Hydroxamic acids: synthesis and adjuvant activity in combinatorial anticancer therapy

Article abstract of DOI:10.1007/s11172-016-1377-0

Monoand disubstituted N-hydroxyamides of dicarboxylic acids were prepared by reaction of dicarboxylic acids or acid anhydrides with hydroxylamine. The use of these compounds in combinatorial cytostatic therapy of implanted tumors with cisplatin or cyclophosphamide totally inhibits metastasis formation in B16 melanoma and Lewis lung carcinoma, and resulted in 100% survival of leukemic animals.

Full text of DOI:10.1007/s11172-016-1377-0

Russian Chemical Bulletin, International Edition, Vol. 65, No. 3, pp. 801—805, March, 2016
801
Hydroxamic acids: synthesis and adjuvant activity
in combinatorial anticancer therapy
B. S. Fedorov, M. A. Fadeev, A. B. Eremeev, N. P. Konovalova, G. N. Bogdanov, L. V. Tatyanenko,
T. E. Sashenkova, and D. V. Mishchenko
Institute of Problems of Chemical Physics, Russian Academy of Sciences,
1 prosp. Akad. N. N. Semenova, 142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 522 3507. Eꢀmail: borisꢀ45@inbox.ru
Monoꢀ and disubstituted Nꢀhydroxyamides of dicarboxylic acids were prepared by reaction
of dicarboxylic acids or acid anhydrides with hydroxylamine. The use of these compounds in
combinatorial cytostatic therapy of implanted tumors with cisplatin or cyclophosphamide totally
inhibits metastasis formation in B16 melanoma and Lewis lung carcinoma, and resulted in
100% survival of leukemic animals.
Key words: hydroxamic acids, reaction of maleic anhydride with hydroxylamine, antimetaꢀ
static and anticancer activity, chemotherapy of cancer.
Today, combinatorial chemotherapy with different
types of cytostatic drugs, alkylating agents, antimetaꢀ
bolites, and platinum complexes is the prevailing approach
in chemotherapy of cancer of internal organs.1,2 However,
cytostatics, due to the lack of cancer cell specificity, cause
side effects throughout the body, and among them the
hematopoietic suppression2 is the most severe and formiꢀ
dable. This is why the attempts are being made to reduce
side effects by using the low toxic adjuvants, which can
provide for the decrease of the therapeutic dose of cytoꢀ
statics while preserving their efficiency.
regression and metastasis suppression. Earlier,5—8 it was
shown that, the zinc atom is included into histon deacetylase,
which is involved into DNA replication and apoptosis.
This observation suggests that the listed above hydroxꢀ
amic acids can deactivate histon deacetylase and, hence,
inhibit the tumor growth and metastasis formation. The
present work is aimed at synthesis and study of adjuvant
properties of hydroxyamides of aliphatic dicarboxylic
acids (hydroxamic acids) in combinatorial cytostatic
therapy with known cytostatic drugs cisplatin and cycloꢀ
phosphamide.
However, discovery of adjuvants for this purpose shows
random nature and cannot be systematized and attributed to
specified classes of compounds. In this connection, beꢀ
tween the end of the ХХ and the beginning of the XXI
century the approach was developed aimed at the research
of so called targeted drugs, which are directed at cell proꢀ
liferation molecules or DNA replication enzymes. With
a view to discover novel compounds with properties listed
above we obtained hydroxyamides of dicarboxylic acids.
In particular, hydroxyamides of oxalic, tartaric, aspartic,
and maleic acids were prepared.3,4 The experimental data
obtained using BDF1 mice evidenced that these hydrꢀ
oxamic acids showed substantial therapeutic effect when
used in combinatorial cytostatic therapy with cisplatin or
cyclophosphamide against Р388 leukemia, В16 melanoꢀ
ma and Lewis lung carcinoma. In this connection, the
research aimed at preparation of biologically active comꢀ
pounds on the basis of metabolically active starting comꢀ
pounds are of great interest. In our publications,3,4 we
explained the presented results by the chelation of the zinc
atom with hydroxamic acids thus causing the tumor growth
Results and Discussion
Recently, the search for new highly efficient adjuvants
has acquired high importance. The development of fundaꢀ
mental research in the field of contemporary medicinal
chemistry necessarily includes the detailed mechanistic
study of biological action of pharmacologically active comꢀ
pounds or their pharmacophore features. In this connecꢀ
tion, the research aimed at synthesis of biologically active
compounds on the basis of metabolically active starting
compounds generates considerable interest. Earlier,9—11
we showed that the functional groups (the ligand) play an
important and, in some case, a crucial role in pharmacoꢀ
logical activity of the complex using the examples of palꢀ
ladium and platinum(IV) complexes with substituted
amides of pyridinecarboxylic acids. Thus, we showed in
publication12 that platinum and palladium complexes with
substituted amides of pyridinecarboxylic acids interact with
sarcoplasmic reticulum Ca2+—Mg2+ATPase and slows
down both the active transport of calcium across the cell
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 0801—0805, March, 2016.
1066ꢀ5285/16/6503ꢀ0801 © 2016 Springer Science+Business Media, Inc.

802
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 3, March, 2016
Fedorov et al.
membrane and ATP hydrolysis the membraneꢀembedded
sarcoplasmic reticulum Ca2+—Mg2+ ATPase.12 It was
shown, that histon deacetylase with a zinc atom, which
plays a catalytic role in metabolism,5—8 takes part in DNA
replication, apoptosis and metastasis formation. In the
view of this, we can anticipate that the considered hydrꢀ
oxamic acids deactivate histon deacetylase by chelating
the zinc atom with hydroxamic acids, thus causing the
tumor growth regression and metastasis suppression.
It should be clarified that in this publication we considꢀ
er dihydroxyamides of oxalic (1) and tartaric acids (2), and
monohydroxyamides of aspartic (3) and maleic acids (4).
N,N´ꢀDihydroxyamide of oxalic acid (1) was syntheꢀ
sized as shown in Scheme 1.
soluble in ethyl acetate and can be removed from the reacꢀ
tion mixture by washing with ethyl acetate. On the conꢀ
trary, sodium chloride and potassium chloride, which are
formed in the reaction of hydroxylamine hydrochloride
and, accordingly, sodium and potassium hydroxides, are
insoluble in ethyl acetate, and additional purification of
the monohydroxamic acid from metal chloride by multiꢀ
ple recrystallizations is needed.
Scheme 4
Scheme 1
NꢀHydroxyamide of maleic acid (4) was prepared by
interaction of maleic anhydride with hydroxylamine in
1,2ꢀdichloroethane in the presence of triethyl amine. The
synthesized compounds were further transferred to the labꢀ
N,N´ꢀDihydroxyamide of tartaric acid (2) was preꢀ
pared as shown in Scheme 2.
N (%)
60
Scheme 2
50
40
30
20
10
However, solubility of compounds 1 and 2 in water
proved to be insufficient for conduction of antiꢀcancer
study. This is why aspartic and maleic acids were convertꢀ
ed to monohydroxyamides leaving one of the carboxyl
groups intact.
NꢀHydroxyamide of aspartic acid (compound 3) was
obtained by interaction of βꢀmethyl ester of aspartic acid
with hydroxylamine hydrochloride in the presence of lithꢀ
ium hydroxide (Scheme 3).
3
CP
3 + CP
N (%)
600
500
400
300
200
100
Scheme 3
3
CP
3 + CP
Fig 1. Increase of antitumor efficiency against Р388) leukemia
upon combination of cyclophosphamide (CP) with aspartic acid
Nꢀhydroxyamide (3): 3 (50 mg kg–1), CP (60 mg kg–1), 3 + CP
(50 + 60 mg kg–1); N is survival rate.
Lithium hydroxide was used because lithium chloride,
which arises from the neutralization reaction, is readily

Hydroxamic acids in anticancer therapy
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 3, March, 2016
803
oratory of experimental chemotherapy of tumors of the
Institute of Problems of Chemical Physics of Russian
Academy of Sciences for examination of antitumor and
antimetastatic activity using BDF1 mice. The experimenꢀ
tal data showed 70—100% inhibition of metastasis formaꢀ
tion in implanted B16 melanoma and Lewis lung carcinoꢀ
ma, when hydroxyamides of aspartic and maleic acids
(3 and 4) were used in combinatorial cytotoxic therapy by
cyclophosphamide or cisplatin (Tables 1—3). Also, the
experiments on animals evidenced that the use of monoꢀ
hydroxyamide of aspartic acid 3 in combinatorial cytoꢀ
static therapy with cyclophosphamide provided 50% surꢀ
vival and the life span of the other 50% of animals inꢀ
creased 5 times (Fig. 1). At the same time, the use of
monohydroxyamide of maleic acid (4) in combinatorial
cytostatic therapy with cisplatin totally inhibited metastaꢀ
sis formation in implanted B16 melanoma and Lewis lung
carcinoma, and provided 100% animal survival in leukeꢀ
Table 1. The effect of compounds 1—4 on the average lifespan (LS) of male BDF1 mice with implanted
Рꢀ388 lymphleukemia
Compound
Single dose/mg kg–1
N60
n
N (%)
LS/day
ILS (%)
Control
cPt
—
1.2
2
1
10
1
10
10
10
1.2 + 1
2 + 1
1.2 + 10
2 + 10
1.2 + 1
2 + 1
1.2 + 10
2 + 10
2 + 10
2 + 10
0
3
3
0
0
5
0
0
0
6
6
6
5
5
6
6
6
6
6
6
3
3
6
6
1
6
6
6
0
0
0
1
1
0
0
0
0
0
0
50
50
0
0
83
0
10.7
22.3
27.3
10.8
10.8
10.7
10.7
10.7
—
—
—
—
57.0
30.0
—
—
—
—
109.4
156.3
1.6
1.6
0.0
0.0
0.0
—
—
—
—
434.4
180.0
—
—
—
1
2
3
4
0
0
cPt+1
100
100
100
83
83
100
100
100
100
100
сPt+2
cPt+3
cPt+4
—
—
—
—
Note. Inoculum: 106 tumor cells in 0.2 mL; intraperitoneal injection. The number of experimental animals
is six. Dosage regimen/days: 1, 3, 5, 7. N60 is the number of survived animals by the 60 day. n is the number
of fallen animals. N is the number of survived animals.
Table 2. Antimetastatic activity of compounds 1—3 administered in combination with cisplatin (сPt) in Lewis lung carcinoma
Compound
Single dose
/mg kg–1
The number of animals
The number of
metastases
MII (%)
Total tumor
weight
Tumor growth
retention (%)
in the
with
per mouse
experiment
metastases
Control
cPt
1
—
12
11
10
11
8*
7*
8*
6*
7*
6*
7*
6*
7*
8*
8*
8*
8
7
8
6
7
6
7
6
7
8
8
8
14.7 1.36
15.7 0.99
11.1 1.99
17.3 1.33
14.3 0.68
16.2 0.87
17.0 1.96
9.8 2.9
6.7 2.4
3 0.34
12.8 0.53
6.3 1.2
—
10.9
—
61.0
24.0
50.0
13.0
58.0
35.0
33.0
54.0
80.0
81.0
100.0
8.8 0.25
9.7 0.45
9.7 0.26
10.4 0.3
9.5 0.51
8.5 0.48
9.1 0.42
8.5 0.37
7.7 0.51
8.9 0.41
9.0 0.47
19.0
11.0
11.0
4.0
13.0
22.0
17.0
22.0
29.0
18.0
13.0
2
10
10
3
сPt+1
2 + 11
2 + 10
2 + 11
2 + 10
2 + 10
сPt+2
сPt+3
Note. Inoculum 106 tumor cells in 0.3 mL; subcutaneous injection. Dosage regimen/days: 2, 4, 6, 8. The animals were sacrificed
on the 20 day after tumor implantation. All groups consisted of eight animals.
* In these groups some animals died before the 20th day after injection.

804
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 3, March, 2016
Fedorov et al.
Table 3. Antimetastatic activity of compounds 1, 2, and 4 administered in combination with cyclophosphamide (CP) in Lewis
lung carcinoma
Compound
Single dose
/mg kg–1
The number of animals
The number of
metastases
per mouse
MII (%)
Total tumor
weight
Tumor growth
retention (%)
in the
with
experiment
metastases
Control
CP
1
—
60
11
10
11
8*
7*
7*
8*
6*
8*
8*
7*
8*
8*
8*
8*
8
7
7
8
6
8
8
7
8
7
5
4
10.4 0.43
*4.4 0.89
*9.7 0.75
11.3 0.75
*9.3 1.5
*6 0.75
*7.0 2.2
*4.3 0.75
*3.4 0.94
*3.6 0.73
*1.5 0.57
*1.7 1.33
—
9.1 0.3*
4.6 0.54
10.2 0.37
9.3 0.3*
9.1 0.52
9.1 0.6*
7.1 1.76
5.3 0.43
4.5 0.28
5.1 0.2
—
57.0
*7.0
*0.0
11.0
42.0
13.0
59.0
68.0
69.0
91.0
88.0
49.0
*0.0
*0.0
*0.0
*0.0
24.0
42.0
21.0
44.0
35.0
56.0
2
10
10
4
CP+1
60 + 11
60 + 10
60 + 11
60 + 10
60 + 10
CP+2
CP+4
5.9 0.27
4.1 1.22
Note. Inoculum 106 tumor cells in 0.3 mL; subcutaneous injection. Dosage regimen/days: 2, 7. The animals were sacrificed on
the 20th day after tumor implantation. All groups consisted of eight animals.
* In these groups animals died before the 20 day after injection.
where М is a molecular mass of 2, [180 – H]+. It is in good
agreement with the molecular mass 180 calculated for 2 with
a molecular formula C4H8N2O6.
mia Рꢀ388. Comparison of the results and, in particular,
the adjuvant properties of hydroxyamides of dicarboxyl
acids under consideration showed that monohydroxyamide
of maleic acid (4) was the most efficient. Notably, the
Animal experiments. Examination of anticancer activity of
new compounds was conducted according to Guidelines13 on
results were obtained using low doses of cisplatin (from 1
male BDF1 hybrid mice (22—24 g) with Р388 lymphleukemia,
to 1.2 mg kg–1), which are not therapeutically effective if
B16 melanoma and mouse Lewis lung cancer LL. Mice were
cisplatin is used alone. Hence, we can resume, that hydrꢀ
oxamic acids 1—4 show pronounced adjuvant (chemoꢀ
senstizing) effect.
kept in cages with free access to food and tap water. Tumors
were implanted according to standard procedure intraperitoꢀ
neally (P388 leukemia), subcutaneously (B16 melanoma) or
intramuscularly (LL carcinoma) (106 tumor cells in isotonic
solution of NaCl in 0.2 mL (Р388 leukemia), 5•106 cells in 0.3 mL
(B16 melanoma and LL carcinoma) inoculum). New chemical
compounds and known cytostatics were injected intraperitoꢀ
neally as aqueous solutions. Dosage regimen of injection (a day
after implantation of the tumor) are listed in Tables. In each
experiment animals with implanted tumor, which did not reꢀ
ceive medication, were used as a control group. Each group
consisted of 6—10 mice. To validate the therapeutic effect of
anticancer therapy we used the number of survived animals for
the 60ꢀday observation period after the beginning of the theraꢀ
py, average life span (LS), and increase of average lifespan (ILS)
as the criteria of efficiency.
The obtained results have thus confirmed the validity
of our approach, which suggests using metabolically active
compounds as the basis for physiologically active groups.
Besides, the obtained results show that the design of antiꢀ
tumor cytostatics and adjuvants on the basis of metaboliꢀ
cally active compounds is an appropriate strategy. At the same
time, the obtained data demonstrated that the effectivity of
search for new biogenic adjuvants and targeted drugs has
promising prospects in experimental chemotherapy of cancer.
Experimental
ILS (%) = 100 (To/Tк – 1),
Identification of obtained hydroxamic acids 1—4 was conꢀ
ducted using the elemental analysis and NMR H spectroscopy
1
where Tо and Tк are the experimental average lifespan (days)
observed for animals of the experimental and the control groups,
respectively, and the number of cured animals (which survived
to the 60ꢀth day of the observation period).
The tumor growth inhibition rate was calculated on the
basis of the regression of tumor growth (TGR) which is calꢀ
culated as
data, which was obtained on an AVANCE III 500 MHz Bruker
Biospin instrument. For compounds 1 and 2 mass spectra were
obtained on a LC/MS 20ꢀ20 Shimadzu instrument (Japan)
equipped with electrospray (ESI) ionization source, with a scan
range 10—2000 m/z. Mass resolution (FWHM) was 0.6. Aqueous
solutions of compounds 1 and 2 were used as analytes. The mass
spectrometer was operated in the positive ion mode in the range
from m/z 10 (150)—600. Compound 1 was detected as the moꢀ
lecular ion m/z 120 Da in good agreement with the calculated
molecular mass of 1 with molecular formula С2H4N2O4.
TGR (%) = [(Vc – Ve)/Vc]•100%,
where Ve and Vc are the average volumes of the tumor (mm3)
observed in the experimental and the control groups, resꢀ
pectively.aaaa
MS analysis of compound 2 showed the base peak at m/z
179. This observation evidences the formation of [M – H]+ ion,

Hydroxamic acids in anticancer therapy
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 3, March, 2016
805
Metastasis inhibition index (MII, %), which shows the
frequency of metastasis, was calculated as
The reaction mixture was heated to the boiling point, kept for
1 h, cooled to 0—5 °C, and CF3COOH (28.5 g, 250 mmol) was
added. The mixture was stirred at 0—5 °C for 30 min, and colorꢀ
less crystals were filtered off, washed with dichloroethane and
dried in air. Compound 4 (12.4 g, 92%) was obtained as a solid,
m.p. 92—93 °C. Found (%): С, 32.45; Н, 3.48; N, 19.64.
C4H6N2O4. Calculated (%): С, 32.87; Н, 3.87; N, 19.18.
1Н NMR (DMSOꢀd6), δ: 11.0 (br.s, 3 Н, СООН, NHOH); 6.24
(br.s, 2 Н, СН=СН).
MII (%) = [((Аc•Вc) – (А•В))/Аc•Вc]•100%,
where Аc and А are the frequency of lung metastases in mice of
experimental and control groups; Вc and В are the average numꢀ
ber of lung metastases in mice of experimental and control
groups. Experimental values of MII found for compound 4 in
Lewis lung carcinoma and B16 melanoma comprised 100%.
Oxalic acid N,N´ꢀdihydroxyamide (1). To a stirred solution
of hydroxylamine hydrochloride (6.95 g, 100 mmol) in methaꢀ
nol (70 mL) a solution of NaОH (4 g, 100 mmol) in methanol
(30 mL) was added. The reaction mixture was stirred for 20 min,
and the precipitated solid NaCl was filtered off. To the resulting
methanolic solution anhydrous oxalic acid (4.25 g, 47 mmol) of
was added. The reaction mixture was stirred for 1 h, white crysꢀ
talline residue was filtered off, washed with methanol (5 mL)
and dried in the air flow for 24 h and for 2 days in a vacuum
desiccator over NaOH. Compound 1 (5 г, 89%) as colorless
crystals, m.p. 185—186.5 °С (decomposes). Found (%): C, 20.21;
H, 3.14; N, 23.40. С2Н4N2О4. Calculated (%): C, 20.0; H, 3.33;
N, 23.3. 1Н NMR (DMSOꢀd6), δ: 4.13 (s, 1 Н, ОН); 8.03
(br.s, 2 Н, NH). Found: m/z 120 [M]+. C2Н4N2O4. Calculated:
М = 120.
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1 М solution of hydroxylamine in dry methanol a solution of
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added upon stirring at ambient temperature, the reaction mixꢀ
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J = 5.6 Hz); 8.75 (br.s, 2 Н, NH); 10.38 (br.s, 1 Н, COOH).
Found: m/z 179 [M – H]+. С4Н8N2O6. Calculated: М = 180.
Aspartic acid Nꢀhydroxyamide hyrdochloride (3). Aspartic acid
βꢀmethyl ester hydrochloride (3 g, 16.5 mmol) and hydroxylꢀ
amine hydrochloride (2.3 g, 33 mmol) were added to methanol
(50 mL), followed by lithium hydroxide (2 g, 83.5 mmol). After
15 min colorless solid residue of aspartic acid monooxamide and
lithium chloride was filtered off, washed with ethyl acetate and
dried in air at room temperature and over P2О5. Compound 3
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composes). Found (%): С, 26.44; Н, 4.67; Сl, 19.02; N, 15.74.
C4H9N2O4Cl. Calculated (%): С, 26.01; Н, 4.98; Cl, 19.24;
N, 15.18. 1Н NMR (DMSOꢀd6), δ: 8.35 (br.s, 6 Н, СООН, NH2,
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Maleic acid Nꢀhydroxyamide (4). Finely powdered hydroxylꢀ
amine hydrochloride 8.34 g (120 mmol) was added to a solution
of maleic anhydride (9.8 g, 100 mmol) in 1,2ꢀdichloroethane
(150 mL) at room temperature, the mixture was cooled to
10—20 °C, and triethylamine (30.4 mL, 220 mmol) was added.
Received October 16, 2015;
in revised form January 19, 2016