Non-secosteroidal Ligands and Modulators

C H A P T E R

78 Non-secosteroidal Ligands and Modulators Keith R. Stayrook 1, Matthew W. Carson 2, Yanfei L. Ma 2, Jeffrey A. Dodge 2 1

Indiana University School of Medicine, Indianapolis, IN, USA 2 Lilly Research Laboratories, Indianapolis, IN, USA

INTRODUCTION The core chemical structures of secosteroids are similar to a steroid except that the two B-ring carbon atoms at positions C9 and C10 of the traditional four steroid rings are not joined. Vitamin D is a secosteroid of utmost physiological importance as vitamin D deficiency causes osteomalacia and rickets and significantly increases risk of osteoporosis, autoimmune disease, infections, various cancers, and cardiovascular disease [1]. Vitamin D is produced from steroidal precursors in the skin via the action of UVB-containing sunlight. Vitamin D produced by the skin as either vitamin D2 or vitamin D3 is physiologically inactive and must undergo a series of successive hydroxylation in the liver, kidney, and/or various target tissues to become the biologically active 1a,25-dihydroxyvitamin D (1a,25(OH)2D3) [2e4] (see Chapter 3). The pleiotropic actions of hormonally active 1a,25(OH)2D3 (calcitriol) are mediated by the nuclear vitamin D receptor (VDR). VDR is a ligand-dependent transcription factor that belongs to the superfamily of steroid/thyroid hormone receptors and classically functions as a heterodimer with its cognate co-receptor retinoid X receptor (RXR) to control expression of genes involved in calcium and phosphorus homeostasis and bone mineral content. However, the scope of vitamin D and VDR biology has expanded to include a wide range of physiological cellular responses including proliferation, differentiation, and immunomodulation. In addition to its welldescribed transrepression and transcriptional regulatory activities, VDR also has the ability to rapidly initiate several key signal transduction pathways in a liganddependent non-genomic manner that does not require its co-receptor partner RXR [5,6]. While driving many cellular activating events through VDR, 1a,25(OH)2D3 levels are regulated by a finely tuned series of regulatory

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10078-2

mechanisms. These regulatory mechanisms include the catabolic inactivation of 1a,25(OH)2D3 via 24-hydroxylase/CYP24A1 enzymatic activity, regulation of 1ahydroxylation via 1a-hydroxylase/CYP27B1 enzymatic activity, and alterations in serum 1a,25(OH)2D3 half-life or target tissue uptake via vitamin-D-binding protein (DBP) binding (see Chapter 5). It is the cooperative complexity of these multifaceted ligand-dependent actions of VDR coupled to regulatory feedback mechanisms that ultimately produce the natural physiological activity of the secosteroid vitamin D and its metabolites. The therapeutic benefit and application of pharmacological doses of 1a,25(OH)2D3 has been used clinically for the treatment of renal osteodystrophy, secondary hyperparathyroidism, psoriasis, osteoporosis, cancer, and autoimmune disease, but its use is limited due to excessive absorption of mineral and pathological tissue hypermineralization [7]. Therefore, the synthesis and design of novel secosteroidal analogs exhibiting beneficial physiologic function with concomitant lower capacity for toxicity has been undertaken in the last two decades. More than 2000 secosteroidal analogs have been synthesized with modifications to the A and/or CD rings or the aliphatic side chains (see Chapters 76, 77, and 81). Many of these molecules display unique VDR modulating behavior in various biological assays and/or preclinical pharmacology models [8]. However, very few of these analogs have displayed the appropriate enhanced therapeutic margin of safety for the treatment of human disease for which they were designed. Furthermore, secosteroidal-based chemical synthesis has proven to be difficult and costly. Upon the identification, development, and proven clinical effectiveness of the synthetic non-steroidal tissue-selective modulators of estrogen receptor, raloxifene and tamoxifen, numerous novel synthetic chemical series for many of the receptors of the nuclear hormone

1497

Copyright Ó 2011 Elsevier Inc. All rights reserved.

1498

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

receptor superfamily have now been pursued and described [9,10]. This chapter is dedicated to highlighting the identification, synthesis, biological and pharmacological activities of non-secosteroidal ligands and/or modulators that are capable of binding in the traditional ligand-binding pocket of VDR to manipulate its physiologic and homeostatic functions in the various target tissues where it is expressed. The two main reasons for pursuing the design and development of novel non-secosteroidal ligands include the need for new and effective pharmacological agents for human disease treatment as well as the identification of research tools to further elucidate the biological function of vitamin D and VDR. To this end, there are relatively few published reports or descriptions of non-secosteroidal VDR ligands by comparison to those described for the classical steroid hormone receptor family. However, it is believed that the unique structural features of nonsecosteroidal ligands may afford advantageous properties that are inherently different from the liabilities surrounding 1a,25(OH)2D3 or its secosteroidal analogs. These properties may include altered DBP binding, reduced 24-hydroxylase metabolism, distinct VDR modulating behavior, or enhanced pharmacokinetic and pharmacodynamic profiles. There are currently five classes of compounds with published data that can be chemically classified as non-secosteroids. These include molecules based on a diarylmethane chemical scaffold (LG190119, VDRM-2), C/D ring-modified scaffolds, bis- and tris-aromatic triols (CD4420), podocarpic acid derivative (VDRL-1), and steroidal compounds such as the secondary bile acid lithocholic acid and/or its derivatives (Fig. 78.2). A review of the structures and activities of the diarylmethanes, C/D ring modified chemistry, bis- and tris-aromatic triols, and podocarpic acid derivative classes are included in this chapter. A review of steroidal lithocholic acid and its derivatives can be found in Chapter 79 and is not included here.

DIARYLMETHANE LIGANDS Identification, Structure, and Biological Characterization The hypothesis that non-secosteroidal analogs could mimic the effects of 1a,25(OH)2D3 (Fig. 78.1) prompted the search for novel chemistry in the hopes of obtaining VDR ligands with improved tissue selectivity relative to calcitriol or secosteroidal analogs. The diarylmethane chemical scaffold was one of the first non-secosteroidal series to be identified and shown to exhibit both in vitro and in vivo vitamin D pharmacology [11]. The original screening hit LG190090 as shown in Figure 78.3 was able to induce activity in a VDR-dependent manner in

20 25 17

C

D

14

OH

H

19

A HO

3

1

OH

FIGURE 78.1 Structure of 1a,25(OH)2D3 (calcitriol). A-, B-, and Drings and carbons mentioned in the text are marked. Aliphatic alcohol side chain is C20eC25.

a cotransfection (CTF) assay using HepG2 cells transfected with a VDRE-based luciferase reporter. Structuree activity relationship (SAR) optimization led to identification of LG190178 (Fig. 78.3), a potent VDR agonist with an EC50 value of 40 nM in the CTF assays coupled with good binding affinity to the vitamin D receptor (Ki ¼ 150 nM) [11]. In biochemical assays measuring SRC peptide coregulator interaction, LG190178 was comparable to 1a,25(OH)2D3 [12]. The individual enantiomers of LG190178 have been prepared and evaluated for VDR binding and transcriptional activity using human osteosarcoma and colon carcinoma cell lines [13]. Overall, one stereoisomer, YR301 (2S,20 R) (Fig. 78.3), demonstrates high binding affinity to VDR (RBA ¼ 28.3%) and strong transcriptional activity in human osteosarcoma cells (MG-63 EC50 ¼ 0.78 nM) and in human colon cells (Caco2 EC50 ¼ 1.8 nM). Interestingly, the remaining stereoisomers (2S,20 R), (2R,20 R), and (2S,20 S), were significantly less active in binding and VDR functional activity indicating ligand stereo-sensitivity of VDR binding pocket with these compounds. Docking studies with all four stereoisomers in VDR indicated that the (2S,20 R)-isomer was the most stable having hydrogen bonds between the 2-OH and Arg274, Ser237 and between the 20 -OH and His305 and His397. Further SAR studies with LG190178 resulted in nitrogen-linked side chains on the diphenylmethane core [14]. One such analog, DPP-1023 (Fig. 78.3), binds with high affinity to VDR (Ki ¼ 52 nM) and displays potent agonism in HL60 cell differentiation with an EC50 of 48 nM. The optically pure isomers of DPP-1023 were obtained and characterized. The (R,R) configuration displays the most potent activity, relative to 1a,25(OH)2D3, having a Ki of 9.5 nM coupled with a >10-fold increase in cellular potency as measured by the induction of HL-60 cell differentiation (i.e., EC50 ¼ 4.1 nM for (R,R)-DPP-1023 compared to 59 nM for 1a,25(OH)2D3). This compound binds to the androgen receptor (AR) with a Ki of 910 nM thereby demonstrating dual VDR-AR activity. Other compounds in this series bind to AR in the 400 nM range while

IX. ANALOGS

1499

DIARYLMETHANE LIGANDS

H OH

O

OH

N OH

O O OH

H HO

VDRM-2

F3C

JN

OH

F F

CF3

F OH F F F

O

O

NH

H OH

OH OH

VDRL-1

CD4420

H OH OH

HO

OH

OH

HO

des CD analogs

CF-ring analogs

FIGURE 78.2 Structures of various non-secosteroid VDR ligands. The five classes of non-secosteroid molecules are represented by the selected structures and include the diarylmethanes (VDRM-2), steroidal (JN), podocarpic acid derivative (VDRL-1), bis- and tris-aromatic triols (CD4420), and deconstructive secosteroidal compounds such as des-CD and CF-ring analogs.

demonstrating anti-androgen activity by inhibiting testosterone-dependent proliferation in SC-3 cells. No in vivo data have been reported to elaborate on the dual intrinsic activity for these ligands. Further elaboration of the SAR within this chemical series finally led to the identification of LG190119 (Fig. 78.3) which demonstrated the first described separation between the induction of certain VDR target genes and serum calcium concentration in mice [17]. LG190119 and LG190178 constitute the first disclosed selective non-secosteroidal vitamin D receptor modulators (VDRMs).

Additional reported synthesis efforts within the diarylmethane chemical series resulted in the identification and description of an analog that demonstrates improved efficacy on bone-related endpoints relative to hypercalcemia in rodent models. Referred to as VDRM2 (Fig. 78.3), it contains a novel amide side chain, displays good affinity for VDR (Ki ¼ 57.8 nM), and is a potent inducer of RXRVDR heterodimerization (EC50 ¼ 7.1 nM). VDRM2 induces osteocalcin-promoter activity with an EC50 of 1.9 nM as compared to 1.3 nM for 1a,25(OH)2D3, while being less potent in calcium channel TRPV6 upregulation

IX. ANALOGS

1500

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

O

O O

Cl

O

O

O

Cl

O

O

LG190090

LG190119

original screening hit

OH

2S

OH

OH

OH

O

O

O

O

2'R OH

OH

YR301

LG190178

O N

O

OH

O

OH

OH

VDRM2

HO

S

O

N

OH OH

H

DPP-1023

HO

O S O O

S OH

O O

LY2109866

LY2108491

FIGURE 78.3 Representative structures of the non-secosteroidal diarylmethane ligand class. Select structures include original screening hit LG190090, diphenylmethanes (LG190119, LG190178, YR301, VDRM2, DPP-1023) and phenyl-thiophenes (LY2108491 and LY2109866).

than 1a,25(OH)2D3 with an EC50 of 37.2 nM compared to that of 0.6 nM for 1a,25(OH)2D3 [15]. Derivatives of the diphenylmethane structure have been identified. In one such series, one of the phenyl groups in LG190119 has been replaced by a thiophene [16]. These ligands, LY2108491 and LY2109866 (Fig. 78.3), contain a traditional hydroxyalkyl side chain on the novel thiophene ring while the adjacent phenyl ring has a sulfate ester in the case of LY2108491 and a carboxylic acid in the case of LY2109866. Both compounds are potent agonists of VDR-RXR heterodimerization in human SaOS-2 cells with an EC50 of 11 nM and 13 nM, respectively. These molecules induce VDRE-dependent expression of a rat osteocalcin

reporter in rat osteoblast cells (ROS17/2.8) with an EC50 of 25 nM and 3 nM. In contrast, LY2108491 and LY2109866 are poor inducers of intestinal vitamin-Dresponsive genes relative to 1a,25(OH)2D3 that include TRPV6, CYP24, and calbindin-D9k, but are potent agonists in keratinocytes and peripheral blood mononuclear cells. Additionally, phenyl-thiophenes with carboxamide side chains have also been disclosed with VDR-modulating activities (US patent 7601850B2). Other analogs related to the diarylmethane chemical scaffold have also been disclosed in the patent literature. These include compounds represented by phenyl-furan, phenyl-benzofuran, and phenyl-benzothiophene (US patents 7468449B2, 7579488B2, 7582775B2).

IX. ANALOGS

1501

DIARYLMETHANE LIGANDS

14

800

750

13

700 12 650

625 mg/cc

s 11.20 mg/dL 11

600 s o o

550

Serum Ca (mg/dl)

The primary goal of identifying and characterizing the biological activities of non-secosteroidal VDR ligands is ultimately to design therapeutics that may serve to treat various disease states sensitive to the benefits of vitamin D physiology. This is manifested by the clinically proven lack of hypercalcemia/hypercalciuria risk while displaying significant disease efficacy at a given dose or exposure of VDR ligand. Currently, no clinical validation of non-secosteroidal VDR ligands demonstrating separation of hypermineralization risk has been reported. Several reports of preclinical validation to this effect have been observed with compounds from the diarylmethane chemical class. Boehm et al. reported that the first non-secosteroidal VDR ligand diphenylmethane LG190119 displays pharmacologic separation as measured by VDR target gene induction versus serum calcium concentrations in vivo. Oral gavage dosing for 3e5 days of LG190119 at 10 or 30 mg/kg/d to Balb/c mice was able to induce 30-fold expression of VDR target gene 24(OH)ase RNA in kidney without significantly increasing serum calcium. Although 1a,25(OH)2D3 5 mg/kg/day was able to induce a similar fold elevation of kidney 24(OH)ase RNA in the same study, it significantly increased serum calcium at this dose [11]. Furthermore, LG190119 was reported to be effective in inhibiting tumor growth while displaying no significant hypercalcemia at the dose used over a 12-week treatment period in an LNCaP-derived prostate tumor xenograft athymic mouse model. This efficacy compared favorably to EB1089, a secosteroidal analog which has been previously shown to be less calcemic but more potent in inhibiting LNCaP cell growth than 1a,25(OH)2D3 in vitro. In the same study, 1a,25(OH)2D3 dosed at 0.5 mg/kg was too low to see significant tumor suppression in this model [17]. The authors claimed that the fewer in vivo calcium effects of their diphenylmethane vitamin D mimics may be attributable to less binding to serum D-binding protein. Sato et al. reported that amide side-chain-containing VDRM2 displays significant pharmacological separation of bone efficacy versus serum calcium in an ovariectomized (OVX), osteopenic rat model. Oral treatment of VDRM2 administered for 8 weeks to 7-month-old, 1-month post-ovariectomized (OVX) rats dose dependently increased bone mineral density of lumbar vertebrae (LVBMD), and increased trabecular bone volume of the proximal tibial metaphyses from OVX control back to Sham levels at a dose of 0.08 mg/kg/day. At doses above 0.03 mg/kg/day, VDRM2 restored bone strength parameters back to Sham levels or above in the vertebra, femoral neck, and femoral midshaft.

Hypercalcemia in those animals was not observed until 4.6 mg/kg/day, indicating a therapeutic safety margin of 57-fold between bone efficacy and hypercalcemia (Fig. 78.4). Histomorphometric analysis showed that VDRM2 increased periosteal bone formation rate resulting in increased cortical bone properties, while functioning as a weak antiresorptive on trabecular bone surfaces. In comparative studies, 1a,25(OH)2D3, ED71, and alfacalcidol displayed a therapeutic safety window of 7.3, 4.9, and 5, respectively, based on the ratio between the Sham level threshold BMD efficacy dose and the hypercalcemic dose from the same animals (Table 78.1). These data suggest that the non-

Vertebral BMD (mg/cc)

In Vivo Characterization of Diarylmethane Ligands

10

9

500 0.001

0.01

1

0.1

0.081 µg/kg

10

100

4.6 µg/kg

FIGURE 78.4 Therapeutic safety window between Sham level threshold bone mineral density efficacy dose and hypercalcemia dose for VDRM2 from the reproducible doseeresponse studies of 8 weeks’ treatment on 7-month-old, 1-month post-ovariectomized (Ovx) rats. Plotted are mean  SEM. Modified from [15].

TABLE 78.1

Comparison of VDRM2 to Vitamin D3 Analogs on Bone Efficacy to Hypercalcemia Ratio (NOEL Based)

Compound (mg/kg/d) VDRM 2 1,25-OH2D3 Alfacalcidol ED-71 LVBMD (mg/cc)*

0.081

0.03

0.046

0.0055

Serum calcium**

4.6

0.22

0.23

0.027

Serum Ca/LVBMD

57

7.3

5

4.9

* Minimal efficacy dose which restores LVBMD from Ovx to Sham levels. ** Minimal hypercalcemia dose which reaches 97.5th percentile of the historical Ovx rat value (11.2 mg/dl). Modified from [15]. LVBMD ¼ lumbar vertebral bone mineral density. Seven-month-old rats were permitted to lose bone due to ovariectomy for 1 month before dosing with compounds for 8 weeks. Data presented were averaged from 2e5 assays for each compound.

IX. ANALOGS

1502

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

secosteroidal compound VDRM2 has an increased safety margin versus its secosteroidal counterpart molecules in osteopenic ovariectomized rats [15]. It remains unknown whether this rodent pharmacological distinction is directly translatable to the human clinical setting. Pharmacology studies with phenyl-thiophene derivatives LY2108491 and LY2109866 (Fig. 78.3) have also been reported. These molecules were found to be significantly less hypercalcemic as compared to 1a,25(OH)2D3 in vivo when administered via oral or topical application routes. In a mouse 6-day oral dosing study, 1a,25(OH)2D3 resulted in a significant increase in blood ionized calcium at a 1 mg/kg/day dose. In contrast, oral dosing of LY2108491 resulted in no significant hypercalcemia up to 3000 mg/kg/day, while LY2109866 treatment similarly showed no evidence of hypercalcemia at a dose of 1000 mg/kg/day [16]. Using a hairless mouse model of epidermal proliferation via a topical application route, Ma et al. calculated the therapeutic indices (TI) of these ligands and compared them head-to-head with 1a,25(OH)2D3. They found that calcitriol had a 0.3 TI ratio between the threshold minimum effective dose (TMED) for hypercalcemia and the TMED of keratinocyte proliferation in the same animal. In contrast, the calculated TI ratio for LY2108491 was found to be greater than 81 and LY2109866 was more than 27. Therefore, LY2108491 and LY2109866 were greater than 270- and 90-fold, respectively, better than calcitriol in terms of the TI in this topically applied surrogate model of psoriasis. This model is regarded as a surrogate in vivo preclinical model of psoriasis since VDR ligands (calcitriol and calcipotriol) and retinoids that inhibit keratinocyte proliferation in psoriatic lesions also induce epidermal proliferation when applied topically to normal skin [16,18,19]. These data indicate that non-secosteroidal phenyl-thiophene derivatives may exhibit less hypercalcemia liability when administered orally but also may extend to topically applied pharmacological settings as well. However, no clinical development information has been reported for these compounds.

C/D-RING MODIFIED LIGANDS Identification, Structure, and Biological Characterization Since the strong calcemic effects of the secosteroid natural hormone calcitriol limit its therapeutic potential, a number of efforts have been carried out to find a safe VDR ligand. Early work in the field involved modifications of either the secosteroid A-ring or the aliphatic side chain [20]. A-ring modified 2MD and

aliphatic alcohol analog calcipotriol exhibit improved margins of safety for bone versus hypercalcemia, but their similarity in structure to calcitriol suggested that the therapeutic index may not have been optimized. To improve upon this, a deconstructive approach of the secosteroid scaffold was undertaken (Fig. 78.5) [21]. These studies commenced with the removal of the C/D rings of calcitriol yielding the retiferols [23]. Retiferols and their 19-nor versions were reported in the patent literature (WO199943646) which revealed that these compounds exhibit selectivity for cell differentiation versus hypercalcemia. Further SAR efforts involved replacement of the fused C/Drings of secosteroids with either a 5- or 6-membered ring [24]. These series of compounds are referred to as the C-ring, D-ring, and E-ring analogs. The C/Dring modification was later expanded to CF-spiro[4.5] decanes and [5.5]undecanes (WO 2006060884 and WO 2006060885). The synthesis of the latter has been reported [25,26]. Investigators have also combined structural elements of the 2-methylenes with the desCD ring scaffold to give a molecule with binding affinity two orders of magnitude greater than 2MD. More recently, it was reported that activity of this compound is improved by increased van der Waals interactions with the central hydrophobic channel of the ligand-binding domain of VDR. This was accomplished by incorporating methyl groups at the C13 position of des-CD-2-methylene-19 norvitamin D compounds [27]. The absolute configuration of C13 was determined by analyzing circular dichromism spectra of g-lactone precursor diastereomers. Biological characterization of C/D-ring modified non-secosteroids clearly demonstrate that full C/Drings are not required for the biological activity of 1a,25(OH)2D3 as C/D-ring replacements in many cases retain full or modulatory 1a,25(OH)2D3 activities in various VDR binding, cellular activity and pharmacological settings. However, for the various reported C/D-ring modified compounds there do appear to be important spatial and geometric requirements that new ring replacements must generate an appropriate spacing of the A-seco B-rings in relationship to the particular side chain used. Several reports including Verstuyf et al. have described in detail the structuree activity relationships of C-ring, D-ring, and E-ring ligand classes and their VDR modulating behavior [21,22,28,29] (see Chapter 76). This was performed by analyzing VDR and DBP binding, in vitro studies using several cell lines, and in vivo calcemia studies in mice. Table 78.2 is a summarized table of biological activities from select ring-modified compounds from each of the C/D- and E-ring SAR efforts [22]. Currently, while these data support the potential therapeutic capacity of C/D-ring modified

IX. ANALOGS

1503

BIS- AND TRIS-AROMATIC TRIOLS

FIGURE 78.5 OH

X

X=CH2 retiferol analog X=H,H 19-nor retiferol analog

6 HO

OH R R

A retiferol and 19-nor retiferol; 19-nor refers to the lack of methylene group at C6. C- and D-ring analogs represent disconnected D- and C-rings, respectively. Ering analogs are retiferols with a gem dimethylcyclopentane spacer element (R ¼ aliphatic alcohol chain). CF-spiro derivatives are C-ring analogs with spiro-fused five- or sixmembered ring spacers between the aliphatic alcohol chain and cyclohexane core. Des-CD-2methylene analogs of 2MD are similar to the retiferol series except that the methylene group on the A-ring is located at the 2-position rather than the C-6. The diastereomeric C-13 methyl substituted analogs are shown.

R

HO

HO

OH

C-ring analogs

HO

OH

D-ring analogs

OH

E-ring analogs

O

OH OH 13

2 HO

OH

OH

HO

(R)-13-methyl (S)-13-methyl

spiro [5.5]undecane

non-secosteroid ligands, no clinical development information has been reported for this compound class.

BIS- AND TRIS-AROMATIC TRIOLS Identification, Structure, and Biological Characterization Bis- and tris-aromatic triols were one of the first non-secosteroidal VDR ligand classes reported. These compounds were originally developed for the treatment of dermatological diseases. The common fragment among the triols is the dibenzyl alcohol located

in the hydrophilic region of the molecule. The synthesis of these tris-aromatics and the phenyl ether-linked bis-aromatic compounds (Fig. 78.6) are covered along with their biological evaluation in the patent literature (WO200138303 and WO2004020379). The biological evaluation of tris-aromatics was conducted in a cotransfected HeLa cell line. The preparation of the bis-aromatic analogs along with their biological characterization on the proliferation of human keratinocytes was reported in these patents as well. Another subseries of the bis-aromatic triols containing a dienyl alcohol chain was reported by Pera¨kyla¨ et al. [30]. The most potent dienyl bis-aromatic

IX. ANALOGS

1504

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

TABLE 78.2

Biological Activities of Various C/D-modified Analogs

Compound

VDR1 binding (%)

HL-602 diff (%)

MCF-73 prolif (%)

Kerat4 prolif (%)

Serum Ca5(%)

Calcitriol

100

100

100

100

100

10

20

30

10

<0.1

60

1000

6000

6000

50

80

85

85

90

0.3

OH

HO

OH

KS 176 KS 176

OH

HO

OH

ZG 1368 ZG 1368

OH

HO

OH

SL 117 SL 117 1

Competitive VDR binding. Differentiation of HL-60 cells (NBT tetrazolium assay). Proliferation of breast cancer MCF-7 cells ([3H]thymidine incorporation assay). 4 Proliferation of human keratinocytes ([3H]thymidine incorporation assay). 5 Determined by intraperitoneal injections for 7 consecutive days in mice. Table modified from [22]. In vitro data expressed as percentage of calcitriol. In vivo data are comparison of doseeresponse curves versus calcitriol (calcitriol ¼ 100%). 2 3

IX. ANALOGS

MISCELLANEOUS NON-SECOSTEROIDS

MISCELLANEOUS NON-SECOSTEROIDS

OH OH O

O

OH

OH

Tris-aromatic triol

1505

OH

OH

Ether-linked bis-aromatic triol

FIGURE 78.6 The tris- and ether-linked bis-aromatic triols. The core is an aromatic ring containing a straight chain hinge-region.

compounds CD4409, CD4420, and CD4528 were evaluated in a variety of in vitro and in vivo assays. In addition, molecular dynamic (MD) simulations demonstrated that these dienyl bis-aromatic ligands occupy the VDR ligand-binding pocket in a similar fashion as calcitriol. During the MD simulation study, distances between the polar anchoring points (Y147, S237, R274, S278, H305, and H397) in the VDR LBD and the hydroxyl groups of the ligand were measured and determined to be comparable to the VDR-bound structure of 1a,25(OH)2D3. The docking of these ligands suggests that the dibenzyl alcohol sits in the same region of the VDR LBD as the cyclohexanediol of 1a,25(OH)2D3 and the branched alcohol interacts with H305 and H397. The combination of MD simulation and single point mutation of the above residues suggests that CD4528 most closely resembles the structure of calcitriol. In VDR biochemical and functional studies, this class of ligands displays the capacity to bind within the VDR ligand-binding pocket and can drive VDReRXR heterodimer complex formation on a DR3 element in gel-shift experiments. In addition, they display potent cellular activity as they induced Gal4-VDR LBD mediated-luciferase activity in Hela cells comparable to 1a,25(OH)2D3 (Fig. 78.7). Furthermore, CD4409, CD4420, and CD4528 demonstrate VDR pharmacological capacity as measured in male BALB/c mice. Interestingly, in 8week-old Balb/c mice, oral gavage with these ligands for 3 or 5 days at non-calcemic doses stimulated higher VDR target gene CYP24 mRNA expression in renal tissue than that of a non-calcemic dose of calcitriol (1 mg/kg) [31]. Representation of summarized functional and pharmacological data generated with CD4409, CD4420, and CD4528 can be found in Fig. 78.7 [31]. However, this non-secosteroidal class continues to be represented by a limited number of peer-reviewed disclosures.

Identification, Structure, and Biological Characterization A survey of the literature reveals that there are a few known singleton non-secosteroids reported. For example, Chen et al. reported on VDRL-1, a novel VDR ligand derived from podocarpic acid (Fig. 78.8) [31]. This compound’s activity was evaluated for VDR binding and transcriptional activity in several cell-based settings. It displays considerably weaker binding affinity as compared to calcitriol (Ki ¼ 1.4 mM versus 0.15 nM for 1a,25(OH)2D3) but is able to drive efficient VDR transactivation at notably higher concentrations. Using computer-assisted molecular docking studies, VDRL-1 conformation and interaction with the VDR LBD was analyzed in both agonist and antagonist modes. VDRL1 bound to the agonist conformation exhibited little strain energy for the top two poses, therefore suggesting that it does not displace helix 12. As is the case with nuclear receptors such as GR, AR, and ER, the position of helix 12 is important for agonist activity due its close proximity to AF-2. Pose 1 docks the ligand so that the hexafluoroisopropanol (HFiP) moiety interacts with H305/397 residues which directly or indirectly influence the position of H12. Pose 2 is flipped relative to pose 1 to allow for the phenolic OH to interact with H305/397 and the HFiP to interact with S237 and R274. The less favorable interaction energies of poses of VDRL-1 in the antagonist conformation of VDR LBD suggest that VDRL-1 likely occupies the same space and interacts with the same polar anchoring points as calcitriol. In addition to mineral and bone homeostasis, VDR biology plays important roles in immunomodulation, antimicrobial defense, xenobiotic detoxification, anticancer actions, control of insulin secretion, and cardiovascular benefits. In a landmark paper, Makishima et al. reported that activated VDR detoxifies bile acid lithocholic acid (LCA) via CYP3A4 activation. This pathway is initiated by the weak binding of LCA or its 3-substituted esters to the VDR [32,33]. LCA-related VDR biology is covered in detail in Chapter 79. However, recent evidence suggests that other non-secosteroidal nutritional ligands exist, albeit with low affinity for VDR, but in high local concentration could function as sensors for extraosseous VDR-mediated pathways. These putative VDR ligands, as illustrated in Figure 78.9, include turmeric-derived curcumin, vitamin E derivative c-tocotrienol, and polyunsaturated acids docosahexaenoic and arachidonic acid. Haussler et al. reported that curcumin, c-tocotrienol, docohexaenoic, and arachidonic acids compete with tritiated calcitriol for VDR with similar affinity as LCA. Curcumin was also shown to activate VDR in a responsive reporter gene assay at

IX. ANALOGS

1506

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

GAL4 luciferase in HeLa EC50, nM (no CYP24 inhib)

GAL4 luciferase in HeLa EC50, nM (+ CYP24 inhib)

Ligand-dep gel shift EC50, nM

calcitriol

1

0.12

0.2

CD4409

8

10

6

CD4420

5

6

4.8

CD4528

1.7

2

9

In vitro profile Compounds

In vivo profile Compounds vehicle

Dose, μg/kg

Treatment, days 5

Kidney CYP24 mRNA induction/serum calcium ratio 1

calcitriol

1

5

6.6

CD4409

500

3

53.6

CD4409

500

5

62.6

CD4420

500

3

0.5

CD4420

500

5

1.2

CD4528

500

3

80.7 2.7

CD4528

50

5

CD4528

150

5

8.5

CD4528

500

5

59.3

F F

OH

F F OH

F

F OH

F S F

CD4409

O

O

OH

F F

F F

CD4528

CD4420

OH

OH

OH

OH

OH

FIGURE 78.7 Structures and summarized data tables of the in vitro and in vivo activities of dienyl bis-aromatic ligands CD4409, CD4420, and CD4528. Data modified from [31].

F3C

O

OH CF3

NH

H

OH

FIGURE 78.8 Merck’s VDRL-1, a podocarpic acid core containing hexafluoro-isopropanol and phenolic anchoring points.

concentrations four orders of magnitude higher than calcitriol [34]. Curcumin and bisdemethoxycurcumin also display significant activity in VDR-mediated nongenomic chloride channel opening in TM4 Sertoli cells [35]. The large volume of the VDR ligand-binding domain coupled with multiple polar residues deep in the pocket may explain how VDR accommodates these nutritional lipophilic acids and phenols. Like LCA, 1a,25(OH)2-lumisterol D3 (JN) is a steroid with demonstrable poor affinity for VDR in competitive binding experiments with 1a,25(OH)2D3. However, JN functions as a highly potent agonist of non-genomic and rapid signaling events mediated by VDR. It has been hypothesized that the impetus for the rapid nongenomic response of JN/VDR is the fact that it prefers to bind in an alternative binding pocket (AP) which contains more hydrophilic and p-bond residues than the genomic pocket (GP). Evidently, the dihydroxy and

IX. ANALOGS

1507

PERSPECTIVES O

O

HO O O

HO

OH

curcumin

O

gamma-tocotrienol O O

OH

OH

ϖ-3-docosahexaenoic acid

ϖ-6-docosahexaenoic acid

O H

OH H OH

H O R

H

R=H lithocholic acid R= -C(O)Me R= -C(O)Et

OH H

HO

JN

FIGURE 78.9 Nutritional and steroidal ligands.

double bonds, as well as the overall planarelinear shape of JN, drive the kinetically driven binding to AP. Alternatively, this is compared to the bowl-shaped calcitriol and many of the other non-secosteroidal ligands described in this chapter which likely prefer the hydrophobic GP. The vitamin D sterol-vitamin D receptor ensemble model described by Mizwicki et al. offers unique dynamic insights into both genomic and rapid-response signaling by these steroidal ligands [5,6,35] (see Chapter 15). Whether other physiological-relevant steroids are capable of this unique VDR activation remain to be determined. Nonetheless, they remain a potentially viable class of ligand for the vitamin D receptor.

PERSPECTIVES The numerous studies performed by both applied and basic researchers into the molecular physiology of vitamin D signaling have greatly enhanced our understanding of this hormone and its metabolites. In particular, the creative work of drug discovery scientists and medicinal chemists in the design and synthesis of vitamin D receptor ligands has paved the way for potential therapeutic exploitation of these compounds in the numerous disease states where classical vitamin D has a proven clinical benefit. The significant hurdle of excessive hypermineralization in vivo at efficacious pharmacologic doses of these compounds continues to hamper their development as useful therapeutic agents. To this end, several hundred secosteroidal analogs have been synthesized and well characterized over the past two decades, with comparably few reports for non-secosteroidal VDR ligands.

Most notably, the diarylmethanes, bis- and trisaromatic triols, and the podocarpic derivative VDRL-I are non-secosteroids not found and based on the core secosteroidal template. By comparison, C/D-ring modified and the newly described steroidal ligands are conceptually derived from classical vitamin D chemistry but claim technical classification as non-secosteroids. Nonetheless, the non-secosteroidal ligands described in this chapter display all of the binding, cellular, and pharmacological characteristics of their secosteroidal counterparts. However, they also display a number of mechanistic and therapeutic properties that may prove valuable including reduced DBP binding, enhanced ADME properties, unique VDR modulating behavior, and scalability of chemical synthesis. Furthermore, many of these ligands demonstrate potential for less calcemic risk while maintaining desired pharmacology in vivo as compared to calcitriol or select analogs. Despite much promise, it remains to be seen whether the non-calcemic behavior of these ligands in preclinical studies using non-human species will readily transfer to the human clinic. In summary, while the number of described and characterized non-secosteroidal chemical scaffolds continues to be small, they continue to hold promise in an everexpanding number of human disease states where vitamin D physiology has demonstrable importance. Additionally, the search and identification of new novel non-secosteroidal chemical scaffolds remains a valuable ongoing yet challenging goal. The growing molecular and structural understanding of the vitamin D receptor ligand-binding pocket in conjunction with availability of superior new research tools will prove useful in this endeavor.

IX. ANALOGS

1508

78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS

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IX. ANALOGS

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