Microbiology

Chemotherapy 17: 130-140 (1972)

Antimicrobial Activity of a Series of

New 5-Nitro-2-furaldehyde Aminoacethydrazones

L. D e g e n , M. S alv aterra , S. V e lla , D. N a r d i and E. M assarani

Research Division, Recordati s.a.s., Milan

Abstract. A series of 5-nitro-2-furaldehyde Key Words

aminoacethydrazones, synthesized in our labo­ Nitrofurans
ratories are screened in vitro and in vivo for 5-Nitro-2-furaldehyde
antibacterial activity. aminoacethydrazones
Because of its good antibacterial activity Antibacterial activity
against gram-negative and gram-positive or­ Urinary excretion in rats
ganisms, its good absorption and its exretion 5-Nitro-2-furaldehyde 4'-methyl-
in high urinary levels in active form, the 5-ni- piperazinoacethydrazone
tro-2-furaIdehyde 4'-methyIpiperazinoacethy- (nifurpipone)
drazone (Rec. 15-0122, nifurpipone) was select­
ed for further studies.

Since the introduction of nitrofurans for the therapy of urinary tract

infections, a large number of chemical derivatives have been prepared
and tested for a better biological activity and more favourable pharma­
cological properties.
In our laboratories during the last years [7, 8] a series of 5-nitro-2-
furaldehyde monoalkylaminoacethydrazones (I), of dialkylaminoacet-
hydrazones (II) and of N'-substituted-N-piperazino-acethydrazones (III)
were synthesized.

OaN-ll j- C H = N-N H CO -CH 2-R R = -NH -alkyl I

XO 7 -N-dialkyl II

-N N -R i III

All compounds were tested for antimicrobial activity in vitro against

a representative series of micro-organisms. Urinary elimination following
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 D egen et al. 131

oral administration in rats was also determined. Compounds with ‘in vi­
tro’ activities were tested in mice against infections with S. pyogenes, E.
coli, S. aureus, and S. typhimurium and some products were also tested
against T. brucei and T. congolense in mice.
Nitrofurantoin was run for comparison ‘in vitro’ and ‘in vivo’ experi­
ments.

Material and Methods

The drugs tested in these experiments are indicated in table I.

Compounds 8, 9, 11, 16 and 17 have been publicated recently by Japanese au­
thors [2] and are mentioned here only for completeness.
In vitro studies. All the micro-organisms used in the in vitro studies described
here were maintained by repeated transfers on nutrient agar and were considered
as laboratory stock cultures. The in vitro studies were carried out against a repre­
sentative spectum of gram-negative and gram-positive bacteria and fungi (see table
I). In all instances, minimal inhibitory concentrations (MIC) were determined by a
serial 1:2 dilution technique. The culture media used were Kirchner medium [5]
with 10°/o bovine serum for M. tuberculosis, fluid thioglycolate medium (USP XV)
for Clostridium and Trichomonas, fluid Sabouraud medium (USP XV) for fungi,
brain heart infusion agar (Difco) for Streptococcus, and nutrient agar (Difco) for
the others. Generally the highest concentration of compound studied was 160 «g/ml
and the least 0.625 ¿ug/ml.
In vivo studies. The animals used in these experiments were:
(a) NMRI albino mice, males and females, from the outbred Recordati line,
weighing 18-20 or 22-24 g for Trypanosomes, and
(b) male Wistar rats from the outbred Recordati line, weighing 250-300 g.
The mice and rats were housed during the experiments in Makrolon® cages at
23 ± 1° C and 55 ± 5% relative humidity, and received Altromin-R® standard diet
and water ad libitum.
(1) Drug elimination studies on rats. Generally, the drugs were given by single
oral tubing dissolved or suspended in water and the urine samples were collected
in metabolic cages. The drugs were assayed by a biological method using B. sub-
tilis ATCC9466 as test organism. USP standard cylinder plate method was used,
slightly modified for our purpose. Each drug was used as its own standard.
(2) Systemic infection with Streptococcus pyogenes C203. The classic method
described by D omagk [1] was used. Groups of 10 or 20 mice were infected i.p.
with 0.5 ml of a culture dilution containing 100-1,000 minimal lethal doses. Cul­
tures were made in brain heart infusion broth (Difco) with 10°/o whole defibrinated
rabbit blood by 4- to 6-hour incubation at 37 °C. Therapy was started immediate­
ly after infection and the drugs were given once daily for 3 days by oral tubing.
Deaths were registered daily and the survivors were observed for a period of 10
days. The statistical significance of results was calculated by the ■/} method.
(3) Local infection. The method described by Hunt [4] was used. Groups of 10
mice were infected in the inner part of the thigh (musculus adductor magnus) of
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132 D egen et al. Antimicrobial Activity of a Series of

Table /. In vitro activities of derivatives of 5-nitro-2-furaldehyde

No. X R Antibacterial and anti­

mycotic activity in vitro
(last inhibitory
concentration, /ig/ml)
l1 2 3

1 ch2 nh-ch3 10 10 40
2 ch2 n h - c h 2- c h 3 10 10 40
3 ch2 N H -C H ^C H ^C H s 20 20 40
4 ch2 NH-CH-CHs 20 20 40
CH3
5 ch2 n h - c h 2- c h 2- c h 2- c h 3 10 10 80
6 ch2 n h - c h 2- c h - c h 3 10 20 80
ch3
7 ch2 n h - c h 2- c h = ch2 10 10 40

8 ch2 n < ^ CH3 10 10 40

n \ ch3
/ C H 2CH3
9 ch2
n \ c h 2c h 3
10 20 80
/C H 2CH2CH3
10 ch2 2.5 40 80
n \ c h 2c h 2c h 3
ch3

/C H -C H 3
11 ch2 20 40 80
N\ C H - C H 3

ch3
/ C H 2CH2CH2CH3
12 ch2 5 80 >160
n \ c h 2c h 2c h 2c h 3
ch3

^ c h 2- c h - c h 3
13 ch2 10 40 >160
n \ c h 2- c h - c h 3

ch3

14 ch2
n / C H 2CH2CH2CH2CH3
20 >160 >160
\ c h 2c h 2c h 2c h 2c h 3
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 New 5-Nitro-2-furaldehyde Aminoacethydrazones 133

amino acethydrazone O 2N -CH = N -N H -C O -X -R

Antibacterial and antimycotic activity in vitro Trichomonas

(last inhibitory concentration, /¿g/ml) vaginalis (last
inhibitory
concentra-
4 5 6 7 8 9 10 11 tion, /rg/ml)

40 2.5 20 1.25 160 >160 >160 >160 50

20 5 20 1.25 80 >160 >160 >160 50
40 20 40 2.5 >160 >160 160 >160 50
40 5 20 1.25 160 >160 >160 >160 50

80 10 10 5 >160 >160 >160 >160 50

40 5 10 5 160 >160 >160 >160 50

40 2.5 5 0.625 >160 >160 >160 >160 50

40 5 40 5 80 20 160 >160 10

80 20 160 20 >160 >160 >160 >160 50

80 10 80 10 160 >160 >160 >160 50

80 10 80 5 80 >160 160 >160 50

>160 20 80 5 80 40 160 >160 50

>160 2.5 40 2.5 160 80 160 80 50

>160 20 80 10 80 40 >160 >160 50

80 20 1.25 5 >160 >160 >160 >160 5

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134 D egen et al. Antimicrobial Activity of a Series of

Table / (continued)

No. X R Antibacterial and anti­

mycotic activity in vitro
(last inhibitory
concentration, //g/ml)
l1 2 3

16 CH2 N 80 80 80
A
17 ch2 N o 80 160 >160
\_

18 ch2 N N -H 80 160 >160

\_
" \
19 ch2 N N-CHa 40 40 160
\_
A
20 c h 2- c h 2 n n-ch3 20 40 >160
\_
A
21 CH N N-CHs 10 40 >160
I \_
ch3

22 CH2 N N -C2H5 10 10 >160

- \
23 ch2 N N -C 3H7 20 20 160

CH3
A I
24 CH2 N N-CH-CHs 10 20 >160
V
A
25 ch2 N N-CiHo 10 40 >160
\.
26 ch2 N N -C i 2H25 102 >160 >160
V
27 ch2 N N-Citronellyl 10 >160 >160
V
A
28 ch2 N N-Geranyl 10 >160 >160
V
A
29 ch2 N N -C H 2CH2OH 80 >160 >160
\.
30 ch2 N N -C H a-^ ^ 40 160 >160
V
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 New 5-Nitro-2-furaldehyde Aminoacethydrazones 135

Table I (continued)

Antibacterial and antimycotic activity in vitro Trichomonas

(last inhibitory concentration, //g/ml) vaginalis (last
inhibitory
concentra-
4 5 6 7 8 9 10 11 tion, /¿g/ml)

80 40 10 2.5 >160 >160 >160 >160 50

160 20 2.5 2.5 >160 >160 >160 >160 50

160 40 160 40 >160 40 80 >160

80 20 2.5 20 160 80 >160 >160 50

>160 10 20 10 160 10 >160 >160

40 10 40 5 80 80 >160 >160

40 5 10 5 160 40 >160 >160 50

80 5 20 5 40 20 >160 >160 50

80 5 10 5 160 10 160 >160 50

80 10 20 5 40 >160 >160 >160

>160 0.6252>160 >160 40 >160 >160 >160

>160 80 10 5 40 1.25 160 >160

>160 10 20 5 40 20 160 >160

>160 160 20 >160 160 40 >160 >160

>160 10 1.25 5 80 20 80 >160

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136 D egen et al. Antimicrobial Activity of a Series of

Table / (continued)

No. X R Antibacterial and anti­

mycotic activity in vitro
(last inhibitory
concentration, /¿g/ml)
I1 2 3

31 CH2 N N -C H 2CH2- ^ y 20 80 160

32 CH2
GO 80 80 80

33 CH2 N N -/ VN 0 2 >160 >160 >160

\__ / \__ /
34 CH2 N N-COCHs 20 80 >160

c 2h 5
35 CH2 N N -c ° n< ; 80 >160 >160
c 2h 5

36 CH2 N
\_
n - c h 2c o - n h - n = ch _r no2
>160 >160 >160

37 Nitrofurantoin 5 40 160
38 Nitrofurazone 20 40 160

1 Strains used: (1) E. coli 100; (2) Salmonella Breslau 1090; (3) Pseudomonas aeru­
ginosa H2; (4) Proteus O; (5) Staphylococcus aureus SG 511; (6) Streptococcus pyogenes
humanus A 88; (7) Bacillus subtilis ATCC 9466; (8) Clostridium novyi ; (9) Mycobacterium
tuberculosis H 37 Ra; (10) Trichophyton mentagrophytes 1236; (11) Candida albicans 28.

one leg with 0.2 ml of a 1:2 dilution of a 6-hour culture of Staphylococcus aureus
742 in brain heart infusion broth (Difco) with 10% whole defibrinated rabbit
blood. Therapy was started immediately after infection and the drugs were given
once daily for 6 days by oral tubing. The diameters of the intact and of the in­
fected leg were measured daily by a calliper and the means of t-test was used to
estimate the significance of the results.
(4) Trypanosomal infection. The technique described by H awking [3] was
used. Groups of 5 mice weighing 22-24 g were infected i.p. and were treated by
subcutaneous route with the compounds on 4 successive days. If the trypanosomes
disappeared from the blood permanently (more than 30 days) the mice were con­
sidered as ‘cured’. If the trypanosomes disappeared and then re-appeared during
the period of observation, the animals were classified as ‘cleared’. If the trypano­
somes multiplied as in controls, the drug was considered as ‘inactive’.
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 New 5-Nitro-2-furaldehyde Aminoacethydrazones 137

Table I (continued)

Antibacterial and antimycotic activity in vitro Trichomonas

(last inhibitory concentration, //g/ml) vaginalis (last
inhibitory
concentra­
4 5 6 7 8 9 10 11 tion, /tg/ml)

160 10 80 10 40 2.5 >160 >160

80 20 40 5 40 40 80 >160

>160 20 2.5 40 80 0.31 >160 >160

160 10 5 20 >160 40 80 >160

160 10 5 10 80 40 80 80

>160 >160 >160 >160 >160 >160 >160 >160

80 10 5 10 40 160 >160 >160 10

80 5 5 10 80 >160 >160

2 In Difco nutrient broth.

(5) Other infections. Some of the products were also tested on other infection
models as E. coli and S. typhimurium peritonitis. For these experiments, groups of
10 mice weighing 18-20 g were infected i.p. with 0.5 ml of a dilution of a 5-hour
broth (+10%> whole rabbit blood) culture at 37 °C containing at least 10 LD50.
Treatment was started 1 h before infection and 2 daily doses were given on the
following days by oral tubing. Mortality was recorded for 8 days and the results
were evaluated using the ■/} or the Litchfield [6] method.
Some products were also tested in vivo on the infection of mice with M yco­
bacterium tuberculosis SG851. Groups of 30 female mice weighing 16-18 g were
infected i.v. with 0.2 ml of a standardized suspension of M. tuberculosis. The
products were administrated once daily, by subcutaneous route starting 1 day after
infection. Isoniazide was run for comparison at the dose of 0.05 mM/kg. The mice
were treated for 45 days. Mortality, body weight and patholgic findings at death
were recorded.
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138 D egen et al. Antimicrobial Activily of a Series of

Results and Discussion

The results are reported in tables I—III.

‘In vitro’ most of the products listed in table I showed a broad anti­
bacterial spectrum comparable to nitrofurantoin (37) and were inactive
against C. novyi and fungi. The monoalkylaminoacethydrazones I (1-7)
exhibited an antibacterial activity higher than 37 whereas the dialkylam-
inoacethydrazones II (8-17) showed an antibacterial activity comparable
to 37. The dimethylamino- (8) and the N-pyrrolidino-derivatives (15)
had some activity against T. vaginalis, as 37. The activities of the piper-
azion acethydrazones III (18-36) varied in dependence of the substituent
(R,) on Nl. With R, = H or hydroxyalkyl (18 and 29) the activity de-

Tabte II. Urinary excretion in rats of active drug (microbiological assay)

Product Dose Drug Product Dose Drug

No. mg/kg' excretion No. mg/kg' excretion
during 24 h during 24 h
% %

1 20 4.2 20 30(b) 0
2 20 10 21 20 18
3 20 2 22 20 20
4 20 3.5 23 20 11
5 164(a) 0 24 20 18
6 31,5 (b) 0 25 20 0
7 156 (a) 0 26 130 (b) 0
8 20 4.5 27 20 0
9 20 0 28 20 0
10 75(c) 0 29 -

11 148 (a) 0 30 100 0

12 136 (a) 0 31 18(b) 0
13 90(b) 0 32 50 (b) 0
14 80 (b) 0 33 20 0
15 20 2.7 34 35(b) 0
16 12(b) 0 35 8(b) 0
17 42(b) 0 36 -

18 30(b) 0 Nitro- 20 37
furantoin
19 20 24

1 The usual dose was 20 mg/kg. In some instances were given 0.5 mM/kg (a), '/to (b)
or '/ 2 (c) of the DLso. - Not done.
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 New 5-Nitro-2-furaldehyde Aminoacethydrazones 139

Table III. Activity against some experimental infections of mice

Intraperitoneal Dose, mM/kgl

infections 0.125 0.250 0.5 0.75

S. pyogenes 192
37
S. typhimurium 23 19
T. brucei 17 37
T. congolense 1 17 37 14

Intramuscular 0.125 0.250 0.5 0.75

infections
S. aureus 7 19
11 33

1 Fraction of the LDso.

2 Number corresponding to the significally active compounds.

creased whereas with = alkyl (19-25) the activity increased with

lengthening of the chain up to 4 C atoms.
With Rt = aralkyl or alkyl group (30-33) the antibacterial activity
was higher against gram-positive bacteria.
Five piperazinoacethydrazones (19, 20, 24, 31 and 33) were also ac­
tive in vitro against M. tuberculosis.
The urinary excretion (table II) of monoalkylaminoacethydrazones
(compounds 1-7) was highest for the ethylaminoderivative (2), and de­
creased with the lengthening of the side chain. The dialkylamino deriva­
tives (compounds 8-14) were scarcely excreted in urine whereas a slight
excretion was observed only for N-dimethyl-amino (8) and N-pyrrolidi-
no-derivative (15). The piperazinoacethydrazones III (compounds
18-36) showed a good urinary excretion when R was an alkyl group
up to 3 C atoms and the highest excretion was observed with the methyl-
piperazino derivative (18).
The results of activities against systemic infections are reported in ta­
ble III.
The ‘in vivo’ experiments reported in table III show that some drugs
are distributed in body tissues in active form and concentration. In fact
compound 19 was significantly active in the systemic infections of mice
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140 D egen et al.

with S. pyogenes and S. typhimurium and 23 was active against the last
infection. Compounds 7, 11, 33, and 19 were active against the local infec­
tion of mice with S. aureus. These results had a more general interest
since usually nitrofurans do not reveal substantial activities against sys­
temic infections, probably because of insufficient blood and tissue concen­
tration to exert antibacterial effects.
Of the drugs with relevant urinary excretion (table II) compound 19
possessed activities comparable to, or better than those of the nitrofur-
anes, run for comparison. Because of its good antibacterial activity against
organisms which can cause urinary tract infections, its good absorption
and resistance against bio-inactivation, its high urinary levels, product
19 was selected for further studies.

References

1 D omagk, G.: Pathologische Anatomie und Chemotherapie der Infektions­

krankheiten (Stuttgart 1947).
2 F ujita , A.; Shinsaku, M., and H ideji , T.: Studies on nitrofuran derivatives. I.
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3 H awking , F.: Experimental chemotherapy, vol. 1, p. 131 (Academic Press, New
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4 H unt , G. A. and M oser , A. J.: Antimicrobial agents and chemotherapy 1962,
p. 87 (Ann Arbor, Michigan 1963).
5 K irchner , O.: Die Leistungsfähigkeit der Tiefenkultur des Tuberkelbazillus bei
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633 (1971).
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Authors’ addresses: Dr. L. D egen , SNAM Progetti, Lab. studi e ricerche, Strada
del Grillo, 00015 - Monterotondo, Rome; Dr. M. Salvaterra, S. Vella, Dr. D. N ar­
d i , Dr. E. M assarani, Research Division, Recordati s.a.s., Via Civitali 1, 1-20148
Milan (Italy)
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