Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Article abstract of DOI:10.1016/j.carres.2004.10.014

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, ¹²⁵I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with ¹²⁵I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).

Full text of DOI:10.1016/j.carres.2004.10.014

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 15–24
Synthesis and radioiodination of some daunorubicin and
doxorubicin derivatives
Senait Ghirmai,a Eskender Mume,a Vladimir Tolmacheva,b and Stefan Sjo¨berga,*
aUppsala University, Department of Chemistry, Organic Chemistry, PO Box 599, BMC, SE751 24 Uppsala, Sweden
bUppsala University, Biomedical Radiation Sciences, Rudbeck Laboratory, S-751 85 Uppsala, Sweden
Received 26 April 2004; received in revised form 25 October 2004; accepted 27 October 2004
Available online 11 November 2004
Abstract—Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by
their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting
the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron
emitter, 125I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of dau-
norubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic este-
rification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic
acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corre-
sponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin
hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with 125I was
performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85–95%) of the radioio-
dinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the acti-
vated phenyl rings providing moderate radiochemical yields (55–75%).
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Daunorubicin; Doxorubicin; Radioiodination; 125I; Chloramine-T
1. Introduction
O
O
OH
11
1
12
10
C
13
Daunorubicin (1a) and doxorubicin (1b) are the most
extensively used anthracycline antitumor antibiotics.
Daunorubicin was the first antibiotic of this class to
show activity against acute leukemia in humans. Later
studies have shown that it also has activity against solid
tumors.1,2 The substitution of one hydrogen atom in the
daunorubicin acetyl side chain with an hydroxyl group
gives doxorubicin. Doxorubicin exhibits antitumor
activity against solid tumors, such as breast and lung
cancers, and is generally known to be more potent than
daunorubicin. Both compounds are known for their
effective intercalation to DNA.
R1
2
3
9
OH
8
4
5
6
OH
7
Daunorubicin (1a): R1 = CH3
Doxorubicin (1b): R1 = CH2OH
O
O
O
'
1
CH3
'
5
O
H3C
4
'
2
'
3
'
NH2
HO
The generation of free radicals following microsomal
or chemical activation of the quinone moiety of the anth-
racyclines is believed to contribute to either their cytotoxi-
city or cardiotoxicity.3 Many derivatives of these
compounds have therefore been synthesized to improve
their cytotoxicity and decrease their cardiotoxicity.4–10
The mechanism of the antibiotic action of these molecules
is related to their ability to intercalate between adjacent
*
Corresponding author. Tel.: +46 18 4713796; fax: +46 18 4713818;
e-mail: ssj@kemi.uu.se
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.10.014

16
S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
O
O
O
OH
OH
O
O
OH
O
11: R1 = COCH3 ; X = I
12: R1 = COCH3 ; X = H
13: R1 = COCH3 ; X = 125
14: R1 = CH(OH)CH3 ; X= I
15: R1 = CH(OH)CH3 ; X= H
16: R1 = CH(OH)CH3 ; X= 125
R1
C
CH2
O C
X
OH
OH
I
O
O
O
O
OH
CH3
H3C
CH3
H3C
2: X = I
3: X = SnMe3
O
O
I
4: X = 125
I
X
NH2
O
HO
NH
HO
CH2
OH
O
C
OH
OH
CH3
OH
Ar
Ar
O
O
O
X
X = I
X = SnMe3
X = 125
5:
6:
7:
X = SnMe3
8:
CH3
H3C
X
O
X = I
X = 125
9:
N
I
10:
I
NH
HO
C
Ar
O
base pairs causing topoisomerase II inhibition, which
leads to the production of hydroxyl free radicals that
are toxic to both normal and tumor cells.11 The problems
that limit the clinical efficacy of these agents are their car-
diotoxicity and multidrug resistance.12 One approach
that possibly improves their therapeutic ability is target-
ing these molecules specifically to the cancer cells.13
In this report, we present the synthesis of some dauno-
rubicin and doxorubicin derivatives as potential candi-
dates for targeted nuclide therapy. The compounds are
esters 2–4, amides 5–10, and amines 11–16.
The precursor compounds are labeled with 125I and
will be loaded into liposomes. We plan to investigate
their efficacy using a two-step targeting technique.14 In
the first step, stabilized liposomes conjugated with a tar-
geting agent will be actively loaded with the labeled
compounds and injected into blood circulation. After
binding to cancer cells, the loaded liposomes will be
internalized, and labeled daunorubicin and doxorubicin
derivatives will be released into the cytoplasm. In
the second step, the compounds, with the help of the
DNA intercalating part, are supposed to bind to the
DNA of the tumor cell. The 125I nuclide emits Auger
electrons that travel short distances, but are highly cyto-
toxic when emitted in close vicinity to DNA. In prelimi-
nary experiments, two of these compounds, 13 and 16,
were found to be taken up by a permeabilized cell line
and transit into the cell nucleus. The biological exper-
iments will be presented elsewhere.
of the hydrochloride of daunorubicin using bromine in
the presence of trimethylorthoformate, as described in
the literature,15 gave 14-bromo-13-dimethyl acetal of
daunorubicin, which upon being stirred in acetone pro-
vided 14-bromodaunorubicin (17), in high yield. The
Stille coupling reaction of 3-iodobenzoic acid and hexa-
methylditin using a palladium(II) catalyst provided an
88% conversion to the corresponding stannyl compound
18, as described in the literature.16
The reaction of 3-iodobenzoic acid with the bromo
compound 17 and K2CO3 at room temperature pro-
duced a good (73%) yield of the ester, 2. Similarly,
synthesis of compound 3 was carried out with the
stannylated compound 18, providing a high yield of
the target. Both esters were purified by flash chromato-
graphy. The purity and identity of the compounds were
confirmed by LC/MS and NMR.
2.1.2. Synthesis of amides 5–10. The amides 5–7 were
synthesized as outlined in Scheme 2. 4-Iodobenzoic acid
and 4-(trimethylstannyl)benzoic acid were reacted with
di-(N-succinimidyl)carbonate in pyridine to yield the
corresponding N-succinimidyl-derivatives (19 and 20,
respectively), as described previously.16 These activated
compounds, 19 and 20, were reacted with daunorubicin
hydrochloride in the presence of a 5-fold molar excess of
triethylamine in DMF to give 74% and 86% yields of
30-N-4-iodo-benzoyldaunorubicin, 5, and 30-N-4-tri-
methylstannylbenzoyldaunorubicin, 6, respectively.
The syntheses of the amides 8–10 are outlined in
Scheme 3. The previously known compound N-succinim-
idyl-5-bromo-nicotinic acid (21),17 was synthesized by the
same procedure as was used for 19, except that 5-bromo-
nicotinic acid was used as the starting material. The acti-
vated stannylated ester 2217 was obtained in a low yield
from 21 using the same method as described for the prep-
aration of 20. N-Succinimidyl 5-(trimethylstannyl)nico-
2. Results and discussion
2.1. Synthesis
2.1.1. Synthesis of esters 2–4. Synthesis of compounds
2–4 was performed as shown in Scheme 1. Bromination

S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
17
O
O
C
O
C
O
O
OH
O
OH
O
O
OH
OH
O
C
CH2Br
CH2
O C
CH3
OH
I
OH
OH
i
ii
O
O
O
O
OH
O
O
OH
O
CH3
CH3
CH3
H3C
O
O
O
H3C
H3C
NH2
NH2
HO
NH2
HO
HO
iii
1a
2
17
O
C
O
O
O
OH
OH
O
OH
OH
O
O
C
CH2
O
C
CH2 O C
125I
SnMe3
OH
iv
OH
O
O
O
O
O
CH3
H3C
CH3
H3C
O
O
CO2H
18
Me3Sn
NH2
NH2
HO
HO
4
3
Scheme 1. (i) (a) CH(OMe)3, MeOH/dioxane (1:2), rt; (b) Br2/CHCl3, 30ꢁC; (c) acetone, rt; (ii) 3-iodobenzoic acid, K2CO3, rt, and (iii) compound 18,
K2CO3, rt; (iv) Na125I, 3% AcOH/MeOH, CAT, Na2S2O5, NaI.
O
C
O
C
O
C
O
O
OH
OH
O
OH
OH
O
O
OH
CH3
CH3
CH3
OH
OH
OH
i
ii
O
O
O
O
O
O
O
OH
CH3
CH3
CH3
O
O
O
H3C
H3C
H3C
O
O
NH
C
NH2
HO
NH
C
HO
HO
5: X = I
6: X = SnMe3
1a
7
125I
O
N
X
O
C
19: X = I
20: X = SnMe3
O
X
O
Scheme 2. (i) For 5: 19, triethylamine, rt, 1.5h; for 6: 20, triethylamine, rt, 1.5h, and (ii) Na125I, 1% AcOH/MeOH, CAT, Na2S2O5.
tinic acid (22) was reacted with daunorubicin hydrochlo-
ride using the same procedure as was used for the prepa-
ration of 6. The crude product was purified by column
chromatography to give a 72% yield of compound 8.
The stannylated precursor was iodinated using NaI in
1% acetic acid/methanol, and chloramine-T (CAT) was
used as an oxidant to produce a 77% yield of 9.
compounds 12 and 15 (as a mixture of two diastereo-
mers). Compound 15, which was formed from reduction
of the carbonyl functional group at position 13 due to
the excess sodium cyanoborohydride, was the major
product. 4-Hydroxybenzaldehyde was iodinated with
I2/Ag2SO4. The reaction afforded a moderate yield of
the monoiodo derivative, 23.18 Compounds 11 and 14
(as a mixture of two diastereomers), were prepared from
4-hydroxy-3-iodobenzaldehyde 23 and daunorubicin
hydrochloride using a procedure similar to that used
for the synthesis of 12 and 15. Similarly, compound 14
was the major product.
2.1.3. Synthesis of amines 11–16. The amino-benzyl
derivatives were synthesized as outlined in Scheme 4.
Compound 12 was prepared by the reductive amination
of 4-hydroxybenzaldehyde with daunorubicin hydro-
chloride using sodium cyanoborohydride. The reaction
was very slow and thus required more than 1equiv
of the reagents. After 3days of reaction at room
temperature, the starting daunorubicin hydrochloride
was totally consumed, according to thin-layer chroma-
tography (TLC). The mixture was purified by chroma-
tography and analyzed by NMR and LC/MS. Two
major fractions were isolated; these were found to be
3. Radiolabeling
3.1. Radioiodination
Compounds 3, 6, 8, 12, and 15 were used as precursors
for the radioiodination. All the compounds were labeled

18
S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
O
C
O
OH
OH
O
C
O
C
O
O
OH
O
O
OH
OH
CH3
CH3
CH3
OH
OH
OH
i
ii
O
O
O
O
O
CH3
O
OH
O
CH3
H3C
CH3
O
H3C
O
O
H3C
O
O
NH
HO
9: X = I
10: X = 125
C
NH2
NH
HO
HO
C
X
SnMe3
1a
8
N
I
N
O
O
C
21: X = Br
X
O
N
iii
22: X = SnMe3
N
O
Scheme 3. (i) Compound 22, triethylamine, rt, 1.5h; (ii) for 9: NaI, chloramine-T, Na2S2O5, rt, 13min; for 10: Na125I, 1% AcOH/MeOH, CAT,
Na2S2O5, NaI; (iii) Pd(PPh3)2Cl2, Sn2Me6, 1,4-dioxane.
O
C
O
OH
OH
O
OH
R
CH3
OH
OH
i
O
O
O
O
O
O
OH
CH3
CH3
O
H3C
O
H3C
I
NH
HO
14: R = CH(OH)CH3
NH2
CH2
OH
11: R = COCH3
HO
1a
O
HO
HO
C
C
ii
O
OH
OH
O
OH
H
R
R
iv
OH
OH
I
iii
O
O
O
O
O
O
O
OH
CH3
CH3
H
O
O
H3C
H3C
23
125I
H
NH
NH
HO
HO
CH2
OH
CH2
OH
12: R = COCH3
15: R = CH(OH)CH3
13: R = COCH3
16: R = CH(OH)CH3
Scheme 4. (i) Aldehyde 23, MeCN–H2O (2:1), 1M solution NaBH3CN/THF, rt, and (ii) 4-hydroxybenzaldehyde, MeCN–H2O (2:1), 1M solution
NaBH3CN/THF, rt; (iii) Na125I, MeOH, CAT, Na2S2O5, NaI; (iv) I2, Ag2SO4, CH2Cl2, rt.
with 125I, and chloramine-T was used as an oxidant. The
labeling was performed with no carrier added. The influ-
Table 1. Radioiodination yield of daunorubicin/doxorubicin
derivatives
Entry
Radioiodinated
compounds
Time (min)
Yield (%) (n = 2)
ence of some parameters, such as amount of oxidant,
amount of substrate, and reaction time on labeling
efficiency was investigated. The acidity of the reaction
media was also found to have a great effect in the cases
of some of the derivatives. The radioiodination yields
are shown in Table 1.
1
2
3
4
5
4
7
5
5
5
5
5
85 0.2
95 0.2
91 0.8
53 1.2
72 1.6
10
13
16
Figure 1a displays a typical radio-TLC chromatogram
of [125I] iodide radioiodination of the model compound,
6, using the final optimized reaction conditions. The
optimized conditions were found to be 80lg of com-
pound 6 in 40lL of 1% acetic acid in methanol solution,
5lL [125I]iodide solution (3.7GBq/mL), and 10lL
chloramine-T (CAT) solution (8mg/mL in methanol).
All radio-TLC analyses were performed with NaI as a
carrier, which was added after labeling to stabilize the
radioiodide by protecting it from oxidation during elu-
tion of the TLC plates. From the TLC analysis, peak

S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
19
crease in yield. Increasing the amount of CAT beyond
80lg had no effect on the labeling yield (Fig. 2b).
The dependence of radiochemical yield on the amount
of substrate is depicted in Figure 2c. To investigate the
influence of substrate (compound 15) concentration on
radiolabeling yield, the reaction was carried out at differ-
ent concentrations in the 1–4mg/mL range. The radio-
labeling yield increased with increasing amounts of
substrate. The best yield (72% for compound 16) was
obtained with a reaction mixture containing at least
80lg of the substrate.
60
50
40
30
20
10
0
0
2
4
6
Reaction time (min)
50
45
40
35
30
25
20
15
10
5
Figure 1. (a) Typical TLC chromatogram of compound 7 using the
general labeling procedure. Peak 1 corresponds to 125I stabilized with
carrier NaI (Rf = 0.3) against oxidation, and peak 2 has the same
retention factor (Rf = 0.8) as the iodinated nonradioactive reference 5,
thus corresponding to radiolabeled compound 7; (b) typical TLC
radiochromatogram of a blank experiment.
0
1 (Rf = 0.3) was found to be Na125I. Peak 2 (Rf = 0.8)
has the same retention factor as the nonradioactive iodi-
nated reference compound 5 and is thus associated with
the labeled compound 7. A blank experiment (Fig. 1b)
was performed using exactly the same conditions, except
that a neat methanol solution was used instead of the
substrate daunorubicin/doxorubicin derivatives.
The dependence of radiochemical yield on various
reaction conditions was investigated using compound
15 as shown in Figure 2a–c. As can be seen from Figure
2a, radiochemical yield is strongly dependent on reac-
tion time, the yield reaching a plateau after an incuba-
tion time of 4min. Further increase in reaction time
was found to have no impact on the labeling yield.
The effect of oxidant concentration was examined by
varying the concentration of CAT. The best result was
obtained with 80lg (0.35lmol) CAT, which is a 5.6:1
molar ratio of CAT to the precursor. Reducing the
amount of CAT to less than 40lg resulted in a sharp de-
0
20
40
60
80
100
µ
Amount of CAT ( g)
80
70
60
50
40
30
20
10
0
0
50
100
150
g)
200
Amount of substrate (
µ
Figure 2. (a) Influence of reaction time on radioiodination yield. Other
conditions were: amount of substrate 15, 40lg; amount of CAT, 80lg;
and total volume, 55lL; (b) influence of CAT on radioiodination yield.
Other conditions were: amount of substrate 15, 40lg; reaction time,
5min; and total volume, 55lL; (c) influence of substrate 15 on the
radioiodination yield. Other conditions were: amount of CAT, 80lg;
reaction time, 5min; and total volume, 55lL. Data in all three figures
represent average values from two experiments.

20
S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
Further improvement of the labeling of the stannyl-
used for comparison and they are the same as those of
their radiolabeled analogues. The positions of standard
chromatography spots were determined visually either
using UV or with the naked eye. The distribution of
radioactivity along the TLC strips (100 · 50mm; elution
path, 80mm) was measured on the CycloneTM storage
phosphor system (Packard Instruments Company Inc.,
ated precursors was associated with acidification of the
reaction media using 1% acetic acid in methanol. The
reaction achieved high to excellent labeling yield of
the corresponding [125I]-iodinated products. The other
compounds were also subjected to the same reaction
conditions to determine the effect of acidity on radio-
chemical yield. However the addition of acid was found
to have very little effect on the labeling of the non-
stannylated precursors.
Downers Grove, US) and analyzed using the OPTI-
TM
QUANT
image analysis software.
4.2. Adriamycin 14-O-3-iodobenzoate (2)
4. Experimental
14-Bromodaunorubicin 17 (15mg, 0.023mmol), and 3-
iodobenzoic acid (11.6mg, 0.047mmol) were dissolved
in acetone under an inert atmosphere. Potassium car-
bonate (9.5mg, 0.069mmol) was added and the reaction
mixture was stirred at rt for 12h. The solvent was evapo-
rated and the crude product was purified by column
chromatography (4:1, CH2Cl2–MeOH) to give com-
pound 2 as a red powder (13mg, 73%). The purity of
the compound was checked by TLC and LC/MS. The
compound was converted to its hydrochloride salt by
adding HCl in ether to a THF solution of the compound
and then stirring at ꢀ20ꢁC for 2h.1H NMR (CD3OD): d
8.39 (s, 1H, Ar), 8.02 (d, 1H, J = 8.1Hz, H-3), 7.96 (d,
1H, J = 8.6Hz, Ar), 7.87 (d, 1H, J = 8.1Hz, H-1), 7.72
(t, 1H, J = 8.1Hz, H-2), 7.35 (d, 1H, J = 8.6Hz, Ar),
7.17 (t, 1H, J = 8.6Hz, Ar), 5.46 (s, 1H, H-10), 5.3–
5.25 (m, 2H, H-14a, H-14b), 5.19 (s, 1H, H-7), 4.15 (q,
1H, J = 6.5Hz, H-50), 4.01 (s, 3H, OCH3), 3.74 (m,
2H, H-30, H-40), 3.24 (d, 1H, J = 20.0Hz, H-10), 3.00
(d, 1H, J = 20.0Hz, H-10), 2.43 (m, 1H, H-8), 2.13 (m,
1H, H-8), 1.97 (m, 1H, H-20), 1.82 (m, 1H, H-20), 1.29
(d, 3H, J = 6.5Hz, CH3); 13C NMR (CD3OD): d
216.9, 178.2, 164.7, 164.7, 161.2, 142.3, 141.0, 138.4,
131.6, 130.4, 129.6, 128.8, 121.8, 119.3, 113.3, 100.1,
93.4, 76.4, 70.3, 67.2, 66.9, 55.9, 34.5, 29.6, 8.8, 15.9.
ESMS: m/z calcd for [C34H32INO12 + H+]: 774.1.
Found: m/z 774.1.
4.1. General methods
4.1.1. Synthesis. 1H and 13C spectra were recorded in
CDCl3 (7.26ppm 1H, 77.0ppm 13C) or CD3OD
(3.35ppm 1H, 49.0ppm 13C) on a Varian Unity 400
spectrometer operating at 400 and 100.6MHz,
respectively. Merck silica gel 60 (230–400mesh) was
used for column chromatography. TLC was performed
using Merck Silica 60 F254 gel. All chemicals were of
p.a. grade and used as supplied by Aldrichꢀ. DMF,
CH2Cl2, 1,4-dioxane, CHCl3, and MeOH were dried
using standard methods.19 An AQA mass spectrometer
and an electrospray positive ionization method in
MeOH solution was used for LC/MS analysis. HRMS
was conducted at the Organisch Chemisches Institut
der Universitaet Muenster, Germany. The acronyms
ÔbenzÕ and ÔpyrÕ are used for benzene and pyridine rings,
respectively.
4.1.2. Radiolabeling and analysis.
[
125I] iodide
(3.7GBq/mL, specific activity 644MBq/lg) was obtained
from Amersham Pharmacia Biotech UK Limited.
MeOH (HPLC grade) was used as supplied. High
quality ELGA water (resistance higher than 18MX/
cm3) was used in preparing aqueous solutions. N-
Chloro-p-toluenesulfonamide sodium salt (chloramine-
T, CAT) and sodium metabisulfite, Na2S2O5 were
purchased from Sigma. Sodium iodide, NaI, pro ana-
lysis was obtained from Merck. The solutions of the
daunorubicin/doxorubicin derivatives, CAT (1, 2, 4,
and 8mg/mL solutions in MeOH), Na2S2O5 (8 and
16mg/mL solutions in H2O), and NaI (10mg/mL solu-
tion in H2O) were always prepared fresh.
Silica gel 60 F254 thin-layer chromatography plates (E.
Merck, Darmstadt, Germany) were used for analysis.
The reaction mixture (1–2lL) was applied to a TLC
plate. A mixture of MeOH (15mL), ether (5mL) and
acetic acid (40lL) was used as eluent for compounds
13 and 16; 4:1:0.002, CH2Cl2–MeOH–HCO2H was used
for compound 4, and a mixture of 5:1, Et2O–MeOH for
compounds 7 and 10. The Rf values for the nonradio-
labeled iododaunorubicin/doxorubicin derivatives were
4.3. Adriamycin 14-O-(3-trimethylstannyl)benzoate (3)
14-Bromodaunorubicin 17 (30.0mg, 0.047mmol), and 3-
trimethylstannylbenzoic acid (26.6mg, 0.093mmol) were
dissolved in acetone under an inert atmosphere. Potas-
sium carbonate (19.3mg, 0.14mmol) was added and
the reaction mixture was stirred at rt for 2h. The solvent
was evaporated and the residue was purified by column
chromatography (4:1, CH2Cl2–MeOH), providing 3
1
(35.0mg, 91%). H NMR (CD3OD): d 8.20 (s with tin
satellites, JH,Sn = 28Hz, 1H, Ar), 8.01 (d, 1H,
J = 7.7Hz, H-3), 7.76–7.66 (m, 3H, H-1, H-2, Ar),
7.46 (t, 1H, J = 8.3Hz, Ar), 7.41 (t, 1H, J = 8.3Hz,
Ar), 5.56 (d, 1H, J = 18.0Hz, H-14a), 5.43 (s, 1H, H-
10), 5.34 (s, 1H, H-14a), 4.98 (s, 1H, H-7), 4.35 (q, 1H,
J = 6.8Hz, H-50), 3.94 (s, 3H, OCH3), 3.70 (br s, 1H,

S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
21
H-40), 3.55 (m, 1H, H-30), 3.04 (d, 1H, J = 20.1Hz, H-
10), 2.72 (d, 1H, J = 20.1Hz, H-10), 2.51 (m, 1H, H-
8), 2.12–1.98 (m, 2H, H-8, H-20), 1.91 (m, 1H, H-20),
1.34 (d, 3H, J = 6.8Hz, CH3), 0.32 (s with tin satellites,
JH,Sn = 28Hz, 9H, Sn(CH3)3); 13C NMR (CD3OD): d
208.6, 187.7, 187.4, 167.9, 162.3, 157.2, 155.9, 144.2,
141.9, 137.7, 137.2, 136.0, 135.4, 135.1, 130.6, 130.1,
129.2, 121.1, 120.3, 112.2, 112.0, 101.3, 77.5, 71.4,
68.3, 68.1, 57.1, 37.0, 33.4, 29.9, 17.1, 9.8. HRMS: m/z
calcd for [C37H41NO12Sn + Na+]: 834.1140. Found:
m/z 834.1151.
flash chromatography using EtOAc–pentane, 70:30 as a
1
mobile phase to give 6 (36.2mg, 86%) as a red solid. H
NMR (CD3OD): d 7.90 (dd, 1H, J = 7.7, 1.1Hz, 1-H),
7.78 (dd, 1H, dd, J = 8.4, 7.7Hz, 2-H), 7.71 (m, 2H,
AA0 part of AA0XX0), 7.53 (m, 2H, XX0 part of
AA0XX0), 7.50 (dd, 1H, J = 8.4, 1.1Hz, 3-H), 5.46
(m, 1H, 10-H), 5.12 (m, 1H, 7-H), 4.41–4.33 (m, 2H,
50-H, 30-H), 3.98 (s, 3H, 4-OCH3), 3.74 (m, 1H, 40-H),
3.05 (d, 1H, J = 18.7, 10-Heq), 2.97 (d, 1H,
J = 18.7Hz, 10-Hax), 2.37 (s, 3H, 14-CH3), 2.36 (m,
1H, 40-OH), 2.22–2.08 (m, 3H, 8-Heq, 8Hax, 20-Heq),
1.82 (m, 1H, 20-Hax), 1.30 (d, 3H, J = 6.5Hz, 50-CH3),
0.32 (s with Sn satellites, 9H, Sn(CH3)3); 13C NMR
(CDCl3): d 212.4, 187.3, 187.0, 167.0, 161.3, 156.7,
156.2, 147.8, 136.2, 136.0, 135.9, 134.7, 134.3, 134.2,
126.3, 120.1, 118.6, 111.8, 111.6, 100.9, 70.2, 69.9,
67.3, 56.9, 46.0, 45.9, 35.4, 33.8, 30.3, 25.1, 17.0, 9.3.
HRMS: m/z calcd for [C37H41NO11Sn + Na+]:
818.1608. Found: m/z 818.1587.
4.4. 30-N-4-Iodo-benzoyldaunorubicin (5)
A 10mg/mL solution of 19 in dry dimethylformamide
(2mL) was added in a 1:1 molar ratio to daunorubicin
hydrochloride (20mg/mL in DMF). A 5-fold molar
excess of triethylamine (17.94mg, 0.18mmol) was
injected into the reaction mixture; which was then
stirred at rt under an argon atmosphere for 1.5h. The
course of the reaction was followed by reversed phase-
HPLC, using a gradient of 80% water–20% acetonitrile
(ACN), changing to 100% ACN over 10min, then held
for 15min (flow rate = 1mL/min, UV monitoring at
254nm). After 1.5h, the reaction was complete. The sol-
vent was evaporated under reduced pressure and the
crude product was purified by flash chromatography
using EtOAc–pentane, 70:30 as a mobile phase to give
4.6. N-Succinimidyl-5-bromo-nicotinic acid (21)
Compound 21 was synthesized via the same procedure
used for 19,16 except that 5-bromo-nicotinic acid was
used as the starting material. The crude product was
purified by flash chromatography using CH3CN–
CH2Cl2 (70:40) as a mobile phase to give 21 as a white
solid. Rf = 0.45 CH3CN–CH2Cl2; 50:20, 1.06g, 69%).
1H NMR spectral data were in accord with those pub-
lished previously.16 13C NMR (CDCl3): d 168.8, 160.0,
156.6, 149.5, 140.3, 123.2 121.1, 25.9.
1
5 (19.9mg, 74%) as a red solid; H NMR (CDCl3): d
13.99, 13.26 (d, 2H, 6,11-OH), 8.03 (dd, 1H, J = 7.8,
1.1Hz, 1-H), 7.77 (dd, 1H, J = 7.8, 8.5Hz, 2-H), 7.72
(m, 2H, AA0 part of AA0XX0), 7.37 (dd, 1H, J = 8.5,
1.1Hz, 3-H), 7.42 (m, 2H, XX0 part of AA0XX0), 5.52
(m, 1H, 10-H), 5.27 (m, 1H, 7-H), 4.44 (s, 1H, 9-OH),
4.37 (m, 1H, 30-H), 4.29 (m, 1H, 50-H), 4.05 (s, 3H, 4-
OCH3), 3.73 (m, 1H, 40-H), 3.24 (dd, 1H, J = 18.9,
1.9Hz, 10-Heq), 2.92 (d, 1H, J = 18.8Hz, 10-Hax), 2.42
(s, 3H, 14-CH3), 2.34 (m, 1H, 40-OH), 2.16–2.14 (m,
2H, 8-Heq, 8Hax), 2.00 (m, 1H, m, 20-Heq), 1.85 (m,
1H, 20-Hax), 1.31 (d, 3H, J = 6.6Hz, 50-CH3); 13C
NMR (CDCl3): d 212.3, 187.3, 186.9, 166.0, 161.3,
156.6, 156.1, 138.0, 135.9, 135.8, 134.7, 134.2, 133.9,
128.8, 121.2, 120.1, 118.6, 111.8, 111.6, 100.9, 98.7,
70.4, 69.9, 67.3, 56.9, 46.1, 46.0, 35.4, 33.7, 30.3, 25.0,
17.0. HRMS: m/z calcd for [C34H32INO11 + Na+]:
780.0918, [C34H32INO11 + K+]: 796.0657. Found: m/z
780.0875, 796.0640, respectively.
4.7. N-Succinimidyl-5-(trimethylstannyl)nicotinic acid
(22)
This compound was synthesized via the same procedure
that was used for the synthesis of 18,16 except that
N-succinimidyl-5-bromo-nicotinic acid 21 (0.59g,
1.97mmol) was used as the starting material. The solu-
tion was stirred at 85ꢁC under an argon atmosphere
for 2h until the catalyst turned black. The solvent was
evaporated under reduced pressure to dryness at rt.
The crude mixture was applied to a flash chromatogra-
phy column and eluted with CH3CN–CH2Cl2 gradient
(100% CH2Cl2 ! 50% CH3CN–20% CH2Cl2) to give
22 (0.15g, 20% yield) as a white crystal (Rf = 0.4
1
CH3CN–CH2Cl2; 50:20); H NMR spectral data were
in accord with those published previously.16 13C NMR
(CDCl3): d 168.8, 160.0, 156.6, 149.5, 140.3, 123.2
121.1, 25.9, ꢀ9.3.
4.5. 30-N-4-Trimethylstannylbenzoyldaunorubicin (6)
The synthesis of 6 was carried out as outlined for 5
except that N-succinimidyl-4-(trimethylstannyl)benzoic
acid (20) was used as the starting material. The course
of the reaction was followed by TLC (EtOAc–pentane,
70:30). The solvent was evaporated under reduced pres-
sure to dryness at rt. The crude product was purified by
4.8. 30-N-5-Trimethylstannyl-nicotinyldaunorubicin (8)
This compound was synthesized using the same proce-
dure used for the synthesis of 6, except that N-succinim-
idyl-5-(trimethylstannyl)nicotinic acid 22 (19.7mg,

22
S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
0.035mmol) was used as the starting material. The crude
product was purified by flash chromatography using
CH3CN–CH2Cl2 (50:50) and then CH3CN–Et3N (99:1)
as mobile phase to give 8 (20.1mg, 72%) as a red solid,
4.10. 30-N-(4-Hydroxybenzyl)daunorubicin (12) and 30-N-
(4-hydroxybenzyl)-13-(R/S)-dihydrodaunorubicin (15)
A mixture of hydrochloride salt of daunorubicin 1a
(25mg, 0.044mmol), and 4-hydroxybenzaldehyde
(16mg, 0.13mmol) was dissolved in acetonitrile–water
(2:1, 6mL). To the stirred solution was added sodium
cyanoborohydride (2.4mg, 0.04mmol, 1M solution in
THF). The reaction mixture was stirred under a nitro-
gen atmosphere at rt in the dark for 24h, and the con-
sumption of the daunorubicin was followed by TLC.
Additional 4-hydroxyaldehyde (22mg) and sodium cya-
noborohydride (4mg) were added successively. The
reaction was stirred for another 2days. The solvent
was evaporated and the crude mixture was purified by
flash chromatography. Two fractions were isolated
and analyzed by NMR and LC/MS. The first fraction
(6mg, 22%) was found to be compound 12. The second
product (21mg, 75%) was analyzed and found to be
compound 15 (as a mixture of two diastereomers).
1
Rf = 0.3 CH2Cl2–CH3CN; 80:20; H NMR (CDCl3): d
14.00, 13.29 (s, 2H, 6,11-OH), 8.80 (m, 1H), 8.69 (m,
1H), 8.11 (m, 1H), 8.06 (dd, 1H, J = 7.7, 1.1Hz, 1-H),
7.78 (dd, 1H, J = 8.5, 7.7Hz, 2-H), 7.37 (dd, 1H,
J = 8.5, 1.1Hz, 3-H), 6.47 (d, 1H), 5.55 (m, 1H, 10-H),
5.30 (m, 1H, 7-H), 4.49 (s, 1H, 9-OH), 4.40 (m, 1H,
30-H), 4.33 (m, 1H, 50-H), 4.07 (s, 3H, 4-OCH3), 3.77
(m, 1H, 40-H), 3.28 (dd, 1H, J = 18.7, 2.0Hz, 10-Heq),
2.98 (d, 1H, J = 18.7Hz, 10-Hax), 2.43 (s, 3H, 14-
CH3), 2.36 (m, 1H, 40-OH), 2.18–2.03 (m, 2H, 8-Heq,
8Hax), 1.96 (m, 1H, 20-Heq), 1.88 (m, 1H, 20-Hax), 1.33
(d, 3H, J = 6.6Hz, 50-CH3), 0.34 (s with Sn satellites,
9H, Sn(CH3)3); 13C NMR (CDCl3): d 212.2, 187.4,
187.0, 165.7, 161.3, 158.1, 156.6, 156.1, 147.4, 142.7
137.7, 135.9, 135.8, 134.7, 134.2, 130.1, 121.3, 120.1,
118.7, 111.8, 111.6, 100.9, 70.4, 69.8, 67.3, 56.9, 46.2,
35.4, 33.8, 30.4, 25.1, 17.0, ꢀ9.2. HRMS: m/z calcd
for [C36H40N2O11Sn + Na+]: 819.1552. Found: m/z
819.1532.
4.10.1. Data for 30-N-(4-hydroxybenzyl)daunorubicin
(12). 1H NMR (CD3OD): d 7.93 (d, 1H, H-3), 7.82
(t, 1H, H-2), 7.56 (d, 1H, H-1), 7.20 (d, 2H, Ar), 6.71
(d, 2H, Ar), 5.46 (br s, 1H, H-10), 5.09 (br s, 1H, H-7),
4.27 (q, 1H, H-50), 3.90 (s, 3H, OCH3), 3.63 (br s, 1H,
H-40), 3.67–3.48 (m, 3H, H-30, H-10eq, H-10ax), 2.99
(m, 2H, CH2), 2.35 (s, 3H, COCH3), 2.33–2.10 (m,
2H, H-8), 2.05–1.90 (m, 2H, H-20), 1.29 (d, 3H, 50-
CH3); 13C NMR (CD3OD): d 213.4, 203.7, 164.3,
160.9, 137.2, 132.3, 121.9, 120.6, 120.5, 116.7, 113.1,
101.3, 97.3, 74.6, 71.8, 68.4, 66.2, 57.1, 54.6, 37.0, 33.2,
4.9. 30-N-5-Iodo-nicotinyldaunorubicin (9)
In a round-bottomed flask, compound 8 (14.0mg,
0.018mmol) was dissolved in 1mL of 1% AcOH/MeOH.
Then 105lL of NaI (5.27mg, 0.35mmol) in MeOH was
added, followed by 0.56mL chloramine-T (0.04g/mL
MeOH). The reaction mixture was stirred vigorously
and the color changed from red to slightly yellow after
13min. After this, the reaction was quenched by the
addition of 0.84mL of sodium metabisulfite (0.04mg/
mL in H2O), which gave the original color, red, again.
The solvent was evaporated under reduced pressure to
dryness at rt. The crude product was purified by flash
chromatography using 100% EtOAc as a mobile phase
29.1,
24.5,
17.1.
HRMS:
m/z
calcd
for
[C34H35NO11 + H+]: 634.2288. Found: m/z 634.2270.
4.10.2. Data for 30-N-(4-hydroxybenzyl)-13-(R/S)-
dihydrodaunorubicin (15). 1H NMR (CD3OD): d 7.77
(m, 2H, H-3, H-2), 7.48 (d, 1H, H-1), 7.01 (2H, Ph),
6.82 (2H, Ph), 6.54 (2H, Ph), 6.37 (2H, Ph), 5.45 (br s,
1H, H-10), 5.00 (br s, 1H, H-7), 4.25 (q, 1H, H-50), 3.98
(s, 3H, OCH3), 3.79 (m, 2H, H-14, H-40), 3.68–3.47 (m,
3H, CH2, H-30), 2.93 (m, 1H, H-10), 2.53 (m, 2H, H-10,
H-8), 2.17 (m, 1H, H-8), 1.96 (m, 2H, H-20), 1.92–1.33
(m, 6H, COCH3, CH3); 13C NMR (CD3OD): d 213.0,
188, 162.4, 159.3, 157.0, 137.0, 136.3, 136.2, 132.2,
132.1, 123.5, 120.9, 120.2, 116.6, 116.4, 112.4, 112.0,
101.1, 74.2, 73.9, 72.7, 71.8, 68.0, 65.9, 57.0, 54.6, 35.9,
33.3, 32.2, 29.2, 28.9, 17.2, 16.8. HRMS: m/z calcd for
[C34H37NO11 + H+]: 636.2445. Found: m/z 636.2418.
1
to give 9 (10.3mg, 77%) as slightly red solid; H NMR
(CDCl3): d 14.00, 13.30 (s, 2H, 6,11-OH), 8.91 (m,
1H), 8.89 (m, 1H), 8.34 (m, 1H), 8.06 (dd, 1H, J = 7.7,
1.1Hz, 1-H), 7.78 (dd, 1H, J = 8.5, 7.7Hz, 2-H), 7.37
(dd, 1H, J = 8.5, 1.1Hz, 3-H), 6.43 (d, 1H), 5.54 (m,
1H, 10-H), 5.32 (m, 1H, 7-H), 4.43 (s, 1H, 9-OH),
4.39–4.30 (m, 2H, 50-H, 30-H), 4.07 (s, 3H, 4-OCH3),
3.74 (m, 1H, 40-H), 3.29 (dd, 1H, J = 19.0, 2.0Hz, 10-
Heq), 2.98 (d, 1H, J = 19.0Hz, 10-Hax), 2.43 (s, 3H,
14-CH3), 2.35 (m, 1H, 40-OH), 2.14–2.04 (m, 2H, 8-
Heq, 8-Hax), 1.96 (m, 1H, 20-Heq), 1.86 (m, 1H, 20-
Hax), 1.33 (d, 3H, J = 6.6Hz, 50-CH3); 13C NMR
(CDCl3): d 212.1, 187.4, 187.0, 163.6, 161.3, 158.5,
156.6, 156.1, 146.5, 143.4, 135.9, 135.8, 134.7, 134.1,
121.3, 120.1, 118.7, 111.8, 111.6, 100.8, 70.4, 69.7,
67.2, 56.9, 46.3, 35.4, 33.7, 30.3, 25.0, 16.9. HRMS:
m/z calcd for [C33H31IN2O11 + Na+]: 781.0870. Found:
m/z 781.0869.
4.11. 30-N-(4-Hydroxy-3-iodobenzyl)daunorubicin (11)
and 30-N-(4-hydroxy-3-iodobenzyl)-13-(R/S)-dihydro-
daunorubicin (14)
To a solution of daunorubicin hydrochloride (30mg,
and
0.053mmol)
4-hydroxy-3-iodobenzaldehyde

S. Ghirmai et al. / Carbohydrate Research 340 (2005) 15–24
23
(39.6mg, 0.16mmol) in a 2:1 ratio mixture of acetonit-
rile and water (8mL) was added 1M THF solution of
sodium cyanoborohydride (3.4mg, 0.053mmol). The
reaction mixture was stirred at rt in the dark under a
nitrogen atmosphere for 24h and the progress of the
reaction was monitored by TLC. Additional aldehyde
(20mg) and sodium cyanoborohydride (7mg) were
added successively and the reaction mixture was stirred
for another 2days. The solvent was evaporated and the
crude mixture was purified by flash chromatography.
The reaction produced 11 (15mg, 37%) and 14 (20mg,
50%, mixture of two diastereomers).
typical labeling experiment, 3.7GBq/lL of [125I] iodide
was used.
To 40lL of MeOH solution of daunorubicin deriva-
tive, 5.0lL aqueous 125I-solution was added, followed
by 10lL CAT solution. The reaction mixture was vor-
texed for 5min, and quenched by the addition of 10lL
sodium metabisulfite solution; 20lL of NaI (10mg/mL
solution in H2O) solution was added to stabilize [125I]-
iodide against oxidation during the TLC analysis. Exact
concentrations of solutions are given in the figure leg-
ends. After preparing the reaction mixture, samples for
radio-TLC analysis (1–2lL) were collected. Blank
experiments were run using exactly the same conditions,
but neat MeOH solution was used instead of the stock
solution of daunorubicin/doxorubicin derivatives. All
blank experiments were tested in both TLC systems.
The crude product was purified using C18 solid phase
extraction, eluting any unreacted [125I] iodide with water
and the reaction product with 100% ethanol.
4.11.1. Data for 30-N-(4-hydroxy-3-iodobenzyl)daunoru-
bicin (11). 1H NMR (CD3OD): d 7.88 (d, 1H, H-3),
7.81 (t, 1H, H-2), 7.72 (s, 1H, Ph), 7.53 (d, 1H, H-1),
7.20 (d, 1H, Ph), 6.76 (d, 1H, Ph), 5.46 (br s, 1H, H-
10), 5.05 (br s, 1H, H-7), 4.28 (m, 1H, H-50), 4.01 (s,
3H, OCH3), 3.86 (br s, 1H, H-40), 3.50 (m, 1H, H-30),
3.22 (m, 1H, H-10), 3.00 (m, 2H, CH2), 2.46 (m, 1H,
H-10), 2.36 (s, 3H, COCH3), 2.01 (m, 2H, H-8), 1.79
(m, 1H, H-20), 1.62 (m, 1H, H-20), 1.30 (d, 3H, 50-
CH3); 13C NMR (CD3OD): d 213.4, 203.7, 162.5,
159.2, 154.9, 142.1, 132.4, 120.5, 120.3, 115.8, 115.7,
101.2, 84.9, 77.5, 73.2, 67.9, 66.1, 57.1, 56.0, 37.0, 31.5,
30.6, 28.7, 24.5, 17.0. HRMS: m/z calcd for [C34H34I-
NO11 + H+]: 760.1255. Found: m/z 760.1286.
Acknowledgements
This work was financially supported by the Swedish
Cancer Foundation and by INTAS (Grant No. 99-
00806).
4.11.2. Data for 30-N-(4-hydroxy-3-iodobenzyl)-13-(R/S)-
dihydrodaunorubicin (14). 1H NMR (CD3OD): d 7.93
(m, 1H, H-3), 7.82 (m, 1H, H-2), 7.64 (s, 1H, Ph),
7.55 (m, 1H, H-1), 7.1 (d, 2 H, J = 7.6Hz, Ph), 6.96
(d, 2 H, J = 7.6Hz, Ph), 6.67 (d, 2 H, J = 7.6Hz, Ph),
6.57 (d, 2 H, J = 7.6Hz, Ph), 5.50 (s, 1H, H-10), 5.10
(br s, 1H, H-7), 4.28 (m, 1H, H-50), 4.02 (s, 3H,
OCH3), 3.83 (br s, 2H, CH2Ph), 3.71 (m, 1H, H-40),
3.67 (m, 1H, H-30), 3.47 (m, 1H, H-13), 3.11 (m, 1H,
H-13), 3.08 (d, 1H, J = 18.7Hz, H-10), 2.99 (d, 1H,
J = 18.7Hz, H-10), 2.71 (d, 1H, J = 18.7Hz, H-10),
2.61 (d, 1H, J = 18.7Hz, H-10), 2.54 (m, 1H, H-8),
2.22 (m, 1H, H-8), 2.15–1.74 (m, 3H, H-8, H-20), 1.35–
1.28 (m, 6H, 14-CH3, 50-CH3); 13C NMR (CD3OD): d
213.0, 188.0, 187.9, 162.5, 159.2, 159.0, 157.1, 157.0,
142.0, 137.2, 136.3, 136.2, 136.1, 136.0, 132.4, 125.0,
121.5, 121.4, 120.6, 120.4, 115.6, 115.4, 112.5, 112.2,
101.1, 84.9, 74.2, 73.9, 73.2, 72.8, 72.3, 57.1, 54.9, 35.9,
33.8, 32.3, 30.6, 28.9, 28.8, 17.2, 17.1, 16.8. HRMS:
m/z calcd for [C34H36INO11 + H+]: 762.1411. Found:
m/z 762.1435.
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and 8mg/mL) was dissolved in MeOH prior to use. In a

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