eth yl 6-Am in o-1-cyclop r o-
p yl-8-m eth yl-7-(4-m eth yl-1-p ip er a zin yl)-4-oxo-1,4-d ih yd -
r oqu in olin e-3-ca r boxyla te (16a ). A stirred solution of 13a
(0.24 g, 0.58 mmol) in 2-methoxyethanol (5 mL) was hydro-
genated over Raney nickel (0.1 g) at room temperature and
atmospheric pressure for 1 h. The mixture was then filtered
over Celite, and the filtrate was evaporated to dryness. The
solid residue was triturated with EtOH giving 16a (0.20 g,
90%): mp 223-225 °C; 1H NMR (CDCl3) δ 0.80-1.55 (7 H, m,
CH2CH3, cyclopropyl CH2), 2.40 (3 H, s, NCH3), 2.60 (3 H, s,
CH3), 2.80-3.60 (8 H, m, piperazine CH2), 3.75-4.00 (1 H, m,
cyclopropyl CH), 4.20 (2 H, bs, NH2), 4.40 (2 H, q, J ) 7 Hz,
CH2CH3), 7.60 (1 H, s, H-5), 8.60 (1 H, s, H-2). Anal.
(C21H28N4O3) C, H, N.
In an analogous procedure, compounds 16b -f,i-n ,p -u ,
17a , and 18a were prepared starting from the corresponding
nitro esters 13b-f,i-n ,p -u , 14a , and 15a , while 19g,h ,v were
prepared from corresponding nitro acids 24g,h ,v.
By a similar procedure intermediate 28 was also obtained
from 10. Compound 28: mp 219-221 °C (92%); 1H NMR
(CDCl3) δ 0.90-1.35 (4 H, m, cyclopropyl CH2), 1.40 (3 H, t, J
) 7 Hz, CH2CH3), 2.70 (3 H, d, J ) 3 Hz, CH3), 3.80-3.95 (1
H, m, cyclopropyl CH), 4.10 (2 H, bs, NH2), 4.35 (2 H, q, J )
7 Hz, CH2CH3), 7.80 (1 H, d, J ) 7.5 Hz, H-5), 8.60 (1 H, s,
H-2). Anal. (C16H17FN2O3) C, H, N.
In an analogous procedure, compounds 24a ,i,j were pre-
pared starting from the corresponding nitro esters 13a ,i,j.
6-Am in o-1-(4-h yd r oxyp h en yl)-8-m eth yl-7-(4-m eth yl-1-
piper azin yl)-4-oxo-1,4-dih ydr oqu in olin e-3-car boxylic Acid
(22a ). A solution of 18a (0.25 g, 0.55 mmol) in 48% HBr (2
mL) was refluxed for 5 h. After cooling, the precipitate was
separated by filtration and solubilized in a minimum amount
of water. The aqueous solution was made basic (pH 7.5-8)
by adding a saturated solution of Na2CO3, and the precipitated
solid was filtered off and dried to give 22a (0.15 g, 63%): mp
>300 °C; 1H NMR (DMSO-d6) δ 1.65 (3 H, s, CH3), 2.25 (3 H,
s, NCH3), 2.40-3.60 (8 H, m, piperazine CH2), 5.40 (2 H, bs,
NH2), 6.90 and 7.35 (each 2 H, d, J ) 8 Hz, aromatic H), 7.50
(1 H, s, H-5), 8.35 (1 H, s, H-2), 8.90 (1 H, bs, OH), 10.00 (1 H,
bs, CO2H). Anal. (C22H24N4O4) C, H, N.
Eth yl 1-Cyclop r op yl-8-m eth yl-7-(4-m eth yl-1-p ip er id i-
n yl)-6-[(tr iflu or oa cetyl)a m in o]-4-oxo-1,4-d ih yd r oqu in o-
lin e-3-ca r boxyla te (25j). The mixture of 16j (0.2 g, 0.5
mmol) and (CF3CO)2O (3 mL) was stirred at room temperature
for 30 min and then poured into water, basified to pH 7 with
a saturated solution of Na2CO3, and extracted with CHCl3. The
combined organic layers were evaporated to dryness to give
25j (0.2 g, 80%) which was hydrolyzed without further
1
purification: mp 248-250 °C; H NMR (CDCl3) δ 0.80-1.20
(7 H, m, cyclopropyl CH2, piperidine CH3), 1.40 (3 H, t, J ) 7
Hz, CH2CH3), 1.55-1.95 (5 H, m, piperidine CH2, CH), 2.65
(3 H, s, CH3), 2.95-3.10 and 3.20-3.40 (each 2 H, m,
piperidine CH2), 3.85-4.00 (1 H, m, cyclopropyl CH), 4.40 (2
E t h yl 6-Am in o-1-cyclop r op yl-8-m et h yl-7-(1,2,3,6-t et -
r a h yd r op yr id in yl)-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r box-
yla te (16o). Ammonium formate (0.4 g, 6.25 mmol) and 10%

444 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Cecchetti et al.
H, q, J ) 7 Hz, CH2CH3), 8.60 (1 H, s, H-5), 9.05 (1 H, s, H-2),
In an analogous procedure, 6-fluoro derivatives 30a ,i were
prepared from 29 by reaction with the appropiate amine.
In Vitr o An tiba cter ia l Activity. The method has been
described previously.13
In h ib it or y E ffect on Su p er coilin g Act ivit y of DNA
Gyr a se. This assay was carried out according to a method
reported previously with slight modifications.14
9.70 (1 H, bs, NH).
1-Cyclop r op yl-6-(m eth yla m in o)-8-m eth yl-7-(4-m eth yl-
1-p ip er id in yl)-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxylic
Acid (26j). A mixture of 25j (0.2 g, 0.4 mmol), CH3I (0.113 g,
0.8 mmol), and K2CO3 (0.1 g, 0.8 mmol) in dry acetone (15 mL)
was refluxed with stirring for 30 min. After removing the
solvent, the residue was taken up in CHCl3 to remove the
inorganic salts. The organic solvent was evaporated to dryness
and the residue dissolved in a mixture of EtOH/20% NaOH
(1:1) (4 mL). The mixture was then refluxed for 2 h. After
cooling, the solution was diluted with water and neutralized
with 2 N HCl. The precipitated solid so obtained was filtered
off, dried, and purified by recrystallization from EtOH to give
log P Ca lcu la tion . The partition coefficient values, log P
(octanol/water), were calculated by MedChem Software Re-
lease 3.515 and are listed as clog P values in Table 4.
In Vivo Effica cy on System ic In fection in Mice. Mouse
protection tests were performed against S. aureus Smith
ATCC19636. Male and female CD-1 mice (Charles River)
weighing 18-22 g were infected intraperitoneally with 0.5 mL
of a bacterial suspension from an overnight culture (in brain-
heart infusion; Difco) diluted in 5% bacteriological mucin
(Difco). The test compounds were solubilized in DMSO.
Subsequent dilutions were performed using 0.5% methocel.
Micronized powders were administered as suspensions in 0.5%
methocel plus 0.0025% of poly(oxyethylene) fatty acid esters
40 (P40). Ciprofloxacin was administered suspended in 0.5%
methocel. Three to five groups, of six mice each, were
subcutaneously or orally treated once, within 10 min after
infection, with each test compound at different dose levels. The
50% effective dose (ED50) and 95% confidence limits were
calculated by the Spearmen-Ka¨rber method20 from the per-
centages of animals surviving to day 7 at each dose.
1
26j (0.10 g, 58%): mp >300 °C; H NMR (DMSO-d6) δ 0.75-
0.85 (2 H, m, cyclopropyl CH2), 1.00 (3 H, bs, piperidine CH3),
1.10-1.25 (2 H, m, cyclopropyl CH2), 1.35-1.75 (5 H, m,
piperidine CH2, CH), 2.60 (3 H, s, CH3), 2.82 (3 H, bd, NHCH3),
2.95-3.25 (4 H, m, piperidine CH2), 4.15-4.30 (1 H, m,
cyclopropyl CH), 5.30 (1 H, bq, NH), 7.05 (1 H, s, H-5), 8.60 (1
H, s, H-2). Anal. (C21H27N3O3) C, H, N.
1-Cyclop r op yl-6-(d im eth yla m in o)-8-m eth yl-7-(4-m eth -
yl-1-p ip er id in yl)-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r boxy-
lic Acid (27j). A mixture of 25j (0.2 g, 0.52 mmol) and
trimethyl phosphate (0.073 g, 0.52 mmol) was heated at 150
°C for 30 min. Then a solution of NaOH (0.062 g, 1.55 mmol)
in water (1 mL) was added, and the mixture was refluxed for
20 min. After cooling, the mixture was diluted with water and
extracted with CHCl3. The combined organic layers were dried
and evaporated to dryness to give a residue which, after
recrystallization with EtOAc, gave 27j (0.15 g, 70%): mp >300
Ack n ow led gm en t. We are grateful to Dr. Claudia
Sissi of Dipartimento di Scienze Farmaceutiche, Uni-
versita` di Padova, for her help in providing the DNA
gyrase data and Mr. Roberto Bianconi for skillful
synthetic assistance.
1
°C; H NMR (CDCl3) δ 0.85-0.95 (2 H, m, cyclopropyl CH2),
1.00 (3 H, d, J ) 5.5 Hz, piperidine CH3), 1.10-1.20 (2 H, m,
cyclopropyl CH2), 1.35-1.90 (5 H, m, piperidine CH2, CH), 2.60
(3 H, s, CH3), 2.75 (6 H, s, NCH3), 3.20-3.40 (4 H, m,
piperidine CH2), 4.00-4.15 (1 H, m, cyclopropyl CH), 7.85 (1
H, s, H-5), 8.85 (1 H, s, H-2), 15.35 (1 H, s, CO2H). Anal.
(C22H29N3O3) C, H, N.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR data of
target acids not reported in the test (6 pages). Ordering
information is given on any current masthead page.
1-Cyclop r op yl-6,7-d iflu or o-8-m et h yl-4-oxo-1,4-d ih y-
d r oqu in olin e-3-ca r boxylic Acid Diflu or obor a te Ch ela te
(29). A solution of NaNO2 (0.45 g, 6.5 mmol) in water (3 mL)
was added portionwise to a solution of 28 (1.5 g, 4.9 mmol) in
6 N HCl (8 mL), maintained at -5 °C. After 20 min at this
temperature, a 50% solution in water of HBF4 (4 mL) was
added. The resulting yellow solid was filtered off, washed
successively with cold water, EtOH, and Et2O, and dried below
50 °C under reduced pressure giving the diazonium tetrafluo-
roborate salt (1.6 g, 72%). This material was thermally
decomposed at 180-200 °C for some minutes (until no fumes
occurred). The obtained dark solid was then extracted with
refluxing EtOAc (2 × 400 mL). The organic solvent was
evaporated to dryness, and the residue was purified by flash
column chromatography eluting with a gradient of cyclohex-
ane/EtOAc (1:1) to EtOAc to give 29 (0.335 g, 21%): mp 195-
Refer en ces
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Activity of MF 5137, a New Potent 6-Aminoquinolone. 5th
International Symposium on New Quinolones, Singapore, Au-
gust 1994; p 11. (b) Cecchetti, V.; Fravolini, A.; Lorenzini, C.;
Tabarrini, O.; Terni, P.; Wise, R.; Xin, T. A New Series of
6-Aminoquinolones with Potent Antimicrobial Activity. XIIIth
International Symposium on Medicinal Chemistry, Paris, France,
September 1994; p 148.
(2) (a) Bonelli, D.; Cecchetti, V.; Clementi, S.; Cruciani, G.; Fravolini,
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1991, 10, 333-343. (b) Bonelli, D.; Cecchetti, V.; Clementi, S.;
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Activity of Quinolones against Staphylococcus aureus. Pharm.
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1
197 °C; H NMR (CDCl3) δ 1.22-1.35 and 1.45-1.60 (each 2
H, m, cyclopropyl CH2), 3.00 (3 H, d, J ) 3 Hz, CH3), 4.45-
4.62 (1 H, m, cyclopropyl CH), 8.30 (1 H, t, J ) 9 Hz, H-5),
9.30 (1 H, s, H-2). Anal. (C14H10BF4NO3) C, H, N.
1-Cyclop r op yl-6,7-d iflu or o-8-m et h yl-4-oxo-1,4-d ih y-
d r oqu in olin e-3-ca r boxylic Acid (30v). A mixture of 29 (0.3
g, 0.92 mmol), 1,2,3,4-tetrahydroisoquinoline (1.27 g, 9.5
mmol), and dry Et3N (0.96 g, 9.5 mmol) in dry CH3CN (6 mL)
was refluxed for 4 days. After cooling, the solvent was
removed and the residue was treated with water, acidified with
6 N HCl to pH 5-6, and extracted with CHCl3. The combined
organic layers were evaporated to dryness, and the residue
was filtered by column chromatography eluting with CHCl3
and then recrystallized from EtOAc to give 30v (0.165 g,
27%): mp 229-232 °C; 1H NMR (CDCl3) δ 0.90-1.05 and
1.20-1.35 (each 2 H, m, cyclopropyl CH2), 2.80 (3 H, s, CH3),
3.00-3.20 and 3.55-3.65 (each 2 H, m, isoquinoline CH2),
4.05-4.20 (1 H, m, cyclopropyl CH), 4.45 (2 H, s, isoquinoline
CH2), 7.05-7.40 (4 H, m, isoquinoline aromatic H), 8.00 (1 H,
d, J ) 11.5 Hz, H-5), 8.90 (1 H, s, H-2), 14.70 (1 H, s, CO2H).
Anal. (C23H21FN2O3) C, H, N.

Studies on 6-Aminoquinolones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 445
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