Synthesis and evaluation of geometric analogs of 1α,25-dihydroxyvitamin D2 as potential therapeutics

Article abstract of DOI:10.1016/j.jsbmb.2015.08.025

An improved convergent strategy was developed for the synthesis of the previously obtained side-chain extended and rigidified analogs of 1α,25-dihydroxyvitamin D₂, PRI-1906 and PRI-1907. New (24Z) geometric isomers of the analogs, PRI-1916 and PRI-1917, were also obtained and identified. These side-chain isomers were separable by flash chromatography, as C-25 alcohols, from the synthetic precursors of PRI-1906 and PRI-1907, respectively. The structures of new analogs were determined by advanced techniques of ¹H and ¹³C NMR, including COSY, HSQC and HMBC sequences. Binding affinities of the geometric analogs PRI-1906 and PRI-1916 and their respective C-26, C-27 homologs PRI-1907 and PRI-1917 for the full-length human vitamin D receptor were determined by a fluorescence polarization competition assay. The binding affinity of (24Z) methyl analog PRI-1906 was much higher than that of (24E) analog PRI-1906, while the affinity of (24Z) ethyl analog PRI-1917 was lower than that of the respective PRI-1907. Investigation of the metabolism of these compounds by human CYP24A1 revealed they are much more resistant to CYP24A1 than 1α,25-dihydroxyvitamin D₂, indicating they could have longer-term biological effects on target tissues.

Full text of DOI:10.1016/j.jsbmb.2015.08.025

Accepted Manuscript
Title: Synthesis and evaluation of geometric analogs of
1␣,25-dihydroxyvitamin D2 as potential therapeutics
Author: Narasimha Rao Bolla Aoife Corcoran Kaori Yasuda
´
Michał Chodynski Krzysztof Krajewski Piotr Cmoch Ewa
Marcinkowska Geoffrey Brown Toshiyuki Sakaki Andrzej
Kutner
PII:
S0960-0760(15)30057-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jsbmb.2015.08.025
SBMB 4488
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date:
Revised date:
Accepted date:
11-6-2015
18-8-2015
24-8-2015
Please cite this article as: Narasimha Rao Bolla, Aoife Corcoran, Kaori Yasuda, Michal
´
Chodynski, Krzysztof Krajewski, Piotr Cmoch, Ewa Marcinkowska, Geoffrey Brown,
Toshiyuki Sakaki, Andrzej Kutner, Synthesis and evaluation of geometric analogs
of 1rmalpha,25-dihydroxyvitamin D2 as potential therapeutics, Journal of Steroid
Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2015.08.025
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Synthesis and evaluation of geometric analogs of 1,25-dihydroxyvitamin D2
as potential therapeutics
Narasimha Rao Bolla a*, Aoife Corcoranb, Kaori Yasudac, Michał Chodyńskia, Krzysztof
Krajewskia, Piotr Cmocha, Ewa Marcinkowskab, Geoffrey Brownd, Toshiyuki Sakakic,
Andrzej Kutnera
aPharmaceutical Research Institute, 8 Rydygiera, 01-793 Warsaw, Poland
bFaculty of Biotechnology, University of Wroclaw, 14a Joliot-Curie, 50-383 Wroclaw, Poland
cDepartment of Biotechnology, Faculty of Engineering, Toyama Prefectural University 5180
Kurokawa, Imizu, Toyama 939-0398, Japan
dSchool of Immunity and Infection, University of Birmingham, Vincent Drive, Edgbaston,
Birmingham, West Midlands B15 2TT, UK
*Corresponding author: Narasimha Rao Bolla, +48 22 456 3930; n.bolla@ifarm.eu
Graphical abstract
fx1
Highlights:
 Analogs of 1,25-dihydroxyvitamin D2 were synthesized by an improved strategy
 New (24Z) geometric isomers, PRI-1916 and PRI-1917, were obtained and identified
 The structures were determined by 1H and 13C NMR, using COSY, HSQC and HMBC
 Binding affinities were determined by a fluorescence polarization competition assay
 New analogs were much more resistant to CYP24A1 than1,25-dihydroxyvitamin D2
A B S T R A C T
An improved convergent strategy was developed for the synthesis of the previously obtained
side-chain extended and rigidified analogs of 1,25-dihydroxyvitamin D2, PRI-1906 and PRI-
1907. New (24Z) geometric isomers of the analogs, PRI-1916 and PRI-1917, were also
obtained and identified. These side-chain isomers were separable by flash chromatography, as
C-25 alcohols, from the synthetic precursors of PRI-1906 and PRI-1907, respectively. The
structures of new analogs were determined by advanced techniques of 1H and 13C NMR,

including COSY, HSQC and HMBC sequences. Binding affinities of the geometric analogs
PRI-1906 and PRI-1916 and their respective C-26, C-27 homologs PRI-1907 and PRI-1917
for the full-length human vitamin D receptor were determined by a fluorescence polarization
competition assay. The binding affinity of (24Z) methyl analog PRI-1906 was much higher
than that of (24E) analog PRI-1906, while the affinity of (24Z) ethyl analog PRI-1917 was
lower than that of the respective PRI-1907. Investigation of the metabolism of these
compounds by human CYP24A1 revealed they are much more resistant to CYP24A1 than
1,25-dihydroxyvitamin D2, indicating they could have longer-term biological effects on
target tissues.
This article is part of a special issue entitled “18th Workshop on Vitamin D”.
Keywords:
analogs of 1,25-dihydroxyvitamin D2
modified Julia olefination
NOE effects
VDR binding affinity
CYP24A1 metabolic stability
1. Introduction
Over several decades numerous laboratories have endeavored to unravel the complexity of
the molecular mechanisms relating to the functions of the vitamin D system [1]. In part, this
has been driven by interests in identifying synthetic analogs with a desired selective activity
profile for use as therapeutics [2] against not only metabolic, bone and skin diseases but also
to treat cancer, cardiovascular disorders, infectious and immune diseases. In regard to these
efforts we have pursued the design, synthesis and biological evaluation [3,4,5] of analogs of
1,25-dihydroxyvitamin D2 (1,25D2, Fig. 1).
Initially, we observed that out of our series of four side-chain extended and branched
analogs of 1,25D2, compounds PRI-1906 and PRI-1907, with two methyl and two ethyl at C-
25, respectively, were able to moderately inhibit proliferation and significantly activate
expression of CYP24A1 mRNA in prostate cancer cells PC-3 [6].

We also reported, for the first time, that inhibition of cellular caspase activity can induce
differentiation of AML blasts and enhance vitamin D-induced cell differentiation of these
cells. Differentiation induced by 1,25-dihydroxyvitamin D3 (1,25D3, Fig. 1) or its analog
PRI-1906 was enhanced by pan-caspase inhibitor QVD (N-(2-quinolyl)-L-valyl-L-aspartyl-
(2,6-difluorophenoxy) methylketone) to a varying degree, depending on the subtype of the
leukemia [7].
Our analogs PRI-1906 and PRI-1907 have increased cell-differentiation activities against
ex-vivo blast cells from patients with acute myeloid leukemia as compared to 1,25D3 [8]. Our
study also revealed a high variability as to the susceptibility of individual patients' blasts to
our vitamin D2 analogs.
Very recently, we have introduced 19-nor modification into the structure of our analogs
PRI-1906 and PRI-1907. We synthesized analogs PRI-5201 and PRI-5202 [9] which have
increased differentiating activity as compared to 1,25D3 and PRI-1907 [10]. In current
attempts to optimize the side-chain geometry of these analogs, for the specific hydrogen
bonding interactions of 25-hydroxyl with the amino acids residues of LBD of VDR [11], we
have now obtained (22E,24Z)-analogs of 1,25D2, PRI-1916 and PRI-1917 (Fig. 2) and tested
them for affinity for VDR. Metabolic stability of our analogs as to degradation by the enzyme
CYP24A1 [12,13] was also determined in order to select the most resistant analog [14] for
further biological evaluation.
2. Results and discussion
2.1. Synthesis of analogs PRI-1906, PRI-1907, PRI-1916 and PRI-1917
Analogs PRI-1906 and PRI-1907 were first synthesized by Julia olefination of C-22
vitamin D phenylsulphone [15].
However, the reductive dehydroxy-desulfonylation of the resulting phenylsulfonyl hydroxy
ester with sodium amalgam was a very inefficient process. We have now synthesized these
analogs by a modified Julia olefination [16,17,18] of C-22 benzothiazoyl sulfone (1, Scheme
1) and the side-chain aldehyde 2 [4]. In this process, the intermediate alkoxide is more
reactive, than for the respective phenylsulfone, and undergoes a Smiles rearrangement to the
respective sulfinate salt. This process is accompanied by the spontaneous elimination of sulfur
dioxide to give the conjugated (22E)-ester 3 directly. Based on the mechanism of a modified
Julia olefination we expected to get, as a result of olefination and Grignard reaction, (22Z)-
isomers 12 and 13 (Fig. 3) as by-products. However, very favorably, ester 3 was obtained as a

single product, not contaminated with its (22Z)-isomer. This was proved by a single signal of
18-CH3 in 1H NMR. Grignard reaction of ester 3 with MeMgBr and EtMgBr, gave the
respective alcohols 4 and 6. Quite unexpectedly, in the course of the Grignard reaction, the
(24Z)-isomers 5 and 7 were also formed, as isomerization by-products.
2.2. Structural assignment of analogs PRI-1916 and PRI-1917
The 1H and 13C NMR spectra of PRI-1917 and PRI-1907, supported by MS
data, indicated [19] that both compounds are geometric isomers. The most significant
differences were observed in the range 7.00-5.00 ppm in the 1H NMR spectra and 140-120
ppm in the 13C NMR spectra. Based on the advanced 2D NMR experiments (including
COSY, HSQC and HMBC sequences) all signals in the 1H and 13C NMR spectra were
assigned to the respective protons and carbon atoms. In the 1H NMR spectrum of PRI-
1907 protons at 5.48 (H-22) and 5.97 ppm (H-23) were of (22E)-geometry, as the coupling
constant 3J(H22-H23) was equal to 15 Hz. The same was the value of the 3J(H22-H23) for
PRI-1917, indicating unequivocally that both, PRI-1907 and PRI-1917 are (22E)-isomers. The
same was also a case for analogs PRI-1906 and PRI-1916. 1D Selective 1H NOESY sequence
allowed us to determine the mutual relation between all protons in the side-chain of analogs
PRI-1907 (Fig. 4) and PRI-1917 (Fig. 5), as well as PRI-1906 and PRI-1916.
Irradiation of H-23 proton at 5.97 ppm in the 1H NMR spectrum of PRI-1907 [19]
gave the NOE enhancement at H-24a proton at 5.28 ppm, whereas irradiation of H-22 proton
at 5.48 ppm resulted in a strong NOE effect at C-28 methyl (1.92 ppm). The same NOE effect
was also observed for H-22 proton at 5.52 ppm and C-28 methyl (1.82 ppm) for PRI-1917. On
the contrary, irradiation of the isolated signal at 7.00 ppm in PRI-1917 did not result in any
NOE effects in this range of 1H NMR shifts. These NOE enhancements clearly indicated
(22E) geometry [20] in both PRI-1907 and PRI-1917, whereas the C-24 geometry was (24E)
and (24Z) for PRI-1907 and PRI-1917, respectively. The same geometries were found for
PRI-1906 and PRI-1916, respectively.
2.3. Human VDR binding assay
Binding affinities of the new analogs PRI-1916 and PRI-1917 for human full-length VDR
were evaluated using a 1,25D3 assay kit and compared to that of 1,25D3, PRI-1906 and PRI-
1907 [10]. In this fluorescence polarization competition assay, VDR was added to a

fluorescent VDR ligand to form a receptor/tracer complex giving a high polarization value.
This complex was then added to individual test compounds to displace the tracer from the
complex. The activity of each compound is shown (Table 1) as a percentage whereby the
activity of 1,25D3 was normalized to 100%. This data was derived from dose-response curves
(data not shown)
2.4. Metabolic resistance of analogs to CYP24A1
The metabolism of each analog by human CYP24A1 (hCYP24A1) was analyzed using the
membrane fraction prepared from the recombinant E. coli cells expressing hCYP24A1 [21].
The conversion ratios of each analog to its metabolites for PRI-1906, PRI-1916, PRI-1907
and PRI-1917 were 2.3, 11, 0.8 and 10%, respectively (Table 2, Fig. 6 an d SI).
Meanwhile, native 1,25D3 and 1,25D2 were sequentially metabolized by CYP24A1 [21,22],
and their conversion ratios were 49% and 39%, respectively. These results indicated that all of
the analogs are more resistant to CYP24A1-dependent metabolism than native 1,25D3 and
1,25D2. In particular, PRI-1906 and PRI-1907 showed a remarkable resistance against
CYP24A1. Since CYP24A1 is transcriptionally induced by VDR agonists in the target tissues
to inactivate them, our analogs, especially PRI-1906 and PRI-1907, should have longer-term
biological effects in the target tissues [23].
The complete evaluation of biological activities of our analogs is underway in this
laboratory, according to the previously outlined scheme [3].
3. Conclusions
We synthesized side-chain extended and homologated (24Z)-analogs PRI-1916 and
PRI-1917 of 1,25D2 and have developed improved method for the synthesis of our leading
1,25D2 analogs PRI-1906 and PRI-1907, by a modified Julia olefination and from the
previously developed C-22 benzothiazoyl sulfone as the advanced intermediate. New analogs
were separable from their geometric isomers by flash chromatography at the level of C-25
alcohols. The structures of side-chains of analogs PRI-1916 and PRI-1917 was assigned by
advanced techniques of 1H and 13C NMR. Binding affinities of the new analogs for the human
full-length VDR were determined using fluorescence polarization competition assay. The
VDR binding affinity of new analogs were lower than that of 1,25D3. Futhermore,
introducing (24Z) geometry increased the affinity for methyl analog PRI-1916, as compared

to (24E) analog PRI-1906, while it decreased the affinity for ethyl analog PRI-1917, as
compared to (24E) analog PRI-1907, meaning that the terminal alkyls at C-25 strongly
influence the binding affinity.
Isomerization of side-chain (24E)-analogs of 1,25D2 (PRI-1906 and PRI-1907) at C24
resulted in the lowered resistance to metabolism by CYP24A1, although (24Z)-analogs (PRI-
1916 and PRI-1917) were still more resistant than native 1,25D2 and 1,25D3. These results
reveal that side-chain extended and rigidified analogs of 1,25D2, especially (24E)-analogs,
may be useful to elicit longer-term biological effects against target tissues.
Acknowledgements
The research leading to these results has received funding from the People Program (Marie
Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 under
Research Executive Agency grant agreement No. 315902. The authors (NBR and AC)
gratefully acknowledge receipt of a Marie Curie Research Associate post. EM, GB and AK
are partners within the Marie Curie Initial Training Network DECIDE (Decision-making
within cells and differentiation entities therapies). This work was presented in part at the 18th
Workshop on Vitamin D, in Delft, The Netherlands, April 21-24, 2015.
References
[1] H.F. DeLuca, Is there more to learn about functional vitamin D metabolism, J. Steroid
Biochem. Mol. Biol. 148 (2015) 3-6.
[2] D. Feldman, A.V. Krishnan, S. Swami, E. Giovannucci, B.J. Feldman, The role of vitamin
D in reducing cancer risk and progression, Nature Rev. Cancer 14 (2014) 342-357.
[3] S. Nadkarni, M. Chodynski, A. Corcoran, E. Marcinkowska, G. Brown, A. Kutner,
Double point modified analogs of vitamin D as potent activators of vitamin D receptor, Curr.
Pharm. Design 21 (2015) 1741-1763.
[4] M. Milczarek, M. Chodynski, B. Filip-Psurska, A. Martowicz, M. Krupa, K. Krajewski.
A. Kutner, J. Wietrzyk, Synthesis and biological activity of diastereomeric and geometric
analogs of calcipotriol, PRI-2202 and PRI-2205, against human HL-60 leukemia and MCF-7
breast cancer cells, Cancers 5 (2013) 1355-1378.
[5] B. Filip, M. Milczarek, J. Wietrzyk, M. Chodynski, A. Kutner, Antitumor properties of
(5E,7E) analogs of vitamin D3, J. Steroid Biochem. Mol. Biol. 121 (2010) 399-402.
[6] H. Baurska, A. Klopot, M. Kielbinski, A. Chrobak, E. Wijas, A. Kutner, E.
Marcinkowska, Structure-function analysis of vitamin D2 analogs as potential inducers of

leukemia differentiation and inhibitors of prostate cancer proliferation, J. Steroid Biochem.
Mol. Biol. 126 (2011) 46-54.
[7] X. Chen-Deutsch, A. Kutner, J. S. Harrison, G.P. Studzinski, The pan-caspase inhibitor Q-
VD-OPh has anti-leukemia effects and can interact with vitamin D analogs to increase HPK1
signaling in AML cells, Leukemia Res. 36 (2012) 884-888.
[8] H. Baurska, M. Kielbinski, P. Biecek, O. Haus, B. Jazwiec, A. Kutner, E. Marcinkowska,
Monocytic differentiation induced by side-chain modified analogs of vitamin D in ex vivo
cells from patients with acute myeloid leukemia, Leukemia Res. 38 (2014) 638-647.
[9] A. Pietraszek, M. Malinska, M. Chodynski, M. Krupa, K. Krajewski, P. Cmoch, K.
Wozniak, A. Kutner; Synthesis and crystallographic study of analogs of 1,25-
dihydroxyergocalciferol, Steroids 78 (2013) 1003-1014.
[10] A. Corcoran, M.A. Bermudez, S. Seoane, R. Perez-Fernandez, M. Krupa, A. Pietraszek,
M. Chodyński, A. Kutner, G. Brown, E. Marcinkowska, Biological evaluation of new vitamin
D2 analogs, J. Steroid Biochem. Mol. Biol. submitted to this issue.
[11] I. Billas, D. Moras, Allosteric Controls of nuclear receptor function in the regulation of
transcription, J. Mol. Biol. 425 (2013) 2317-2329.
[12] M. Takano, D. Sawada, K. Yasuda, M. Nishikawa, A. Takeuchi, K. Takagi, K. Horie,
G.S. Reddy, T.C. Chen, T. Sakaki, A. Kittaka, Synthesis and metabolic studies of 1α,2α,25-,
1α,4α,25- and 1α,4β,25-trihydroxyvitamin D3, J. Steroid Biochem. Mol. Biol. 148 (2015) 34-
37.
[13] M. Takano, S. Ohya, K. Yasuda, M. Nishikawa, A. Takeuchi, D. Sawada, T. Sakaki, A.
Kittaka, Synthesis and biological activity of 1α,2α,25-trihydroxyvitamin D3: active metabolite
of 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3 by human CYP3A4, Chem. Pharm.
Bull. 62 (2014) 182-184.
[14] T. Sakaki, K. Yasuda, A. Kittaka, K. Yamamoto, T.C. Chen, CYP24A1 as a potential
target for cancer therapy, Anticancer Agents Med. Chem. 14 (2014) 97-108.
[15] M. Chodynski, J. Wietrzyk, E. Marcinkowska, A. Opolski, W. Szelejewski, A. Kutner,
Synthesis and antiproliferative activity of side-chain unsaturated and homologated analogs of
1,25-dihydroxyvitamin D2, (24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol
and congeners, Steroids 67 (2002) 789-798.
[16] A. Glebocka, R.R. Sicinski, L.A. Plum, M. Clagett-Dame, H.F. DeLuca, New 2-
alkylidene 1α,25-dihydroxy-19-norvitamin D3 analogues of high intestinal activity: synthesis
and biological evaluation of 2-(3'-alkoxypropylidene) and 2-(3'-hydroxypropylidene)
derivatives, J. Med. Chem. 49 (2006) 2909-2920.

[17] A. Kutner, Convergent synthesis of vitamin D analogs, Przem. Chem. 85 (2006) 322-329
(Pol).
[18] A. Kutner, J. Martynow, M. Chodynski, W. Szelejewski, H. Fitak, M. Krupa, Process of
preparation of 24-alkyl analogs of cholecalciferol and intermediate compounds, US 7915241
B2 (2011); CA 2003-2481909.
[19] 1H and 13C NMR data of new analogs:
9, PRI-1916: 1H NMR (600 MHz, CD3OD, 25.8 °C, TMS) δ = 6.988 (d, J = 15.6 Hz, 1H, 23-
H), 6.324 (d, J = 11.4 Hz, 1H, 7-H), 6.084 (d, J = 10.8 Hz, 1H, 6-H), 5.523 (dd, J1 = 15 Hz, J2
= 9 Hz, 1H, 22-H), 5.377 (s, 1H, 24a-H), 5.286 (s, 1H, 19E-H), 4.897 (s, 1H, 19Z-H), 4.349
(t, J1 = J2 = 6 Hz, 1H, 1-H), 4.126 (t, J1 = J2 = 5.4 Hz, 1H, 3-H), 1.774 (s, 3H, 28-CH3), 1.352
(s, 6H, 26-CH3, 27-CH3), 1.078 (d, J = 7.2 Hz, 3H, 21-CH3), 0.601 (s, 3H, 18-CH3); 13C NMR
(600 MHz, CD3OD, 25.8 °C, TMS): δ =149.79 (C=CH2, C-10), 142.33 (C=C H2, C-8),
139.29 (C=CH2, C-5), 136.39 (C=CH2, C-24a), 135.78 (C=CH2, C-23), 133.72 (C=CH2, C-
24), 126.84 (C=CH2, C-22), 124.83 (C=CH2, C-6), 119.05 (C=CH2, C-7), 112.07 (C=CH2,
C-19), 77.10 (C-OH, C-25), 71.46 (C-OH, C-1), 67.38 (C-OH, C-3), 57.72 (C-CH2, C-14),
57.59 (C-CH2, C-17), 46.97 (C-CH2, C-4), 46.15 (C-CH2, C-13), 43.70 (C-CH2, C-2), 42.26
(C-CH3, C-20), 41.72 (C-CH2, C-12), 29.98 (C-CH2, C-9), 28.88 (C-CH2, C-16), 24.60 (C-
CH2, C-15), 23.25 (C-CH2, C-11), 21.29 (C-21), 21.19 (C-28), 12.96 (C-18), 12.69 (C-26),
12.68 (C-27). HRMS: calc‘d for C29H44O3 [M+Na]+, 463.3188 found: 463.3190.
11, PRI-1917: 1H NMR (600 MHz, CD3OD, 25.8 °C, TMS): δ = 6.997 (d, J = 15.6 Hz, 1H,
23-H), 6.321 (d, J = 11.4 Hz, 1H, 7-H), 6.082 (d, J = 11.4 Hz, 1H, 6-H), 5.515 (dd, J1 = 15.6
Hz, J2 = 8.4 Hz, 1H, 22-H), 5.284 (s, 1H, 24a-H), 5.168 (s, 1H, 19E-H), 4.896 (s, 1H, 19Z-H),
4.347 (t, J1 = 6, J2 = 5.4 Hz, 1H, 1-H), 4.123 (t, J1 = 6.6 Hz, J2 = 4.8 Hz, 1H, 3-H), 1.811 (s,
3H, 28-CH3), 1.441-1.372 (m, 6H, 26-CH3, 27-CH3), 1.078 (d, J = 7.2 Hz, 3H, 21-CH3),
0.601 (s, 3H, 18-CH3). 13C NMR (600 MHz, CD3OD, 25.8 °C, TMS): δ = 149.79 (C=CH2,
C-10), 142.42 (C=C H2, C-8), 138.00 (C=CH2, C-5), 135.73 (C=CH2, C-24a), 135.00
(C=CH2, C-23), 133.58 (C=CH2, C-24), 127.84 (C=CH2, C-22), 124.85 (C=CH2, C-6), 119.02
(C=CH2, C-7), 112.07 (C=CH2, C-19), 77.32 (C-OH, C-25), 71.48 (C-OH, C-1), 67.39 (C-
OH, C-3), 57.87 (C-CH2, C-14), 57.60 (C-CH2, C-17), 46.96 (C-CH2, C-4), 46.15 (C-CH2,
C-13), 43.69 (C-CH2, C-2), 42.24 (C-CH3, C-20), 41.74 (C-CH2, C-12), 35.49 (C-CH3, C-
26), 35.35 (C-CH3, C-27), 29.98 (C-CH2, C-9), 28.79 (C-CH2, C-16), 24.61 (C-CH2, C-15),
23.27 (C-CH2, C-11), 21.90 (C-21), 21.31 (C-28), 12.68 (C-18), 8.59 (C-26’), 8.522 (C-27’).
HRMS: calc‘d for C31H48O3 [M+Na]+, 491.3501 found: 491.3502.

[20] J. Wietrzyk, M. Chodynski, H. Fitak, E. Wojdat, A. Kutner, A. Opolski; Antitumor
properties of diastereomeric and geometric analogs of vitamin D3, Anti-Cancer Drugs 18
(2007) 447-457.
[21] T. Kusudo, T. Sakaki, D. Abe, T. Fujishima, A. Kittaka, H. Takayama, S. Hatakeyama,
M. Ohta, K. Inouye, Metabolism of A-ring diastereomers of 1α,25-dihydroxyvitamin D3 by
CYP24A1, Biochem. Biophys. Res. Commun. 321 (2004) 774-782.
[22]. N. Urushino, K. Yasuda, S. Ikushiro, M. Kamakura, M. Ohta, T. Sakaki, Metabolism of
1α,25-dihydroxyvitamin D2 by human CYP24A1, Biochem. Biophys. Res. Commun. 384
(2009) 144-148.
[23]. T. Sakaki, K. Yasuda, A. Kittaka, K. Yamamoto, T. C. Chen, CYP24A1 as a potential
target for cancer therapy, Anticancer Agents Med. Chem. 14 (2014) 97-108.
Scheme 1. Convergent synthesis of side-chain (22E,24E)-analogs of 1α,25-
dihydroxyvitamin D2 (8, PRI-1906 and 10, PRI-1907) and (22E,24Z)-analogs of 1α,25-
dihydroxyvitamin D2 (9, PRI-1916 and 11, PRI-1917).
Fig. 1. Structures of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2.
Fig. 2. Structures of side-chain homologated and unsaturated analogs of 1,25-
dihydroxyvitamin D2.
Fig. 3. Structures of hypothetic (22Z)-analogs of 1,25-dihydroxyvitamin D2.
Fig. 4. Structure of 10 (PRI-1907) showing NOE assignments for characteristic protons in the
side-chain.
Fig. 5. Structure of 11 (PRI-1917) showing NOE assignments for characteristic protons in the
side-chain.
Fig. 6. HPLC profiles of 1,25D3, 1,25D2, analogs PRI-1906 and PRI-1916 and their
metabolites generated by human CYP24A1. The peaks marked with arrows indicate putative
metabolites. Profiles for PRI-1907 and PRI-1917 are included in the supporting information
(SI).

Table 1
Binding affinity of analogs for the full-length human vitamin D receptor in a fluorescence
polarization competition assay.
Compound
1,25D3
PRI-1906
PRI-1916
PRI-1907
PRI-1917
6.172e-09
IC50
2.232e-09
5.561e-008
6.048e-09
6.848 e-008
Relative binding
affinitya
38
100
4
37
3
aPotency of 1,25D3 normalized to 100
Table 2
Metabolic conversion of active forms of vitamins D and of analogs by human CYP24A1
Compound
Metabolic
1,25D3
1,25D2
PRI-1906 PRI-1916
2.3 11
PRI-1907
PRI-1917
49
39
0.8
10
conversiona (%)
aData represent the mean of at least 2 independent experiments.