Nitration of camptothecin with various inorganic nitrate salts in concentrated sulfuric acid: A new preparation of anticancer drug 9- nitrocamptothecin

Article abstract of DOI:10.1055/s-1998-2207

The nitration reactions of camptothecin (1) with 19 commonly used inorganic nitrate salts and a combination of two or more different nitrate salts in concentrated sulfuric acid are discussed. A new preparation of a promising anticancer drug 9-nitro-camptothecin (4) with a combination of potassium nitrate and thallium(I) nitrate as the nitrating reagents in concentrated sulfuric acid is described.

Full text of DOI:10.1055/s-1998-2207

PAPERS
1724
Nitration of Camptothecin with Various Inorganic Nitrate Salts in Concentrated
Sulfuric Acid: A New Preparation of Anticancer Drug 9-Nitrocamptothecin
Zhisong Cao,* Kim Armstrong, Marcus Shaw, Eddie Petry, Nick Harris
The Stehlin Foundation for Cancer Research and St. Joseph Hospital Cancer Laboratory, 1918 Chenevert Street, Houston, Texas 77003, USA
Tel. +1(713)7565750; Fax +1(713)7565783; E-mail: zcao@pipeline.com
Received 1 May 1998; revised 16 June 1998
Abstract: The nitration reactions of camptothecin (1) with 19
commonly used inorganic nitrate salts and a combination of two
or more different nitrate salts in concentrated sulfuric acid are dis-
which uses a nitric acid/sulfuric acid system as nitrating
cussed. A new preparation of a promising anticancer drug 9-nitro-
camptothecin (4) with a combination of potassium nitrate and
Literature procedure for preparing compound 4 from
camptothecin (1) is a conventional nitration reaction,
reagents.4 When following this procedure to prepare 4 in
our laboratory, a poor yield (from 3–7%) of the pure prod-
uct 4 (good for patient-using) was obtained. This mixed
acid procedure gave 9-nitrocamptothecin (4) and the inac-
tive isomer 12-nitrocamptothecin (5) in a ratio of ca. 1:3
and about 20 other unidentified byproducts. Compound 5
was the major product (~60%). The unfavorable low ratio
of 4 to 5 contributed to low yield of the active compound
4. Too many other byproducts complicated the process of
separation and purification, which decreased the yield of
4 as well. Thus, it is necessary to find an effective proce-
dure to increase the yield of the anticancer compound 4 in
order to meet the tremendous demand of clinical trials and
laboratory uses (Scheme 1).
thallium(I) nitrate as the nitrating reagents in concentrated sulfuric
acid is described.
Key words: anticancer drugs, camptothecin, nitration, 9-nitro-
camptothecin
Inhibitors of topoisomerase I, camptothecin (1)1 and some
of its semisynthetic derivatives such as topotecan (2),2
irinotecan (CPT-11) (3),3 and 9-nitrocamtothecin (4),4
have exhibited strong antitumor activity against various
experimental tumor models including human lung, colon,
and mammary tumor lines.5–10 The water soluble com-
pounds 2 and 3 are now commercially available as solu-
tions and injectable for intravenous (i.v.) treatment. The
water insoluble 9-nitrocamptothecin (4), a nitration prod-
uct of camptothecin, has been thoroughly studied both in
vitro and in vivo in our laboratory,11–20 demonstrating
very potent antitumor activity against many different
types of human cancers growing as xenografts in Swiss
nude mice. Phase 1 human clinical trial with 4 was con-
cluded by the Stehlin Foundation for Cancer Research.
Phase 2 clinical trial with this drug against human pancre-
atic cancer is being conducted by the same institute and a
very good response rate has been observed. Compound 4
has been approved by FDA to market as an orphan drug.
Inorganic nitrate salts have been used in the presence of
strong mineral acids to nitrate organic compounds. Nu-
merous examples may be found primarily in the patent lit-
erature. Olah reviewed the field of organic nitration reac-
tions using a variety of inorganic nitrate salts as nitrating
agents.21 Except for strong inorganic acids, some organic
acids and the corresponding anhydrides such as acetic ac-
id, acetic anhydride, trifluoroacetic acid, and trifluoro-
acetic anhydride are also frequently employed as nitration
reaction media.22–25
Employing inorganic nitrate salts as nitrating agents to ni-
trate anticancer agent camptothecin has not been reported
before. In this paper we wish to report our results of the
nitration of camptothecin with various inorganic nitrate
salts in concentrated sulfuric acid.
In order to select a suitable reaction media for the nitration
of camptothecin with various inorganic nitrate salts, the
following solvents, acetic acid, acetic anhydride, trifluoro-
acetic anhydride, and concentrated sulfuric acid, were at-
tempted while using potassium nitrate as nitrating agent.
The results are shown in Table 1. When acetic acid and
acetic anhydride were used as reaction medias no nitration
reaction occurred. The starting camptothecin (CPT, 1)
was recovered in 100% yield in both cases. In trifluoro-
Scheme 1

SYNTHESIS
December 1998
1725
Table 1. Nitration of Camtothecin with KNO3 in Different Solventsa
Solvent
Yield (%)
Ratio
9NC 4
12NC 9
CPT 1
Byproducts
9NC/12NC
Total Nitration (%)
HOAc
Ac2O
TFAA
H2SO4
0
0
4
0
0
8
100
100
23
0
0
65
6
0:0
0:0
1:2
1:2.2
0
0
12
68
21
47
26
a Camptothecin: (0.5 g), KNO3 (0.5 g), solvent (30 mL), r.t., 24 h.
acetic anhydride, the nitration reaction happened, but the value was also observed for each of these eight salts,
HPLC analyses of crude reaction mixture showed that the which increases difficulty for separation and purification.
total nitration [9-nitrocamptothecin (%) + 12-nitrocamp- The remain salts in Table 2, NH4NO3, AgNO3, Ba(NO3)2,
tothecin (%)] was only 12% in which only 4% was 9-ni- Pb(NO3)2, and Al(NO3)3, belong to another category
trocamptothecin (9NC, 4). The majority was unidentified which gave 9-nitrocamptothecin (4) the values of 18 ±
byproducts. The HPLC analyses of unseparated reaction 1%. Thus, TlNO3 and KNO3, as nitrating agents, are bet-
products for the nitration of camptothecin with KNO3 in ter than others for the nitration of camptothecin under
concentrated sulfuric acid showed a total nitration yield of these reaction conditions.
68% with a 9NC yield of 21%. The byproducts in this case
The nitration of camptothecin with a combination of two
were only 6%. Thus concentrated sulfuric acid was thor-
different inorganic nitrate salts was attempted. The results
oughly employed as the media for all nitration reactions to
are shown in Table 3. The comparison of Table 3 with
be discussed.
Table 2 shows that some combination systems are better
As shown in Table 1, nitration of camptothecin (1) with than when the two nitrates are used separately and some
inorganic nitrate salt KNO3 gave 9-nitrocamptothecin combinations are not as good as when they are used indi-
(9NC, 4), 12-nitrocamptothecin (12NC, 5), and many oth- vidually. For example, the combination of LiNO3 and
er byproducts. A good nitration reaction should be one Co(NO3)2 gave a much lower 9NC value (11%), a lower
that gives a higher percentage total nitration yield with a total nitration value (41%), an unfavorable ratio of 9NC to
better ratio of percentage 9-nitrocamptothecin (4) to per- 12NC (11%:30%, i.e., 1:2.7), and a much higher value of
centage 12-nitrocamptothecin (5), and a lower percentage byproducts (57%). The following combination systems,
of byproducts.
KNO3/TlNO3, KNO3/Zn(NO3)2, and KNO3/Sr(NO3)2,
showed improved results: higher %9NC values, more fa-
vorable ratios of %9NC to %12NC. The %total nitration
values for these three systems are matchable with those in
Table 2 with the corresponding individual salt as nitrating
agent. The result obtained from KNO3/TlNO3 system is
especially encouraging: a better ratio of %9NC to %12NC
(1:1.4) and a higher 9NC value (29%) when compared
with the values with KNO3 or TlNO3 as a nitrating agent
separately. The KNO3/Hg(NO3)2 system showed the best
ratio of %9NC to %12NC (1:1.2), but the total nitration
value was low (46%) and the value of byproducts was
high (42%). Employment of a combination of three or
more inorganic salts as the nitrating reagents was also at-
tempted. The results are summarized in Table 4. All these
combinations gave 9NC a 21–26% value. However, as
shown in Table 3 the KNO3/TlNO3 combination is still
the best one under our reaction conditions.
The results of the nitration reaction of camptothecin with
19 commonly used inorganic nitrate salts in concentrated
sulfuric acid are summarized in Table 2. All these nitrat-
ing reagents gave positive reactions, but the results were
different. It is apparent that BiONO3, Cr(NO3), and
La(NO3) are not good nitrating agents under these reac-
tion conditions. The 9NC values for these three salts were
not good and were only 12 ± 1%. However, TlNO3
showed good results: a better ratio of 9NC to 12NC
(24%:49%, i.e., ~1:2.0), a higher total nitration value
(73%), and a lower byproducts value (12%). KNO3 also
showed good results: the lowest byproducts value (6%),
and the highest total nitration value (75%). But the ratio of
9NC to 12NC (23%:52%, i.e., 1:2.3) was not as good as
with TlNO3 as nitrating agent. Other salts such as LiNO3,
Cu(NO3)2, Hg(NO3)2, Ca(NO3)2, Zn(NO3)2, Mg(NO3)2,
Co(NO3)2, Sr(NO3)2, and Fe(NO3)3 gave about the same
results. All these 8 salts gave 9NC a value 20–23% and a Thus, the KNO3/TlNO3 system was chosen to be studied
good %total nitration value, but a higher %byproducts further. Table 5 summarizes the results of the nitration of

SYNTHESIS
Papers
Table 2. Nitration of Camptothecin with Various Nitrates in Sulfuric Acida
1726
Nitrate Salt
Yield (%)
9NC
Ratio
12NC
CPT
Byproducts
9NC/12NC
Total Nitration (%)
NH4NO3
KNO3
LiNO3
AgNO3
TlNO3
BiONO3
Cu(NO3)2 2.5 H2O
Hg(NO3)2 H2O
Ca(NO3)2 4 H2O
Ba(NO3)2
Zn(NO3)2 6 H2O
Mg(NO3)2 6 H2O
Co(NO3)2 6 H2O
Sr(NO3)2
Pb(NO3)2
Al(NO3)3 9 H2O
Fe(NO3)3 9 H2O
Cr(NO3)3 9 H2O
La(NO3)3 6 H2O
19
23
22
19
24
11
22
22
20
17
21
23
21
22
18
17
23
13
11
41
52
38
43
49
17
53
43
49
54
44
45
41
36
50
46
51
28
34
25
19
0
18
15
48
1
1
1
3
19
9
0
1
13
1
0
15
6
1:2.2
1:2.3
1:1.7
1:2.3
1:2.0
1:1.5
1:2.4
1:2.0
1:2.5
1:3.2
1:2.1
1:2.0
1:2.0
1:1.6
1:2.8
1:2.7
1:2.2
1:2.2
1:3.1
60
75
60
62
73
28
75
65
69
71
65
68
62
58
68
63
74
41
45
40
20
12
24
24
35
30
20
16
23
38
41
19
36
26
13
54
•
•
•
•
•
•
•
•
•
46
2
•
a For each reaction: Camptothecin (0.5 g, 0.0014 mol), H2SO4 (20 mL), nitrate (0.0070 mol), r.t., 72 h.
Table 3. Nitration of Camptothecin with a Combination of Two Different Nitrates in Sulfuric Acida
b
A(NO3)m/B(NO3)n
Yield (%)
9NC
Ratio
12NC
CPT
Byproducts
9NC/12NC
Total Nitration (%)
K/Cuc
K/Tl
K/Hg
K/Ca
K/Ba
K/Zn
K/Sr
K/Pb
K/Al
K/Fe
Li/Hg
Li/Cu
Li/Co
Ag/Cr
Cu/Fe
Hg/Fe
NH4/Cu
24
29
21
18
20
26
29
24
25
18
17
22
11
18
17
23
17
50
41
25
42
39
50
45
49
50
41
38
47
30
37
33
45
49
6
8
12
3
0
10
3
12
2
0
17
10
2
20
22
42
37
41
14
23
15
23
41
26
21
57
44
49
30
32
1:2.1
1:1.4
1:1.2
1:2.3
1:2.0
1:1.9
1:1.6
1:2.0
1:2.0
1:2.3
1:2.2
1:2.1
1:2.7
1:2.1
1:1.9
1:2.0
1:2.9
74
70
46
60
59
56
74
73
75
59
55
69
41
55
50
68
66
1
1
2
2
a For each reaction: Camptothecin (4.0 g, 0.0115 mol), H2SO4 (100 mL), r.t., 72 h
b Molar ratio of A(NO3)m to B(NO3)n: 2.0:1.0
c
•
K/Cu represents the corresponding KNO3/Cu(NO3)2 2.5 H2O, same applied to the other combinations.

SYNTHESIS
December 1998
1727
Table 4. Nitration of Camptothecin with a Combination of Three or More Nitrates in Sulfuric Acida
Reagents
Yield (%)
9NC
Ratio
12NC
CPT
Byproducts
9NC/12NC
Total Nitration (%)
K/Tl/Cuc
K/Tl/Zn
K/Tl/Pb
K/Cu/Fe
K/Cu/Hg/Zn
K/Li/Cu/Hg/Fe
K/Li/Ag/Cu/Hg/Fe
K/Li/Zn/Cu/Hg/Fe
K/Zn/Cu/Hg/Fe
25
25
25
22
24
21
24
23
26
44
46
46
48
57
44
50
44
50
10
0
5
6
2
12
4
7
21
29
24
24
17
23
22
30
20
1.0:1.8
1.0:1.8
1.0:1.8
1.0:2.2
1.0:2.4
1.0:2.1
1.0:2.1
1.0:1.9
1.0:1.9
69
71
71
70
81
65
74
67
76
4
a For each reaction: Camptothecin (4.0 g, 0.0115 mol), H2SO4 (100 mL), r.t., 72 h.
b Molar ratio for all nitrates in a combination of the reagents is 1 to 1 to 1.
c
•
K/Tl/Cu represents the corresponding KNO3/TlNO3/Cu(NO3)2 2.5 H2O, same applied to other reagents.
Table 5. Nitration of Camptothecin with Different Ratios of a Combination of KNO3 and TlNO3 in Sulfuric Acida
Ratio
KNO3/TlNO3
Yield (%)
9NC
Ratio
12NC
CPT
Byproducts
9NC/12NC
Total Nitration (%)
3.3:1.0
2.0:1.0
1.4:1.0
1.3:1.0
1.2:1.0
1.0:1.0
1.0:1.3
1.0:1.5
1.0:2.1
26
29
27
28
27
26
27
26
25
47
41
46
49
50
49
49
47
38
5
8
10
6
22
22
17
17
15
14
13
15
28
1.0:1.8
1.0:1.4
1.0:1.7
1.0:1.8
1.0:1.9
1.0:1.9
1.0:1.8
1.0:1.8
1.0:1.5
73
70
73
77
77
75
76
73
63
8
11
11
12
9
a For each reaction: Camptothecin (6.0 g, 0.0172 mol), H2SO4 (100 mL), r.t. 72. h.
camptothecin with a combination of KNO3 and TlNO3 creased from 23.5 ± 0.5% to 27 ± 2% and the ratio of
with different ratios in concentrated sulfuric acid. Once %9NC to %12NC increased from 1.0:2.15 ± 0.15 to
again, Table 5 shows that a higher %9NC value and a 1.0:1.65 ± 0.25. The molar ratios of KNO3 to TlNO3
improved ratio of %9NC to %12NC are obtained when a seem to have no influence on %total nitration, but it does
combination of KNO3 and TlNO3 are used as the nitrat- have an effect on %byproducts. A higher %byproducts
ing reagents. For example, when only KNO3 was used as value makes the process of separation and purification
a nitrating agent the HPLC analysis of the reaction mix- more difficult. A combination with a ratio range of
ture showed that 9NC was 23% of the total area and 1.0:1.0 to 1.0:1.5 of KNO3 to TlNO3 was chosen for the
12NC was 52% of the same total area, the %9NC/ nitration reactions. The lowest %byproducts values were
%12NC ratio in this case was 1.0:2.3; when only TlNO3 observed under these conditions. Generally, a combina-
was used as a nitrating agent the same analysis showed a tion with a ratio of 1.0 to 1.3 of KNO3 to TlNO3 is em-
value of 24% for 9NC and 49% for 12NC. The ratio ployed as the nitrating reagent in our laboratory for the
%9NC/%12NC in this case was 1.0:2.0. When these two preparation of 9-nitrocamptothecin (4). A consistent
nitrating reagents were used combined the 9NC value in- yield of 20 ± 1% of pure 4 is obtained.

SYNTHESIS
Papers
1728
a
Table 6. Effects of the Volumes of Concentrated Sulfuric Acid on the Nitration of Camtothecin with a Combination of KNO3 and TlNO3
H2SO4 (mL)
Yield (%)
9NC
Ratio
12NC
CPT
Byproducts
9NC/12NC
Total Nitration (%)
100
200
300
400
29
25
28
29
41
49
53
45
8
15
9
22
11
10
16
1.0:1.4
1.0:2.0
1.0:1.9
1.0:1.6
70
74
81
74
10
a Camptothecin (4.0 g), KNO3 (2.0 g), TlNO3 (4.0 g), r.t., 72 h.
stirring. The suspension was extracted with CH2Cl2 (3 × 200 mL).
The combined extracts were dried (anhyd Na2SO4, 20 g, 6 h). After
removal of CH2Cl2 by a rotary evaporator, the residue was refluxed in
petroleum ether for 4 h. After filtration and drying in air for 4 h the
product was obtained as gray white powders. HPLC analysis showed
no nitration indicative of camptothecin with KNO3 in acetic acid. The
starting camptothecin was recovered 100%.
Table 6 shows the effects of the volumes of concentrated
sulfuric acid on the nitration of camptothecin with a com-
bination of KNO3 and TlNO3. The better results are ob-
tained when 300 mL of concentrated sulfuric acid is used
as the reaction media while the amount of starting camp-
tothecin is 4 grams.
Thus, the nitration procedure described in this report for
the preparation of anticancer agent 9-nitrocamptothecin
(4) from camptothecin (1) with a combination of two in-
organic salts such as KNO3 and TlNO3 as nitrating re-
agents is superior to the conventional literature method (a
mixed HNO3/H2SO4 procedure4). The yield of pure 9-ni-
trocamptothecin (4) is improved from 5 ± 2% to 20 ± 1%.
The concentrated sulfuric acid is recommended for the ni-
tration reaction of camptothecin with all those inorganic
salts discussed above.
Reaction of Camptothecin (1) with KNO3 in Acetic Anhydride:
Camptothecin (1) was nitrated and worked-up in the same way as the
reaction in HOAc. The HPLC analysis of the reaction product showed
100% recovery of the starting camptothecin.
Reaction of Camptothecin (1) with KNO3 in Trifluoroacetic An-
hydride:
Camptothecin was nitrated and worked up in the same manner as in
HOAc. The HPLC analysis data for the reaction mixture is shown in
Table 1.
Nitration of Camptothecin (1) with KNO3 in Concentrated Sulfu-
ric Acid:
Dry N2 was routinely used as the reaction atmosphere in all reactions.
All glassware was baked at 70 + 10°C for a minimum of 2 h before
used. Mps were obtained with a MEL–TEMP melting point apparatus
and are uncorrected. The 1H NMR spectra of approximately 10% (w/
v) solution in CDCl3 were obtained at 270.05 MHz with a JEOL GX-
270 WB NMR spectrometer. Chemical shifts are reported in parts per
million (δ scale), employing TMS as an internal standard. In reporting
the NMR data, we have used the following abbreviations: coupling
constants in Hz (J), singlet (s), doublet (d), triplet (t), broad singlet
(bs), multiplet (m), and etc. MS were recorded using a VG ZAB–SEQ
mass spectrometer (VG Analytical Co., England) with a resolution of
10000. Routinely used solvents such as CHCl3 and CH2Cl2 were dried
and freshly distilled. Silica gel (230–400 mesh, Aldrich) for column
chromatography was used for all product separations. Eastman chro-
magram (silica gel with fluorescent indicator on polyethylene) sheets
were employed in TLC operations. The numbering system used in
reporting NMR data is shown in structure 1 of Table 1. Camptothecin
was from The People’s Republic of China and used as purchased. All
other inorganic nitrate salts were from Aldrich Chemical Co. (Mil-
waukee, WI) and used as purchased.
Camptothecin (1) (0.50 g, 0.0014 mol) and KNO3 (0.50 g, 0.0050 mol)
were added to concd H2SO4 (30 mL) in a 100-mL round-bottomed
flask equipped with a magnetic stirrer all at once. The mixture was
stirred at r.t. for 1 d and then poured onto ice-water (500 mL) slowly
while stirring. The yellow suspension was extracted with CH2Cl2 (3
× 200). The combined extracts were dried (anhyd Na2SO4, 24 h). The
CH2Cl2 was removed by a rotary evaporator and the residue was re-
fluxed in petroleum ether for 4 h. After cooling to r.t. the mixture was
filtrated and the yellow powders was dried in air for 1 d. The HPLC
analysis data for the reaction mixture (yellow powder) is shown in
Table 1.
Nitration of Camptothecin (1) with Various Nitrates in Sulfuric
Acid; Typical Procedure:
Camptothecin (1) (0.50 g, 0.0014 mol) and NH4NO3 (0.56 g, 0.0070
mol) were added to concd H2SO4 (20 mL) in a 100-mL round-bot-
tomed flask equipped with a magnetic stirrer. The mixture was stirred
at r.t. for 72 h after which it was poured onto ice-water (500 mL)
while stirring, extracted with CH2Cl2 (3 × 200 mL). The combined
extracts were washed with water (2 × 100 mL). The two washes were
combined and extracted with CH2Cl2 (2 × 100). All extracts
(~600 mL + ~200 mL) were combined, dried (Na2SO4, 20 g, 4 h) and
then evaporated to give crude reaction products as yellow powders
which comprised of 9NC (19%), 12NC (41%), unreacted camptothe-
cin (1) (25%), and many other byproducts (15%). The results of
HPLC analyses for all these reactions are shown in Table 2.
Reaction of Camptothecin (1) with KNO3 in Acetic Acid;Typical
Procedure:
To HOAc (30 mL) in a 100-mL round-bottomed flask equipped with
a magnetic stirrer were added camptothecin (1) (0.50 g, 0.0014 mol)
and KNO3 (0.50 g, 0.0050 mol). The mixture was stirred at r.t. for
24 h and poured onto ice-water (500 mL) portion-by-portion while

SYNTHESIS
December 1998
1729
Nitration of Camptothecin (1) with a Combination of Two Differ-
ent Nitrates in Sulfuric Acid; Typical Procedure:
1H NMR: δ = 1.05 (3H, t, J = 7.40 Hz, C19-methyl protons), 1.92 (2H,
m, C18-methylene protons), 3.82 (1H, s, C20-OH), 5.40 (2H, s, C5-
methylene protons), 5.55 (2H, dd, J = 14.21, 14.21 Hz, C17-methyl-
ene protons), 7.70 (1H, s, C14-H), 7.92 (1H, t, J = 8.40 Hz, C11-H),
8.48 (1H, d, J = 8.35 Hz, C10-H), 8.55 (1H, d, J = 8.35 Hz, C12-H),
9.36 (1H, s, C7-H).
Camptothecin (1) (4.0 g, 0.0115 mol) was added to concd H2SO4
(100 mL) in a 250-mL three-necked flask. The suspension was stirred
with a mechanical stirrer until the most of camptothecin went to solu-
tion (~15 to 30 min). To this solution KNO3 (2.32 g, 0.0230 mol) and
Cu(NO3)2•2.5 H2O (2.67 g, 0.0115 mol) were added all at once. The
mixture was stirred at r.t. for 72 h and poured onto ice-water
(1500 mL) while stirring. The yellow suspension in water was ex-
tracted with CH2Cl2 (4 × 500 mL). The combined extracts were dried
(anhyd Na2SO4, 8 h). The Na2SO4 was removed by filtration. After
removal of CH2Cl2 by a rotary evaporator, the crude reaction products
were obtained as yellow powders, containing 9NC (24%), 12NC
(50%), unreacted camptothecin (6%), and other byproducts (20%).
The results of HPLC analyses for all these nitration reactions are
shown in Table 3.
MS: m/z (%) = 393 (M+, 100), 364 (M – C2H5, 35), 349 (48), 334 (25),
320 (25), 293 (35), 274 (8), 262 (8), 246 (15), 234 (6), 218 (20), 205
(8), 190 (9), 177 (5), 164 (3), 151 (3), 137 (5), 123 (4), 109 (5), 95 (5),
75 (3), 60 (23).
HRMS: calcd for C20H15N3O6 393.0960. Found 393.0961.
HPLC Procedure for Purity Analysis of 9-Nitrocamptothecin (4):
Instrumentation: The HPLC system consisted of a Beckman 421 con-
troller with two 110A pumps and a 2 mL injection loop. The UV de-
tector was a SPD-110AV model (Shimadzu, Kyoto, Japan). The
HPLC detector was set to monitor the UV absorbance at 220 nm. The
integrating software used for the analyses was EZChrome (Shimadzu,
Japan) and FLO-ONE\beta (Radiomatic Instruments, Meridian, CT).
A C-8 Microsorb was from Rainin Instruments (Woburn, MA).
HPLC analysis: Reverse phase HPLC analysis of the samples was
carried out by using a MeCN/HOAc/H2O mobile phase system. Anal-
yses were carried out at r.t. with a flow rate 1 mL/min. The solution
with a concentration of approximately 0.1 mg/mL of 9NC in MeCN
was prepared by dissolving it in the solvent. A 300 µL portion of this
solution was taken and added to 700 µL solution of 0.1% HOAc/H2O.
After shaking for ~10 s, 100 µL of this solution was injected through
a 2 mL loop onto column and chromatographed with 70% H2O with
0.1% HOAc and 30% MeCN as the mobile phase for the period of
first 5 min, and then the gradient of the mobile phase was program-
matically increased to 100% MeCN over a period of 4 min. A com-
plete HPLC spectrum was obtained in 15 min. The purity of 9NC was
determined by measuring the UV peak areas at 254 nm and calculat-
ing the percentage associated with the 9NC peak. The retention time
of 9-nitrocamptothecin under these conditions is approximately
6.5 min.
Nitration of Camptothecin (1) with a Combination of Three or
More Inorganic Nitrates in Sulfuric Acid; Typical Procedure:
Camptothecin (1) (4.0 g, 0.0115 mol) was suspended in concd H2SO4
(100 mL) in a 250-mL round-bottomed flask equipped with a me-
chanical stirrer. After stirring for ~30 min (until camptothecin was
almost dissolved), a combination of KNO3 (1.16 g, 0.0115 mol),
TlNO3 (3.10 g, 0.0116 mol), and Cu(NO3)2•2.5 H2O (2.67 g.
0.0115 mol) was added all at once. The mixture was stirred at r.t. for
72 h and then poured onto ice-water (1500 mL) while stirring. The
yellow suspension was extracted with CH2Cl2 (4 × 500 mL). The
combined extracts were dried (anhyd Na2SO4), filtered, and evaporat-
ed. The crude reaction products were obtained as yellow powders,
containing 9NC (25%), 12NC (44%), unreacted camptothecin (10%),
and other byproducts (21%). The results of HPLC analyses for all
these nitration reactions are shown in Table 4.
Nitration of Camptothecin (1) with Different Ratios of a Combi-
nation of KNO3 and TlNO3 in Sulfuric Acid; Typical Procedure:
Camptothecin (1) (6.0 g, 0.0172 mol) was added to concd H2SO4
(100 mL) in a 250-mL three-necked flask equipped with a mechanical
stirrer. After stirring at r.t. for ~30 min, a combination of KNO3
(1.74 g, 0.0172 mol) and TlNO3 (4.58 g, 0.0172 mol) was added all at
once. The mixture was stirred at r.t. for 72 h and then poured onto ice-
water (1500 mL) while stirring. The yellow suspension was extracted
with CH2Cl2 (4 × 500 mL). The combined extracts were dried (anhyd
Na2SO4, 8 h). After removal of CH2Cl2 by a rotary evaporator, the
crude reaction products were obtained as yellow powders, containing
9NC (26%), 12NC (49%), unreacted camptothecin (11%), and other
byproducts (14%). The results of HPLC analyses for all these reaction
are shown in Table 5.
HPLC Procedure for the Analyses of Crude Reaction Products:
The HPLC procedure for the analyses of crude nitration reaction
products is the same as the one for the purity analysis of 9-nitrocamp-
tothecin.
Financial support from the Stehlin Foundation for Cancer Research
and The Friends of The Stehlin Foundation is gratefully acknowl-
edged.
(1) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail,
A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888.
(2) Kingsburg, W. D.; Boehm, L. C.; Jakas, D. R.; Holden, K. G.;
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Nitration of Camptothecin (1) with a Combination of KNO3 and
TlNO3 in Various Volumes of Sulfuric Acid:
The general procedure was the same as above. The results was shown
in Table 6.
9-Nitrocamptothecin (4):
(3) Sawada, S.; Matsuoka, S.; Nokata, K.; Nagata, H.; Furuta, T.;
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Camptothecin (1) (4.0 g, 0.0115 mol) was added to concd H2SO4
(300 mL) in a 1000-mL three-necked flask. After stirring for ~15 min,
KNO3 (2.0 g, 0.0198 mol) and TlNO3 (5.0 g, 0.0188 mol) were added
all at once. The mixture was stirred at r.t. for 72 h and then poured
onto ice-water (3500 mL) while stirring. The yellow suspension was
extracted with CH2Cl2 (1 × 1500 mL and 2 × 900 mL). The combined
extracts were dried (anhyd Na2SO4, 8 h). After filtration, the solvents
were removed by a rotary evaporator. The residue was chromato-
graphically separated. The crude 9-nitrocamptothecin was allowed to
reflux in abs EtOH for 2–4 h. Pure product 4 was obtained by repre-
cipitation from EtOH to give bright yellow crystals; yield: 20%; mp
268°C.

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