Glycosyl and polyalcoholic prodrugs of lonidamine

Article abstract of DOI:10.1016/j.bmcl.2008.02.046

Polyhydric alcohol derivatives of the anticancer agent lonidamine (LND) have been synthesized. The increased water solubility showed by prodrugs 4, 7, and 25 together with their log P values (2.19, 2.55, and 2.54, respectively) and chemical stability migh

Full text of DOI:10.1016/j.bmcl.2008.02.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2445–2450
Glycosyl and polyalcoholic prodrugs of lonidamine
G. Giorgioni,a,* S. Ruggieri,a A. Di Stefano,b P. Sozio,b B. Cinque,c
L. Di Marzio,b G. Santonid and F. Claudia
a
c
`
Dipartimento di Scienze Chimiche, Universita di Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
b
`
Dipartimento di Scienze del Farmaco, Universita G. D’Annunzio, Via dei Vestini, 66100 Chieti, Italy
`
Dipartimento di Medicina Sperimentale, Universita di L’Aquila, Via Vetoio Coppito 2, 67100 L’Aquila, Italy
d
` `
Dipartimento di Medicina Sperimentale e Sanita Pubblica, Universita di Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy
Received 4 January 2008; revised 15 February 2008; accepted 17 February 2008
Available online 5 March 2008
Abstract—Polyhydric alcohol derivatives of the anticancer agent lonidamine (LND) have been synthesized. The increased water sol-
ubility showed by prodrugs 4, 7, and 25 together with their logP values (2.19, 2.55, and 2.54, respectively) and chemical stability
might be beneficial for prodrugs absorption after oral administration. Moreover, the new prodrugs undergo enzymatic hydrolysis
in plasma and release LND demonstrating that they are promising candidates for in vivo investigations.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Lonidamine (LND) 1, a drug used in the treatment of sev-
eral neoplasia (i.e., lung, breast, prostate, and brain), was
first synthesized in 1976 with the aim of obtaining an anti-
spermatogenic agent,1 and it was only later that the anti-
neoplastic activity of this agent was discovered. The
mechanism of action of LND does not involve protein
or nucleic acid synthesis. LND acts by reducing both
the oxygen consumption and the glucose utilization of
neoplastic cells by inhibiting the mitochondria-bound
glycolytic enzyme hexokinase that is usually absent in
normal cells.2 Lactate efflux and intracellular accumula-
tion are also lowered.3 LND strongly potentiates the ther-
apeutic efficacy of other antineoplastic drugs, for
example, cis-platin,4 adriamicyn5, and alkylating agents.6
ported even over a long-term treatment period.7 How-
ever, pancreatic and hepatic toxicity were observed in
dogs receiving LND by intravenous injection whereas,
in the same studies, oral administration of LND was de-
void of such toxicity.8 The bioavailability of LND after
oral administration might be limited by its extremely
low water solubility (17 mg/L).8 Attempts to increase
LND water solubility have been performed by preparing
inclusion complexes with b-cyclodextrins,9 and solid dis-
persions of LND in both PVP and PEG 4000.10 Such
preparations induced a considerable improvement in
LND water solubility as well as an improvement in
LND bioavailability after oral administration. Such re-
sults confirmed the hypothesis that bioavailability after
oral administration and water solubility is strictly
correlated.10
Cl
To improve LND water solubility we designed and syn-
thesized prodrugs by conjugation of LND with hydro-
philic molecules such as carbohydrates and polyhydric
alcohols to form water soluble esters which can be cleft
by chemical and enzymatic hydrolysis in the living body.
Hydroxyl groups can modulate the hydrophilicity of the
conjugated compound and may affect both its absorp-
tion after oral administration and its bioavailability.
Cl
HO
N
N
O
1
Due to its particular mechanism of action, LND is de-
void of the usual side effects induced by antiproliferative
agents and no serious adverse reactions have been re-
The choice of carbohydrates was based on: (a) several
antitumor antibiotics such as bleomycin, mithramycin,
and anthracycline, which contain a glycosidic moiety
embedded in their structure; (b) glycosyl promoiety11
Keywords: Lonidamine; Anticancer; Prodrug; Polyalcoholic; Polyhy-
dric; Synthesis.
*
Corresponding author. Tel.: +39 0737 402368; fax: +39 0737
637345; e-mail: gianfabio.giorgioni@unicam.it
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.046

2446
G. Giorgioni et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2445–2450
Table 1. Derivatives of LND
Acetonide
Intermediate
Product
R
OH
O
O
O
O
O
O
O
HO
O
O
O
O
R
O
O
OH
HO
R
OH
2
3
4
O
O
HO
OH
OH
R
O
O
HO
R
O
HO
OH
O
O
O
7
OH
O
6
HO
O
O
O
HO
R
OH
R
O
R
O
5
O
R
O
HO
OH
O
O
9
8
O
R
O
OH
R
O
HO
O
O
N
N
O
O
OH
12
O
10
11
O
O
OH
OH
Cl
R
O
O
R
O
HO
Cl
HO
O
O
O
15
14
O
HO
OH
O
O
O
HO
13
R
O
O
R
O
OH
O
O
R
R
16
17
OH
O
OH
R
R
O
O
O
HO
HO
OH
20
19
O
O
O
OH
O
R
O
O
R
R
R
18
O
HO
OH
22
21
OH
O
O
HO
HO
OH
O
O
O
O
O
O
O
O
O
O
O
R
OH
R
25
23
24

G. Giorgioni et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2445–2450
2447
which might work as a transporter to enhance intestinal
absorption12; (c) the observation that tumor cells have
higher than normal glucose metabolism13 probably due
to an overexpression of the glucose transporter
(GLUT-1)14; (d) since glucose and galactose are actively
transported through the blood–brain barrier, their inclu-
sion in prodrugs could help the antitumor agent reach
tumor cells in the central nervous system.
no significant reduction in cell viability was observed.
In addition, the effect of these drug concentrations on
the cell cycle was investigated. The cells were incubated
for 12 h at two different drug concentrations and then
analyzed with a flow cytometry.21 The 50 lM pro-
drugs-treated SK-OV showed a cell cycle profile compa-
rable to LND-treated cells (Fig. 1A), however a
decreased number of cells in the S-compartment was evi-
dent after 12 h exposure to 25 at 50 lM dose. A similar
decreased cell number was observed after the treatment
with prodrugs at 150 lM dose (Fig. 1B). Interestingly,
for 25 at 150 lM dose, the cell depletion in S-phase
was associated to a concomitant accumulation of cell
percentage in G1-phase. These results indicated that 25
at 150 lM dose blocked the cell cycle at G1/S transition.
The synthesis of the new prodrugs (Table 1) was carried
out by condensation of glucofuranose and mannitol di-
acetonides or glycerol, xylofuranose, and threitol aceto-
nides with LND in the presence of N-ethyl-N0-(3-
dimethylaminopropyl)carbodiimide
hydrochloride
(EDC) and 4-(dimethylamino)pyridine (DMAP) to give
the esters 3, 6, 8, 11, 14, 16, 19, and 21.15 Mannitol,
xylofuranose, and threitol acetonides gave a mixture of
mono- and bis-adducts, which were separated by col-
umn chromatography using a 1:1 mixture of ethyl ace-
tate/hexane. As an exception, the galactose ester 2416
was synthesized from the reaction of 1,2:5,6-di-O-iso-
propylidene-a-D-galactopyranose with 1-(3,5-dichlo-
robenzyl)-1H-indazole-3-carbonyl chloride.17 The
hydrolysis of the acetonide protecting group was carried
out using trifluoroacetic acid to give the target com-
pounds 4, 7, 9, 12, 15, 17, 20, 22, and 25.18
The physical–chemical properties such as water solubil-
ity and the octanol/water partition coefficient of the new
compounds were evaluated (see Table 2).22 Glucose,
mannitol, and galactose conjugates (4, 7, and 25)
showed water solubility from 5 to 8 times higher than
LND. Such monoconjugates bear in their structure five
or four hydroxyl groups. The mono-adducts, 12, 15, and
20, bearing only two or three hydroxyls, as well as the
bis-adducts 9 and 17 showed lower solubility than
LND. Threitol derivative 22 was insoluble.
With the aim to verify whether the new prodrug mole-
cules are endowed with cytotoxic activity, in vitro sulfo-
rhodamine B (SRB) assay on human PC-3 prostate
cancer cells was carried out,19 according to the National
Cancer Institute protocol.20 In such a test, LND and its
conjugates did not show significant cytotoxicity up to
100 lM dose. Moreover, LND did not show measurable
growth inhibition activity, whereas compounds 7 and 25
showed a GI50 = 3.16 lM. Furthermore, the effects of
the derivatives on ovarian carcinoma cell line (SK-OV)
were evaluated treating SK-OV for 12 h at three differ-
ent concentrations (50, 150, 300 lM) of drugs in the
presence of 0.01% DMSO. The cell viability was depen-
dent on the concentration of 7 (at 300 lM % inhibi-
tion ꢀ 30%) and 25 (at 300 lM % inhibition ꢀ60%),
whereas no cell viability reduction was detectable after
the cell treatment with both the other prodrugs and
LND. However, at doses 50 and 150 lM of all drugs
All LND polyhydroxyesters, except 22, showed higher
logP values than LND. Compounds 4, 7, and 25 dem-
Table 2. Measured water solubility and logP values
Compound
Water solubilitya (mg/L)
logPa
4
118.3 ( 8.3)
137.3 ( 8.4)
0.92 ( 0.12)
0.51 ( 0.06)
1.3 ( 0.20)
3.3 ( 0.30)
1.2 ( 0.09)
Insoluble
2.19 ( 0.34)
2.55 ( 0.28)
3.52 ( 0.36)
3.96 ( 0.32)
3.40 ( 0.24)
2.86 ( 0.14)
3.04 ( 0.18)
—
7
9
12
15
17
20
22
25
LND
84.3 ( 8.4)
17 ( 0.6)
2.54 ( 0.20)
1.30 ( 0.05)
a The reported values are the means of three experiments. The SEM are
reported in parentheses.
A
B
100
G1
100
75
50
25
0
G1
S
S
*
G2/M
G2/M
75
50
25
0
*
*
*
7
*
LND
7
25
4
LND
4
25
Figure 1. Effect of drugs on cell cycle distribution. Cell cycle analysis by propidium iodide and flow cytometry after cell treatment with 50 lM (A)
and 150 lM (B) drugs. The results represent means SEM of three independent experiments. *P 6 0.05 when compared with LND-treated cells.

2448
G. Giorgioni et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2445–2450
Table 3. Kinetic data for chemical hydrolysis of prodrugs 4, 7, and 25 at 37 ꢁC
Compound
pH 1.3a
pH 7.4a
t1/2 (h)
Kobs (hꢁ1
)
t1/2 (h)
Kobs (hꢁ1
)
4
7
>>120
>>120
>>120
—
—
—
44.07 ( 1.32)
80.70 ( 1.61)
114.03 ( 0.57)
0.0068 ( 2.0 · 10ꢁ4
0.0037 ( 7.5 · 10ꢁ5
0.0026 ( 1.3 · 10ꢁ5
)
)
)
25
a The reported values represent means of three experiments. The SEM are reported in parentheses.
The prodrugs 4, 7, and 25 were evaluated for their chem-
ical stability in both pH 1.3 buffer (non enzymatic gas-
tric environment simulation) and pH 7.4 at 37 ꢁC
(Table 3).22 The kinetics of chemical hydrolysis were
determined (Fig. 2). The results showed that the tested
prodrugs were extremely stable in acidic media and were
not hydrolyzed 120 h after their administration. Quite
good stability at pH 7.4 was also observed. Thus, com-
pounds 4, 7, and 25 potentially pass unchanged through
the stomach and might be adsorbed at the intestinal le-
vel. Moreover, it has been reported that for good
absorption after oral administration a logP P 2 is re-
quired.23 Compounds 4, 7, and 25 showed logP values
of 2.19, 2.55, and 2.54, respectively. These findings, ta-
ken together with the improved water solubility and
the observed chemical stability, suggest that derivatives
4, 7, and 25 might be candidate drugs with good absorp-
tion after oral administration.
4
2.0
1.8
1.6
1.4
1.2
7
25
0
20
40
60
80
100
120
Time (h)
Figure 2. First order kinetic plots for hydrolysis of prodrugs 4, 7, and
25 in phosphate buffer of pH 7.4 at 37 ꢁC.
The enzymatic stability of compounds 4, 7, and 25 (Ta-
ble 4) at 37 ꢁC in 80% rat plasma and in 80% human
plasma was studied.24 Figure 3 shows the first order ki-
netic plots for hydrolysis of the prodrugs.
onstrated aqueous solubility and lipophilicity. Conju-
gates 9, 12, 15, 17, and 20 with a logP > 2.6 bearing only
two or three hydroxyl groups exhibited high lipophilicity
and poor aqueous solubility.
Table 4. Rate constants for the hydrolysis of prodrugs 4, 7, and 25 in 80% rat plasma and in 80% human plasma at 37 ꢁC
Compound
Rat plasmaa
Human plasmaa
t1/2 (min)
Kobs (minꢁ1
)
t1/2 (min)
Kobs (minꢁ1
)
4
7
32.06 ( 0.96)
130.32 ( 2.61)
59.73 ( 2.99)
0.0094 ( 2.8 · 10ꢁ3
0.0023 ( 4.6 · 10ꢁ5
0.0050 ( 2.5 · 10ꢁ4
)
)
)
42.64 ( 3.84)
360.02 ( 7.20)
123.88 ( 2.48)
0.0071 ( 6.4 · 10ꢁ4
0.0008 ( 1.7 · 10ꢁ5
0.0024 ( 4.9 · 10ꢁ5
)
)
)
25
a The reported values represent the mean of three experiments. The SEM are reported in parentheses.
4
7
4
7
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
25
25
Human Plasma
Rat Plasma
0
50
100
Time (min)
150
0
50
100
150
200
Time (min)
Figure 3. First order kinetic plots for hydrolysis of prodrugs 4, 7, and 25 in 80% rat and human plasma at 37 ꢁC.

G. Giorgioni et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2445–2450
2449
13. Gatenby, R. A.; Gillies, R. J. Int. J. Biochem. Cell Biol.
2007, 39, 1358.
It was observed that prodrugs 4, 7, and 25 underwent
faster hydrolysis in rat plasma than in human plasma
and compound 7 proved to be the most stable prodrug
(t1/2 = 360.02 min). Comparing the rate of hydrolysis
in buffer at pH 7.4 and in both rat and human plasma,
it can be argued that an enzymatic hydrolysis occurs in
plasma. In human plasma glucose conjugate 4 was
quickly hydrolyzed (t1/2 = 42.6 min) and could generate
high levels of LND in the blood. Prodrugs 7 and 25
were more slowly hydrolyzed; galactose derivative 25
(t1/2 ffi 2 h) might be a substrate for the transporter
GLUT-1. Further pharmacokinetic and biological stud-
ies are in progress to investigate whether an increased
delivery of LND to the tumor cells occurs.
14. Brown, R. S.; Wahl, R. I. Cancer 1993, 72, 2979.
15. General method for the preparation of LND-esters 3, 6, 8,
11, 14, 16, 19, and 21: To a solution of LND (0.45 g,
1.4 mmol) and the proper acetonide in anhydrous DMF
(4 ml), EDC (0.34 g, 1.7 mmol) and DMAP (0.27 g,
2.2 mmol) were added. The mixture was stirred over-
night at room temperature. A 2 N HCl solution (10 ml)
was added and the mixture extracted with CH2Cl2 (3·
5 ml). The organic extracts were washed with 2 N HCl
solution, and water, then dried over anhydrous sodium
sulfate, filtered, and rotary evaporated to give the
product.
Data for (3aR,5R,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxo-
lan-4-yl)-2,2-dimethyl-dihydro-5H-furo[3,2-d][1,3]dioxol-
6-yl
1-(3,5-dichlorobenzyl)-1H-indazole-3-carboxylate
In conclusion we have reported the effects of the conju-
gation of LND with polyhydric molecules on water sol-
ubility. Higher water solubility was obtained when LND
was conjugated with glucose, galactose and mannitol.
The measured physical–chemical properties might be
beneficial for prodrugs absorption after oral administra-
tion. Moreover, the new prodrugs undergo enzymatic
hydrolysis in plasma and release LND demonstrating
that they are promising candidates for in vivo
investigations.
(3): mp 136–137 ꢁC from ethanol. Yield 73%. 1H
NMR (d) (DMSO-d6): 8.05 (d, 1H), 7.90 (d, 1H), 7.68
(d, 1H), 7.54 (t, 1H), 7.42 (m, 2H), 7.13 (d, 1H), 6.02 (d,
1H), 5.87 (m, 2H), 5.36 (d, 1H) 4.78 (d, 1H), 4.28 (m,
2H), 3.96 (m, 2H), 1.45, 1.33, 1.28, 1.14 (four s, 12H).
Data for 1,2-bis(2,2-dimethyl-1,3-dioxolan-4-yl)-2-hydro-
xy-ethyl 1-(3,5-dichlorobenzyl)-1H-indazole-3-carboxyl-
ate (6): foam. Yield 19%. 1H NMR (d) (DMSO-d6):
8.20 (d, 1H), 7.82 (d, 1H), 7.70 (d, 1H), 7.52 (t, 1H),
7.32 and 7.43 (m, 2H), 6.98 (d, 1H), 5.88 (s, 2H), 5.83
(d, 1H), 5.48 (dd, 1H), 4.50 and 4.35 (q, 1H), 1.30, 1.24
and 1.16 (three t, 12H).
Data for 1,2-bis(2,2-dimethyl-1,3-dioxolan-4-yl)ethane-
1,2-diyl bis(1-(3,5-dichlorobenzyl)-1H-indazole-3-carbox-
ylate) (8): foam. Yield 13%. 1H NMR (d) (DMSO-d6):
8.22 (d, 2H), 7.86 (d, 2H), 7.70 (d, 2H), 7.53 (t, 2H),
7.35–7.18 (m, 4H), 6.90 (d, 2H), 5.92 (s, 4H), 5.75 (d,
2H), 4.42–4.28 (m, 4H), 1.22 (s, 12H).
Data for (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 1-(3,5-
dichlorobenzyl)-1H-indazole-3-carboxylate (11): mp 121–
122 ꢁC from ethyl acetate. Yield 87%. 1H NMR (d) (DMSO-
d6): 8.13 (d, 1H), 7.80 (d, 1H), 7.63 (d, 1H), 7.49 (t, 1H), 7.34
(m, 2H), 6.90(d, 1H), 5.84 (s, 2H), 4.38 (m, 3H) 4.07and 3.78
(two m, 2H), 1.18 and 1.12 (two s, 6H).
Data for ((5R,6S,6aR)-6-hydroxy-2,2-dimethyl-dihydro-
5H-furo[3,2-d][1,3]dioxol-5-yl)methyl 1-(3,5-dichloroben-
zyl)-1H-indazole-3-carboxylate (14): oil. Yield 24%. 1H
NMR (d) (DMSO-d6): 8.14 (d, 1H), 7.85 (d, 1H), 7.71 (d,
1H), 7.52 (t, 1H), 7.38 (m, 2H), 6.93 (d, 1H), 5.90 (m, 3H),
5.53 (d, 1H), 4.46 (m, 4H), 4.13 (m, 1H) 1.40 and 1,24 (two s,
6H).
Acknowledgments
This work was supported by the CARIMA Foundation,
and by the University of Camerino. Lonidamine was a
generous gift from Aziende Chimiche Riunite Angelini
Francesco (ACRAF), Rome, Italy.
References and notes
1. Corsi, G.; Palazzo, G.; Germani, C.; Scorza Barcellona,
P.; Silvestrini, B. J. Med. Chem. 1976, 19, 778.
2. Floridi, A.; Paggi, M. G.; D’Atri, S.; De Martino, C.;
Marcante, M. L.; Silvestrini, B.; Caputo, A. Cancer Res.
1981, 41, 4661.
3. Fanciulli, M.; Valentini, A.; Bruno, T.; Citro, G.; Zupi,
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C. E.; Martin, L.; Maroun, J. A.; De Santis, S. J.
Anticancer Res. 1990, 10, 923.
Data for (5R,6S,6aR)-5-((1-(3,5-dichlorobenzyl)-1H-inda-
zole-3-carbonyloxy)methyl)-2,2- dimethyl-dihydro-5H-
furo[3,2-d][1,3]dioxol-6-yl
1-(3,5-dichloro-5-methylben-
5. Zupi, G.; Greco, C.; Laudonio, N.; Benassi, m.; Solves-
trini, B.; Caputo, A. Anticancer Res. 1986, 6, 1245.
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8. Price, G. S.; Page, R. L.; Riviere, J. E.; Cline, J. M.;
Thrall, D. E. Cancer Chemother. Pharmacol. 1996, 38,
129.
1
zyl)-1H-indazole-3-carboxylate (16): foam. Yield 19%. H
NMR (d) (DMSO-d6): 8.07 (dd, 2H), 7.85 (t, 2H), 7.72 (d,
2H), 7.43 (m, 6H), 6.98 and 6.87 (two d, 2H), 6.14 (d, 1H),
5.84 (s, 4H), 5.58 (d, 1H) 4.86 (d, 1H), 4.69 (m, 3H), 1.50 and
1.28 (two s, 6H).
Data for ((4R,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)methyl1-(3,5-dichlorobenzyl)-1H-indazole-
3-carboxylate (19): oil. Yield 30%. H NMR (d) (DMSO-
1
d6): 8.15 (d, 1H), 7.84 (d, 1H), 7.70 (d, 1H), 7.52 (t, 1H), 7.37
(m, 2H), 6.96 (d, 1H), 5.85 (s, 2H), 4.86 (t, 1H), 4.48 (m, 2H),
4.17 (m, 1H), 3.97 (m, 1H), 3.60 (t, 2H), 1.55 and 1.30 (two s,
6H).
Data for ((4R,5R)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)
bis(methylene) bis(1-(3,5-dichlorobenzyl)-1H-indazole-3-
carboxylate) (21): foam. Yield 22%. 1H NMR (d)
(DMSO-d6): 8.14 (d, 2H), 7.85 (d, 2H), 7.66 (d, 2H), 7.52
(t, 2H), 7.35 (m, 4H), 6.96 (d, 2H), 5.84 (s, 4H), 4.52 (m, 6H),
1.38 (s, 6H).
9. Palmieri, G. F.; Wehrle, P.; Martelli, S. Drug Dev. Ind.
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Andersen, R.; Frokjaer, S. Eur. J. Pharm. Sci. 2004, 21, 3.

2450
G. Giorgioni et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2445–2450
16. Method for the preparation of 1-(3,5-dichlorobenzyl)-1H-
indazole-3-carboxylic acid (5R,5aS,8aS,8bR)-2,2,7,7-tet-
ramethyl-tetrahydro-bis[1,3]dioxolo[4,5-b;40,50-d]pyran-
5- ylmethyl ester (24): To a solution of 1,2:5,6-di-O-
isopropylidene-a-D-galactopyranose (0.24 g, 0.92 mmol)
in dry THF (5 ml), dry triethylamine (0.3 ml, 1.8 mmol)
and 1-(3,5-dichlorobenzyl)-1H-indazole-3-carbonyl chlo-
ride (0.31 g, 0.92 mmol) were added. The reaction mixture
was stirred for 3 h at room temperature, then the solvent
was removed under reduced pressure. The residue was
dissolved in dichloromethane, the organic solution was
washed with brine, then dried over anhydrous sodium
sulfate. After filtration of the solid, the solvent was
removed under reduced pressure, the hard foam residue
was dissolved in dichloromethane and filtered through a
small pad of silica gel. The solvent was removed in vacuo
and the residue recrystallized from cyclohexane. Mp 119–
120 ꢁC. Yield 97%. 1H NMR (d) (DMSO-d6): 8.18 (d, 1H),
7.86 (d, 1H), 7.71 (d, 1H), 7.53 (t, 1H), 7.37 (m, 2H), 6.98
(d, 1H), 5.88 (s, 2H), 5.53 (d, 1H), 4.68 (dd, 1H), 4.40 ( m,
4H), 4.20 ( m, 1H), 1.32, 1.42 and 1.50 (three s, 12H).
17. Preparation of 1-(3,5-dichlorobenzyl)-1H-indazole-3-
carbonyl chloride: Thionyl chloride (2 ml) was added
portionwise to 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-
boxylic acid (0.5 g). The mixture was warmed for 5 min to
80 ꢁC. The excess of SOCl2 was removed under reduced
pressure. The resulting solid was recrystallized from
AcOEt. Mp 144–145 ꢁC. Yield 81%. H1 NMR (DMSO-
d6) d 8.15 (d, 1H), 7.82 (d, 1H), 7.70 (d, 1H), 7.45 (m, 3H),
6.95 (d, 1H), 5.86 (s, 2H).
7.65 (d, 1H), 7.36 (m, 4H), 6.81 (d, 1H), 5.84 (s, 2H), 4.56
(m, 2H), 4.04 (m, 1H), 3.68 (d, 2H).
Data for [(2R,3R,4R)-3,4,5-trihydroxy-tetrahydrofuran-2-
yl]methyl 1-(3,5-dichlorobenzyl)-1H-indazole-3-carboxyl-
ate (15): mp 66–69 ꢁC. Yield 77%. (a:b isomer = 2:3 based
1
1
on H NMR). H NMR (d) (DMSO-d6 + D2O): 8.15 (m,
1H), 7.80 (d, 1H), 7.65 (m, 1H), 7.50 (t, 1H), 7.37 (m, 3H),
6.97 (d, 1H), 5.80 (s, 2H), 5.18 and 4.96 (two d, 1H,
J0 = 3.62, J00 = 1.28), 4.40 (m, 3H), 4.05 (m, 1H), 3.78 (m,
1H).
Data for (2R,3R,4R)-2-((1-(3,5-dichlorobenzyl)-1H-inda-
zole-3-carbonyloxy)methyl)-4,5-dihydroxy-tetrahydrofu-
ran-3-yl 1-(3,5-dichlorobenzyl)-1H-indazole-3-carboxylate
(17): mp 159–162 ꢁC from ethanol. Yield 77%. (a:b
1
1
isomer = 1:1 based on H NMR). H NMR (d) (DMSO-
d6): 7.98 (m, 2H), 7.75 (t, 2H), 7.68 (s, 2H), 7.49 (m, 2H),
7.30 (m, 4H), 6.88 (m, 2H), 6.62 (d, 1H), 5.78 (s, 4H), 5.45
(m, 2H), 5.22 and 4.16 (two d, 1H, J0 = 2.8, J00 = 1.06),
4.57 (m,4H).
Data for (2R,3R)-2,3,4-trihydroxybutyl 1-(3,5-dichlorob-
enzyl)-1H-indazole-3-carboxylate (20): mp 120–123 ꢁC
from ethylacetate. Yield 57%. 1H NMR (d) (CD3OD):
8.13 (d, 1H), 7.62 (d, 1H), 7.50 (m, 2H), 7.38 (t, 1H), 7.24
(m, 1H), 6.82 (d, 1H), 5.92 (s, 2H), 4.54 (m, 2H), 4.08 (m,
1H), 3.65 (m, 3H).
Data for (2R,3R)-2,3-dihydroxybutane-1,4-diyl bis(1-(3,5-
dichlorobenzyl)-1H-indazole-3-carboxylate) (22): mp 199–
200 ꢁC from chloroform. Yield 63%. 1H NMR (d)
(DMSO-d6): 8.10 (d, 2H), 7.81 (d, 2H), 7.68 (d, 2H),
7.49 (t, 2H), 7.34 (m, 4H), 6.92 (d, 2H), 5.83 (s, 4H), 5.15
(d, 2H), 4.42 (m, 4H), 4.04 (m, 2H).
18. General method for the preparation of compounds 4, 7, 9,
12, 15, 17, 20, 22, and 25: A solution of the LND-
acetonide conjugate (0.1 g) in TFA (0.9 ml) and water
(0.1 ml) was stirred for 5 min at room temperature. Water
(5 ml) was added and the mixture extracted three times
with dichloromethane. The organic extracts were washed
with water, dried over sodium sulphate, filtered, and
evaporated to give the product.
Data for ((2R,3R,4S,5R)-3,4,5,6-tetrahydroxy-tetrahydro-
1-(3,5-dichlorobenzyl)-1H-inda-
2H-pyran-2-yl)methyl
zole-3-carboxylate (25): mp 176–178 ꢁC from ethanol.
Yield 79%. (a:b isomer = 3:4 based on 1H NMR). 1H
NMR (d) (DMSO-d6 + D2O): 8.14 (d, 1H), 7.82 (d, 1H),
7.68 (d, 1H), 7.54 (t, 1H), 7.39 (m, 2H), 6.98 (m, 1H), 5.85
(s, 2H), 4.97 and 4.30 (two d, 1H, J0 = 3.4, J00 = 7.4), 4.42
(m, 2H), 4.12 and 3.83 (two m, 1H), 3.77 e 3.70 (two d, 1H,
J0 = 3.4; J00 = 7.4).
Data for (4S,5R)-2,3,5-trihydroxy-6-(hydroxymethyl)-tet-
rahydro-2H-pyran-4-yl 1-(3,5-dichlorobenzyl)-1H-inda-
zole-3-carboxylate (4): foam. Yield 59%. (a:b
isomer = 2:3 based on 1H NMR). 1H NMR (d) (CD3OD):
8.25 (d, 1H), 7.48 (m, 5H), 7.22 (dd, 1H), 6.83 (d, 1H), 5.84
(s, 1H), 5.61 and 5.32 (two t, 1H, J = 9.53), 5.21 (d, 1H,
J = 3.66), 4.66 (d, 1H, J = 7.7), 3.85 (m, 5H), 3.50 (m, 1H).
Data for 1,2,4,5,6-pentahydroxyhexan-3-yl 1-(3,5-dichlo-
robenzyl)-1H-indazole-3-carboxylate (7): foam. Yield
19. For experimental details see Quaglia, W.; Santoni, G.;
Pigini, M.; Piergentili, A.; Gentili, F.; Buccioni, M.;
Mosca, M.; Lucciarini, R.; Amantini, C.; Nabissi, M. I.;
Ballarini, P.; Poggesi, E.; Leonardi, A.; Giannella, M.
J. Med. Chem. 2005, 48, 7750.
20. Grever, M. R.; Shepartz, S. A.; Chabner, B. A. The
National Cancer Institute: Cancer Drug Discovery and
Development Program. Semin. Oncol. 1992, 19, 622.
21. Brisdelli, F.; Coccia, C.; Cinque, B.; Cifone, M. G.; Bozzi,
A. Mol Cell. Biochem. 2007, 296, 137.
22. For experimental details see Farghaly, A. O. Eur. J. Med.
Chem. 1998, 33, 123.
23. Yalkowsky, S. H.; Morozowich, W. In A Physical
Chemical Basis for the Design of Orally Active Prodrugs;
Ariens, E. J., Ed.; Drug Design; Academic Press: New
York, 1980; Vol. 9, p 121.
1
80%. H NMR (d) (CD3OD): 8.34 (d, 1H), 7,66 (d, 1H),
7.48 (m, 3H), 7.25 (m, 1H), 6.86 (d, 1H), 5.88 (s, 2H), 5.56
(d, 1H, J = 7.55), 4.12 (m, 2H), 3.69 (m, 5H).
Data for 1,2,5,6-tetrahydroxyhexane-3,4-diyl bis(1-(3,5-
dichlorobenzyl)-1H-indazole-3-carboxylate) (9): foam.
1
Yield 92%. H NMR (d) (DMSO-d6): 8.33 (d, 2H), 7.84
(d, 2H), 7.72 (d, 2H), 7.50 (t, 2H), 7.38 (m, 2H), 7.20 (m,
2H), 6.88 (d, 2H), 5.90 (s, 4H), 5.72 (m, 2H), 5.0, 4.52 and
4.10 (3bs, 4H), 3.76 (m, 2H), 3.42 (m, 4H).
Data for 2,3-dihydroxypropyl 1-(3,5-dichlorobenzyl)-1H-
indazole-3-carboxylate (12): mp 125–128 ꢁC from ethyl-
24. For experimental details see Mahfouz, N. M.;
Tarek, A.; Ahmed, K. D. Eur. J. Med. Chem. 1988,
33, 675.
1
acetate. Yield 86%. H NMR (d) (CD3OD): 8.24 (d, 1H),