Synthesis and biological evaluation of a new vitamin D2 analogue

Article abstract of DOI:10.1016/j.bmcl.2012.07.095

A new vitamin D₂ analogue was synthesized using the Julia-Kocienski olefination. It has antiproliferative effects on cell lines from squamous cell carcinomas of colon and head and neck, but is also as hypercalcaemic as calcitriol in vivo.

Full text of DOI:10.1016/j.bmcl.2012.07.095

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6276–6279
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a new vitamin D2 analogue
Zoila Gándara a, Manuel Pérez a, Débora G. Salomón b, María J. Ferronato b, María E. Fermento b,
Alejandro C. Curino b, María M. Facchinetti b, Generosa Gómez a, Yagamare Fall a,
⇑
a Departamento de Química Orgánica, Facultad de Química, Universidad de Vigo, 36200 Vigo, Spain
b Laboratorio de Biología del Cáncer, Instituto de Investigaciones Bioquímicas Bahía Blanca, Centro Científico Tecnológico Bahía Blanca, CONICET, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A new vitamin D2 analogue was synthesized using the Julia–Kocienski olefination. It has antiproliferative
effects on cell lines from squamous cell carcinomas of colon and head and neck, but is also as hypercal-
caemic as calcitriol in vivo.
Received 12 June 2012
Revised 27 July 2012
Accepted 30 July 2012
Available online 7 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D2 analogues
Calcitriol
Cancer
Hypercalcemia
Cellular proliferation
Julia–Kocienski olefination
1a,25-Dihydroxyvitamin D3 (1b, calcitriol) (Fig. 1), the active
hormone metabolized from vitamin D3 (1a), acts as a regulator in
calcium and phosphate homeostasis.1 Apart from these classic
activities, calcitriol has proved to inhibit cellular proliferation
and to induce cellular differentiation.2 However, therapeutic use
of these latter effects of 1b is hampered by the hypercalcaemic side
effects of effective doses. This has stimulated a search for ana-
logues that combine a relatively weak systemic effect on calcium
metabolism with potent regulatory effects on cell differentiation
and proliferation.
R2
R2
H
H
HO
R1
HO
1a,
R1
2a, R1 = R2 = H
2b,
R1 = R2 = H
The metabolism and biological activity of the non-natural vita-
min ergocalciferol (2a, vitamin D2) appear to parallel those of vita-
R1 = R2 = OH
1b, R1 = R2 = OH
3
min D3, and it is in fact marketed as a treatment for refractory
Figure 1. Structures of vitamine D3 (1a) and vitamine D2 (2a) and their metabolites
1b and 2b.
rickets. In particular, 2a undergoes double hydroxylation to
1a
,25-dihydroxyvitamin D2 (2b),4 which like 1b induces cell differ-
entiation and inhibits the proliferation of a number of tumour cell
lines, including leukaemia cells5 (in spite of which, very few syn-
vitamin D2 analogues. Here we describe the synthesis of vitamin
D2 analogue 3 (Fig. 2), the 22Z side chain of which was recently
obtained serendipitously by means of a Julia–Kocienski olefin-
ation,12 together with assays of its antiproliferative activities in
various cancer cell lines and its hypercalcaemic effects in vivo.
For the synthesis of 3 we started from alcohol 4 (Scheme 1),
which is readily obtained in large quantities from vitamin D2 using
the procedures originally described by Calverley13 and later modi-
fied by Choudhry.14 TPAP oxidation of 4 afforded aldehyde 5 in
95% yield, and Julia–Kocienski olefination of 5 with sulfone 6 gave
a 65% yield of ester 7, which upon reaction with ethyl magnesium io-
dide in ether at 0°C yielded alcohol 8. Removal of the silyl protecting
theses of either 1
to date).6
a,25-(OH)2-D2 or 25-(OH)-D2 have been reported
The vitamin D2 analogue paricalcitol has been shown to be less
hypercalcaemic than calcitriol in vitro7 and to have antiprolifera-
tive activity,8,9 and in clinical trials it has proved to be partially
effective against certain cancers.10,11 These findings imply that
the search for members of the vitamin D family with low hyper-cal-
caemic activity and a high therapeutic index should not overlook
⇑
Corresponding author.
E-mail address: yagamare@uvigo.es (Y. Fall).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.095

Z. Gándara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6276–6279
6277
the effects of analogue 3 with those elicited by the natural hormone
calcitriol. As shown in Figure 3, we observed a significant decrease
in cell count after treatment with analogue 3, in the human squa-
mous cell carcinoma (A) and the human colorectal cancer (B) cell
lines. The antiproliferative effect exerted by analogue 3 in HN12
cells was similar to that elicited by calcitriol. The half maximal
inhibitory concentration (IC50) was 5.05 nM and 4.68 nM for ana-
logue 3 and calcitriol, respectively. In HCT116 cells, the IC50 was
0.76 nM and 0.01 nM for analogue 3 and calcitriol, respectively.
No effect on cell proliferation was observed in the mammary cancer
(C and D) and the glioma (E) cell lines.
OH
H
3
HO
OH
Figure 2. Structure of vitamine D2 analogue 3.
It is known that cancer cell lines display a range of sensitivities
to the anti-proliferative effects of calcitriol and its derivatives; the
reason for this is largely unknown and could result from defects in
any component in the VDR signaling pathway including VDR and
24-hydroxylase (CYP24A1).15 Calcitriol action is limited by its
catabolism, which occurs mainly by CYP24A1 resulting in
groups of 8 with TBAF in THF afforded a 98% yield of triol 9, and
photoisomerization of 9 using anthracene as sensitizer finally gave
the target analogue 3 in 90% yield. The overall yield of this five-step
synthetic sequence was an excellent 44%.
In order to test the biological activity of analogue 3, we first
analyzed the antiproliferative effects of this compound in several
cancer cell lines. For this purpose we performed cell count follow-
ing treatment of human squamous cell carcinoma (HN12), glioblas-
toma (T98G), mammary carcinoma (human T47D and murine LM3)
and colorectal cancer (HCT116) cell lines with analogue 3, calcitriol
or vehicle. We performed 120 h dose-response analyses comparing
1a,24,25 (OH)3-D3, a metabolite with substantially lower affinity
for the vitamin D receptor VDR. Although this enzyme is located
primarily in liver, it has been demonstrated to be expressed by
many tissues.15 Augmented expression of CYP24A1 has been
shown to be detrimental to calcitriol antiproliferative effects. In
prostate cancer cell lines, it has been demonstrated that enzyme
expression was inversely correlated to the antiproliferative effects
displayed by the cells.16 In this regard, analogue 3 may be degraded
by cells displaying high expression of CYP24A1, which could ac-
count for the lack of activity observed in some of the cell lines, sim-
ilarly to what occurs with calcitriol treatment. It has also been
reported that splice variants of the enzyme may have implications
for the antitumorigenic effects of the hormone and analogues.17
As already stated, vitamin D and many of its analogues induce
hypercalcemia, a fact that limits the therapeutic efficacy of these
compounds for cancer treatment. Therefore, analyses of the calce-
mic effects need to be performed for each novel analogue synthe-
sized. Because of its significant in vitro antiproliferative activity in
human squamous cell carcinoma and in colorectal carcinoma, ana-
logue 3 was then evaluated for its calcemic effects in vivo. Previous
pharmacokinetic studies performed in normal mice indicated that
O
OH
H
H
i
ii
H
5
4
O
S
O
N
S
O
OMe
TBSO
OTBS
6
TBSO
OTBS
calcitriol at 0.125 lg/mouse (approximately 5 lg/kg body weight)
results in a Cmax > 10.0 ng/mL and AUC > 40.0 ng h/mL18, exceeding
the concentration needed for calcitriol anti-tumor activity in
vitro.19 Other studies using either calcitriol or vitamin D analogues
O
OMe
iii
OH
at doses lower than 5
mor burden in animal models of cancer.20–22 Therefore we chose
the dose of 5 g/kg body weight that have antitumor effects
lg/kg body weight showed a reduction in tu-
iv
H
H
l
7
in vivo as a starting concentration for the studies of calcemia, with
the idea of increasing the doses in escalating experiments, pro-
vided no toxic effects were observed. Mice were divided into three
groups (n = 5/group) and given a daily intraperitoneal injection of
8
TBSO
OTBS
TBSO
OTBS
calcitriol, analogue 3 or vehicle at 5 lg/kg body weight for 4 days.
Blood was collected prior to dose administration, then at 24, 48, 72
and 96 h post-treatment. The health condition of the animals was
recorded. Plasma calcium levels were measured by reading the
absorbance of metallochromic indicator arsenazo III, as previously
described.23 As observed in Figure 4, analogue 3 induced hypercal-
cemia starting at 24 h of compound delivery, similar to that elicited
by calcitriol. Moreover, mice treated with analogue 3 or calcitriol
died after 4 days of treatment. Together with the high calcium
plasma levels we observed, in the calcitriol- treated mice and the
analogue 3-treated ones, the appearance of conjunctivitis, chills
and thirst which are symptoms and signs that have been associ-
ated to hypercalcemia.24 Additionally, an increase in haematocrit
was observed (50 at 72 hours; normal levels range from 39 to
4725), which is a sign of intoxication.26
OH
OH
H
H
v
3
9
HO
OH
HO
OH
Scheme 1. Reagents and conditions: (i) TPAP, NMO, CH2Cl2, molecular sieves (95%);
(ii) 6, LiHMDS, THF, À78 °C (65%); (iii) EtMgI, Et2O, 0 °C (80%); (iv) TBAF, THF (98%);
Vitamin D2 analogues such as paricalcitol have been shown to
(v) anthracene, Et3N, hm, CH2Cl2, MeOH (90%).
be less calcemic than calcitriol in in vitro studies7 and have been

6278
Z. Gándara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6276–6279
140
120
100
80
140
A
B
120
100
80
***
***
***
*
60
60
***
*
***
40
40
20
0
***
20
***
-8
***
0
-11
-11
-11
-10
-9
-7
-11
-10
-9
-8
-7
log(agonist)
log(agonist)
140
120
100
80
140
120
100
80
C
E
D
60
60
*
*
40
40
20
20
0
0
-10
-9
-8
-7
-11
-10
-9
-8
-7
log(agonist)
log(agonist)
140
120
100
80
Isopropanol
Analogue 3
Calcitriol
60
40
20
0
-10
-9
-8
-7
log(agonist)
Figure 3. Dose-response effects of analogue 3 on cellular survival and its comparison with calcitriol. (A) Human head and neck squamous cell carcinoma HN12. (B) Human
colorectal carcinoma HCT116. (C) Murine mammary adenocarcinoma LM3. (D) Human mammary adenocarcinoma T47D and (E) Human glioma T98G. Cells were exposed to
the indicated doses of vehicle (isopropanol), analogue 3 ⁄o⁄r⁄ calcitriol over a total time of 120 h. Cellular survival was expressed as percentage of the vehicle. Data points
represent means SD from five replicates; ⁄p 6 0,05 and
p 6 0001, with respect to vehicle. The experiments were repeated at least three times for each cell line.
demonstrated to have antiproliferative activity.8,9 Clinical trials
showed a partial response of paricalcitol in cancer10,11 and there-
15
***
14
Vehicle
Analogue 3
Calcitriol
fore new vitamin D2 analogues should be assayed for antitumoral
activity with the aim to identify one with low hypercalcemic ef-
fects and high therapeutic index. The study of the biologic effects
of analogue 3 showed that although it exerts antiproliferative ef-
fects in colon and head and neck squamous cell carcinomas, it dis-
plays similar hypercalcemic effects to those elicited by calcitriol.
13
***
***
12
11
10
9
***
***
***
8
7
Acknowledgments
6
5
4
This work was supported financially by the Spanish Ministry of
Foreign Affairs and Cooperation (PCI A/030052/10) and the Xunta
de Galicia (INCITE845B-2010/020 and INCITE08PXIB314255PR)
and the ANPCyT from Argentina. The work of the NMR and MS divi-
sions of the research support services of the University of Vigo
(CACTI) is also gratefully acknowledged. Zoila Gándara and Manuel
Pérez thank the Xunta de Galicia for an Angeles Alvariño contract.
3
2
1
0
0
24
48
72
96
Time (h)
Figure 4. Plasma calcium levels in mice in response to daily intraperitoneal
injections of isopropanol (vehicle), analogue 3 or calcitriol during a period of 4 days.
Animals were injected with
5
lg/kg body weight of compounds, and plasma
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treatment due to hypercalcemia. Values are means SDs from five animals in each
group. The experiment was repeated two times. ⁄⁄⁄p 6 0.0001, with respect to
vehicle.
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