Synthesis and in-vitro antibacterial activity of 7-(3-aminopyrrolo[3,4-c] pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives

Article abstract of DOI:10.1002/ardp.201000160

A series of novel 7-(3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro- 4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives was designed, synthesized and characterized by ¹H-NMR, MS and HRMS. These fluoroquinolones were evaluated for their in-vitro antibacterial activity against representative Gram-positive and Gram-negative strains. Generally, all of the target compounds display rather weak potency against the tested Gram-negative strains, but most of them exhibit good potency in inhibiting the growth of S. aureus including methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis including methicillin-resistant S. epidermidis (MRSE) (MIC: 0.125-8 μg/mL). In particular, the compound 9g is 2 to 32 fold more potent than gemifloxacin (GM), moxifloxacin (MX), gatifloxacin (GT), and levofloxacin (LV) against S. pneumoniae 08-3, K. pneumoniae 09-23, and P. aeruginosa ATCC27853, 4 to 32 fold more potent than MX, GM, and LV against K. pneumoniae 09-21, and more active than or comparable to the four reference drugs against P. aeruginosa 09-32. A series of novel fluoroquinolone derivatives was designed and synthesized. All target compounds display rather weak potency against the tested Gram-negative strains, but most of them exhibit good potency in inhibiting the growth of Staphylococcus aureus and Staphylococcus epidermidis. In particular, the compound 9g is the most promising drug.

Full text of DOI:10.1002/ardp.201000160

Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
523
Full Paper
Synthesis and In-vitro Antibacterial Activity of 7-(3-
Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic Acid Derivatives
Xin Guo1,2, Xiao-Guang Bai1, Yi-Liang Li1,3, Zhi-Jiao An1, Le-Xing Xu1, Li-you Han1, Ming-Liang Liu1,
Hui-Yuan Guo1, and Yu-Cheng Wang1
1 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing, P.R. China
2 College of Pharmaceutical Sciences, Southwest University, Chongqing, P.R. China
3 Tianjin Institute of Pharmaceutical Research, Tianjin Key Laboratory of Molecular Design & Drug
Discovery, Tianjin, P.R. China
A series of novel 7-(3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid derivatives was designed, synthesized and characterized by H-NMR, MS and HRMS.
These fluoroquinolones were evaluated for their in-vitro antibacterial activity against representative
Gram-positive and Gram-negative strains. Generally, all of the target compounds display rather weak
potency against the tested Gram-negative strains, but most of them exhibit good potency in inhibiting
the growth of S. aureus including methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus
epidermidis including methicillin-resistant S. epidermidis (MRSE) (MIC: 0.125–8 mg/mL). In particular, the
compound 9g is 2 to 32 fold more potent than gemifloxacin (GM), moxifloxacin (MX), gatifloxacin (GT),
and levofloxacin (LV) against S. pneumoniae 08-3, K. pneumoniae 09-23, and P. aeruginosa ATCC27853, 4 to
32 fold more potent than MX, GM, and LV against K. pneumoniae 09-21, and more active than or
comparable to the four reference drugs against P. aeruginosa 09-32.
Keywords: Antibacterial activity / Fluoroquinolone / Structure–activity relationship
Received: May 30, 2010; Revised: August 6, 2010; Accepted: August 20, 2010
DOI 10.1002/ardp.201000160
Introduction
protein–DNA complex thus disrupting normal DNA replica-
tion, inducing oxidative damage, and triggering cell-death
mechanisms. DNA gyrase appears to be the primary target for
quinolones in Gram-negative bacteria such as Escherichia coli,
while topoisomerase IV is the primary target in Gram-positive
bacteria such as Staphylococcus aureus [3, 4].
Since the discovery of nalidixic acid by Lesher et al. in 1962 [1],
the quinolones have evolved into an important class of anti-
bacterial agents used mainly for the treatment of respiratory
tract infections (RTI), urinary tract infections (UTI), sexually
transmitted diseases (STD), gastrointestinal and abdominal
infections, skin and soft tissue infections, and infections of
the bone and joints among many other uses [2]. These anti-
biotics act by binding to the quinolone-resistance-determin-
ing region (QRDR) in the catalytic domain of the
topoisomerase II (DNA gyrase) or IV complex with DNA.
Cell death is induced by trapping the topoisomerase
Although most of the quinolones currently on the market
or under development are generally characterized by a broad
antimicrobial spectrum, their activity against clinically
important Gram-positive cocci including Staphylococci,
Streptococci, and Enterococci is relatively moderate, which has
not only limited their use in infections caused by these
organisms, but is also believed to be one of the reasons for
the rapidly developing quinolone resistance. Thus, recent
efforts have been directed toward the synthesis of new qui-
nolones that can provide improved Gram-positive antibacte-
rial activity, while retaining the good Gram-negative activity
of early fluoroquinolones, such as ciprofloxacin and ofloxa-
cin [5].
Correspondence: Yu-Cheng Wang, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, P.R. China,
E-mail: wyc9999@gmail.com
Fax: þ86 10 63165263
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Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
Figure 1. The structure of some quinolones containing an aminopyrrolidine residue at the C-7 position.
Structure–activity relationship (SAR) studies of quinolone
antibacterial agents showed that the basic group at the C-7
position is the most adaptable site for chemical change and an
area that greatly influences their potency, spectrum, and
safety [6]. In general, 5- and 6-membered nitrogen heterocycles
including piperazinyl, pyrrolidinyl, and piperidinyl type side
chains have been proven to be the optimal substituents [7].
The introduction of the pyrrolidine derivatives to the qui-
nolones resulted in a dramatic improvement of Gram-
positive activity compared to piperazinyl analogs. Many
some fourth generation cephalosporins, such as cefoselis
[13], was fused with a pyrrolidine to offer the 3-amino-
2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole which can be readily
obtained from the corresponding 3-cyano-4-oxopyrrolidine in
a single operation. A series of novel fluoroquinolone com-
pounds containing these bicyclic amines at the 7-position
were designed and synthesized. Our primary objective was to
optimize the potency of these compounds against Gram-
positive and Gram-negative organisms.
new fluoroquinolones used currently in the clinic, including Results and discussion
tosufloxacin, sitafloxacin, trovafloxacin, gemifloxacin (GM),
and moxifloxacin (MX), contain an aminopyrrolidine residue
at the C-7 position [8–12] (Fig. 1). In this study, additional
attempts have been made to modify the pyrrolidinyl moiety.
The 3-aminopyrazole function group at the 3-positions of
Chemistry
The synthesis of the novel fluoroquinolone derivatives 8a–g
and 9a–g is outlined in Scheme 1. Addition reaction of ethyl-
glycinate hydrochloride 1 with acrylonitrile in the presence of
Scheme 1. Synthesis route of compounds 8a–g and 9a–g.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
New pyrrolo[3,4-c]pyrazolylquinolone derivatives
525
Table 1. Structures and physical data of novel fluoroquinolones 8a–g and 9a–g.
O
F
COOH
H2N
R
X
N
N
R1
N
N
Compound
8a
R
R1
X
Yield (%)
Mp (8C)a)
>300
ESI-MS (M þ H)þ
H
N
67
371
8b
8c
8d
H
H
H
2,4-F2-C6H3
C2H5
2-F-C2H4
N
CF
CF
72
56
84
283–285 (dec)
260–263 (dec)
256–258 (dec)
443
376
394
8e
H
CF
39
43
18
83
273–275 (dec)
239–241 (dec)
244–245 (dec)
214–216 (dec)
388
436
400
385
8f
H
COCHF2
COCH3
N
8g
9a
H
CH3
9b
9c
9d
CH3
CH3
CH3
2,4-F2-C6H3
C2H5
2-F-C2H4
N
CF
CF
91
77
68
236–237 (dec)
223–225 (dec)
243–245 (dec)
457
390
408
9e
CH3
CF
69
267–268 (dec)
402
9f
9g
a)
CH3
CH3
COCHF2
COCH3
47
26
220–223 (dec)
231–232 (dec)
450
414
Melting points are uncorrected.
sodium hydroxide gave the secondary amine 2, which was
subsequently treated with di-tert-butoxycarbonyl dicarbonate
(Boc2O) to produce the Boc-protected cyano ester 3. The com-
pound 3 was cyclized to the cyano ketone 4 by sodium hydride
in refluxing toluene, with an overall yield of 66% for the three
steps. The cyano ketone 4 was treated with hydrazine or
methylhydrazine in ethanol to give the Boc-protected bicyclic
amines 5a,b which upon deprotection afforded the key inter-
mediates 6a,b by pumping hydrogen chloride gas [14–16].
Finally, the target compounds 8a–g and 9a–g were
obtained by the coupling reaction of the intermediates
6a,b with various boric chelates containing quinolone and
naphthyridone cores 7, and then hydrolysis of chelating
groups according to well-established literature procedures
[17–19]. Table 1 shows the novel fluoroquinolone analogs,
yields of the final coupling step, and melting points of the
purified compounds.
Antibacterial activity
The novel fluoroquinolones 8a–g and 9a–g were evaluated for
their in-vitro antibacterial activity against representative
Gram-negative and Gram-positive strains using standard
techniques [20]. Minimum inhibitory concentration (MIC)
is defined as the concentration of the compound required
to give complete inhibition of bacterial growth and MICs of
the synthesized compounds along with the standard drugs
GM, MX, gatifloxacin (GT), and levofloxacin (LV) for compari-
son are reported in Table 2.
The novel fluoroquinolones 8a–g and 9a–g display generally
rather weak potency against the tested Gram-negative
strains, but most of them exhibit good potency in inhibiting
the growth of S. aureus including methicillin-resistant
Staphylococcus aureus (MRSA) and Staphylococcus epidermidis including
methicillin-resistant S. epidermidis (MRSE) (MIC: 0.125–8 mg/mL).
In particular, the most active compound 9g is 2 to 32 fold
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X. Guo et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
Table 2. In-vitro antibacterial activity of compounds 8a–g and 9a–g.
strains
Compd. MIC (mg/mL) a)
9a 9b 9c 9d
0.25 0.25 0.25 0.125 0.25 0.25 0.125 0.5
8a
8b
8c
8d
8e
8f
8g
9e
9f
9g
MX
GM
GT
LV
S.a.1
S.a.2
S.a.3
S.a.4
S.e.1
S.e.2
S.e.3
S.e.4
S.e.5
S.p.1
S.p.2
S.p.3
E.fa.1
E.fa.2
E.fm.1
E.fm.2
E.c.1
E.c.2
E.c.3
E.c.4
E.c.5
K.p.1
K.p.2
K.p.3
P.a.1
P.a.2
P.a.3
P.a.4
P.a.5
2
16
8
8
8
8
8
8
8
2
8
4
8
8
4
4
32
4
4
32
8
8
8
8
8
8
0.5 0.125 0.125 0.125 0.125 0.06 0.125
0.5
2
2
2
2
2
2
2
0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.25 0.25 0.25 0.25 0.25
>128 0.25 0.25 0.25 0.25 0.25 0.125
0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.25 0.25 0.25 0.25 0.25
1
1
4
1
1
1
1
1
0.25 0.25 0.25 0.25 0.125 0.125
0.25 0.25 0.125 0.06 0.06 0.125
0.125 0.125 0.06 0.125 0.06 0.125
0.125 0.25 0.25 0.125 0.25 0.25
0.125 0.25 0.125 0.06 0.25 0.25
0.25 0.25 0.06 0.03 0.125 0.25
0.5
0.5
128
64
4
0.25
0.25 0.25 0.25
2
0.25 0.25
32
8 4
0.25 0.25
0.125
16
0.5
0.25 0.125 0.25 0.25
64
0.5
0.25 0.125 0.25 0.25
8
0.06 0.125 0.25 0.125
32
32
32
0.25 0.25 0.25 0.25 0.25 0.25 0.125
0.25 >128 >128 32
4
3
2
2
8
8
4
16
2
16
8
>128 32
16
16
>128 >128 >128 >128 64
1
1
16
8
8
4
32
0.25 0.25
2
16
0.25
2
2
0.25
8
4
4
16
8
0.25
16 >128 0.25
32 >128 0.25
>128 >128 >128 128 >128 >128 64
>128 >128 >128 128 >128 >128 64
>128 >128 >128 >128 >128 >128 64
>128 >128 >128 >128 128
>128 >128 >128 >128 128
>128 >128 >128 >128 128
>128 >128 >128 >128 128
64
1
2
2
2
2
2
2
2
2
2
1
1
2
16
16
2
16
4
16
16
16
32
2
8
8
2
0.5
2
8
4
2
4
2
8
8
2
1
128 >128
64 >128
128 >128
32
64
>128 >128 32
>128 >128 >128 >128 128
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
>128 >128 >128 64
128
128 >128 128
128 >128
4
8
128
>128
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
16
16
>128 >128 >128 >128 >128 >128 64
>128 >128 >128 128 16 64 128 >128 >128 >128 128
>128 >128 >128 >128 >128 64 >128 >128 >128 128
128 128 >128 128
>128 16 >128
>128 >128 >128 128
128
4
64
128
4
32
32
8
128
4
64
8
0.5
>128
0.5
0.125 16
0.06
2
2
16
4
16
16
4
16
4
8
>128 >128 >128 128
>128 >128 >128 128
16
16
8
0.25
8
8
1
4
4
0.25 0.25
>128 >128 >128 128 >128 >128 128 >128 >128 >128 128
>128 >128 >128 128 >128 >128 64 >128 >128 >128 32
>128 >128 >128 >128 32 64 16 >128 16 >128
32
32
8
64
0.5
2
2
0.25
0.5
0.5
0.5
1
0.5
2
0.5
8
a)
b)
The values were reproduced in three experiments. Abbreviations: S.a.1, Staphylococcus aureus ATCC259223; S.a.2, methicillin-
resistant Staphylococcus aureus 08-1; S.a.3, methicillin-sensitive Staphylococcus aureus 08-1; S.a.4, methicillin-sensitive Staphylococcus aureus
08-2; S.e.1, methicillin-resistant Staphylococcus epidermidis 09-2; S.e.2, methicillin-resistant Staphylococcus epidermidis 09-3; S.e.3, methi-
cillin-resistant Staphylococcus epidermidis 09-4; S.e.4, methicillin-sensitive Staphylococcus epidermidis 09-3; S.e.5, methicillin-sensitive
Staphylococcus epidermidis 09-6; S.p.1, Streptococcus pneumoniae 08-2; S.p.2, Streptococcus pneumoniae 08-3; S.p.3, Streptococcus pneumoniae
08-4; E.fa.1, Enterococcus faecalis 08-10; E.fa.2, Enterococcus faecalis 08-12; E.fm.1, Enterococcus faecium 08-2; E.fm.2 Enterococcus faecium 08-7; E.c.1,
Escherichia coli ATCC 25922; E.c.2, Escherichia coli 08-21; E.c.3, Escherichia coli 08-22; E.c.4, Escherichia coli 08-23; E.c.5, Escherichia coli 08-24; K.p.1,
Klebsiella pneumoniae 09-21; K.p.2, Klebsiella pneumoniae 09-22; K.p.3, Klebsiella pneumoniae 09-23; P.a.1, Pseudomonas aeruginosa ATCC27853;
P.a.2, Pseudomonas aeruginosa 09-32; P.a.3, Pseudomonas aeruginosa 09-33; P.a.4, Pseudomonas aeruginosa 09-34; P.a.5, Pseudomonas aeruginosa
09-35.
more potent than the reference drugs MX, GM, GT, and LV
against S. pneumoniae 08-3, K. pneumoniae 09-23 and P. aeruginosa
ATCC27853, 4 to 32 fold more potent than MX, GM, and LV
against K. pneumoniae 09-21, and more active than or compa-
rable to the four reference drugs against P. aeruginosa 09-32.
Generally, the activity of the quinolone nuclei in this study
is in the order of 1-(2-fluoroethyl)-8-fluoroquinolone > 1-
cyclopropyl-8-methoxylquinolone > 1-cyclopropyl-8-fluoro-
quinolone ꢀ 1-cyclopropyl-8-difluoromethoxylquinolone >
1,8-naphthyridone
dine ꢀ 1-ethyl-8-fluoroquinolone > 1-(2-fluoroethyl)-8-fluoro-
quinolone 1-cyclopropyl-8-fluoroquinolone for fluoro-
ꢀ
1-(2,4-difluorophenyl)-1,8-naphthyri-
ꢀ
quinolones containing 3-amino-2-methyl-pyrrolo[3,4-c]pyra-
zol-5(2H,4H,6H)-yl substitution at C-7 position. In addition,
fluoroquinolones containing 3-aminopyrrolo[3,4-c]pyrazol-
5(2H,4H,6H)-yl substitution at C-7 position appear to be less
than corresponding 2-methyl analogs.
1-cyclopropyl-1,8-naphthyridone
ꢀ
1-(2,4-difluorophenyl)- Conclusion
1,8-naphthyridine ꢀ 1-ethyl-8-fluoroquinolone for fluoroqui-
nolones containing 3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl
substitution at C-7 position; 1-cyclopropyl-8-fluoroquinolone >
1-cyclopropyl-8-difluoromethoxylquinolone > 1-cyclopropyl-
In summary, we report herein the synthesis of a series of
novel 7-(3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives. The
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Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
New pyrrolo[3,4-c]pyrazolylquinolone derivatives
527
dissolved in ethanol (50 mL) was stirred for 12 h at room
temperature to give the Boc-protected bicyclic compound 5a.
To the reaction mixture containing compound 5a was pumped
dried hydrochloride gas at room temperature for 0.5 h, and then
stirred for another 0.5 h at the same temperature. The resulting
solid was collected by suction, and dried in vacuo to give the title
compound 6a (1.0 g, 51%) as a white solid. 1H-NMR (DMSO-d6,
400 MHz) dH 4.13 (2H, s, CH2), 4.28 (2H, s, CH2). ESI-MS: m/z 125
(M þ H)þ.
antibacterial activity of the newly synthesized compounds
was evaluated. Results reveal that most of the target com-
pounds have good activity S. aureus including MRSA and
S. epidermidis including MRSE. However, all of them display
generally rather weak potency against the tested Gram-nega-
tive strains. The reduced activity might be due to the con-
jugated effect between the introduced new double bond and
the amino group possessing a lone pair of electrons on the
nitrogen, hindering the amino group from participating in
hydrogen bonding with the drug target, which in case of
quinolone is DNA gyrase.
3-Amino-2-methyl-2,4,5,6-tetrahydro-pyrrolo[3,4-c]-
pyrazole hydrochloride 6b
The title compound was obtained in a similar manner as for the
preparation 6a (23%) as a white solid. 1H-NMR (DMSO-d6, 400MHz)
dH 3.69 (3H, s, CH3), 4.20 (2H, s, CH2),4.41 (2H, s, CH2). ESI-MS: m/z
139 (M þ H)þ.
Experimental
All chemical reagents and solvents used in this study were
purchased from Beihua Fine Chemicals Company (Beijing,
China). Melting points were determined in open glass capillaries
and are uncorrected. 1H-NMR spectra were recorded on a Varian
Mercury-400 or an INOVA-500 spectrometer using tetramethyl-
silane as internal standard. Electron spray ionization (ESI) mass
spectra and high resolution mass spectra (HRMS) were recorded
on a MDSSCIEX Q-Tap mass spectrometer. Merck silica gel
ART5554 60F254 plates were used for analytical TLC. Column
chromatography was carried out on silica gel HG/T2354-92 made
in Haiyang Chemical Company (Qingdao, China).
General procedure for the synthesis of 7-(3-aminopyrrolo-
[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid derivatives 8a–g
and 9a–g
A mixture of 7 (1.0 mmol), 6a,b (1.5 mmol), triethylamine
(8.0 mmol), and dry acetonitrile (20 mL) was stirred at
25ꢁ608C under an atmosphere of nitrogen for 1ꢁ48 h and
detected with TLC (ethyl acetate as the mobile phase). The result-
ing solid was collected by suction, and dried in vacuo to give the
title compounds 8a–g and 9a–g.
tert-Butyl 3-cyano-4-oxopyrrolidine-1-carboxylate 4
A mixture of ethylglycinate hydrochloride 1 (167.4 g, 1.2 mol),
sodium hydroxide (48.0 g, 1.2 mol), and methanol (600 mL) was
stirred for 0.5 h, and then acrylonitrile (79.5 g, 1.5 mol) was
added dropwise over a period of 40 min at room temperature.
The reaction mixture was heated to 658C and stirred for 3 h to
give the secondary amine 2, which was pure enough to be used
for the next step without further purification.
To the reaction mixture containing the amine 2 was added
(Boc)2O (218.0 g, 1.0 mol) at room temperature, stirred at 55–
608C for 1 h and filtered. The filtrate was concentrated under
reduced pressure. The residue was diluted with ethyl acetate
(400 mL), washed with water and then saturated saline, dried
over anhydrous sodium sulfate and filtered. The filter was concen-
trated under reduced pressure to afford the cyano ester 3 as a
colorless oil.
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxylic acid 8a
The title compound was obtained as a white solid (23%). 1H-NMR
(DMSO-d6, 400 MHz) dH 1.11–1.22 (4H, m, 2 cyclopropyl CH2),
3.74–3.76 (1H, m, cyclopropyl CH), 4.43–4.81 (6H, m, 2 CH2 and
NH2), 5.14 (1H, br, NH), 8.03 (1H, d, J ¼ 12.8Hz, C5-H), 8.59
(1H, s, C2-H), 15.36 (1H, br, COOH); HRMS-ESI: m/z calcd.
for C17H16FN6O3 (M þ H)þ: 371.12679. Found 371.12655.
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-(2,4-
difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 8b
1H-NMR (DMSO-d6, 400 MHz) dH 3.87–4.05 (4H, m, 2 CH2), 4.75
(2H, br, NH2), 5.09 (1H, br, NH), 7.33–7.37 (1H, m, ph-1H), 7.60–
7.63 (1H, m, ph-1H), 7.79–7.82 (1H, m, ph-1H), 8.09 (1H, d, J ¼ 12.8
Hz, C5-H), 8.80 (1H, s, C2-H), 15.27 (1H, br, COOH); HRMS-ESI:
m/z calcd. for C20H14F3N6O3 (M þ H)þ: 443.10795. Found
443.10891.
To a refluxing suspension of sodium hydride (70%, 41.1 g,
1.2 mol) in dry toluene (600 mL) was added dropwise a solution
of the cyano ester 3 dissolved in dry toluene (200 mL) under over a
period of 0.5 h. The reaction mixture was stirred for 1 h at the same
temperature, cooled to room temperature and then water (400 mL)
was added slowly. The aqueous layer was separated and adjusted
to pH 7 with 10% glacial acetic acid. The solid obtained was filtered,
washed twice with water and dried in vacuo to give the title com-
1
pound 4 (190.0 g, 75%) as a white solid, mp: 160–1618C. H-NMR
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-ethyl-
6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(CDCl3, 400 MHz) dH 1.47 (9H, s, Boc-9H), 3.64–4.06 (4H, m, pyrro-
lidine), 4.34–4.38 (1H, m, pyrrolidine). ESI-MS: m/z 209 (M – H)–.
8c
1H-NMR (DMSO-d6, 400 MHz) dH 1.22 (3H, t, J ¼ 6.8Hz, NCH2CH3),
4.56 (2H, q, J ¼ 6.8 Hz, NCH2CH3), 4.70–5.12 (7H, m, 2 CH2 and
NH2 and NH), 7.81 (1H, d, J ¼ 14.8 Hz, C5-H), 8.86 (1H, s, C2-H),
15.13 (1H, br, COOH); HRMS-ESI: m/z calcd. for C17H16F2N5O3
(M þ H)þ: 376.12212. Found 376.12131.
3-Amino-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole
hydrochloride 6a
A solution of tert-butyl 3-cyano-4-oxopyrrolidine-1-carboxylate 4
(2.1 g, 10.0 mmol) and hydrazine hydrochloride (1.1 g, 10.0 mol)
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Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-(2-
fluoroethyl)-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid 8d
carboxylic acid 9c
1H-NMR (DMSO-d6, 400 MHz) dH 4.69–4.88 (4H, m, 2 CH2), 4.95–
5.07 (7H, m, CH2 CH2F and NH2 and NH), 7.82 (1H, d,
J ¼ 14.4 Hz, C5-H), 8.80 (1H, s, C2-H), 15.21 (1H, br, COOH);
HRMS-ESI: m/z calcd. for C17H15F3N5O3 (M þ H)þ: 394.11270.
Found 394.11090.
1H-NMR (DMSO-d6, 400 MHz) dH 1.43 (3H, t, J ¼ 6.4 Hz, NCH2CH3),
3.51 (3H, s, CH3), 4.55 (2H, q, J ¼ 6.8 Hz, NCH2CH3), 4.70–4.86
(6H, m, 2 CH2 and NH2), 7.79 (1H, d, J ¼ 14.4 Hz, C5-H), 8.86
(1H, s, C2-H), 15.04 (1H, br, COOH); HRMS-ESI: m/z calcd.
for C18H18F2N5O3 (M þ H)þ: 390.13777. Found 390.13416.
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-(2-fluoroethyl)-6,8-difluoro-4-oxo-1,4-
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-
cyclopropyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-
dihydroquinoline-3-carboxylic acid 9d
carboxylic acid 8e
1H-NMR (DMSO-d6, 400 MHz) dH 3.50 (3H, s, CH3), 4.69–4.83
(4H, m, 2 CH2H), 4.86–4.94 (6H, m, CH2 CH2F and NH2), 7.82
(1H, d, J ¼ 14.4 Hz, C5-H), 8.81 (1H, s, C2-H), 14.94 (1H, br, COOH);
HRMS-ESI: m/z calcd. for C18H16F3N5O3Na (M þ Na)þ: 430.11029.
Found 430.10827.
1H-NMR (DMSO-d6, 400 MHz) dH 1.13–1.17 (4H, m, CH2CH2), 3.89–
3.94 (1H, m, CH), 4.69–4.73 (4H, m, 2 CH2), 4.79 (2H, br, NH2), 5.07
(1H, br, NH),7.76 (1H, d, J ¼ 14.4 Hz, C5-H), 8.63 (1H, s, C2-H), 15.24
(1H, br, COOH); HRMS-ESI: m/z calcd. for C18H16F2N5O3 (M þ H)þ:
388.12212. Found 388.11966.
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-cyclopropyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-
cyclopropyl-6-fluoro-8-difluoromethoxy-4-oxo-1,4-
3-carboxylic acid 9e
dihydroquinoline-3-carboxylic acid 8f
1H-NMR (DMSO-d6, 400 MHz) dH 1.16–1.17 (4H, m, CH2CH2), 3.51
(3H, s, CH3), 4.10–4.11 (1H, m, CH), 4.70–4.87 (6H, m, 2 CH2 and
NH2), 7.77 (1H, d, J ¼ 14.0 Hz, C5-H), 8.64 (1H, s, C2-H), 14.90 (1H,
br, COOH); HRMS-ESI: m/z calcd. for C19H17F2N5O3Na (M þ Na)þ:
424.11971. Found 424.11852.
1H-NMR (DMSO-d6, 400 MHz) dH 1.00–1.18 (4H, m, CH2CH2),
4.05–4.09 (1H, m, CH), 4.57–4.60 (6H, m, 2 CH2 and NH2), 5.07
(1H, br, NH), 6.88 (1H, t, J ¼ 73.2 Hz, OCHF2), 7.87 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.75 (1H, s, C2-H), 15.24 (1H, br, COOH);
HRMS-ESI: m/z calcd. for C19H16F3N5O4Na (M þ Na)þ:
458.10521. Found 458.10554.
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-cyclopropyl-6-fluoro-8-difluoromethoxy-4-oxo-1,4-
7-(3-Aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-1-
cyclopropyl-6-fluoro-8-methoxyl-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid 9f
1H-NMR (DMSO-d6, 400 MHz) dH 1.00–1.18 (4H, m, CH2CH2), 3.52
(3H, s, CH3), 4.05–4.09 (1H, m, CH), 4.55–4.73 (6H, m, 2 CH2 and
NH2), 6.90 (1H, t, J ¼ 73.6 Hz, OCHF2), 7.89 (1H, d, J ¼ 13.6 Hz, C5-
H), 8.75 (1H, s, C2-H), 14.81 (1H, br, COOH); HRMS-ESI: m/z calcd.
for C20H19F3N5O4 (M þ H)þ: 450.13891. Found 450.13566.
dihydroquinoline-3-carboxylic acid 8g
1H-NMR (DMSO-d6, 400 MHz) dH 1.00–1.14 (4H, m, CH2CH2),
3.59 (3H, s, OCH3), 4.15–4.20 (1H, m, CH), 4.53–4.56 (6H, m, 2
CH2 and NH2), 5.05 (1H, br, NH), 7.72 (1H, d, J ¼ 13.6 Hz,
C5-H), 8.67 (1H, s, C2-H), 15.07 (1H, br, COOH); HRMS-ESI:
m/z calcd. for C19H18FN5O4Na (M þ Na)þ: 422.12405. Found
422.12722.
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-cyclopropyl-6-fluoro-8-methoxyl-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid 9g
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-
1H-NMR (CDCl3, 400 MHz) dH 1.02–1.25 (4H, m, CH2CH2), 3.66 (s,
3H), 3.72 (3H, s, CH3), 4.03–4.07 (1H, m, CH), 4.63–4.79 (6H, m, 2
CH2 and NH2), 7.88 (1H, d, J ¼ 13.6 Hz, C5-H), 8.81 (1H, s, C2-H),
14.81 (1H, br, COOH); HRMS-ESI: m/z calcd. for C20H21FN5O4
(M þ H)þ: 414.15776. Found 414.16054.
naphthyridine-3-carboxylic acid 9a
1H-NMR (DMSO-d6, 400 MHz) dH 1.11–1.27 (4H, m, CH2CH2), 3.52
(3H, s, CH3), 3.65–3.76 (1H, m, CH), 4.66–5.00 (6H, m, 2 CH2 and
NH2), 8.03 (1H, d, J ¼ 12.8 Hz, C5-H), 8.59 (1H, s, C2-H), 15.35 (1H,
br, COOH); HRMS-ESI: m/z calcd. for C18H18FN6O3 (M þ H)þ:
385.14244. Found 385.14036.
Antibacterial activity
Compounds 8a–g and 9a–g were evaluated for their in-vitro anti-
bacterial activity using conventional agar-dilution method in
comparison to the reference drugs. Drugs (10.0 mg) were dis-
solved in 0.1 N sodium hydroxide solution and water (10 mL).
Further progressive twofold serial dilution with melted Mueller-
Hinton agar was performed to obtain the required concen-
trations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and
0.03 mg/mL. Petri dishes were incubated with 104 colony form-
ing units (cfu) and incubated at 358C for 18 h. The MIC was the
lowest concentration of the test compound, which resulted in no
visible growth on the plate.
7-(2-Methyl-3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-
yl)-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylic acid 9b
1H-NMR (DMSO-d6, 400 MHz) dH 3.46 (3H, s, CH3), 3.82–4.18
(4H, m, 2 CH2), 4.94 (2H, br, NH2), 7.32–7.36 (1H, m, ph-1H),
7.58 (1H, s, ph-1H), 7.79–7.85 (1H, m, ph-1H), 8.01 (1H, d,
J ¼ 12.8 Hz, C5-H), 8.80 (1H, s, C2-H), 15.10 (1H, br, COOH);
HRMS-ESI: m/z calcd. for C21H16F3N6O3 (M þ H)þ: 457.12360.
Found 457.11910.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 523–529
New pyrrolo[3,4-c]pyrazolylquinolone derivatives
529
This work was supported by the ‘‘National S&T Major Special Project on
Major New Drug Innovation’’ (2009ZX09301 -003).
[9] M. L. Liu, L. Y. Sun, Y. G. Wei, H. Y. Guo, Chin. J. Pharm. 2003,
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[11] M. L. Liu, H. Y. Guo, Chin. J. Pharm. 2000, 31, 564–565.
The authors have declared no conflict of interests.
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www.archpharm.com