Synthesis and biological activity of 1a,2a,25-Trihydroxyvitamin D3: Active metabolite of 2a-(3-Hydroxypropoxy)-1a,25-dihydroxyvitamin D3 by human CYP3A4

Article abstract of DOI:10.1248/cpb.c13-00646

Our previous studies revealed that recombinant human CYP3A4 converted 2a-(3-hydroxypropoxy)-1a,25- dihydroxyvitamin D3 (O2C3), which was a more potent binder to vitamin D receptor (VDR) than the natural hormone, 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3, 1), to 1a,2a,25-trihydroxyvitamin D3 (2). Here, we synthesized 2 using the Trost Pd-mediated coupling reaction between an A-ring precursor and a CD-ring bromoolefin and evaluated its preliminary biological activity. We found that metabolite 2 from O2C3 was still active as a VDR ligand while maintaining human VDR binding affinity (27.3% of 1a,25(OH)2D3) and HL-60 cell differentiation activity (62% of 1a,25(OH)2D3).

Full text of DOI:10.1248/cpb.c13-00646

182
Note
Chem. Pharm. Bull. 62(2) 182–184 (2014)
Vol. 62, No. 2
Synthesis and Biological Activity of 1α,2α,25-Trihydroxyvitamin D3:
Active Metabolite of 2α-(3-Hydroxypropoxy)-1α,25-dihydroxyvitamin D3
by Human CYP3A4
Masashi Takano,a Saori Ohya,a Kaori Yasuda,b Miyu Nishikawa,b Akiko Takeuchi,c
,b
,a
Daisuke Sawada,a Toshiyuki Sakaki,* and Atsushi Kittaka*
a Faculty of Pharmaceutical Sciences, Teikyo University; Itabashi-ku, Tokyo 173–8605, Japan: b Department of
Biotechnology, Faculty of Engineering, Toyama Prefectural University; Imizu, Toyama 939–0398, Japan: and c Teijin
Institute for Bio-medical Research, Teijin Pharma Ltd.; Hino, Tokyo 191–8512, Japan.
Received August 14, 2013; accepted November 1, 2013
Our previous studies revealed that recombinant human CYP3A4 converted 2α-(3-hydroxypropoxy)-1α,25-
dihydroxyvitamin D3 (O2C3), which was a more potent binder to vitamin D receptor (VDR) than the natural
hormone, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, 1), to 1α,2α,25-trihydroxyvitamin D3 (2). Here, we syn-
thesized 2 using the Trost Pd-mediated coupling reaction between an A-ring precursor and a CD-ring bro-
moolefin and evaluated its preliminary biological activity. We found that metabolite 2 from O2C3 was still
active as a VDR ligand while maintaining human VDR binding affinity (27.3% of 1α,25(OH)2D3) and HL-60
cell differentiation activity (62% of 1α,25(OH)2D3).
Key words 1α,2α,25-trihydroxyvitamin D3; vitamin D receptor; cell differentiation; CYP3A4; CYP24A1
The active metabolite of vitamin D3, 1α,25-dihydroxy- (PMB) protecting group was removed by 2,3-dichloro-5,6-
vitamin D3 [1α,25(OH)2D3, 1], plays important roles in cellular dicyano-p-benzoquinone (DDQ) to give 7, and the methoxy-
growth, differentiation, apoptosis, and immune responses, in methyl (MOM) group was introduced instead for the next
addition to its classical major roles in calcium homeostasis bromination reaction using N-bromosuccinimide (NBS). NBS
and bone mineralization.1–4) Actually, 1 and several synthetic treatment for the resulting O-MOM-protected bezylidene ac-
analogs of 1 have been used clinically in the treatment of bone etal gave bromide 8. Activated Zn-reduction in the presence
diseases, secondary hyperparathyroidism, psoriasis, and osteo- of NaBH3CN produced alcohol 9, which was converted to
porosis.1,5) Although 1 is inactivated by CYP24A1-dependent epoxide 10 via tosylation followed by tetrabutylammonium
catabolism via C-24 hydroxylation to calcitroic acid for excre- fluorideꢀ(TBAF)ꢀtreatment.ꢀTrimethylsilylꢀ(TMS)–ethynylationꢀ
tion from the body,1) it was found that some 2α-substituted ac- of 10 afforded enyne 11, and subsequent solvolysis in K2CO3–
tive vitamin D analogs were highly resistant to CYP24A1, for MeOH and O-silylation provided enyne 12 (Chart 1).
example, the kcat/Km value of 2α-(3-hydroxypropoxy)-1α,25-
Theꢀ CD-ringꢀ bromoolefinꢀ 1320) and enyne 12 obtained
dihydroxyvitamin D3 (O2C3), which showed 1.8-times greater above were connected using Pd-catalyst to give the coupling
bindingꢀ affinityꢀ forꢀ vitaminꢀ Dꢀ receptorꢀ (VDR)ꢀ thanꢀ 1,6–10) product 14.21) Desilylation by TBAF and deacetalization under
was only 3% of that for 1.11)ꢀCYP24A1ꢀisꢀtheꢀspecificꢀenzymeꢀ acidic conditions gave the target molecule 222) (Chart 2). The
induced by the VDR-ligand (1 or its analog) complex in the isolatedꢀ productꢀ wasꢀ re-purifiedꢀ byꢀ HPLCꢀ toꢀ testꢀ biologicalꢀ
target tissue and inactivates 1 and its analogs; therefore, activity.
CYP24A1-resistant ligands would have long-term biological
Theꢀ bindingꢀ affinityꢀ ofꢀ theꢀ newꢀ analogꢀ 2 for the human
effects on the target tissues.12) On the other hand, CYP3A4 vitamin D receptor (hVDR)23) and induction activity of
is a broad-spectrum drug-metabolizing P450 enzyme,13) but 1 HL-60ꢀ cellꢀ differentiation24) are shown in Table 1. The new
and its analog 2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin analog 2 was still active, like its 2β-diastereoisomer 4,14) and
D3 (O1C3) are not primary substrates for CYP3A4.12) Recently, these results were different from 4-OH analogs of 1α,4α,25-
however, we demonstrated that O2C3 was metabolized by trihydroxyvitamin D3 and 1α,4β,25-trihydroxyvitamin D3,
CYP3A4 and converted to 1α,2α,25-trihydroxyvitamin D312) which were very weak agonistic ligands for hVDR (for hVDR
(2, Fig. 1). Its 2β-isomer (4) is a known compound and shows bindingꢀ affinity:ꢀ 0.9%ꢀ andꢀ 2.9%ꢀ ofꢀ theꢀ naturalꢀ hormoneꢀ 1,
potent 1α,25(OH)2D3-like activities,14) and we report here the respectively).25) The 2α-OH analog 2 showed lower bind-
synthesis of a new 2α-hydroxylated analog 2 using the Trost ingꢀ affinityꢀ forꢀ hVDRꢀ thanꢀ thatꢀ ofꢀ theꢀ naturalꢀ hormoneꢀ 1,
Pd-mediated coupling reaction between an A-ring precursor and X-ray cocrystallographic analysis of 2α-methyl-1α,25-
12ꢀ andꢀ aꢀ CD-ringꢀ bromoolefinꢀ 13 to evaluate its preliminary dihydroxyvitamin D3, which was a better binder for hVDR
biological activity.15–18)
than 1,ꢀ inꢀ theꢀ ligandꢀ bindingꢀ domainꢀ (LBD)ꢀ ofꢀ hVDRꢀ ex-
The A-ring precursor 12 for Trost coupling was prepared plained the 2α-methyl group of 2ꢀwasꢀfittedꢀinꢀtheꢀhydrophobicꢀ
from the known epoxide 519)ꢀ inꢀ 11ꢀ stepsꢀ (Chartꢀ 1).ꢀ Briefly,ꢀ pocketꢀ formedꢀ byꢀ Phe150,ꢀ Leu233,ꢀ andꢀ Ser23710); therefore,
treatment of 5 with p-methoxybenzyl alkoxide with heating the 2α-OH group of 2 at the same position as the 2α-methyl
gave methyl 3-O-(p-methoxybenzyl)altropyranoside 6, which groupꢀ wouldꢀ notꢀ beꢀ suitableꢀ forꢀ theꢀ pocketꢀ toꢀ bindꢀ theꢀ LBDꢀ
had the 2α-hydorxy group (steroidal numbering) required in of hVDR. The 2β-OH group with the different direction in the
target molecule 2. After 2-O-silylation, the p-methoxybenzyl LBDꢀmayꢀnotꢀbeꢀdisturbedꢀinꢀbinding.14)
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
© 2014 The Pharmaceutical Society of Japan
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀꢀtsakaki@pu-toyama.ac.jp;ꢀ
akittaka@pharm.teikyo-u.ac.jp

February 2014
183
Fig. 1. Structures of 1α,25-Dihydroxyvitamin D3 (1) and 2-Oxygenated Analogs 2–4
Chart 1. Synthetic Route to A-Ring Precursor 12
Chart 2. Synthesis of 1α,2α,25-Trihydroxyvitamin D3 (2)
Tableꢀ 1.ꢀ RelativeꢀBindingꢀAffinityꢀforꢀhVDRꢀandꢀHL-60ꢀCellꢀDifferentiationꢀActivityꢀofꢀ1–4
Compound
hVDRꢀbindingꢀaffinitya)
HL-60ꢀcellꢀdifferentiationa,b)
1α,25(OH)2D3 (1)
1α,2α,25(OH)3D3 (2)
O2C3 (3)
100
100
62
27.3
180c)
110d)
55
44d)
1α,2β,25(OH)3D3 (4)
a) The potency of 1α,25(OH)3D3 is normalized to 100. b) Relative activity is calculated at EC50. c)ꢀReferencesꢀ6ꢀandꢀ7.ꢀd) Reference 14.

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11) Abe D., Sakaki T., Kusudo T., Kittaka A., Saito N., Suhara Y.,
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13)ꢀ ZhuꢀJ.,ꢀDeLucaꢀH.ꢀF.,ꢀArch. Biochem. Biophys., 523, 30–36 (2012).
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15) Synthesis of the A-ring precursor of 2ꢀwasꢀreported,ꢀsee:ꢀPosnerꢀG.ꢀ
H., Nelson T. D., J. Org. Chem., 56, 4339–4341 (1991).
16) Previously, synthesis of 1α,2α,25-trihydroxy-19-norvitamin D3 was
reported,ꢀsee:ꢀSicinskiꢀR.ꢀR.,ꢀPerlmanꢀK.ꢀL.,ꢀDeLucaꢀH.ꢀF.,ꢀJ. Med.
Chem., 37,ꢀ3730–3738ꢀ(1994).ꢀSeeꢀalsoꢀrefs.ꢀ17ꢀandꢀ18.
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4,ꢀ671–674ꢀ(2013).
Conclusion
We synthesized a new analog of active vitamin D3,
1α,2α,25-trihydroxyvitamin D3 (2), which was the major
metabolite of O2C3 by CYP3A4, to study its biological ac-
tivity. 2ꢀ showedꢀ moderateꢀ hVDRꢀ bindingꢀ affinityꢀ andꢀ potentꢀ
HL-60ꢀ cellꢀ differentiationꢀ activityꢀ (EC50 68nM:ꢀ 62%ꢀ activ-
ity of 1). The data demonstrate that 2 and 4 with 2α- and
2β-stereochemistries of the 2-OH group on the active vitamin
DꢀskeletonꢀmaintainꢀVDRꢀbindingꢀandꢀHL-60ꢀcellꢀdifferentia-
tion activity. This is a totally different effect from that of the
4-OH group.25) Although O2C3 was resistant to CYP24A1, its
CYP3A4 metabolite 2 was further metabolized by CYP24A1,
similar to 1. The kcat/Km value of hCYP24A1 for 2 was 60%
of that for 1.12) Further biological testing is underway in our
laboratories.
Acknowledgments This work was supported in part by a
Grant-in-Aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (No. 25860011 to M.T.) as
well as a Grant-in-Aid from the Japan Society for the Promo-
tion of Science (No. 24590021 to A.K.).
21) Trost B. M., Dumas J., Villa M., J. Am. Chem. Soc., 114, 9836–9845
(1992).
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λmax 266.5nm, λminꢀ227.5ꢀnm;ꢀIRꢀ(neat)ꢀ3379,ꢀ2927,ꢀ2858,ꢀ1724,ꢀ1643,ꢀ
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1462,ꢀ 1377,ꢀ 1284,ꢀ 1219,ꢀ 1142,ꢀ 1072,ꢀ 918ꢀcm−1 1H-NMR (400MHz,
;
CDCl3) δ:ꢀ0.53ꢀ(s,ꢀ3H),ꢀ0.93ꢀ(d,ꢀJ=6.3ꢀHz,ꢀ3H),ꢀ1.21–1.76ꢀ(m,ꢀ27H),ꢀ
1.96–2.01ꢀ(m,ꢀ2H),ꢀ2.16–2.26ꢀ(m,ꢀ2H),ꢀ3.70–3.72ꢀ(m,ꢀ1H),ꢀ4.41ꢀ(ddd,ꢀ
J=7.1,ꢀ13.7,ꢀ14.1ꢀHz,ꢀ1H),ꢀ4.42ꢀ(ddd,ꢀJ=7.1,ꢀ13.7,ꢀ14.2ꢀHz,ꢀ1H),ꢀ5.01ꢀ
(d, J=1.7ꢀHz,ꢀ1H),ꢀ5.27ꢀ(d,ꢀJ=0.98Hz, 1H), 5.96 (d, J=11.2Hz, 1H),
6.40 (d, J=12.0Hz, 1H). 13C-NMR (100MHz, CDCl3) δ:ꢀ12.1,ꢀ18.8,ꢀ
20.8,ꢀ 22.2,ꢀ 23.5,ꢀ 27.6,ꢀ 29.2,ꢀ 29.4,ꢀ 29.7,ꢀ 36.1,ꢀ 36.4,ꢀ 40.5,ꢀ 41.3,ꢀ 44.4,ꢀ
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assay kit (Polarscreen Vitamin D Receptor Competitor Assay, Red,
Cat. No. PV4569) purchased from Invitrogen.
24)ꢀ Potencyꢀ ofꢀ inductionꢀ ofꢀ HL-60ꢀ cellꢀ differentiationꢀ wasꢀ testedꢀ asꢀ
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