Kinetic investigation on aqueous decomposition of 2- chloroethylnitrososulfamide

Article abstract of DOI:10.1016/j.bmcl.2005.10.080

The kinetics decomposition of 2-chloroethylnitrososulfamides (CENS) was studied in aqueous buffered solutions with pH ranging from 0 to 14. The study was monitored by RP-LC-MS and conventional UV spectrophotometry. The reaction proceeded via a pseudo-firs

Full text of DOI:10.1016/j.bmcl.2005.10.080

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1021–1027
Kinetic investigation on aqueous decomposition
of 2-chloroethylnitrososulfamide
a
b,
*
Achour Seridi,a,b Mekki Kadri,a,* Mohamed Abdaoui, Jean-Yves Winum and
Jean-Louis Monterob
aLaboratoire de Chimie Applique´e, Universite´ de Guelma, BP 401, 24000 Guelma, Algeria
bLaboratoire de Chimie Biomole´culaire UMR 5032, Universite´ Montpellier II, ENSCM,
8 rue de lÕEcole Normale, 34296 Montpellier, France
Received 11 October 2005; revised 20 October 2005; accepted 24 October 2005
Available online 10 November 2005
Abstract—The kinetics decomposition of 2-chloroethylnitrososulfamides (CENS) was studied in aqueous buffered solutions with pH
ranging from 0 to 14. The study was monitored by RP-LC–MS and conventional UV spectrophotometry. The reaction proceeded
via a pseudo-first-order kinetic with significant correlation coefficient. The major decomposition products from CENS after incu-
bation in phosphate buffer were isolated and identified by NMR and mass spectrometry. The results indicate that the mechanism
pathway involves a denitrosation of the CENS and competitive hydrolysis with nucleophilic attack on the sulfur atom and forma-
tion of sulfamate compounds.
ꢀ 2005 Elsevier Ltd. All rights reserved.
2-Chloroethylnitrososulfamides (CENS) are promising
O
O
O
O
alkylating agents which have focused our interest over
the past decade due to their better biological profile
compared to their structural parent, the chloroethylni-
trosoureas (CENU).1–8 Indeed, in contrast to CENU,
this class of compounds does not have the ability to gen-
erate carbamoylating species which are known to be in-
volved in the toxicity of CENU.3
S
S
Cl
Cl
N
N
N
N
N
N
O
O
1
2
O
O
S
Cl
N
N
N
O
To progress to clinical evaluation, information are need-
ed on the stability and mechanism of decomposition of
CENS. So, to gain insight into the stability of this family
of compounds, we have carried out the first experimen-
tal investigation on the fragmentation of CENS in aque-
ous media by LC–MS and conventional UV
spectrophotometry.
3
Figure 1. CENS used in this study.
In a first approach, we monitored by TLC the decompo-
sition of the different CENS in buffer solution. The first
observation indicated that CENS lost their nitroso
group to give two compounds with different Rf.
For this study, three CENS of interest were selected
(Fig. 1) considering their better activity in vitro on
A549 and MCF7 cell lines, compared to CENU.9–11
The kinetics of decomposition for the three different
compounds were experimentally determined by UV
spectrophotometry, analyzing evolution of the spectra
according to the time of 10ꢀ4–10ꢀ5 M solutions in buffer
solutions with pH ranging from 0 to 14. Unless other-
wise stated, the reactions were monitored at 291 K, the
ionic strength being adjusted (0.2 M) with addition of
KCl.
Keywords: 2-Chloroethylnitrososulfamide; Alkylating agent; Antican-
cerous compounds; Kinetic decomposition; LC–MS; UV spectroscopy.
*
Corresponding authors. Tel./fax: +213 3721 5855 (M.K.); tel./fax:
+33 467 144 344 (J.-Y.W.); e-mail addresses: mekadri@yahoo.fr;
winumj@univ-montp2.fr
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.080

1022
A. Seridi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1021–1027
0.8
For the decomposition of CENS, three distinct types of
reactivity can be distinguished. First in basic solution,
the decomposition of the compounds 1, 2 and 3 takes
place in only one step with hypsochromic shift and
appearance of an isobestic point (Fig. 2).
t=30h
t=0
(1)
0.6
0.4
0.2
0
For all the compounds studied at acidic pH interval, the
decomposition of the CENS occurred via independent
step with a decreasing of absorbance in time. This obser-
vation can be attributed to the loss of chromophoric ni-
troso group.
Abs
t=20
days
t=196h
The way of decomposition of the three compounds in
neutral pH interval was quite different. First, we ob-
served a slow decrease in absorbance with hypsochromic
shift and apparition of new absorption band which in-
crease according to the time. The reaction in neutral
pH is more complex. As we can see in Figure 4, the
decomposition occurred probably via two competitive
steps. The slow decrease in absorbance value can be
attributed to the denitrosation phenomena which corre-
spond to the reaction in acidic media (Fig. 3). The in-
220
240
260
280
300
Wavelength (nm)
Figure 4. Evolution of 10ꢀ4 M solution of compound 1 at pH 7.4.
crease in absorbance and hypsochromic shift
accompanied with apparition of isobestic point near
254 nm was similar to the decomposition in basic medi-
um (Fig. 2).
0.8
For the decompositions taking place in only one step,
the values of the observed rate coefficients (Kobsd) have
been determined graphically using the function
ln [(A ꢀ A )/(A0 ꢀ A )] = f(t) for the acidic medium
7 h
6 h
5 h
4 h
(1)
1
1
3 h
2 h
1 h
0.6
and ln A = f(t) for the basic medium. Linearization of
the experimental data showed that these decompositions
are excellent pseudo-first-order reactions. The coeffi-
cients of linear regression are superior to 0.98 for all
compounds. Absolute values of the slopes represent
the rate constant of the reaction of the pseudo-first-or-
der. For neutral pH, the decomposition reactions of
the compounds 1, 2 and 3 are competitive and the treat-
ment of kinetic data revealed pseudo-first-order. To in-
crease the precision of calculation of the rate
constants, a sufficient number of experimental points sit-
uated in the first time of half-reaction of each process
have been used. For the first stage 20 cycles of 90 s have
been recorded and 25 cycles of 900 s for the second. For
all pH values, the rate constant (Kobsd) and the times of
half-life have been determined.
Abs
0 h
0.4
0.2
0
220
240
260
280
300
Wavelength (nm)
Figure 2. Evolution of 10ꢀ4 M solution of compound 1 at pH 8.6.
0.8
The relation between the logarithms of the observed that
pseudo-first order rate constants and the pH are repre-
sented in the following Figure 5.
(2)
0.6
The U-shaped profile is a characteristic of the reaction
catalyzed in acidic or basic medium as already noted
for analogues compounds.12–20
t=0mn
0.4
t=2mn
t=4mn
Abs
t=6mn
t=8mn
For each compound, three distinct regions of reactivity
can be distinguished. For the three compounds, the
maximum stability was observed for the pH interval
ranging from 5 to 8. As shown in Figure 5, the presence
of H3O+ or OHꢀ catalyzes the hydrolysis of these
compounds.
t=10m
t=12m
0.2
t=14m
t=16m
t=24h
250
0
220
300
350
400
For the studied compounds and for a given pH, R1 and
R2 have a moderate influence on the values of the
decomposition constants.
Wavelength[nm]
Figure 3. Evolution of 10ꢀ4 M solution of compound 2 at pH 0.14.

A. Seridi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1021–1027
1023
-2,5
-3,0
The kinetic of decomposition of compounds 1, 2 and 3
was monitored at pH 7.4; T = 37 ꢁC by RP-LC–MS.34
The evolution of the chromatograms is depicted in Fig-
ure 6. We observed a disappearance of CENS with
formation of two main products (Table 1).
1
2
3
-3,5
-4,0
-4,5
The first single signal corresponds to the starting prod-
uct. After decomposition, there are two components
eluting earlier at 2 and 3 min. The main decomposition
products were tentatively identified by LC–MS; data
are shown in Table 2.
Initially, compounds 1, 2 and 3 have been identified by
HPLC–MS. They have shown, respectively, a retention
time at 9.26, 12.44 and 5.5 min detected with UV-PDA
detector at k = 253.46, 251.44 and 244.64 nm. Their
mass spectrum revealed an ion molecular peak at
278.12 [M+Na]+ for compound 1, 390.01 [M+Na]+ for
compound 2 and 352.44 [M+H]+ for compound 3.
-5,0
-5,5
0
2
4
6
8
10
12
14
After decomposition, the total ion chromatograms
(TIC) contain at least two components with nearly iden-
tical retention times for all compounds used in this
pH
Figure 5. Plot of log Kobsd versus pH.
Figure 6. Evolution according to time of HPLC chromatograms of compound 3.

1024
A. Seridi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1021–1027
Table 1. Rate constants and half-life of decomposition of CENS at different pH
pH
0.1
2.1
4.1
6.2
7.4
8.6
9.25
10.0
11.5
13.0
1
2
3
K(obsd) · 104, sꢀ1
t(1/2), min
6.114
19
0.876
132
0.137
868
0.057
2003
0.065
1777
0.125
917
0.183
632
0.350
329
0.858
134
2.185
53
K(obsd) · 104, sꢀ1
t(1/2), min
14.00
8
1.960
59
0.420
273
0.150
750
0.137
841
0.142
817
0.163
705
0.200
576
0.355
325
0.865
133
K(obsd) · 103, sꢀ1
t(1/2), min
1.857
62
0.521
221
0.218
530
0.145
786
0.260
444
0.419
275
0.537
215
1.002
115
2.433
48
5.480
21
Table 2. Mass spectral and HPLC chromatographic characterization of the products resulting from aqueous decomposition of CENS after 72 h in
phosphate buffer at 37 ꢁC
Compound
UV detection
ESI mass detection m/z (positive mode)
Tr (min)
UV data (nm)
Tr (min)
Masse
1
3.33
2.58
253.26
236.47
3.32
2.59
279.15
227.15
2
3
9.33
3.14
2.17
251.46
265.46; 226.46
268.26
9.26
3.02
2.09
390.01
279.06; 301.02 101.79
339.12
3.25
1,74
265.44
256.46
3.15
2.07
279.15; 301.09
345.29
study. LC-ESI-MS data in Table 2 show that both com-
pounds eluting near 2 min have been detected, respec-
tively, by an on-line UV-PDA detector at k = 236.4,
268.26 and 256.44 nm for the decomposition of com-
pounds 1, 2 and 3. Their mass spectrum showed, respec-
tively, a molecular ion peak at m/z 227 [M+H]+,
m/z = 339.12 (M+H)+ and m/z = 345.29 [M+Na]+. They
were identified as denitrosated CENS, for example, the
chloroethylsulfamide.
experimental data
fitted data
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
The situation of the second product B is somewhat more
complex; ESI-MS detection data showed the same val-
ues for the three compounds with m/z = 279 [M+H]+
and m/z = 301 [M+Na]+. The problem was to identify
the common species which is eluted near 3 min. It
should be noted that this product of decomposition
was stable. After isolation, it was identified on the basis
of the NMR and mass spectrum.
-20
0
20
40
60
80
100
120
140
160
time hours
Figure 7. Plot of peak area versus time of decomposition of
compound 2.
The kinetic parameters of the decomposition of the
CENS under aqueous condition were determined by fol-
lowing the disappearance of HPLC–UV peak of the
starting compound and plotting the peak area of each
CENS versus time (Fig. 7). The experimental data were
fitted using an exponential model (data not shown). The
half-life values were calculated by a mathematical model
illustrating pseudo-first-order kinetics: C = C0 exp
(ꢀKt), where C0 represents the initial concentration of
the CENS, C, the concentration at time t, and t time
of incubation (hours). The observed rate constants and
half-lives, calculated by ln2/K, in aqueous solution are
given in Table 3.
Table 3. Decomposition kinetic parameters of CENS at pH 7.4;
T = 37 ꢁC
a
t1/2 min
Compound
Kobsd minꢀ1
r2
1
0.0060
0.0016
0.0339
0.0133
0.0144
0.997
0.983
0.989
—
115.0
424.5
2
3
20.40
52.0033
48.0014
BCENU
CCNU
—
a The variability of triplicate samples was no more than 4%.
stable; however, compound 3 is somewhat not stable
and its half-life exhibited a comparable value with the
CENU.
The rate constants and half-life determined and com-
pared to some CENU14–33 showed that CENS are more

A. Seridi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1021–1027
1025
HPLC analysis of the residue obtained after complete
decomposition of CENS showed the formation of two
degradation products (Fig. 7). These products have been
tentatively identified using the following data:
Product (C) was identified comparing the chromato-
graphic feature (Fig. 8) of the synthesized product.
The NMR and mass spectrometry showed that this
compound is the chloroethylsulfamide. Product (B)
Figure 8. HPLC chromatograms: 1—Evolution of decomposition reaction of compound 3 after 72 h of incubation in phosphate buffer pH 7.4;
T = 37 ꢁC. 2—Synthetic compound C (N-chloroethylsulfamide).
O
O
Cl
S
N
R1
N
products
O
N
O
OH
R2
O
S
Cl
N
N
pH=7.4
B
D
R1
N
R2
O
O
O
S
Cl
N
+ HNO2 +
H+
R1
N
H
R2
C
Scheme 1. Proposed mechanism of decomposition.

1026
A. Seridi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1021–1027
O
O
S
Cl
N
N
R1
N
R2
OH
H2O
O
O
O
O
S
Cl
S
Cl
H3O+
N
R1
N
N
N
R1
N
H
+ HNO2 + H+
+
R2
R2
O
O
O
S
Cl
N
N
R1
N
R2
OH
Scheme 2. Proposed mechanism of denitrosation.
`
`
´
was identified as sulfamate by ESI-MS and NMR
spectrometry.34
des Affaires Etrangeres et Ministere Algerien de lÕEns-
´
eignement Superieur et de la Recherche Scientifique
(Ph.D. fellowship A.S.).
We can propose the following decomposition mecha-
nism (Scheme 1) in aqueous phosphate buffer. The phe-
nomena implied that the decomposition proceeded via
two competitive steps.
References and notes
1. Kadri, M.; Dahoui, N.; Abdaoui, M.; Winum, J. Y.;
Montero, J.-L. Eur. J. Med. Chem. 2004, 39, 79.
The formation of compounds C and B is the result of the
denitrosation and competitive hydrolysis of the CENS.
The compound D could not be detected because of its
high instability in the experimental conditions. Lown
and co-workers12–33 have suggested these species as
intermediate in the decomposition of chloroethyl nitro-
soureas under physiological conditions. It has been pos-
tulated to be the ultimate electrophile responsible for the
biological activity of CENU.
`
2. Winum, J.-Y.; Bouissiere, J.-L.; Passagne, I.; Evrard, A.;
Montero, V.; Cuq, P.; Montero, J.-L. Eur. J. Med. Chem.
2003, 38, 319.
3. Passagne, I.; Evrard, A.; Winum, J.-Y.; Depeille, P.; Cuq,
P.; Montero, J.-L.; Cupissol, D.; Vian, L. J. Pharmacol.
Exp. Ther. 2003, 307, 816.
4. Abdaoui, M.; Dewynter, G.; Toupet, L.; Montero, J.-L.
Tetrahedron 2000, 56, 2427.
5. Abdaoui, M.; Dewynter, G.; Aouf, N. E.; Favre, G.;
Morere, A.; Montero, J.-L. Bioorg. Med. Chem. 1996, 4,
1227.
6. Abdaoui, M.; Dewynter, G.; Montero, J.-L. Tetrahedron
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7. Abdaoui, M.; Dewynter, G.; Aouf, N. E.; Montero, J.-L.
Phosphorus, Sulfur and Silicon 1996, 118, 39.
8. Dewynter, G.; Abdaoui, M.; Regainia, Z.; Montero, J.-L.
Tetrahedron Lett. 1996, 52, 14217.
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10. Lown, J. W.; Chauhan, S. M. S. J. Org. Chem. 1981, 46,
5309.
11. Naghipor, A.; Ikonomou, M. G.; Kebarle, P.; Lown, J. W.
J. Am. Chem. Soc. 1990, 112, 3178.
The denitrosation mechanism of CENS takes place
through the initial protonation of N-chloro ethyl-
nitrososulfamide and subsequent nucleophilic attack on
the protonated oxygen (Scheme 2). The alkaline hydroly-
sis step occurs via a nucleophilic attack of OHꢀ on the sul-
fur atom to form sulfamate species
B
and
chloroethyldiazohydroxide D. The same process has been
noted for the same type of nitroso compounds such as
MNTS (N-methyl-N-nitroso-p-toluene sulfonamide)
and MNNG (N-methyl-N-nitro-N-nitrosoguanidine).32
On the other hand, the spectrophotometric study indi-
cates that in acidic media the decomposition occurs
via independent step and corresponds to the denitrosa-
tion reaction. In basic pH, compounds undergo hydroly-
sis but not denitrosation.
12. Lown, J. W.; McLaughlin, L. W.; Plambeck, J. A.
Biochem. Pharmacol. 1978, 28, 2115.
13. Colvin, M.; Brundertt, R. In Nitrosoureas: Current Status
and New Developments; Prestayko, A. W., Crooke, S. T.,
Baker, L. H., Carter, S. K., Schein, P. S., Eds.; Academic
Press: New York, 1981, p 43.
14. Sariban, E.; Erickson, L. C.; Kohn, K. W. Cancer Res.
1984, 44, 1352.
15. Lasker, P. A.; Ayres, J. W. J. Pharm. Sci. 1977, 66,
1072.
16. Garret, E. R. J. Am. Pharm. Assoc. Sci. Ed. 1960, 49, 767.
17. Den-Brok, M. W. J.; Nuijen, B.; Hillebrand, M. J. X.;
Lutz, C.; Optiz, H. G.; Beijnen, J. H. J. Pharm. Biomed.
Anal. 2005, 38, 686.
In conclusion, this study constitutes the first decomposi-
tion study of three CENS in aqueous buffer solutions. It
revealed that these compounds decompose via a general
acido-basic catalysis as previously noted for nitrosoure-
as. The LC–MS study at physiological pH and temper-
ature showed a formation of at least two products
which have been identified.
18. Garret, E. R.; Goto, S. Chem. Pharm. Bull. 1973, 21, 1811.
19. Brundrett, R. B.; Cowens, J. W.; Colvin, M.; Jardine, L. J.
Med. Chem. 1976, 19, 958.
Acknowledgments
20. Snyder, J. K.; Stock, L. M. J. Org. Chem. 1980, 45, 1990.
21. Snyder, J. K.; Stock, L. M. J. Org. Chem. 1980, 45,
4494.
This research was supported in part by the Franco-Alge-
`
rian Intergouvernemental Program, Ministere Franc¸ais

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22. Carminati, A.; Barascut, J. L.; Naghipour, A.; Lown, J.
W.; Imbach, J. L. Biochem. Pharmacol. 1989, 38, 2253.
23. Montgomery, J. A.; James, R. A.; McCaleb, G. S.; Kirk,
M. C.; Johnstan, T. P. J. Med. Chem. 1967, 10, 668.
24. Montgomery, J. A.; James, R. A.; McCaleb, G. S.; Kirk,
M. C.; Johnstan, T. P. J. Med. Chem. 1975, 18, 568.
25. Golding, B. T.; Bleasdale, C.; McGinnis, J.; Muller, S.;
Rees, H. T.; Rees, N.; Farmer, P. B.; Watson, W. P.
Tetrahedron 1997, 53, 4063.
26. Blans, P.; Vigroux, A. Chem. Eur. J. 1999, 5, 1526.
27. White, E. H.; Ryan, T. J.; Hahn, B. S.; Erikson, R. H. J.
Org .Chem. 1984, 49, 4860.
28. White, E. H.; Reefer, J.; Erikson, R. H.; Dzadsik, P. M. J.
Org .Chem. 1984, 49, 4872.
29. White, E. H.; Depinto, J. T.; Polito, A. J.; Bauer, I.;
Rooswell, D. F. J. Am. Chem . Soc. 1988, 110, 3708.
30. White, E. H.; Li, M.; Lu, S. J. Org .Chem. 1992, 57, 1252.
31. Morrica, P.; Fidente, P.; Seccia, S. Biomed. Chromatogr.
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32. GarciaRio, L.; Leis, J. R.; Moreira, J. A.; Araujo, E.;
Norberto, F.; Ribeiro, L. J. Org .Chem. 2003, 68, 4330.
33. Lown, J. W.; Koganty, R. R.; Tew, K. D.; Imbach, J. L.
Biochem . Pharmacol. 1985, 34, 1015.
compounds were detected using Waters (Micromass ZQ)
single quadrupole mass spectrometer equipped with
electrospray ionisation (ESI) source. The MS was
operated in the positive ion mode. General procedure
for analysis of products of aqueous decomposition of
chloroethylnitrososulfamides: A solution 2.10ꢀ5 mol dmꢀ3
of the chloroethylnitrososulfamides in 100 mM sodium
phosphate buffer (pH 7.4) was incubated at 37 ꢁC in vial
equipped with Teflon septum. Each compound was
dissolved in the minimum of acetonitrile and diluted
with the phosphate buffer. The mixture was vortexed for
10 s and the aliquots were removed at intervals and
injected for analysis into RP-HPLC-MS system.
Isolation of the products resulting from the decomposition
of the CENS: A mixture of CENS (200 mg) and pH 7.4
phosphate buffer 2% acetonitrile 50 ml was stirred at
37 ꢁC in reaction vials equipped with Teflon septum.
The reaction was monitored by TLC and showed the
appearance of two new spots of different Rf. The
reaction was stirred until total disappearance of the
starting material and then diluted with dichloromethane.
The mixture was washed with saturated aqueous solu-
tion of NaCl. The organic phase was separated and
dried on anhydrous sodium sulfate and concentrated
under reduced pressure to lead to a yellow oil. The
products of the decomposition were then separated on a
silica gel column eluted with dichloromethane. Com-
pound 1 (B) 1H NMR (250 MHz, CDCl3): d 1.55 (m,
6H, CH2 cycl); 2,74 (q, 2H, N-CH2); MS ESI+ 25 eV
m/z = 187.29 [M+Na]+ (100%). Compound 1 (C): 1H
NMR (250 MHz, CDCl3): d 4.62 (t, 1H, NH); 3.64 (t,
2H, Cl-CH2); 3.40 (q, 2H, N-CH2); 3.32 (t,4H, N-CH2
cycl); 1.62–1.50 (m, 6H, CH2 cycl); MS ESI+ 20 eV:
34. Experimental information: UV spectrophotometry: The
kinetics were monitored spectrophotometrically with a
UV–vis double beam spectrophotometer Jasco (Japan)
model (V-530) connected to
a PC computer with
spectral analysis program and equipped with cell com-
partment thermostated by a Jasco EHCT temperature
controller. Sealed quartz cells were used for each
experiment. HPLC system: The HPLC system consisted
of Waters model 2695 separation module with auto-
sampler and a PDA Waters 996 photodiode array
detector (starting from 220 to 400 nm). The chromato-
graphic apparatus is controlled by Masslynx version 3.5
software package. The separation of products was
achieved on a RP-C18 column (150*2 mm id, 5 lm),
protected with guard column with the same materials.
The column temperature was set at 25 ꢁC. The mobile
phase consisted of two solvents: A (water 0.1% formic
acid) and solvent B (acetonitrile 0.1% formic acid). The
flow rate was 0.25 ml/min. A linear gradient elution was
performed as follows: 0 min, 40% A, 60% B; 10–20 min,
100% B; 20.10 min, 40% A, 60%; 20.10 to 30 min, 40%
A, 60% B. The volume of injection into the column was
50 ll, and the injector syringe was washed with 50/50
methanol–water grade HPLC before each injection. The
1
m/z = 227.24 [M+H]+ (85%). Compound 2 (B) H NMR
(250 MHz, CDCl3): d 3.86 (s, 4H, 2CH2Bn); 7.26 (m,
10H, 2ArH); MS ESI+ 25 eV: m/z = 300.08 [M+Na]+
(65%). Compound 2 (C): 1H NMR (250 MHz, CDCl3):
d 7.45 (m, 10H, 2ArH), 4.35 (t, 1H, NH); 4.35 (s, 4H,
2CH2Bn); 3.60 (t, 2H, ClCH2); 3.25 (q, 2H, N-CH2);
MS ESIꢀ 20 eV: m/z = 337.99 [MꢀH]ꢀ (100%). Com-
pound 3 (B) 1H NMR (250 MHz, CDCl3): d 2.57 (q,
2H, N-CH2); 1.44 (m, 20H, CH2 cycl); MS ESI+ 40 eV:
m/z = 283.18 [M+Na]+ (100%). Compound 3 (C) 1H
NMR (250 MHz, CDCl3): d 4.45 (t, 1H, NH); 3.70 (t,
2H, ClNH2); 3.35 (q, 2H, N-CH2); 3.25 (m, 2H, 2N-
CH); 1.75–1.00 (m, 20H, CH2 cycl); MS ESI+ 40 eV:
m/z = 345.29 [M+Na]+ (100%).