HETEROCYCLES

Review | Special issue | Vol. 86, No. 1, 2012, pp. 69-88
Received, 12th March, 2012, Accepted, 6th April, 2012, Published online, 11th April, 2012.
DOI: 10.3987/REV-12-SR(N)1

■ Surveying the Effects of Eldecalcitol and Related Analogs from a Biological Perspective

Noboru Kubodera*

International Institute of Active Vitamin D Analogs, 35-6, Sankeidai, Mishima, Shizuoka 411-0017, Japan

Abstract

In the previous review paper, explorative and developmental researches of eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3), an analog of active vitamin D3, calcitriol (1α,25-dihydroxyvitamin D3), were introduced. Eldecalcitol possesses potent effects on bone disease such as osteoporosis. The completion of a phase III clinical trial of eldecalcitol for bone fracture prevention in comparison with alfacalcidol (1α-hydroxyvitamin D3), prodrug of calcitriol, produced excellent results. Although clinically, eldecalcitol showed greater potency than calcitriol/alfacalcidol, the detailed physiological properties and mechanism of action of the enhanced activity of eldecalcitol toward bone remains to be clarified. To explore structure-activity relationships, related analogs of eldecalcitol have been synthesized with inherent biological background of each targeted analogs. These include epimeric analogs at 1, 2, 3, and 20 positions, nor analog at 19 position and deoxy analogs at 1 and 25 positions. This review discusses eldecalcitol and related analogs in a biological perspective. The synthetic features of analogs are also outlined.

CONTENTS
1. Introduction
2. Epimeric analogs at 1, 2, 3, and 20 positions
2-1. 20-Epieldecalcitol
2-2. 3-Epieldecalcitol
2-3. 1-Epieldecalcitol
2-4. 1,3-Diepieldecalcitol
2-5. 2-Epieldecalcitol and 2,20-diepieldecalcitol
3. Nor analog at 19 position and deoxy analogs at 1 and 25 positions
3-1. 19-Noreldecalcitol
3-2. 25-Deoxyeldecalcitol
3-3. 1-Deoxyeldecalcitol
4. Conclusion
5. Acknowledgments
6. References

1. INTRODUCTION
It is well-established that vitamin D3 (cholecalciferol 1) ingested into foods or synthesized in the skin is metabolized to 25-hydroxyvitamin D3 (calcifediol 2) in the liver, which is further hydroxylated at the 1α position in the kidney to produce the active form, 1α,25-dihydroxyvitamin D3 (calcitriol 3).1 Calcitriol (3) is well recognized as a potent regulator of calcium and phosphorous metabolism while also possessing regulatory effects on cell proliferation and differentiation processes.2 In Japan, calcitriol (3) and its synthetic prodrug, 1α-hydroxyvitamin D3 (alfacalcidol 4), which is also activated to 3 in the body (liver and bone), have been widely used for the treatment of osteoporosis for more than a quarter-century.3 Calcitriol (3) and alfacalcidol (4) have been recognized as very safe

medicines that show mild or moderate increase in bone mineral density (BMD) in osteoporotic patients. There exists intense interest in obtaining active vitamin D3 analogs more potent than 3 and 4 towards increasing BMD and preventing bone fracture with less calcemic activity for treating osteoporosis. 1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (eldecalcitol 5, developing code; ED-71), which possesses a hydroxypropoxy substituent at the 2β position of the A-ring of 3, is such an analog that shows potent effects on bone therapy.4-6 Recent completion of a phase III trial of 5 for bone fracture prevention and BMD increase in comparison with alfacalcidol (4) produced excellent results.7,8 The marketing of eldecalcitol (5) with the sales name of Edirol as an excellent medicine for the treatment of osteoporosis has started very recently in Japan by Chugai Pharmaceutical Co., Ltd (Figure 1).

Figure 2 illustrates the basic relationship between calcemic activity (serum calcium) and the targeted effect on bone (BMD increase) with cholecalciferol (1), calcitriol (3)/alfacalcidol (4), and eldecalcitol (5).9 The potent effect on bone is highest with 5 followed by 3 and 4 and then 1, at doses that induce approximately the same level of serum calcium (Figure 2). The question is what is the different mode-of-action between calcitriol (3)/alfacalcidol (4) and eldecalcitol (5). To explore structure-activity relationships, related analogs of eldecalcitol (5) have been synthesized by Chugai group or other groups with inherent biological background of each targeted analogs. These include epimeric analogs at 1, 2, 3, and 20 positions, nor analog at 19 position and deoxy analogs at 1 and 25 positions. This review discusses eldecalcitol (5) and related analogs, 20-epieldecalcitol (6), 3-epieldecalcitol (7), 1-epieldecalcitol (8), 1,3-diepieldecalcitol (9), 2-epieldecalcitol (10), 2,20-diepieldecalcitol (11), 19-noreldecalcitol (12), 25-deoxyeldecalcitol (13), and 1-deoxyeldecalcitol (14), from a biological perspective (Figure 3). The synthetic features of the analogs are also outlined.

2. EPIMERIC ANALOGS AT 1, 2, 3, AND 20 POSITIONS
2-1. 20-EPIELDECALCITOL (6)10,11
It has been reported that 20-epicalcitriol, a diastereomer of calcitriol (3), which possesses an inverted C21 methyl substituent at the 20 position of the side chain of 3, shows remarkably enhanced biological activities compared to parent compound 3.12 For example, 20-epicalcitriol exhibits 18 times the potency of induction of differentiation in human myeloid leukemia cells (HL-60).13 Furthermore 20-epicalcitriol shows 50 times the inhibition of proliferation in human histiocytic lymphoma cells (U937)14 and a 4.5 fold increase in osteocalcin concentration in human osteosarcoma cells (MG-63)15 compared to calcitriol (3). These findings prompted our interest in an analog of eldecalcitol (5) epimerized at the 20 position and its biological responses. We, therefore, synthesized 20-epieldecalcitol (6) and investigated preliminary biological activities using HL-60, U937, and MG-63 compared to 5.

The synthesis of 20-epieldecalcitol (6) was envisioned using the Trost coupling reaction of A-ring fragment 22 with C/D-ring fragment 37.16,17 First, the required A-ring fragment 22 was synthesized from C2-symmetrical epoxide 15 based upon the methodology that has been previously established by us (Scheme 1).18 Thus, cleavage of 15 with 1,3-propanediol in the presence of t-BuOK gave diol 16 in 86% yield. After protection of the primary hydroxy group to give pivalate 17 in 88% yield, cleavage of the benzyl ether moiety in 17 and subsequent protection of the resulting 1,2-diol as the acetonide gave alcohol 18 in 87% overall yield. Swern oxidation of 18 and subsequent Grignard reaction of the resulting aldehyde with vinylmagnesium bromide followed by pivaloylation of the alcohol afforded dipivalate 19 as an epimeric mixture (R/S=3/2). Without separation of the epimeric mixture, the acetonide moiety in 19 was cleaved quantitatively to give diol 20. Exposure of 20 to Mitsunobu conditions afforded epoxide 21 in 77% yield. The acetylene unit was successfully installed by the regioselective epoxide opening of 21 with lithium TMS acetylide in the presence of BF3-OEt2 to provide enyne 22 as the A-ring fragment in 36% yield after protecting group exchange from pivalate to TBS ether. The accompanied (S)-isomer 23, which consists of the requisite stereochemistry to obtain 1-epieldecalcitol (8), was separated in 24% yield by simple column chromatography (Scheme 1). Next, we performed the synthesis of C/D-ring fragment 37 from the Inhoffen-Lythgoe diol (24), which is obtained by ozonolysis of vitamin D2.19 Based on the reported route to 37 from 24,20,21 we developed a convenient approach for the facile introduction of the C23 – C27 side chain moiety as shown in Scheme 2. Upon treatment of A-ring fragment 22 and C/D-ring fragment 37 with Pd(PPh3)4 and Et3N, the coupled product 38 was obtained in 42% yield. Deprotection of the TBS group with 47% HF afforded 20-epieldecalcitol (6) in 73% yield (Scheme 2).10,11

The results of preliminary in vitro biological evaluation of 20-epieldecalcitol (6) in comparison with eldecalcitol (5) and calcitriol (3) are summarized in Table 1. As anticipated, 20-epieldecalcitol (6) showed greatly enhanced activity toward the induction of HL-60 differentiation (6085.9/49.6=122.7 times), inhibition of U937 proliferation (738.74/4.15=178.0 times), and increase in osteocalcin concentration in MG-63 (2980/15=198.7 times), compared to eldecalcitol (5).10,11 Based on these encouraging in vitro results, we are very interested in in vivo biological activity of 20-epieldecalcitol (6) on bone.

2-2. 3-EPIELDECALCITOL (7)22,23
It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by calcitriol (3).24 Interestingly during the clinical development of eldecalcitol (5), serum intact PTH in osteoporotic patients did not change significantly upon treatment with 5, although the reason remains unclear.25 Brown group reported that epimerization of calcitriol (3) at the 3 position plays a major role in hormone activation and inactivation, especially in parathyroid cells.26 It has been also reported that 3-epicalcitriol, an epimer of calcitriol (3) at the 3 position, shows equipotent and prolonged activity compared to 3 at suppressing PTH secretion.27 Since eldecalcitol (5) has a bulky hydroxypropoxy substituent at the 2 position, epimerization of 5 at the adjacent and sterically hindered 3 position might be prevented. This could be the reason why eldecalcitol (5) showed weak potency in PTH suppression during clinical studies. Therefore, we have significant interest in eldecalcitol (5) epimerization at the 3 position and the biological potency of 3-epieldecalcitol (7) in suppressing PTH production.
The synthesis of 3-epieldecalcitol (7) was also accomplished using the Trost coupling methodology. As shown in Scheme 3, the preparation of the A-ring fragment 47 began with inversion of the C3 configuration of alcohol 16 which was obtained from C2-symmetrical epoxide 15 during the synthesis of 20-epieldecalcitol (6). Thus, reaction of 16 with p-(NO2)PhCO2H in the presence of diethylazodicarboxylate (DEAD) and Ph3P gave the p-nitrobenzoate 39 in 84% yield. After hydrolysis of 39 (86%) and subsequent

acetonide 41 formation (88%), Swern oxidation of 41 followed by Grignard reaction of the resulting aldehyde produced alcohol 42 as an epimeric mixture (S/R=3/2) in 66% yield. To separate this epimeric mixture, 42 was subjected to lipase-catalyzed acetylation using vinyl acetate and Novozyme. As a result, the R-epimer preferentially underwent acetylation to give acetate 43 and S-42 (R/S=1/20) in 40% and 57% yields, respectively. Acetate 43 was converted to A-ring fragment 47 by a similar reaction sequence as in the preparation of the A-ring fragment 22 for 20-epieldecalcitol (6). The A-ring fragment 47 was allowed to react with C/D-ring fragment 48, obtained from the Inhoffen-Lythgoe diol by known method, in the condition of the Trost coupling reaction to give the desired coupling product 49. Deprotection of the TES groups afforded 3-epieldecalcitol (7) (Scheme 3).22,23
The results of preliminary in vitro biological evaluation of 3-epieldecalcitol (7) in comparison with eldecalcitol (5), calcitriol (3), and 3-epicalcitriol are summarized in Table 2. 3-Epieldecalcitol (7) showed only slight inhibition of PTH secretion in cultured bovine parathyroid cells compared to eldecalcitol (5). In our assays, 3-epicalcitriol did not show greater activity than calcitriol (3) in suppressing PTH secretion. The inhibitory potency of analogs were calcitriol (3) > eldecalcitol (5) > 3-epicalcitriol >> 3-epieldecalcitol (7), and well-responsible for affinity to human recombinant vitamin D receptor (VDR) as also shown in Table 2. Regarding the affinity to human vitamin D binding protein (DBP) which was previously known in the rat DBP case, eldecalcitol (5) showed more potent affinity than calcitriol (3). This increase in DBP affinity is due to the existence of a hydroxypropoxy substituent at the 2β position and was also observed in the 3-episeries – 3-epicalcitriol: 8.3 and 3-epieldecalcitriol (7): 113.1 – as shown in Table 2.22,23 Eldecalcitol (5) and 3-epieldecalcitol (7) appear to be inherently weak agents toward PTH suppression. This should be examined further with in vivo studies using renal insufficient animal models such as 5/6 nephrectomized rats showing high level of serum PTH.28 Nevertheless, the less potent activity of eldecalcitol (5) toward PTH suppression compared to calcitriol (3) might be a beneficial characteristic of 5 for treating osteoporosis.

2-3. 1-EPIELDECALCITOL (8)29,30
Although the detailed mode of action of enhanced activity of eldecalcitol (5) beyond calcitriol (3) and alfacalcidol (4) toward bone remains to be clarified, the long duration of 5 in the blood stream arises from its strong affinity for DBP (2-fold~4-fold in comparison with 3) and might explain, in part, the enhanced biological effects of 5. We, therefore, were highly interested in an analog with strong affinity for DBP. It was reported that the epimerization of calcitriol (3) at the 1 position remarkably enhances the affinity for DBP. Norman and co-workers reported that 1-epicalcitriol shows a 65.7-fold increase in affinity for DBP as comparered to 3.31 These findings stimulated our interest in the biological profile of epimeraized eldecalcitol at the 1 position namely, 1-epieldecalcitol (8), including DBP affinity and its effects on bone.

As previously mentioned, in our preparation of A-ring fragment 22 for convergent route to 20-epieldecalcitol (6), epimeric epoxide 21 produced the (R)-isomer 22 as separable diastereomeric mixture along with (S)-isomer 23 in a 3:2 ration (Scheme 1). The A-ring fragment 51, obtained from 21, possesses the requisite stereochemistry for 1-epieldecalcitol (8) (Scheme 4). Thus, the Trost coupling reaction of 51 with excess bromomethylene 48 gave triene 52 which was desilylated to 1-epieldecalcitol (8) in 37% yield from 51 (Scheme 4).30
As anticipated, 1-epieldecalcitol (8) showed enhanced affinity for DBP (1.6-fold in comparison with eldecalcitol) (Table 3). Further in vivo biological evaluations of 8 using ovariectomized rats model for osteoporosis in comparison with eldecalcitol (5) would be highly interesting.

2-4. 1,3-DIEPIELDECALCITOL (9)32
With completion of the synthesis of 3-epieldecalcitol (7) and 1-epieldecalcitol (8) and to further explore structure-activity relationships between eldecalcitol (5) and related analogs, we focused significant attention on the epimer of 5, at both 1 and 3 positions of the A-ring, namely 1,3-diepieldecalcitol (9). The synthesis of the A-ring fragment 57 of 1,3-diepieldecalcitol (9) started from the alcohol S-42 which was obtained from the previous lipase-catalyzed acetylation of 41 as the unreacted (S)-isomer. The alcohol S-42 possesses the requisite stereochemistry at positions 1, 2 and 3 of the A-ring that comprises 9. Acetylation of S-42 gave acetate 53 in 80% yield which was converted to A-ring fragment 57 by a similar reaction sequence to the preparation of the A-ring fragment 47 for 3-epieldecalcitol (7). The A-ring fragment 57 was allowed to react with C/D-ring fragment 48 under the Trost coupling conditions to give the coupled product 58, which was desilylated to 1,3-diepieldecalcitol (9) (Scheme 5).
Although 1,3-diepieldecalcitol (9), in combination with others, is anticipated to enhance our understanding of the mode-of-action of medicinally important eldecalcitol (5), the detailed biological characterization of 9 in comparison with eldecalcitol (5), 3-epieldecalcitol (7) and 1-epieldecalcitol (8) remains to be clarified.32

2-5. 2-EPIELDECALCITOL (10) AND 2,20-DIEPIELDECALCITOL (11)13
2-Epieldecalcitol (10) and 2,20-diepieldecalcitol (11) were synthesized by Kittaka group during their own modification studies on A-ring part of calcitriol (3) to obtain analogs with high VDR affinity.13 Methyl α-D-glucoside (59) was converted to the known epoxide 60.33 Treatment of 60 with 1,3-propanediol in the presence of t-BuOK at 110 °C followed by O-silylation afforded protected methyl 3-O-(3-hydroxypropoxy)altropyranoside (61) in 90% yield, in which the chiralities of C2, C3, and C4 satisfy the 3β, 2α, and 1α stereochemistry of the targeted molecules, 10 and 11. NBS treatment of benzylidene acetate 61 produced bromide 62 in 91% yield. Reaction of 62 with activated zinc powder and NaBH3CN provided diol 63 in 86% yield. Diol 63 was converted to epoxide 64 through sulfonylation of the primary hydroxy moiety followed by LiHDMS treatment in 60% yield.

Ethnylation of 64 using lithium TMS acetylide in the presence of BF3-OEt2 in THF and subsequent solvolysis in K2CO3/MeOH supplied enyne 65 in 90% yield. Persilylation with TBSOTf afforded the desired product enyne 66, quantitatively. The seco-steroidal structure was constructed using the Trost coupling cyclization strategy with C/D-ring fragment 48 or 20-epiC/D-ring fragment 37. Subsequent deprotection of the resulting products, 67 and 68, furnished 2-epieldecalcitol (10) and 2,20-diepieldecalcitol (11) in 32% and 57% yields, respectively (Scheme 6).
The VDR binding affinities and potencies of induction of HL-60 differentiation of the synthesized analogs, 10 and 11, are summarized in Table 4 in comparison with those of calcitriol (3). In inducing HL-60 differentiation, 2-epieldecalcitol (10) exhibited a rather lower effect while 2,20-diepieldecalcitol (11) showed quite a high potency, compared to calcitriol (3).13

3. NOR ANALOG AT 19 POSITION AND DEOXY ANALOGS AT 1 AND 25 POSITIONS
3-1. 19-NORELDECALCITOL (12)34
19-Noreldecalcitol (12) was synthesized by DeLuca group as an analog of 19-norseries of calcitriol (3) in the hope of obtaining a selective activity profile that exhibits high potency in inducing differentiation of malignant cells with very low or no bone calcification activity. DeLuca group considered that 19-noreldecalcitol (12) might retain potent bone formation activity without hypercalcemia resulting from bone calcium mobilization.34
The cyclohexanone derivative 70 was prepared from commercially available (-)-quinic acid (69).35,36 The 4-hydroxy group of 70 was protected as TMS ether in 95% yield. Peterson reaction of 71 with TMSCH2CO2Me in the presence of LDA gave a 3:1 mixture of the two isomeric cyclohexylidene esters 72 and 73 in 91% yield. The formation of the isomeric mixture was the result of the newly created axial chirality of the methyl 2-(4-hydroxycyclohexylidene)ethanoate system. Isomeric esters 72 and 73 were reduced to the allylic alcohols 74 and 75 which were easily separated by preparative HPLC. The separated alcohol 75 was transformed to the A-ring phosphine oxide 76 by in situ tosylation and conversion into corresponding phosphine followed by oxidation in ca. 60% overall yield. Wittig-Horner coupling of 76 with the protected Windaus-Grundmann ketone (77) gave 19-nor type compound 78 in 54% yield. The TMS protecting group in 78 was selectively hydrolyzed under carefully controlled condition to alcohol 79 in 38% yield, which was converted to ether 80 by alkylation with Br(CH2)3OTBS. Finally deprotection of 80 with TBAF gave 19-noreldecalcitol (12) in 21% yield from 79 (Scheme 7).

DeLuca group mentioned that 19-noreldecalcitol (12) possessed intestinal calcium transport activity but much less than that of calcitriol (3) and 12 showed also bone calcium mobilizing activity.34

3-2. 25-DEOXYELDECALCITOL (13)4
25-Deoxyeldecalcitol (13) was synthesized during our exploratory research for eldecalcitol (5).4 As previously described, alfacalcidol (4) was launched as a prodrug of calcitriol (3) in Japan by Chugai and Teijin in 1981. Scheme 8 depicts the synthetic route to alfacalcidol (4) from inexpensive cholesterol (81) as a starting material, in which α-epoxide 92 served as a key intermediate for the introduction of biologically important 1α hydroxy moiety of 4 by hydride reduction of 92.37 The start of an industrial scale production of alfacalcidol (4) provided us an abundant amount of α-epoxide 92. Treatment of 92 with 1,3-propanediol in the presence of t-BuOK resulted in stereo and regioselective introduction of a hydroxypropoxy group into 2β position to give 94, which was then converted to 25-deoxyeldecalcitol (13) by irradiation using a high pressure mercury lamp followed by thermal isomerization in 14% yield (Scheme 8).4

Table 5 compares the plasma calcium levels in rats (Sprague-Dawley rats) on a low calcium (0.003%) and vitamin D deficient diet after oral administration of calcitriol (3), alfacalcidol (4), eldecalcitol (5), and 25-deoxyeldecalcitol (13) (6.25µg/kg/day for 5 days, respectively). 25-Deoxyeldecalcitol (13) significantly increased plasma calcium levels, which reached an almost normal range.4 Although the structural relationship between 25-deoxyeldecalcitol (13) and eldecalcitol (5) corresponds to that between alfacalcidol (4) and calcitriol (3), the possible hydroxylation of 13 at 25 position in liver or bone to produce 5, such as metabolic conversion from 4 to 3, has not been investigated until now.

3-3. 1-DEOXYELDECALCITOL (14)38
Considering the metabolic pathway of cholecalciferol (1) and biological effects of 1, an idea was recently presented that calcitriol (3) is responsible for calcemic activity whereas the strong binding of calcifediol (2) to DBP and therefore long existence of 2 in blood are responsible for an anabolic effect on bone resulting in BMD increase.39 Since the structural relationship between 3 and 2 corresponds to that between eldecalcitol (5) and 1-deoxyeldecalcitol (14), we have been very interested in the biological action of 14, e.g. possible hydroxylation of 14 to 5 in the kidney, affinity for DBP, duration in blood stream, and anabolic effect on bone.
The requisite A-ring fragment 102 for the synthesis of 1-deoxyeldecalcitol (14) corresponds to the deoxygenated enyne of (R)-isomer 22 and (S)-isomer 23 in Scheme 1. The epimeric alcohol 95, prepared from C2-symmetrical epoxide 15 as shown in Scheme 1, was acetylated to acetate 96, which was converted to diol 98 after several steps. Mitsunobu reaction of 98 afforded epoxide 99 in 71% yield. Reaction of 99 with lithium TMS acetylide gave the enyne 100 in 86% yield, which was converted to the A-ring fragment 102 by saponification and subsequent protection of the hydroxy groups in 101 as their TES ether. Based on the Trost coupling methodology involving A-ring fragment 102 and C/D-ring fragment 48, 1-deoxyeldecalcitol (14) was obtained in 10% yield after desilylation of the resulting coupled product 103 (Scheme 9). The detailed biological action of recently synthesized 14 in comparison with eldecalcitol (5) will be investigated and reported in due course.

4. CONCLUSION
There are still many challenges ahead in attempting to gain a full understanding of the mode-of-action of eldecalcitol (5) with the objective of developing an even more effective and sophisticated pharmaceutical product. This demands the need for new improvements to achieve a more effective and safer vitamin D3 analog for osteoporosis based on the assessment of its limitations. Nevertheless, it is expected that eldecalcitol (5), a promising new medicine, will contribute to the treatment of patients with osteoporosis.40

5. ACKNOWLEDGMENTS
The author would like to express his sincere appreciation to Professor David A. Horne of the Department of Molecular Medicine, Beckman Research Institute at City of Hope for helpful suggestions and English editing. Thanks are also due to Drs. Katsuhito Miyamoto and Yoshiyuki Ono of Chugai Pharmaceutical Co., Ltd, for their reading of the manuscript and suggestions.

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