Synthesis and evaluation of several new (2-chloroethyl)nitrosocarbamates as potential anticancer agents

Article abstract of DOI:10.1021/jm990417j

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an

Full text of DOI:10.1021/jm990417j

1484
J . Med. Chem. 2000, 43, 1484-1488
Syn th esis a n d Eva lu a tion of Sever a l New (2-Ch lor oeth yl)n itr osoca r ba m a tes a s
P oten tia l An tica n cer Agen ts
Robert C. Reynolds,* Anita Tiwari, J oyce E. Harwell, Deborah G. Gordon, Beverly D. Garrett, Karen S. Gilbert,
Steven M. Schmid,† William R. Waud, and Robert F. Struck
Drug Discovery and Drug Development Divisions, Southern Research Institute, 2000 Ninth Avenue South,
Birmingham, Alabama 35255-5305
Received August 16, 1999
Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer
alkylating agents. These compounds were designed with carrier moieties that would either
act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of
five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28
(melanoma), and SNB-7 (CNS). Several agents showed good activity with IC50 values in the
range of 1-10 µg/mL against at least two of the cell lines. One compound, carbamic acid, (2-
chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against
intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned
compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid,
(2-chloroethyl)nitroso-2,3,4,6-tetra-O-acetyl-1-R,â-D-glucopyranose ester (24) were evaluated
against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds
were active in vivo.
Sch em e 1
In tr od u ction
The observed activity of ethyl (2-chloroethyl)-N-nitro-
socarbamate,1 and the similarity of this class of alky-
lating agent to the clinically used N-(2-chloroethyl)-N-
nitrosourea anticancer drugs, has led us to investigate
a number new of N-(2-chloroethyl)-N-nitrosocarbamates
for evaluation as potential anticancer alkylating agents.
Several such compounds, although only modestly water
soluble, have shown significant increases in life span
and/or curative benefits in mice implanted with P388
leukemia.2 On the basis of this activity, seven new
target compounds were synthesized in order to ascertain
different approaches for optimizing solubility and activ-
ity of the N-(2-chloroethyl)-N-nitrosocarbamates. Among
these approaches, N-(2-chloroethyl)-N-nitrosocarbam-
ates were conjugated to small, water-soluble hetero-
cycles (11, 20), a carbohydrate (24), or substituted
benzylic functions with the potential for in situ bioac-
tivation (3, 8, 9, 18) for evaluation as potential anti-
cancer alkylating agents. Several carbohydrate-derived
alkylating agents have been reported.3 On the basis of
the activity of these known agents and the potential for
selective uptake of carbohydrate-alkylating agents,4
compound 24 was synthesized and screened. The benzyl-
substituted N-(2-chloroethyl)-N-nitrosocarbamates (3, 8,
9, 18) were designed as potential prodrugs whereby
deacetylation5 (3, 18) or bioreduction of the nitro func-
tion6 (8, 9) would lead to potential “self-immolative”
agents as previously reported.7 The new agents were
evaluated against a panel of human tumor cell lines in
Sch em e 2
vitro, and three were also evaluated in vivo against a
murine leukemia and/or a murine sarcoma.
Ch em istr y
Selective acetylation of the phenolic group of 4-hy-
droxybenzyl alcohol (Scheme 1) by the method of Para-
disi et al.8 gave 1. This intermediate was reacted with
2-chloroethyl isocyanate and nitrosated by standard
methods9 to give the target compound 3 in good overall
yield. The commercially available nitrobenzyl alcohols
(4, 5) were carbamoylated and nitrosated (Scheme 1)
to yield the target compounds 8 and 9. 5-Hydroxypy-
rimidine10 and commercially available 3-hydroxypyri-
dine were likewise carbamoylated and nitrosated
(Scheme 2) to yield the target heterocycle-conjugated
(2-chloroethyl)-N-nitrosocarbamates (11, 20).
* Corresponding author: Dr. Robert C. Reynolds, Manager, Medici-
nal Chemistry, Southern Research Institute, 2000 Ninth Ave. S.,
Birmingham, AL 35255-5305. Tel: 205/581-2454. Fax: 205/581-2877.
E-mail: reynolds@sri.org.
† Present address: Preclinical Products, Ilex Oncology, Inc., 11550
IH 10 West, Suite 300, San Antonio, TX 78230-1064.
10.1021/jm990417j CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/22/2000

(2-Chloroethyl)nitrosocarbamates as Anticancer Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1485
Sch em e 3
to the higher toxicity and mutagenicity of this class of
alkylating agent relative to the substituted N-(2-chlo-
roethyl)-N-nitrosoureas.12 Although these data are re-
ported only for simple, linear, alkyl-substituted N-(2-
chloroethyl)-N-nitrosocarbamates, the lack of in vivo
activity and the toxicity profile of the agents reported
herein suggest that these agents may also be subject to
rapid hydrolysis in vivo.
Exp er im en ta l Section
Ch em istr y. Examinations by TLC were performed on
Analtech precoated (250 µm) silica gel GF plates. Column
chromatographic purifications were done with silica gel (Merck,
60 A, 230-400 mesh for flash chromatography). Evaporations
were performed with a rotary evaporator; higher boiling
solvents (DMF, Me2NAc, Me2SO) were removed in vacuo (<1
mm, bath to 35 °C) and more volatile solvents with a water
aspirator. Products were dried in vacuo (<1 mm) at 22-25 °C
over P2O5 and NaOH pellets. Analytical results indicated by
element symbols were within (0.4% of the theoretical values,
and where solvents are indicated in the formula, their presence
was confirmed by 1H NMR. Spectral determinations and
elemental analyses were performed in the Molecular Spec-
troscopy Section of the Southern Research Institute under the
direction of Dr. J . M. Riordan. The 1H NMR spectra were
determined with a Nicolet NMC 300 NB spectrometer using
Me4Si as internal reference. Chemical shifts (δ) listed for
multiplets were measured from the approximate centers, and
relative integrals of peak areas agreed with those expected
for the assigned structures. Mass spectra were recorded on a
Varian MAT 311A mass spectrometer in the fast-atom-
bombardment (FAB) mode. Infrared spectra were recorded
with a Nicolet 10 DX spectrometer using samples in KBr disks.
NOCl was prepared fresh for each reaction by the method of
Becham.9 Note that all target compounds were carefully dried
and stored under argon at -20 °C until used for screening.
Sch em e 4
Intermediate 12 (Scheme 3) was prepared by selective
acetylation11 of the benzyl hydroxyl group of 2-hydroxy-
benzyl alcohol. The phenolic hydroxyl group was sub-
sequently blocked as the THP ether, and deacetylation
exposed the benzylic hydroxyl group for further elabora-
tion. The THP derivative 14 was then carbamoylated,
deprotected, acetylated, and nitrosated to yield the
target 18. Finally, commercially available 2,3,4,6-tetra-
O-benzyl-1-R,â-D-glucopyranose was reacted with 2-chlo-
roethyl isocyanate, deblocked by hydrogenolysis, per-
acetylated, and nitrosated with NOCl to give 24 (Scheme
4). All samples were characterized by FAB-MS, 1H
NMR, and IR spectroscopy and TLC and CHN analysis
prior to evaluation.
P r ep a r a tion of Ca r ba m ic Acid , (2-Ch lor oeth yl)-4-a ce-
toxyben zyl Ester (2). Compound 18 [2.1 g (11.5 mmol)] was
dissolved in 10 mL of anhydrous CH2Cl2. The reaction was
cooled to 0 °C. Et3N (106 µL) and then 2-chloroethyl isocyanate
1.07 mL (12.6 mmol) were added slowly. The reaction mixture
was stirred for 48 h at ambient temperature and concentrated
to dryness with a rotary evaporator. The resulting oil was
dissolved in a minimum of CH2Cl2 and purified by column
chromatography (silica gel 230-400 mesh) using CH2Cl2. The
desired fractions were collected, concentrated and dried in
vacuo over P2O5 to give an oil that solidified in the freezer:
1
yield 3.1 g (90.3%); mp 61-63 °C; MS m/z 272 (M + H)+; H
NMR (CDCl3) δ 2.31 (S, 3H, CH3), 3.54 (q, 2H, CH2N), 3.61 (t,
2H, CH2Cl), 5.1 (S, 2H, CH2O), 5.16 (bs, 1H, NH), 7.08 (m,
2H, H-3,5), 7.37 (m, 2H, H-2,6); IR cm-1 913, 1040, 1144, 1192,
Resu lts a n d Discu ssion
Several of the new agents showed good in vitro
activity with IC50 (see Table 1) values in the range of
1-10 µg/mL against at least two of the human tumor
cell lines. Three compounds (3, 9, and 24) were selected
for further study in vivo against either intraperitoneally
(ip) implanted P388 murine leukemia (3, Table 2) or
subcutaneously (sc) implanted M5076 murine sarcoma
(3, 9, and 24, Table 2) in mice. Although these three
compounds showed significant activity in vitro, they
were not active in vivo but demonstrated notable
toxicity. Target 3 was frankly toxic at a single dose of
50 mg/kg (LD80) but was well-tolerated at a dosage of
25 mg/kg/dose (LD10 or less) and also at a dosage of 12.5
mg/kg/dose for 5 days. Compounds 9 and 24 were more
toxic, yielding LD100 at dosages of 10 and 5 mg/kg/dose
for 5 days, respectively. It has been reported that the
substituted N-(2-chloroethyl)-N-nitrosocarbamates are
rapidly hydrolyzed at the ester function in vivo leading
1206, 1222, 1269, 1437, 1510, 1550, 1704, 1756. Anal. (C12H14
ClNO4) CHN.
-
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-4-a cetoxyben -
zyl Ester (3). Compound 2 [1.61 g (5.9 mmol)] was dissolved
in 15 mL of anhydrous pyridine. The reaction mixture was
cooled to -20 °C, and freshly prepared nitrosyl chloride (5.7
mL) was added dropwise. The resulting solution was stirred
at -20 °C for 2 h, and the reaction mixture was then poured
into ice-cold water. The mixture was extracted with CH2Cl2
(2 × 100 mL), and the extract was dried over MgSO4 and
concentrated to dryness. The residual yellow oil was purified
by column chromatography (silica gel 230-400 mesh, elution
with CH2Cl2). The desired fractions were combined, concen-
trated and dried in vacuo over P2O5 to give a yellow oil that
solidified in the freezer: yield 1.6 g (89.8%); mp 48-50 °C;
1
MS m/z 301 (M + H)+; H NMR (CDCl3) δ 2.31 (S, 3H, CH3),
3.45 (t, 2H, CH2Cl), 4.08 (t, 2H, CH2N), 5.5 (S, 2H, CH2O),
7.13 (m, 2H, H-3,5), 7.5 (m, 2H, H-2,6); IR cm-1 910, 1076,
1139, 1153, 1217, 1347, 1369, 1394, 1510, 1760. Anal. (C12H13
ClN2O5) CHN.
-

1486 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Reynolds et al.
Ta ble 1. Cytotoxicity of Selected Drugs in the Core in Vitro Tumor Panel
IC50 values in µg/mLa (IC50 molar value)
CAKI-1
(renal)
DLD-1
(colon)
NCI-H23
(lung)
SK-MEL-28
(melanoma)
SNB-7
(CNS)
agent
name
MW (g)
300.69
3
carbamic acid, (2-chloroethyl)-N-nitroso-4-
acetoxy-1-benzyl ester
2 × 101
(7 × 10-5
3
2 × 101
(7 × 10-5
5
6
1 × 101
3 × 101
(1 × 10-4
1
)
)
)
)
)
)
)
)
(2 × 10-5
)
(3 × 10-5
1
)
)
)
)
)
)
)
)
)
)
)
8
carbamic acid, (2-chloroethyl)-N-nitroso-4-
nitro-1-benzyl ester
carbamic acid, (2-chloroethyl)-N-nitroso-2-
nitro-1-benzyl ester
carbamic acid, (2-chloroethyl)-N-nitroso-5-
pyrimidine ester
carbamic acid, (2-chloroethyl)-N-nitroso-3-
pyridine ester
287.65
287.65
230.60
229.62
1
(3 × 10-6
3
(1 × 10-5
4 × 10-1
(1 × 10-5
6
(2 × 10-5
)
)
)
(3 × 10-6
4 × 10-1
(1 × 10-7
3 × 101
(1 × 10-4
(3 × 10-6
9
6
1
(2 × 10-5
4 × 101
(2 × 10-4
(1 × 10-5
(3 × 10-6
6
11
20
24
18
2
(3 × 10-5
(9 × 10-6
(3 × 10-5
3 × 101
(1 × 10-4
1
>3 × 101
(>1 × 10-4
>3 × 101
(>6 × 10-5
3 × 101
>3 × 101
(>1 × 10-4
2 × 101
>3 × 101
(>1 × 10-4
3
>3 × 101
(>1 × 10-4
1 × 101
)
)
)
)
)
carbamic acid, (2-chloroethyl)-N-nitroso-2,3,4,6- 482.82
tetra-O-acetyl-1-D-glucopyranose ester
carbamic acid, (2-chloroethyl)-N-nitroso-2-
(4 × 10-5
1 × 101
(3 × 10-5
>5b
)
)
(6 × 10-6
)
(2 × 10-5
2 × 101
(7 × 10-5
1b
)
)
(2 × 10-6
300.69
1 × 101
6
acetoxy-1-benzyl ester
(1 × 10-4
>5b
)
(3 × 10-5
)
(2 × 10-5
Ara-C cytosine arabinoside
279.68
1 × 10-1 b
1 × 10-1 b
2b
9 × 10-2 b
a
b
72-h exposure to compound. IC50 values are in µM.
Ta ble 2. Summary of the in Vivo Antitumor Activity of Carbamic Acid, (2-Chloroethyl)-N-nitroso-4-acetoxy-1-benzyl Ester (3)
optimal ip dosage
(eLD10) (mg/kg/dose)
median % ILS
(dying mice only)
net log
cell kill
T-Cb
(days)
tumor-free
surv/total
murine tumora
schedule
day 1
days 10-14
ip P388 leukemia
25
12.5
+4
-
-0.3
-
-
3.0
0/5
0/5
sc M5076 sarcomac
a
CD2F1 mice were implanted ip with 106 murine P388 leukemia cells, whereas B6C3F1 mice were implanted sc with 5 × 106 murine
b
M5076 sarcoma cells. The difference in the median of times poststaging for tumors of the treated (T) groups to double in mass twice
compared to the median of the control (C) group. c Both compounds 9 and 24 were evaluated as well and were inactive (data not shown).
Ca r ba m ic Acid , (2-Ch lor oeth yl)-4-n itr oben zyl Ester
(6). Compound 6 was prepared from 4-nitrobenzyl alcohol (4)
(2.0 g, 13.05 mmol) over a period of 86 h using the procedure
described for the preparation of 2: yield after column chro-
matography (CHCl3) 2.97 g (88%); mp 73-75 °C; MS m/z 259
(M + H)+; 1H NMR (CDCl3) δ 3.58 (q, 2H, CH2N), 3.65 (t, 2H,
CH2Cl), 5.21 (s, 2H, CH2O), 5.26 (bs, 1H, NH), 7.52 (d, 2H,
H-3,5), 8.23 (d, 2H, H-2,6); IR cm-1 740, 847, 1016, 1075, 1157,
1259, 1273, 1312, 1350, 1373, 1458, 1510, 1548, 1605, 1690.
Anal. (C10H11ClN2O4) CHN.
under the same conditions as those described for the prepara-
tion of 2. The product was purified using column chromatog-
raphy (99:1 CH2Cl2:MeOH): yield 508 mg (71.2%); mp 97-98
°C; MS m/z 202 (M + H)+; 1H NMR (CDCl3) δ 3.7 (m, 4H,
CH2CH2Cl), 5.61 (bs, 1H, NH), 8.66 (s, 2H, H-4,6), 9.7 (S, 1H,
H-2); IR cm-1 620, 1256, 1265, 1277, 1418, 1559, 1572, 1737,
1746. Anal. (C7H8ClN3O2‚0.3H2O) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-5-p yr im id in e
Ester (11). Compound 11 was prepared from 10 (440 mg, 2.18
mmol) by the similar procedure as previously described for the
preparation of 3. The product was purified using column
chromatography (99:1 CH2Cl2:MeOH), and a yellow oil was
obtained that solidified in the freezer: yield 70 mg (14%); mp
Ca r b a m ic Acid , (2-Ch lor oet h yl)-2-n it r ob en zyl E st er
(7). The same procedure as described for 6 was used to prepare
7 from 2-nitrobenzyl alcohol (5) (2.25 g, 14.69 mmol): yield
1
1
3.42 g (90%); mp 67-69 °C; MS m/z 259 (M + H)+; H NMR
78-80 °C; MS m/z 231 (M + H)+; H NMR (CDCl3) δ 3.57 (t,
(CDCl3) δ 3.58 (q, 2H, CH2N), 3.65 (t, 2H, CH2Cl), 5.3 (bs, 1H,
NH), 5.55 (s, 2H, CH2O), 7.66 (m, 1H, H-3) 7.64 (m, 2H, H-4,5),
8.2 (d, 1H, H-6); IR cm-1 656, 724, 749, 1003, 1073, 1153, 1197,
1263, 1273, 1318, 1348, 1436, 1465, 1520, 1545, 1693. Anal.
(C10H11ClN2O4) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-4-n itr oben zyl
Ester (8). The general procedure previously described for 3
was used to prepare 8 using 6 (2.96 g, 11.44 mmol). In this
case, the reaction was stirred at -20 °C for 2 h and at -10 °C
for 3 h. After column chromatography (6:4 hexanes:CH2Cl2),
a yellow oil was obtained that solidified in the freezer: yield
3.0 g (90%); mp 27-29 °C; MS m/z 288 (M + H)+; 1H NMR
(CDCl3) δ 3.7 (t, 2H, CH2Cl), 4.13 (t, 2H, CH2N), 5.62 (s, 2H,
CH2O), 7.65 (d, 2H, H-3,5), 8.68 (d, 2H, H-2,6); IR cm-1 767,
2H, CH2Cl), 4.20 (t, 2H, CH2N), 8.87 (s, 2H, H-4,6), 9.21 (s,
1H, H-2); IR cm-1 719, 746, 972, 1078, 1092, 1125, 1224, 1301,
1411, 1517, 1571, 1770. Anal. Calcd for C7H7ClN4O3: C, 36.46;
H, 3.05; N, 24.29. Found: C, 36.84; H, 3.12; N, 23.61.
Ben zen em eth an ol, 2-[(Tetr ah ydr o-2H-pyr an -2-yl)oxy]-,
Aceta te (13). A solution of 2-acetyl salicylate11 (7.3 g, 40.00
mmol) and dihydropyran (5.29 mL) in anhydrous CH2Cl2 (9
mL) containing pyridinium p-toluenesulfonate (PPTS; 1.094
g; 4.3 mmol) was stirred at room temperature for 4.5 h. The
solution was diluted with ether and washed with brine solution
to remove the catalyst. The organic layer was dried over
MgSO4 and concentrated to dryness. The residual product was
purified by column chromatography (silica gel 230-400 mesh)
and was eluted first with cyclohexane followed by 11:1 cyclo-
hexane:EtOAc. The desired fractions were combined, concen-
trated, and dried in vacuo over P2O5 to give an oil that
solidified in the freezer: yield 8.82 g (88%); mp 47-49 °C; MS
m/z 251 (M + H)+; 1H NMR (CDCl3) δ 1.70 (m, 3H, two H-4’s,
one H-3 of THP ring), 1.87 (m, 2H, H-2 of THP ring), 2.17 (m,
1H, H-3 of THP ring), 2.11 (s, 3H, CH3), 3.61 (m, 1H, H-5 of
THP ring), 3.88 (m, 1H, H-5 of THP ring), 5.21 (S, 2H, CH2-
O), 5.29 (t, 1H, H-1 of THP ring), 6.98 (dt, 1H, H-5), 7.14 (dd,
1H, H-3), 7.26-7.35 (m, 2H, H-4, H-6).
856, 1077, 1146, 1302, 1346, 1395, 1524, 1751. Anal. (C10H10
ClN3O5) CHN.
-
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-2-n itr oben zyl
Ester (9). The same procedure previously described in 3 and
8 was used to prepare 9 from 7 (3.00 g, 11.59 mmol), and the
reaction was stirred at -20 °C for 2 h and at -10 °C for 5 h.
After column chromatography (6:4 hexanes:CH2Cl2) a yellow
syrup was obtained: yield 3.10 g (93.09%); MS m/z 288 (M +
H)+; 1H NMR (CDCl3) δ 3.51 (t, 2H, CH2Cl), 4.15 (t, 2H, CH2N),
5.97 (s, 2H, CH2O), 7.56 (m, 1H, H-4), 7.74 (m, 2H, H-3,5),
8.25 (m, 1H, H-6); IR cm-1 729, 789, 1075, 1153, 1303, 1343,
1396, 1425, 1528, 1755. Anal. (C10H10ClN3O5) CHN.
Ben zen em eth a n ol, 2-[(Tetr a h yd r o-2H-p yr a n -2-yl)oxy]-
(14). Compound 13, 2.23 g (8.9 mmol), in 20 mL of ethanol
was saponified with 19 mL of ethanolic NaOH (10 mL of 2 N
NaOH in 10 mL of EtOH) for 1 h at 18 °C. The resulting
solution was evaporated to dryness and the product extracted
Ca r b a m ic Acid , (2-Ch lor oet h yl)-5-p yr im id in e E st er
(10). 5-Hydroxypyrimidine10 (340 mg, 3.5 mmol) was reacted

(2-Chloroethyl)nitrosocarbamates as Anticancer Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1487
with ether (50 mL), washed with water, dried over MgSO4 and
concentrated to dryness. The residue was applied to a column
of silica gel (230-400 mesh) and eluted with cyclohexanes-
EtOAc (6:1). Fractions containing the product 14 were com-
bined, concentrated and dried in vacuo to give an oil: yield
1.7 g (91.9%); MS m/z 209 (M + H)+.
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2-[(tetr a h yd r o-2H-p y-
r a n -2-yl)oxy]ben zyl Ester (15). The same procedure was
previously described in 2 was used to prepare 15 from 14 (1.68
g, 8.0 mmol). After column chromatography (6:1 cyclohexanes-
EtOAc), an oil was obtained: yield 2.48 g (98%); MS m/z 314
(M + H)+; 1H NMR (CDCl3) δ 1.64 (m, 3H, two H-4’s, one H-3
of THP ring), 1.86 (m, 2H, H-2’s of THP ring), 2.00 (m, 1H,
H-3 of THP ring), 3.49 (m, 5H, H-5 of THP ring and CH2CH2-
Cl), 3.87 (dt, 1H, H-5 of THP ring), 5.15 (bs, 1H, NH), 5.23 (s,
2H, CH2-O), 5.28 (bt, 1H, H-1 of THP ring), 6.98 (t, 1H, H-5),
7.15 (d, 1H, H-3), 7.24-7.35 (m, 2H, H-4, H-6); IR cm-1 756,
922, 965, 1022, 1037, 1124, 1202, 1240, 1457, 1493, 1532, 1705,
1721, 2946. Anal. (C15H20ClNO4) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2-h yd r oxy-1-ben zyl
Ester (16). To a solution of 15 [7.25 g (23.10 mmol)] in
anhydrous ethanol (30 mL) was added 611 mg (2.4 mmol) of
PPTS. The reaction mixture was stirred at 50 °C for 4.5 h and
concentrated to dryness. The residue was chromatographed
(CHCl3). The desired fractions were collected, concentrated,
and dried in vacuo over P2O5 to give an oil that solidified upon
freezing: yield 4.35 g (82%); mp 65-67 °C; MS m/z 330 (M +
H)+; 1H NMR (CDCl3) δ 2.32 (1bs, 1H, OH), 3.62 (m, 2H, CH2-
N), 3.69 (m, 2H, -CH2-Cl), 4.60 (s, 2H, -CH2-O), 5.61 (bs, 1H,
NH), 7.11 (dd, 1H, J ) 0.6 Hz, J ) 8 Hz, H-3), 7.26 (dt, 1H,
H-5), 7.34 (dt, 1H, H-4), 7.48 (dt, 1H, J ) 0.8 Hz, J ) 6 Hz,
H-6); IR cm-1 1050, 1182, 1226, 1249, 1271, 1487, 1540, 1711,
3319. Anal. (C10H12ClNO3) CHN.
reaction was carried out at -20 °C for 0.5 h followed by -10
°C for an additional 2 h. After column chromatography (CH2-
Cl2), a yellow oil was obtained: yield 1.62 g (56.6%); MS m/z
230 (M + H)+; 1H NMR (CDCl3) δ 3.57 (t, 2H, CH2Cl), 4.20 (t,
2H, CH2-N), 7.44 (ddd, 1H, H-5, J ) 0.6 Hz, 4.7 Hz, 8.4 Hz),
7.72 (ddd, 1H, H-4, J ) 1.4 Hz, 2.8 Hz, 8.4 Hz), 8.61 (dd, 1H,
H-6, J ) 1.4 Hz, 4.7 Hz), 8.68 (brd, 1H, J ) 2.8 Hz); IR cm-1
704, 750, 795, 813, 959, 971, 1071, 1097, 1110, 1186, 1209,
1302, 1356, 1376, 1431, 1521, 1525. Anal. (C8H8ClN2O3‚
0.12H2O) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2,3,4,6-tetr a -O-ben zyl-
1-r,â-D-glu cop yr a n ose Ester (21). Compound 21 was pre-
pared from 2,3,4,6-tetra-O-benzyl-D-glucopyranose (2.5 g, 4.6
mmol), 2-chloroethyl isocyanate (0.42 mL) and Et3N (125 µL)
by the same procedure as described for the preparation of 2.
The mixture was allowed to react for 24 h. The product was
purified by column chromatography (CHCl3): yield 2.6 g (87%);
mp 144-146 °C; MS m/z 646 (M + H)+; 1H NMR (CDCl3) δ
3.48-3.96 (m’s, 10H, CH2CH2 and H-2, H-3, H-4, H-5), 4.44-
4.99 (m, 8H, four C6H5 CH2’s), 5.09 (t, 0.75H, NHâ), 5.26 (t,
0.25H, NHR), 5.54 (d, 0.75H, H-1â, J ) 8.1 Hz), 6.24 (d, 0.25H,
H-1R, J ) 3.5 Hz) 7.12 (m, 2H, aromatic H’s), 7.25-7.35 (m,
18H, aromatic H’s); IR cm-1 693, 716, 748, 1003, 1018, 1029,
1051, 1072, 1085, 1106, 1119, 1151, 1270, 1453, 1558, 1702,
2874, 2917, 3030, 3319. Anal. (C37H40ClNO7‚0.02CHCl3) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2,3,4,6-tetr a -O-h yd r ox-
yl-1-r,â-D-glu cop yr a n ose Ester (22). To a suspension of
compound 21 [3.62 g (5.6 mmol)] in 150 mL of anhydrous
ethanol was added 5% Pd/C (1.8 g). The suspension was
hydrogenated at atmospheric pressure at 40 °C for 24 h,
filtered and concentrated to dryness. The residue in a mini-
mum of MeOH was applied to a column packed with silica gel
(230-400 mesh). The column was first eluted with CHCl3
followed by CHCl3:MeOH (6:1). The desired fractions were
combined, concentrated and dried in vacuo over P2O5: yield
1.36 g (85%); mp 127-129 °C; MS m/z 286 (M + H)+; 1H NMR
(Me2SO-d) δ 3.04-3.68 (m, H-2, H-3, H-4, H-5, H-6, CH2CH2),
4.53 (t, 0.3H, 6-OHR), 4.60 (t, 0.7H, 6-OHâ), 4.94, 4.98, 5.07,
5.10, 5.19 (five d’s, 3H, 2-OH, 3-OH, 4-OH of R and â anomers),
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2-a cetoxy-1-ben zyl Es-
ter (17). Compound 16, 4.33 g (18.85 mmol), was dissolved in
anhydrous pyridine (12 mL), and the mixture was cooled to 0
°C. Acetic anhydride (1.77 mL) was added dropwise. The
resulting solution was stirred overnight at ambient temper-
ature. The mixture was poured into ice-cold water, and the
product was extracted with CH2Cl2. The organic layer was
dried over Na2SO4 and concentrated to dryness, and the
residual oil was purified by column chromatography (CHCl3)
5.26 (d, 0.7H, H-1â, J 1,2 ) 8.0 Hz), 5.83 (d, 0.3H, H-1R, J 1,2
)
3.3 Hz), 7.54 (t, 0.3H, NHR), 7.64 (t, 0.7H, NHâ); IR cm-1 1078,
1713. Anal. (C9H16ClNO7‚0.1C2H5OH) CHN.
1
to give an oil: yield 4.66 g (91%); MS m/z 272 (M + H)+; H
Ca r ba m ic Acid , (2-Ch lor oeth yl)-2,3,4,6-tetr a -O-a cetyl-
1-r,â-D-glu cop yr a n ose Ester (23). Compound 22, 800 mg
(2.8 mmol), was dissolved in 5 mL of anhydrous pyridine, and
the resulting solution was cooled in an ice bath. Acetic
anhydride (0.96 mL) was added dropwise, and the solution was
stirred at ambient temperature overnight. The reaction mix-
ture was poured into ice-cold water, the product was extracted
with CH2Cl2 (2 × 30 mL), and the organic layer was dried over
MgSO4 and concentrated to dryness. The crude product was
chromatographed (CHCl3). The desired fractions were col-
lected, concentrated and dried in vacuo over P2O5: yield 930
NMR (CDCl3) δ 2.09 (s, 3H, CH3), 3.63 (m, 2H, CH2N), 3.69
(m, 2H, -CH2Cl), 5.12 (s, 2H, CH2-O), 5.55 (bs, 1H, NH), 7.18-
7.26 (m, 2H, H-4, H-6), 7.37 (t, 1H, H-5), 7.43 (d, 1H, H-3); IR
cm-1 762, 952, 1028, 1041, 1122, 1184, 1222, 1246, 1310, 1363,
1381, 1438, 1455, 1490, 1532, 3327, 3332. Anal. (C12H14ClNO4‚
0.1H2O) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-2-a cetoxy-1-
ben zyl Ester (18). The same procedure as described for the
preparation of 3 was used to prepare 18 from 17 (2.25 g, 8.28
mmol). The reaction was carried out at -20 °C for 0.5 h
followed by -10 °C for an additional 2 h. The product was
purified using column chromatography (CH2Cl2) to give a
yellow oil: yield 2.39 g (95.6%); MS m/ z 301 (M + H)+; 1H
NMR (CDCl3) δ 2.02 (s, 3H, CH3), 3.57 (t, 2H, CH2Cl), 4.20 (t,
2H, CH2N), 5.18 (s, 2H, CH2O), 7.33-7.38 (m, 2H, H-4, H-6),
7.24-7.55 (m, 2H, H-3, H-5); IR cm-1 754, 1072, 1123, 1141,
1181, 1228, 1377, 1742, 1764. Anal. (C12H13ClN2O5) CHN.
1
mg (73.2%); mp 52-54 °C; MS m/z 460 (M + Li)+; H NMR
(CDCl3) δ 2.02, 2.03, 2.04, 2.05, 2.09, 2.10 (six s’s, 12H, R, â
CH3’s), 3.51-3.67 (m, 4H, -CH2CH2-), 3.84 (ddd, 0.3H, H-5â, J
) 2.2 Hz, 4.3 Hz, 9.9 Hz), 4.07-4.14 (m, 1.7H, H-5R, H-6′â,
H-6′R), 4.28 (dd, 0.3H, H-6R, J 5,6 ) 4.5, J 6,6′ ) 12.8 Hz), 4.32
(dd, 0.7H, H-6â, J 5,6′ ) 4.4 Hz, J 6,6′ ) 12.5 Hz), 5.07-5.18 (m,
2H, H-2R, H-2â, H-4R, H-4â), 5.26 (t, 0.7H, H-3â, J 2,3 ) J 3,4
)
9.4 Hz), 5.32 and 5.34 (two t’s, 1H, NH), 5.47 (t, 0.3H, H-3R,
J 2,3 ) J 3,4 ) 9.9 Hz), 5.67 (d, 0.7H, H-1â, J 1,2 ) 8.3 Hz), 6.25
(d, 0.3H, H-1R, J 1,2 ) 3.7 Hz); IR cm-1 1039, 1077, 1117, 1165,
1235, 1369, 1438, 1534, 1755, 3377, 3412. Anal. (C17H24ClNO11‚
0.05CHCl3) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-2,3,4,6-tetr a -O-
a cetyl-1-r,â-D-glu cop yr a n ose Ester (24) Usin g 23 (912 m g,
2.0 m m ol). The same procedure as described for the prepara-
tion of 3 was used to prepare 24. The reaction was carried out
at -20 °C for 1.5 h followed by -10 °C for an additional 1.5 h.
After column chromatography (CH2Cl2), a pale, yellow foam
was obtained upon drying in vacuo: yield 458 mg (47.2%); mp
38-40 °C; MS m/z 489 (M + Li)+; 1H NMR (CDCl3) δ 2.03,
2.04, 2.05, 2.06, 2.09, 2.10 (six s’s, 12H, R and â, CH3’s), 3.44-
Ca r ba m ic Acid , (2-Ch lor oeth yl)-3-p yr id in e Ester (19).
Compound 19 was prepared using the procedure as described
for the preparation of 2 from 3-hydroxypyridine (1.42 g, 14.9
mmol). The mixture was allowed to react for 24 h. After column
chromatography (CHCl3), an oil was obtained that solidified
upon freezing: yield 2.53 g (84.6%); mp 47-49 °C; MS m/z
201 (M + H)+; 1H NMR (CDCl3) δ 3.63-3.73 (m, 4H, -CH2CH2-
), 5.66 (bs, 1H, NH), 7.33 (dd, 1H, H-5, J ) 5.1 Hz, J ) 8.3
Hz), 7.46 (ddd, 1H, H-4, J ) 5.1 Hz, J ) 8.3 Hz), 8.48 (m, 2H,
H-2, H-6); IR cm-1 703, 1026, 1188, 1216, 1247, 1426, 1476,
1534, 1744, 3322, 3327. Anal. (C8H9ClN2O2) CHN.
Ca r ba m ic Acid , (2-Ch lor oeth yl)n itr oso-3-p yr id in e Es-
ter (20). The same procedure as described for the preparation
of 3 was used to prepare 20 from 19 (2.5 g, 12.4 mmol). The

1488 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
3.55 (m, 2H, CH2Cl), 3.94-4.36 (m, 5H, H-5R,â, H-6R,â, H-6′R,â
CH2-N), 5.17-5.53 (m, 2.5H, H-2R, H-4R, H-2â, H-3â, H-4â), 5.52
(t, 0.5H, H-3R, J ) 9.6 Hz, J ) 10.1 Hz), 5.99 (d, 0.5H, H-1â,
J 1,2 ) 7.9 Hz), 6.63 (d, 0.5H, H-1R, J 1,2 ) 3.6 Hz); IR cm-1 1039,
1221, 1756. Anal. (C17H23ClN2O12) CHN.
Reynolds et al.
29 (11), 3262-3273. (d) Tsujihara, K.; Ozeki, M.; Morikawa, T.;
Arai, Y. A New Class of Nitrosoureas, I. Synthesis and Antitu-
mor Activity of 1-(2-chloroethyl)-3,3-disubstituted-1-nitrosoureas
having a Hydroxyl Group at the â-Position of the Substituents.
Chem. Pharm. Bull. 1981, 29 (9), 2509-2515. (e) Tsujihara, K.;
Ozeki, M.; Morikawa, T.; Kawamori, M.; Akaike, Y.; Arai, Y. A
New Class of Nitrosoureas. 4. Synthesis and Antitumor Activity
of Disaccharide Derivatives of 3,3-Disubstituted 1-(2-Chloro-
ethyl)-1-nitrosoureas. J . Med. Chem. 1982, 25, 441-446. (f)
Morikawa, T.; Tsujihara, K.; Takeda, M.; Arai, Y. A New Class
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of Nitrosoureas. V. Structure of Isomeric L-Arabinosylureas and
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Biologica l Eva lu a tion . 1. In Vitr o Eva lu a tion of Cy-
totoxicity. Cell lin es: The cell lines CAKI-1 (renal), DLD-1
(colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7
(CNS) were propagated using standard tissue culture tech-
niques in RPMI 1640 media with 10% FBS, and 2 mmol
L-glutamine at 37 °C with 5% CO2 and humidity. For each
experiment, cells were dispersed with trypsin/EDTA, sus-
pended at 5 × 104/mL, and seeded in 96-well plates at 5 × 103
µL. Compounds were dissolved in 100% DMSO, diluted in
complete media, and then added to replicate wells in 100-µL
samples. For each concentration level, eight samples were
prepared. Controls included mock treatment, positive control
(doxorubicin, 200 µM), and vehicle control of media diluted
with DMSO. The plates were incubated as described for 72 h
and analyzed with either the XTT assay13 or the neutral red
dye uptake procedure.14 The data were processed using Lotus
1-2-3 to calculate the IC50 values (Table 1).
2. In Vivo Eva lu a tion of An titu m or Activity. For the
in vivo evaluation of the sensitivity of transplantable murine
tumors to the compound, CD2F1 mice were implanted ip with
106 P388 leukemia cells, whereas B6C3F1 mice were implanted
sc with 5 × 106 M5076 sarcoma cells. Tumor implantation day
was designated day 0. In each experiment, the compound(s)
was administered ip at several dosage levels.
(5) Mizen, L.; Burton, G. The Use of Esters as Prodrugs for Oral
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(12) (a) Aukerman, S. L.; Brundrett, R. B.; Hilton, J .; Hartman, P.
E. Effect of Plasma and Carboxylesterase on the Stability,
Mutagenicity, and DNA Cross-Linking Activity of Some Direct-
Acting N-Nitroso Compounds. Cancer Res. 1983, 43, 175-181.
(b) Brundrett, R. B.; Aukerman, S. L. Enzymatic Inactivation
of N-Nitroso Compounds in Murine Blood Plasma. Cancer Res.
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(13) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
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M. R. Evaluation of a Soluble Tetrazolium/formazan Assay for
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V.; Cummings, K. B.; Borden, E. C. Synergistic Antiproliferative
For P388 leukemia, antitumor activity (Table 2) was as-
sessed on the basis of percent (%) median ILS (increase in life
span) and net log cell kill. Calculations of net log cell kill were
made using the tumor doubling time that was based on
historical data. To assess tumor cell kill at the end of
treatment, the survival time difference between treated and
control groups was adjusted to account for regrowth of tumor
cell populations that may occur between individual treatments.
For M5076 sarcoma, antitumor activity (Table 2) was
assessed on the basis of delay in tumor growth (T-C). The delay
in tumor growth is the difference in the median of times
poststaging for tumors of the treated (T) and control (C) groups
to double in mass two times. Drug deaths and any animal
whose tumor failed to attain the evaluation size were excluded.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health, National Cancer Institute,
Grant CA34200.
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J M990417J