D, its metabolites
a review
of the structural
for biological D. A.
Procsal,3
and
W.
requirements
activity’2 H.
Okamura,4
and
A.
W. Norman3
It has become increasingly apparent that modifications in the structure of vitamin D can lead to analogs that are more effective than the parent compound in the treatment of various vitamin D-related disorders (1-3). Comparable advances have also been made in understanding the mode of action of vitamin D (4, 5). Together these clinical and biochemical studies have stimulated interest and made possible a detailed analysis of the structurefunction relationships that characterize this regulator of calcium metabolism. It is the purpose of this article to review pertinent studies (6-18) with emphasis on recent work from this laboratory directed toward a systematic analysis of the chemical and biological properties of vitamin D3, its major metabolites, and a number of structural analogs. Structure
of vitamin
D3
Figure 1 traces the history in the development of our understanding of the structure of vitamin D. Structure I in Figure 1 is a representation of the initial formulation of the steroidal structure of the vitamin established in the early 1930’s (19, 20). Its early chemistry has been reviewed comprehensively by Fieser and Fieser (21). These studies established that vitamin D results when the B-ring of its provitamin [7-dehydrocholesterol (D3) or ergosterol (D2)] is cleaved by a photochemically catalyzed reaction. Vitamin D3 is, therefore, a secosteroid with the chemical name of 9,10-secocholesta-5,7,10 (19)triene-33-ol. It should be noted that Structure 1 does not describe the stereochemistry of the molecule. This was established some 20 years ago by elegant X-ray crystallography performed by Crowfoot and Dunitz (22)
and
The American
Hodgkin Journal
et al. of
analogs:
Clinical
(23)
employing
Nutrition
an
29: NOVEMBER
analog of vitamin D2. The results of these studies (Structure 2) established that the diene system extending from C-5 to C-8 is coplanar and transoid (as opposed to the cisoid configuration shown in Structure I). The 5,6-double bond is considered cis (Z) in relation to the A-ring by virtue of the fact that the C-7 is cis to C-lO, rather than to C-4. The x-ray crystallographic data also indicated that the A-ring is in a single chair conformation. This feature, however, is rarely incorporated in the planar Structure 1. Recently Knobler et al. (24) reported x-ray crystallographic data for another vitamin D analog indicating an opposite chair conformation is that reported by Crowfoot-Hodgkin’s group. This difference was the result of the fact that the A-ring can be frozen in either of two chair conformations, depending upon the nature of these analogs as they exist in their crystalline form. The existence of different A-ring conformations is a consequence of the open ring structure of vitamin D. It is this structure that imparts upon the A- and seco-B6 rings a degree of conformational mobility not found in other steroids in which the A, B, C, and D rings exist as a fused array. Recent studies in our laboratory (25-27) and independently in that of Lamar and Budd ‘From the Departments of Biochemistry and Chemistry, University of California, Riverside, California 92502. 2 This work was supported in part by United States Public Health AM-l6,595.
Service
Grant
AM-09012,
AM-14,750,
and
Department
of Biochemistry. ‘Department of Recipient of a USPHS Career Research Development Award, IKD-AM-l3,654. 6 The term “seco” is used to designate cleavage of one
Chemistry.
or more rings of the cyclopentanoperhydrophenanthrene steroid skeleton. Thus vitamin D, where the 9-10 carbon bond of the B ring is broken, is designated as a seco-B steroid.
1976,
pp. 1271-1282.
Printed
in U.S.A.
1271
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Vitamin
1272
PROCSAL
equatorial configuration, while 45% will have the la-hydroxyl in the axial orientation. This distribution is governed by a dynamic equilibrium between the two chair forms such that the A-ring of any one molecule may flip from one chair conformation to the other some one million times per second. It is clear that alterations in this ratio can be biased in favor of one conformer over the other as a consequence of the introduction of different substituent groups into the A-ring (Fig. 2). In view of the profound influence these conformational effects may have on the structure of the A-ring, it is clear that the experimentalist interested in defining structure-function rela-
2
OH CH3
OH
FIG. ofvitamin I resulted
5 I
.
6
Evolution of conformational D. The structure for vitamin from
the
original
structure
representations D3 represented determination.
AL.
in The
structure depicted in 2 was deduced from x-ray crystallographic analysis. Structures 3 and 4 illustrate the rapid equilibration between the two A-ring chair conformations, while structures 5 and 6 show the same relationship for la,25-(OH)2-D3. The (e) and (a) refer, respectively, to the equatorial and axial orientations of the indicated hydroxyl group (i.e., either the 3i or extended
la-hydroxyl).
(28) have focused on the conformational mobility of the molecule as it exists in solution. In both instances, high resolution PMR spectroscopy was employed to demonstrate that the A-ring of vitamin D and of several related seco steroids exists in solution as a pair of dynamically equilibrating chair conformers (Structures 3 and 4 of Figure 1). This observation becomes particularly significant in the light of the fact that for each conformational inversion every equatorial position becomes axial and every axial position becomes equatorial. Furthermore, the equilibrium constant between the two chair conformers will be dependent upon the nature and location of substituent groups on the A-ring as deduced from model studies on cyclohexane (reviewed by Eliel et al. (29)). For example, as shown in Structures 5 and 6 (Figure 1) the la-hydroxyl of la,25-dihydroxyvitamin (I a,25-(OH)2-D3), depending upon the chair conformer, will be either equatorial or axial. In fact, analysis of the conformational ratio of la,25-(OH)2-D3 indicates an equatorial to axial ratio (e/a ratio) of 55/45 (25, 26). That is to say, in a population of la,25-(OH)2-D3 molecules in solution, 55% will have the la-hydroxyl in the
tionships should be aware of this important consideration. In determining structure-function relationships, an area of primary concern is the choice and design of an appropriate experimental model system to evaluate vitamin D related biological response(s). Currently, several assays are available to assess the biological activity in vivo. Included in this group are assays designed to measure in vivo intestinal calcium transport (30), bone calcium mobilization (31), and the ability to cure rachitic lesions (32). In addition, various in vitro techniques are also at hand including the measurement of the rate of intestinal calcium and phosphate transport employing Ussing transport blocks (33, 34), the induction of a vitamin D-dependent calcium binding protein in an embryonic chick intestine organ culture (35) and the measurement of calcium mobilization in cultured bone tissue (36, 37). Choice and application of these systems to structurefunction studies raises many problems, some of which are listed in Table I. For the purposes of this article, attention will be focused primarily on the assessment of the structural features required to stimulate intestinal calcium absorption. This system was chosen because stimulation of intestinal calcium transport is one of the principal effects of vitamin D action and because its biochemical mechanisms are better understood than those underlying the action of vitamin D on bone. Natural
metabolites
To date, four and occurring biologically
perhaps active
five naturally metabolites of
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R
ET
VITAMIN
D, ITS
METABOLITES
AND
1273
ANALOGS
CH3
-‘90/tO
(I)
(9
110(a)
B H
G H
-.-IO/9O CH3(o)
OH (0)
70/30
H(e)
H[H (0)
..-
H3C (o)/ HOft
H--’
H3C (6)
e)
-=
c,3
OH
-“10/90
98/2 H
H
I
H0’
H
)o)
T
30/7O ‘50/50
HC)oI
H
FIG. 2. Dynamically equilibrating A-ring chair conformations. The A-ring structures for the following compounds are depicted. A) vitamin D3; B) la-OH-D3; C) 3-deoxy-la,25-dihydroxyvitamin D3; D) 3-deoxy-3a-methyl-lahydrovitamin D3; E) 3-deoxy-33-methyl-la-hydroxyvitamin D3; F) DHV3-II; G) IOR,l9-dihydrovitamin D3; H) DHT3; I) lOR,l9-dihydro-5E-vitamin D3. Shown below each pair of conformers is a theoretically derived population ratio indicating the relative amounts of each conformer in solution.
vitamin D3 have been identified. The first was 25-hydroxyvitamin D3 (25-OH-D3) (38), which was more active than vitamin D3 in stimulating intestinal calcium transport and bone calcium mobilization in vivo. In addition, this compound stimulated bone calcium mobilization, under conditions in vitro where vitamin D3 was devoid of activity (39). Soon thereafter, Blunt and DeLuca (40) achieved the chemical synthesis of 25-OH-D3 and proposed that this metabolite was the biologically active form of vitamin D3 (41). However, Norman’s laboratory had already reported the existence of a more polar metabolite (42) which was then shown to have greater biological activity than 25-OH-D3 (43). Subsequently, this more polar metabolite was isolated and identified as la,25-(OH)-D3 (44-46) and is now recognized as the most potent metabolite of vitamin D (47-49). Complete chemical synthesis of la,25-
(OH)-D3 was first achieved by Semmleret al. (50), with a yield of less than 0.005%; more recently by Barton and coworkers (51), and Uskokovic and associates who have reported a synthesis with good yield (Dr. M. Uskokovic, Hoffman LaRoche, Nutley, N. J., personal communication). Two other dihydroxy metabolites of vitamin D3, 24,25-dihydroxyvitamin D3 (24,25(OH)2-D3) (52) and 25,26-dihydroxyvitamin D3 (25,26-(OH)2-D3) (53), also circulate in the intact animal and, when given to vitamin D-deficient animals, stimulate intestinal calcium transport to some extent. However, their physiological role is not known. A trihydroxy metabolite, l,24,25-(OH)3D3, first detected under in vitro conditions, has now been produced biosynthetically with the aid of a kidney homogenate system (54). Although 1,24,25-(OH)3D3 will stimulate intestinal calcium transport, it is less active
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cHo)
A
1274
Problems
ET
I of
assay
Which
bioresponse
Should
the assay
of new
should
vitamin
D metabolites
be measured?
(ICA”
or analogs or BCM”
or other).
be in vivo or in vitro? Method of dose administration? (oral, IP,5 or ICb). Problems related to the physiology and biochemistry of vitamin Steroid structural requirements (metabolism-transport-interaction 25-Hydroxylation and/or 1-hydroxylation 5,6-cis versus 5,6-trans vitamin D compounds Equilibrium of A-ring conformers Side chain effects Effects of missing, altered, or added functional groups Consequences of catabolism ‘ICA,
intestinal
calcium
absorption;
BCM,
bone
calcium
than la,25-(OH)2-D3 on a weight basis (54). Whether l,24,25(OH)3D3 occurs in vivo is as yet uncertain; Friedlander and Norman (55) have failed to detect it in the chicken. Vitamin D3 is hydroxylated in the liver at the 25-position (56, 57) and then in the kidney at the 1 position (58-60). Attempts to assess the relative importance of these steps have shown that the la-hydroxyl group is required for normal biological activity. Thus, nephrectomized rats cannot convert 25-OH-D3 to la,25-(OH)2-D3, since they lack the renal enzyme, 25-hydroxy-cholecalciferol1a-hydroxylase. Administration of vitamin D3 or 25-Ol-I-D3 to such animals failed to evoke the biological responses obtained with la,25(OH)2-D3 (58, 61, 62). Moreover, administration of either 25,26-(OH)2-D3 (61) or 24,25-(OH)2-D3 (53) cannot stimulate intestinal calcium transport in nephrectomized rats; this further indicates that hydroxylation in the la position is required for these compounds to be biologically active. Synthetic
AL.
analogs
of la,25-(OH)2-D3
Once lct,25-(OH)2-D3 was recognized as the biologically active form of vitamin D3, it became feasible to search for structural analogs with the objective of defining the structural requiremnts for biological activity. Our laboratory currently has on hand some 30 analogs, many of which are shown in Figure 3. All have been synthesized in our laboratory with the exception of la,25-(OH)2D3, the R and S epimers of 24,25-(OH)2-D3 and la,24,25-(OH)3-D3 (kindly supplied by M.
D:
mobilization.
with
target
b
tissue
receptors)
IP, intraperitoneal;
IC,
intracardial.
Uskokovic), 25-OH-D2 (generously provided by H. F. DeLuca), and 25-hydroxydihydrotachysterol3 25-OH-DHT3 (a gift from P. Bell). The reader is asked to refer to Figure 3 whenever the structure of these analogs is discussed. One of the earliest vitamin D analogs shown to possess biological activity is dihydrotachysterol2 (63). This compound was first isolated by von Werder (64) as the product following the reduction of the C10-C19 double bond of tachysterol2, an isomer of vitamin D2 produced by the ultraviolet irradiation of ergosterol (21). The corresponding dihydrotachysterol3 (C8H17 side chain) was prepared some years later (11). DHT3 is a member of the 5,6-trans (SE) series of vitamin D analogs, so named because the A-ring is rotated 180 C about the 5,6-double bond, thus orienting the 3fl-hydroxyl in a position geometrically equivalent to the la-hydroxyl of la,25-(OH)2-D3. Members of the 5,6-trans series of analogs are, therefore, often referred to as pseudo-la-hydroxy analogs. In the chick the dose of DHT3 required to achieve a biological response is 20 times that of a physiological dose of vitamin D3 (30). In the body, DHT3 is further metabolized to 25-OH-DHT3 (65, 66), a compound that has also been synthesized (53, 66). Both 25-OHDHT3 and DHT3 retain their biological activity in nephrectomized rats (67, 68). This indicates that the pseudo-la-OH groups can fulfill to a substantial degree the structural requirement for a la-OH group. The synthesis of 5,6-trans-vitamin D3 was first achieved by utilizing an iodine-catalyzed
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TABLE
PROCSAL
VITAMIN A
03 AtC
D, IT
AND
ANALOGS
1275
METABOLITES
5Z (5,6-ca)
0.25-1014)303
24R,
25’(OH)3D3
25C.26-(OH)303
0.24R.25-(0H)303
ANALOGS
,c
H
OH 3-d-ia-0H03
100HD3
H3C
3-d-3aMs-b-0H03
3d-.25(014)203
oc?ov-D3
i0.OH-sPD3
3-d-3-
3d3.3M,2
Ms-Io-0H03
160H03
o14v3-m
9Cp0HV3fl
H0.f
r
HOT
004
02
OHV3-D
110,000 > 10,000 >10,000 90 600 > 10,000 >10,000 >10,000 > 10,000 >10,000 >
10,000
The data shown are the concentrations of analog required to reduce the binding of 3H-la,25-(OH)2-D3 to its intestinal cytosol-chromatin receptor system by 50%. The assay was conducted as described by Procsal et al. (95).
±
Ia,25-dihydroxycholesterol standard deviation.
and
filipin.
Each
point
(OH)2-D3. The dramatically decreased ability of either 25-OH-D3 or la-OH-D3 to compete with la,25-(OH)2-D3 emphasizes the great importance that must be attributed to the presence of both 25 and la hydroxyl groups in order for a compound to attain biological activity. The 100 times greater ability of 3-D-la,25-(OH)2-D3 to compete than either la-OH-D3 or 25-OH-D3 argues strongly that the requirement for the 3j3-OH group is relatively less stringent than for either the 25-OH-D3 or la-OH groups. The importance of the 25-OH group for biological activity in the intestine is brought out by the greatly enhanced ability of 25-OHDHT3, 25-OH-5,6-t-D3, and 3-D-Ia,25(OH)2-D3 to compete as compared to their non-25-hydroxylated counterparts (DHT3 ,5, 6-t-D3 and 3-D-la-OH-D3, respectively). It is interesting to note that 25-OH-DHT3 is a significantly better competitor than 25-OH5,6-t-D3. This suggests that a C-l9 methyl group in these 5,6-trans analogs permits the analog to interact more effectively with the receptor system than a C- 19 methylene group. Our laboratory has recently succeeded (18) in introducing a hydroxyl into the 19 position of the 5,6-cis analogs lOS,19-dihydrovitamin D3 (DHV3-II) and DHV3-III and the 5,6trans analogs DHT3 and DHV3-IV. It was reasoned that the presence of an hydroxyl at
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FIG. 5. Competition of vitamin D3 analogs with la,25-dihydroxyvitamin D3 for the chick intestinal receptor system. Increasing concentrations of nonradioactive analogs were incubated for 45 mm at 25 C with a reconstituted cytosol-chromatin receptor system in the presence of 2.0 x 10’ M tritiated la,25-(OH)2D3. The percentage of maximum radioactivity tightly bound to the chromatin (radioactivity bound in the absence of analog equals 100%) is plotted as a function of the relative concentration of analog and tritiated la,25-(OH)2D3. Non-radioactive compounds are denoted as follows: (.-#{149}) Ia,25-(OH)2-D3; (U.-), 3-D-la,25-(OH)2-D3; (-), 25-OH-DHT; (D-D). 25-OH-5,6-t-D3; (A-A), 25-OH-D3; (O-O), la-OH-D3; (V-V). 24,25-(OH)2-D3 (R or 5)
VITAMIN
D,
ITS
METABOLITES
Conformational A
theory
further refinement of the requirements for biological activity has recently been proposed by Okamura and co-workers (18, 105). Their model also takes into account the chair-like nature of the A-ring and proposes that the chair conformer in which the la-OH is equatorial is the prefered biologically active
ANALOGS
1279
form. The evidence to support this model is largely derived by a comparison ofthe significant biological activity of DHT3 (e/a ratio of 91/2) with the inactivity of its structural congener DHV3-IV (e/a ratio of 50/50); (24-26). If one were to assume (4, 8, 13) that the mode ofaction of la,25-(OH)2-D3 is like that of other classical steroids, then one might expect that the ultimate magnitude of the response produced by the natural steroid and related analogs would be the same, but that different concentrations would be required to produce the maximum response. In the light of the elegant studies of Samuels and Tomkins (106) the situation does not appear to be 8
Unpublished
DeLuca
(personal
studies,
by
communication),
us,
as well indicate
as
by
H.
that
the
F. R
epimer of 24,25-(OH)2-D3 is more active biologically than the S epimer. Recently Tanaka et al. (I 13) have reported that biosynthesized 24,25-(OH)2-D3 comigrates with 24R,25-(OH)2-D3 in a chromatography system which can separate the R and S epimers. 9 Abbreviations: vitamin D3, (D3); 25-hydroxyvitamin D3 (25-OH-D3); lct,25-dihydroxyvitamin D3, (la,25(OH)2-D3); 24R,25-dihydroxyvitamin D3, (24R,25(OH)2-D3); 25,26-dihydroxyvitamin D3 (25,26-(OH )D7); la,24R,25-trihydroxyvitamin D3. (la,24R,25(OH)3-D3); la-hydroxyvitamin D3, ( la-OH-D3); 3-
,
deoxyla-hydroxyvitamin D3, (3-d- la-OH-D3); 3deoxy- la,25-dihydroxyvitamin D3, (3-d- la,25-(OH )2D3 ); 3-deoxy-3a-methylI a-hydroxyvitamin D3 (3-d-3aMe- la-OH-D3); 3-deoxy-3f3-methylla-hydroxyvitamin D3, (3-d-3fl-Mela-OH-D3): 3-deoxy-3,3-dimethylIahydroxyvitamin D3, (3-d-3,3-(Me)2la-OH-D3); Iahydroxy vitamin D3, (3-d-3,3-(Me)2la-OH-D3); la-hydroxy-epi-vitamin D3, (Ia-OH-epi-D3); 20,21, 22,23,24,25,26,27-octanovitamin D3, (octanor-D3); vitamin D2, (D2); 105, 19-dihydrovitamin D3, (DHV3II or the lOS-a isomer); IOR, 19-dihydrovitamin D3, (DHV3-III or the IOR-a isomer); 19-hydroxy-IOS, 19dihydrovitamin D3, (l9-OH-DHV3-II); 19-hydroxybR, 19-dihydrovitamin D3, (19-OH-DHV 3-Ill). 245,25-dihydroxyvitamin D3, (24S,25-(OH)2-D3); 24homo-25-hydroxyvitamin D3, (24-homo-25-OH-D3); 24-nor-25-hydroxyvitamin D3, (24-nor-25-OH-D3); 23,24-dinor-25-hydroxyvitamin D3, (23,24-dinor-25OH-D3); 22,23,24-trinor-25-hydroxyvitamin D3, (22,23,24-trinor-25-OH-D3); 20,21 ,22,23,24-pentanor25-hydroxyvitamin D3, (20,21 ,22,23,24-pentanor-25OH-D3); SE-vitamin D3, (5E-D3 or 5,6-trans-D3); 25hydroxy-5E-vitamin D3, (25-OH-S E-D3); dihydrotachysterol3, (DHT3 or the lOS-b isomer: also IOS,l9-dihydro-SE-D3); 25-hydroxydihydrotachysterol3, (25-OHDHT3); IOR,19-dihydro-5E-vitamin D3, (DHV3-IV, the bR-b isomer or IOR,l9-dihydro-SE-D3); 19-hydroxydihydrotachysterol3, (19-OH-DHT3); 19-hydroxydihydrovitamin D3-IV, (19-OH-DHV3-IV); dihydrotachysterol2, (DHT2); dihydrovitamin D2-IV, (DHV2IV); isovitamin D3, (iso-D3); isotachysterols, (iso-T3); tachysterol3, (T3).
,
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the 19 position might, to some limited degree, satisfy the requirement for a pseudo 3-OH group in the 5,6-trans series, or the I-OH in the 5,6-cis series. However, no significant competition was observed for any of these compounds in our competitive binding assay. It may be that these compounds will first have to be hydroxylated in the 25 position before their relative ability to bind to the receptor can be evaluated. The insertion of an hydroxyl group into the 24 position of 25-OH-D3 to give either the R or S epimer of 24,25-(OH)2-D3 significantly reduces the ability of the compound to compete with lcv,25-(OH)2-D3. Under these assay conditions, it was not possible to discern any significant difference between the two epimers.8 The naturally occurring epimer of 24, 25-(OH)2-D3 has been shown to have its 24OH in the R configuration (113). In general, there is a remarkable correlation between the relative binding of analogs in the chick intestinal receptor system, and their relative ability to stimulate intestinal calcium absorption. The following conclusions may therefore be drawn with respect to the structural features required for vitamin D-like biological activity in the intestinal cell: 1) All biologically active compounds must have a la-hydroxyl or its geometric equivalent and a 25-hydroxyl group. 2) The presence of a hydroxyl at a geometrically equivalent position to the 3fl-hydroxyl of la,25-(OH)2-D3 is not an absolute requirement. 3) A 5,6-cis geometry of the A-ring is preferred to a 5,6-trans geometry. A comparison of the biological and binding activities of 25-OHDHT3 with those of 25-OH-5,6-t-D3 suggests that in compounds of the 5,6-trans series a C-19 methyl is preferred over a C-19 methylene group. 4) Altering the length or modifying the side chain, including the insertion of a hydroxyl group as in 24,25-(OH)2-D3 or 25,26-(OH)2D3, significantly decreases the activity of a compound.
AND
1280
PROCSAL
AL.
la,25-(OH)2-D3 are already being used clinically to treat patients who suffer from vitamm D-related diseases, principally renal osteodystrophy (108-Ill). As our understanding of the structural basis for biological activity increases, it may become possible to synthesize analogs with predetermined activities or that can act as antivitamins (1 12). The availability of such compounds will undoubtedly serve as a further stimulus for this rapidly expanding area of research. References 1. COBURN,
L. HARTENBOWER AND A. W. J. Med. 121: 22, 1974. NORMAN, A. W., AND H. HENRY. Clin. Orthopaed. Related Res. 98: 258, 1974. HOLICK, M. F., AND H. F. DELUCA. Ann. Rev. Med. 25: 349, 1974. NORMAN, A. W., AND H. HENRY. Rec. Prog. Hormone Res. 30: 431, 1974. OMDAHL, J. L., AND H. F. DELUCA. Physiol. Rev. 53: 327, 1973. HAUSSLER, M. R., J. F. MYRTLE AND A. W. NORMAN. J. Biol. Chem. 243: 4055, 1968. LUND, J., AND H. F. DELUCA. J. Lipid Res. 7: 739, 1966. HAU5SLER, M. R. Nutr. Rev. 32: 257, 1974. HOLICK, M. F., AND H. F. DELUCA. Adv. Steroid Biochem. and Pharmacology 4: III, 1974. SCHNOES, H. K., AND H. F. DELUCA. Vit. Hormones 32: 385, 1975. HAVINGA, E. Experientia 29: 1181, 1973. NORMAN, A. W. Biol. Rev. 43: 97, 1968. NORMAN, A. W. Vit. Hormones 32: 325, 1975. NORMAN, A. W., W. H. OKAMURA AND R. M. WING. Proc. 5th Parathyroid Conf., Amsterdam NORMAN.
2. 3. 4. S. 6. 7. 8. 9. 10. II.
12. 13. 14.
Excerpta
IS.
NORMAN, AND
R.
J. W.,
D.
Western
1974. A. W., D. A. PROCSAL, M. WING. J. Steroid Medica,
W. H. OKAMURA Biochem. 6: 461,
1975. 16. OKAMURA, W. H., M. L. HAMMOND, M. R. PIRI0, R. M. WiNG, A. REGO, M. N. MITRA AND A. W. NORMAN. Proc. 2nd Workshop on Vit. D and Problems Related to Uremic Bone Disease, Edited by A. W. Norman et al. Berlin: Walter de Gruyter, 1975, pp. 259 278. 17. NORMAN, A. W., W. H. OKAMURA, E. J. FRIEDLANDER, H. L. HENRY, R. L. JOHNSON, M. N. MITRA, D. A. PROCSAL AND W. WECKSLER. Proc. XI Eur. Symp. on Calcified Tissues. Copenhagen: F.A.D.L. Pub. Co.. 1975. 18. NORMAN, A. W., R. L. JOHNSON, T. W. OSBORN, D. A. PR0cSAI., S. C. CAREY, M. L. HAMMOND, M. N. MITRA, M. R. PIRI0, A. REGO, R. M. WING AND W. H. OKAMURA. Clin. Endocrinol. In press. 19. WINDHAUS, A., 0. LINSERT, A. LUTTRINGHAUS, G. WEIDLICH AND J. LIEBIGS. Annal. Chem. 492: 226, 1932. 20. ASKEW, F. A., R. B. BOURDILLON, H. M. BRUCE, R. K. ST. L. CALLOW, J. PHILPOT AND T. A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1271/4649688 by East Carolina University Health Sciences Library user on 16 January 2019
quite so simple. These investigators measured the induction of the enzyme, tyrosine aminotransferase, in hepatoma tissue culture (HTC) cells in tissue culture, utilizing analogs of glucocorticoids as inducers. It became apparent that there existed at least three levels of maximum response, each level being related to a structural class of the analogs. The three classes were: optimal inducers, i.e., those that gave the largest magnitude of response: suboptimal inducers, i.e., those that generated only a fraction of the response of the optimal inducers; and a third class of analogs, which were inactive. Samuels and Tomkins proposed that this classification resulted from the fact that the cytoplasmic glucocorticoid receptor in the HTC cells exists as two allosteric species. If, moreover, only one of the two allosteric species was capable of being “activated” and transported to the nucleus to induce the biological response, then the relative magnitude of response produced by the steroids was the result of their relative affinity for the two forms of the cytoplasmic receptor. Conceivably such a situation may exist also for the vitamin D metabolites and their analogs. The problem is even more complicated in the light of the rapid equilibration in solution of the two conformers of the seco-steroids. Each of these two conformers may have a different affinity for the two presumed allosteric forms of the cytoplasmic receptor for la,25-(OH)2-D3. In this connection it is interesting that the natural hormone, la,25-(OH)2-D3 may be a suboptimal inducer, since the analog, 3-D-laOH-D3 can produce a greater maximum response (107). It follows logically from what has been said that the intracellular sequence of receptor events must contain an irreversible step. Otherwise, the “inactive conformer” could be transformed back to the “active conformer.” Experimental support for our model requires further comparisons of the biological activities of a number of structural stereoisomers that differ in the relative proportion of A-ring conformers. Studies of this type are currently under way. The structure-function studies described here have made it possible to design and construct a number of analogs for clinical use. Compounds such as la-OH-D3 and
ET
VITAMIN Proc.
York: 1959. 22.
24. 25.
26.
27.
28.
pp.
AND New
127-130,
D.,
AND
J.
D.
DUNITZ.
SI.
96:
52.
7317, 1974. E. N., N. L. ALLINGER,
G.
MORRISON.
Conformational
Interscience
Publications,
A.
L. ANGYAL Analysis, Chap.
40:
Lett.
4147,
R. H. J. Am.
M.
HESSE, Chem.
M.
Soc.
PECHET
95:
2748,
M. F., H. K. SCHONES, H. F. DELUCA, I. T. BOYLE AND T. SUDA. Biochemistry
HOLICK,
SUDA,
R.
1972.
T., AND
H.
F. M.
DELUCA,
F. HOLICK.
H.
K.
Y. 9: 4776,
SCHNOES,
Biochemistry
M. F., A. KLEINER-BOSSALLER, H. K. P. M. KASTEN, I. T. BOYLE AND H. F. DELUCA: J. Biol. Chem. 248: 6691, 1973. FRIEDLANDER, E. J., AND A. W. NORMAN. Arch. Biochem. Biophys. 170: 731, 1975. HORSTING, M., AND H. F. DELUCA. Biochem. Biophys. Res. Commun. 36: 251, 1969. PONCHON, G., AND H. F. DELUCA. J. Clin. Invest. 48: 1273, 1969. FRASER, D. R., AND E. KODICEK. Naure 228: 764, 1970. NORMAN, A. W., R. J. MIDGETT, J. F. MYRTLE AND H. G. NOWICKI. Biochem. Biophys. Res. Commun.42: 1082, 1971. GRAY, R., I. BOYLE AND H. F. DELUCA. Science 172, 1232, 1971. LAM, H-Y., H. K. SCHNOES, H. F. DELUCA ANDT. C. CHEN. Biochemistry 12: 4851, 1973. WONG, R. G., A. W. NORMAN, C. R. REDDY ANt) J. W. COBURN. J. Clin. Invest. SI: 1287, 1972. LIU, S. H., AND H. I. CHU. Medicine 22: 103, 1943. VON WERDER, F. Hoppe-Seyler’s Z. Physiol. Chem. 260: 1 19, 1939. HALLICK, R. B., AND H. F. DELUCA. J. Biol. Chem. 246: 5733, 1971. LAWSON, D. E. M., AND P. A. BELL. Biochem. J. 142: 37, 1974. HALLICK, R. B., AND H. F. DELUCA. J. Biol. Chem. 247: 91, 1972. HARRISON, H. E., AND H. C. HARRISON. J. Clin. Invest. 51: 1919, 1972. VERLOOP, A., A. L. KOEVOET AND E. HAVINGA. Rec. Tray. Chim. Pays-Bas. 74: 1125, 1955. INHOFFEN, H. H., G. QUINKERT, H. J. HESS AND H. HIRSCHFELD. Chem. Ber. 90: 2544, 1957. HOLICK, M. F., M. GARABEDIAN AND H. F. DELUCA. Biochemistry II: 2715, 1972. GAGNON, R., G. W. OGDEN, G. JUST AND M. KAYE. Can. J. Physiol. Pharmacol. 52: 272, 1974. HOLICK, M. F., E. J. SEMMLER, H. K. SCHNOES AND H. F. DELUCA. Science 180: 190, 1973. BARTON, D. H. R., R. H. HESSE, M. M. PECHET AND E. RIZZARDO. J. Am. Chem. Soc. 95: 2748, 1973. HARRISON, R. G., B. LYTHGOE AND R. W. WRIGHT. Tetrahedron Lett. 37: 3649, 1973. CORK, D. J., M. R. HAUSSLER, M. J. PITT, E. RIZZARDO, R. H. HESSE AND M. M. PECHET. Endocrinology 94: 1337, 1974. HAUSSLER, M. R., J. E. ZERWEKH, R. H. HESSE, E. RIZZARDO AND M. M. PECHET. Proc. NatI. Acad. Sci. U.S. 70: 2248, 1973. KANEKO, C., S. YAMADA, A. SUGIMOTO, Y. EGUCHI, M. ISHIKAWA, T. SUDA, M. SUZUKI, J. HOLICK,
SCHNOES,
55. 56. 57.
59.
60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.
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46. NORMAN, A. W., J. F. MYRTLE, R. J. MIDGETT, H. G. N0WICKI, V. WILLIAMS ANt) G. POPJAK. Science 173: 51, 1971. 47. NORMAN, A. W. J. Nutr. 102: 1243, 1971. 48. TANAKA, Y., H. FRANK AND H. F. DELUCA. J. Nutr. 102: 1569, 1975. 49. HAUSSLER, M. R., D. W. BOYCE, E. T. LITTLEDIKE AND H. RASMUSSEN. Proc. NatI. Acad. Sci. U.S. 68: 177, 1971. 50. SEMMLER, E. J., M. F. HOLICK, H. K. SCHNOES
Tetrahedron
1970. 54.
K.
Pharmacol.
D. H. R., E. RIZZARDO.
TANAKA
2, 1967.
A. AND A. W. NORMAN. Biochem. 18: 2347, 1969. 31. COATES, M. E., AND E. J. HOLDSWORTH. Brit. J. Nutr. 15: 131, 1961. 32. Association of Official Agricultural Chemists Methods of Analysis 5th ed. 1940. pp. 371373. 33. ADAMS, T. H., AND A. W. NORMAN. J. Biol. Chem. 245: 4421, 1970. 34. WALLING, M. W., AND D. V. KIMBERG. Am. J. Physiol. 225: 415, 1973. 35. CORRADINO, R. A. Science 179: 402, 1973. 36. REYNOLDS, J. J., AND J. T. DINGLE. Calcif. Tissue Res. 12: 295, 1973. 37. RAISz, L. G. J. Clin. Invest. 44: 103, 1965. 38. BLUNT, J. W., H. F. DELUCA AND H. K. SCHNOES. Biochem. 7: 3317, 1968. 39. TRUMMEL, C. L., L. G. RAISZ, J. W. BLUNT AND H. F. DELUCA. Science 163: 1450, 1969. 40. BLUNT, J. W., AND H. F. DELUCA. Biochemistry 8: 671, 1969. 41. DELUCA, H. F. Am. J. Clin. Nutr. 22: 412, 1969. 42. HAUSSLER, M. R., J. F. MYRTLE AND A. W. NORMAN. J. Biol. Chem. 243: 4055, 1968. 43. MYRTLE, J. F., AND A. W. NORMAN. Science 171: 79, 1971. 44. HOLICK, M. F., H. K. SCHNOES, H. F. DELUCA, T. SUDA AND R. J. COUSINS. Biochemistry 10: 2799, 1971. 45. LAWSON, D. E. M., D. M. FRASER, E. KODICEK, H. R. MORRIS AND D. H. WILLIAMS. Nature 230: 228, HIBBERD,
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53.
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York:
30.
AND
1932.
Steroids.
1948.
HODGKIN,
Soc.
29.
B 109: 488,
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AND
CROWFOOT,
608, 23.
Roy.
L. F., AND M. FIESER. In: Rheinhold Publishing Corp.,
FIESER,
ITS
75. 76.
77.
78.
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WEBSTER.
21.
D,
PROCSAL
1282
80.
81.
82.
83.
84.
85. 86. 87. 88. 89. 90. 91. 92. 93.
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AND
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109.
A. S., J. W. COBURN, G. R. FRIEDMAN, S. G. MASSRY AND A. W.
BRICKMAN,
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106. SAMUELS. H. H., AND C. M. TOMKINS. J. Mol. Biol. 52: 57, 1970. 107. NORMAN, A. W., M. N. MITRA, W. H. OKAMURA AND R. M. WING. Science 188: 1013, 1975.
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H. C., AND A. W. NORMAN. J. Biol. Chem. 248: 5967, 1973. 98. BRUMBAUGH, P. F., AND M. R. HAUSSLER. J. Biol. Chem. 249: 1251, 1974. 99. BRUMBAUGH, P. F., AND M. R. HAUSSLER. J. Biol. Chem. 249: 1258, 1974. 100. TSAI, H. C., AND A. W. NORMAN. Biochem. Biophys. Res. Commun. 54: 622, 1973. 101. CORRADINO, R. A. Science 179: 402, 1972. 102. FREUND, T. S., AND F. BRONNER. Science 190: 1975. 103. MYRTLE, J. F., AND A. W. NORMAN. Science 171: 79, 1971. 104. BRUMBAUGH, P. F., D. H. HAUSSLER, K. M. BURSAC AND M. R. HAUSSLER. Biochemistry 13: 4091, 1974. 105. OKAMURA, W. H., A. W. NORMAN AND R. M. WING. Proc. NatI. Acad. Sci. U.S. 71: 4194, 1974.
publication). 94. HOLICK.
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S. SASAKI. Steroids 23: 75, 1974. A. S., J. W. COBURN, G. R. FRIEDMAN, W. H. OKAMURA, S. G. MASSRY AND A. W. NORMAN. J. Clin. Invest. 57: 1540, 1976. BRICKMAN, A. S., G. R. FRIEDMAN, J. W. COBLRN, M. N. MITRA, S. G. MASSRY, W. H. OKAMURA AND A. W. NORMAN. Clin. Res. 23: I 19A, 1975. ZERWEKH, J. E., P. F. BRUMBAUGH, D. H. HAUSSLER, D. S. CORK AND M. R. HAUSSLER. Biochemistry 13: 4097, 1974. OKAMURA, W. H., M. N. MITRA, R. M. WING AND A. W. NORMAN. Biochem. Biophys. Res. Commun. 60: 179, 1974. LAM, H. Y., B. L. ONISKO, H. K. SCHNOES AND H. F. DELUCA. Biochem. Biophys. Res. Commun. 59: 845, 1974. OKAMURA, W. H., M. N. MITRA, D. A. PROCSAL AND A. W. NORMAN. Biochem. Biophys. Res. Commun. 65: 24, 1975. MCDONALD, F. G. J. Biol. Chem. 114: IXV, 1936. ROSENBERG, H. R. Chemistry and Physiology of Vitamins. New York, 1945. DIMROTH, K., AND J. PALAND. Ber. dt. Ver. Volkshyg. 72: 187, 1939. HASLEWOOD, G. A. D. Biochem. J. 33: 454, 1939. CHEN, P. 5., AND H. BOSMANN. J. Nutr. 83: 133, 1964. BOUCHER, R. V. J. Nutr. 27: 403, 1944. HUNT, R. 0., F. G. GARCIA AND D. M. HEGSTED. Lab. Animal Care 17: 222, 1967. JOHNSON, R. L., AND W. H. OKAMURA. Federation Proc. 34: 894, 1975. JOHNSON, R. L., S. C. CAREY, A. W. NORMAN AND W. H. OKAMURA. J. Org. Chem. (submitted for KAKUTA
79.
ET
a review
of the structural
for biological D. A.
Procsal,3
and
W.
requirements
activity’2 H.
Okamura,4
and
A.
W. Norman3
It has become increasingly apparent that modifications in the structure of vitamin D can lead to analogs that are more effective than the parent compound in the treatment of various vitamin D-related disorders (1-3). Comparable advances have also been made in understanding the mode of action of vitamin D (4, 5). Together these clinical and biochemical studies have stimulated interest and made possible a detailed analysis of the structurefunction relationships that characterize this regulator of calcium metabolism. It is the purpose of this article to review pertinent studies (6-18) with emphasis on recent work from this laboratory directed toward a systematic analysis of the chemical and biological properties of vitamin D3, its major metabolites, and a number of structural analogs. Structure
of vitamin
D3
Figure 1 traces the history in the development of our understanding of the structure of vitamin D. Structure I in Figure 1 is a representation of the initial formulation of the steroidal structure of the vitamin established in the early 1930’s (19, 20). Its early chemistry has been reviewed comprehensively by Fieser and Fieser (21). These studies established that vitamin D results when the B-ring of its provitamin [7-dehydrocholesterol (D3) or ergosterol (D2)] is cleaved by a photochemically catalyzed reaction. Vitamin D3 is, therefore, a secosteroid with the chemical name of 9,10-secocholesta-5,7,10 (19)triene-33-ol. It should be noted that Structure 1 does not describe the stereochemistry of the molecule. This was established some 20 years ago by elegant X-ray crystallography performed by Crowfoot and Dunitz (22)
and
The American
Hodgkin Journal
et al. of
analogs:
Clinical
(23)
employing
Nutrition
an
29: NOVEMBER
analog of vitamin D2. The results of these studies (Structure 2) established that the diene system extending from C-5 to C-8 is coplanar and transoid (as opposed to the cisoid configuration shown in Structure I). The 5,6-double bond is considered cis (Z) in relation to the A-ring by virtue of the fact that the C-7 is cis to C-lO, rather than to C-4. The x-ray crystallographic data also indicated that the A-ring is in a single chair conformation. This feature, however, is rarely incorporated in the planar Structure 1. Recently Knobler et al. (24) reported x-ray crystallographic data for another vitamin D analog indicating an opposite chair conformation is that reported by Crowfoot-Hodgkin’s group. This difference was the result of the fact that the A-ring can be frozen in either of two chair conformations, depending upon the nature of these analogs as they exist in their crystalline form. The existence of different A-ring conformations is a consequence of the open ring structure of vitamin D. It is this structure that imparts upon the A- and seco-B6 rings a degree of conformational mobility not found in other steroids in which the A, B, C, and D rings exist as a fused array. Recent studies in our laboratory (25-27) and independently in that of Lamar and Budd ‘From the Departments of Biochemistry and Chemistry, University of California, Riverside, California 92502. 2 This work was supported in part by United States Public Health AM-l6,595.
Service
Grant
AM-09012,
AM-14,750,
and
Department
of Biochemistry. ‘Department of Recipient of a USPHS Career Research Development Award, IKD-AM-l3,654. 6 The term “seco” is used to designate cleavage of one
Chemistry.
or more rings of the cyclopentanoperhydrophenanthrene steroid skeleton. Thus vitamin D, where the 9-10 carbon bond of the B ring is broken, is designated as a seco-B steroid.
1976,
pp. 1271-1282.
Printed
in U.S.A.
1271
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Vitamin
1272
PROCSAL
equatorial configuration, while 45% will have the la-hydroxyl in the axial orientation. This distribution is governed by a dynamic equilibrium between the two chair forms such that the A-ring of any one molecule may flip from one chair conformation to the other some one million times per second. It is clear that alterations in this ratio can be biased in favor of one conformer over the other as a consequence of the introduction of different substituent groups into the A-ring (Fig. 2). In view of the profound influence these conformational effects may have on the structure of the A-ring, it is clear that the experimentalist interested in defining structure-function rela-
2
OH CH3
OH
FIG. ofvitamin I resulted
5 I
.
6
Evolution of conformational D. The structure for vitamin from
the
original
structure
representations D3 represented determination.
AL.
in The
structure depicted in 2 was deduced from x-ray crystallographic analysis. Structures 3 and 4 illustrate the rapid equilibration between the two A-ring chair conformations, while structures 5 and 6 show the same relationship for la,25-(OH)2-D3. The (e) and (a) refer, respectively, to the equatorial and axial orientations of the indicated hydroxyl group (i.e., either the 3i or extended
la-hydroxyl).
(28) have focused on the conformational mobility of the molecule as it exists in solution. In both instances, high resolution PMR spectroscopy was employed to demonstrate that the A-ring of vitamin D and of several related seco steroids exists in solution as a pair of dynamically equilibrating chair conformers (Structures 3 and 4 of Figure 1). This observation becomes particularly significant in the light of the fact that for each conformational inversion every equatorial position becomes axial and every axial position becomes equatorial. Furthermore, the equilibrium constant between the two chair conformers will be dependent upon the nature and location of substituent groups on the A-ring as deduced from model studies on cyclohexane (reviewed by Eliel et al. (29)). For example, as shown in Structures 5 and 6 (Figure 1) the la-hydroxyl of la,25-dihydroxyvitamin (I a,25-(OH)2-D3), depending upon the chair conformer, will be either equatorial or axial. In fact, analysis of the conformational ratio of la,25-(OH)2-D3 indicates an equatorial to axial ratio (e/a ratio) of 55/45 (25, 26). That is to say, in a population of la,25-(OH)2-D3 molecules in solution, 55% will have the la-hydroxyl in the
tionships should be aware of this important consideration. In determining structure-function relationships, an area of primary concern is the choice and design of an appropriate experimental model system to evaluate vitamin D related biological response(s). Currently, several assays are available to assess the biological activity in vivo. Included in this group are assays designed to measure in vivo intestinal calcium transport (30), bone calcium mobilization (31), and the ability to cure rachitic lesions (32). In addition, various in vitro techniques are also at hand including the measurement of the rate of intestinal calcium and phosphate transport employing Ussing transport blocks (33, 34), the induction of a vitamin D-dependent calcium binding protein in an embryonic chick intestine organ culture (35) and the measurement of calcium mobilization in cultured bone tissue (36, 37). Choice and application of these systems to structurefunction studies raises many problems, some of which are listed in Table I. For the purposes of this article, attention will be focused primarily on the assessment of the structural features required to stimulate intestinal calcium absorption. This system was chosen because stimulation of intestinal calcium transport is one of the principal effects of vitamin D action and because its biochemical mechanisms are better understood than those underlying the action of vitamin D on bone. Natural
metabolites
To date, four and occurring biologically
perhaps active
five naturally metabolites of
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R
ET
VITAMIN
D, ITS
METABOLITES
AND
1273
ANALOGS
CH3
-‘90/tO
(I)
(9
110(a)
B H
G H
-.-IO/9O CH3(o)
OH (0)
70/30
H(e)
H[H (0)
..-
H3C (o)/ HOft
H--’
H3C (6)
e)
-=
c,3
OH
-“10/90
98/2 H
H
I
H0’
H
)o)
T
30/7O ‘50/50
HC)oI
H
FIG. 2. Dynamically equilibrating A-ring chair conformations. The A-ring structures for the following compounds are depicted. A) vitamin D3; B) la-OH-D3; C) 3-deoxy-la,25-dihydroxyvitamin D3; D) 3-deoxy-3a-methyl-lahydrovitamin D3; E) 3-deoxy-33-methyl-la-hydroxyvitamin D3; F) DHV3-II; G) IOR,l9-dihydrovitamin D3; H) DHT3; I) lOR,l9-dihydro-5E-vitamin D3. Shown below each pair of conformers is a theoretically derived population ratio indicating the relative amounts of each conformer in solution.
vitamin D3 have been identified. The first was 25-hydroxyvitamin D3 (25-OH-D3) (38), which was more active than vitamin D3 in stimulating intestinal calcium transport and bone calcium mobilization in vivo. In addition, this compound stimulated bone calcium mobilization, under conditions in vitro where vitamin D3 was devoid of activity (39). Soon thereafter, Blunt and DeLuca (40) achieved the chemical synthesis of 25-OH-D3 and proposed that this metabolite was the biologically active form of vitamin D3 (41). However, Norman’s laboratory had already reported the existence of a more polar metabolite (42) which was then shown to have greater biological activity than 25-OH-D3 (43). Subsequently, this more polar metabolite was isolated and identified as la,25-(OH)-D3 (44-46) and is now recognized as the most potent metabolite of vitamin D (47-49). Complete chemical synthesis of la,25-
(OH)-D3 was first achieved by Semmleret al. (50), with a yield of less than 0.005%; more recently by Barton and coworkers (51), and Uskokovic and associates who have reported a synthesis with good yield (Dr. M. Uskokovic, Hoffman LaRoche, Nutley, N. J., personal communication). Two other dihydroxy metabolites of vitamin D3, 24,25-dihydroxyvitamin D3 (24,25(OH)2-D3) (52) and 25,26-dihydroxyvitamin D3 (25,26-(OH)2-D3) (53), also circulate in the intact animal and, when given to vitamin D-deficient animals, stimulate intestinal calcium transport to some extent. However, their physiological role is not known. A trihydroxy metabolite, l,24,25-(OH)3D3, first detected under in vitro conditions, has now been produced biosynthetically with the aid of a kidney homogenate system (54). Although 1,24,25-(OH)3D3 will stimulate intestinal calcium transport, it is less active
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/11/1271/4649688 by East Carolina University Health Sciences Library user on 16 January 2019
cHo)
A
1274
Problems
ET
I of
assay
Which
bioresponse
Should
the assay
of new
should
vitamin
D metabolites
be measured?
(ICA”
or analogs or BCM”
or other).
be in vivo or in vitro? Method of dose administration? (oral, IP,5 or ICb). Problems related to the physiology and biochemistry of vitamin Steroid structural requirements (metabolism-transport-interaction 25-Hydroxylation and/or 1-hydroxylation 5,6-cis versus 5,6-trans vitamin D compounds Equilibrium of A-ring conformers Side chain effects Effects of missing, altered, or added functional groups Consequences of catabolism ‘ICA,
intestinal
calcium
absorption;
BCM,
bone
calcium
than la,25-(OH)2-D3 on a weight basis (54). Whether l,24,25(OH)3D3 occurs in vivo is as yet uncertain; Friedlander and Norman (55) have failed to detect it in the chicken. Vitamin D3 is hydroxylated in the liver at the 25-position (56, 57) and then in the kidney at the 1 position (58-60). Attempts to assess the relative importance of these steps have shown that the la-hydroxyl group is required for normal biological activity. Thus, nephrectomized rats cannot convert 25-OH-D3 to la,25-(OH)2-D3, since they lack the renal enzyme, 25-hydroxy-cholecalciferol1a-hydroxylase. Administration of vitamin D3 or 25-Ol-I-D3 to such animals failed to evoke the biological responses obtained with la,25(OH)2-D3 (58, 61, 62). Moreover, administration of either 25,26-(OH)2-D3 (61) or 24,25-(OH)2-D3 (53) cannot stimulate intestinal calcium transport in nephrectomized rats; this further indicates that hydroxylation in the la position is required for these compounds to be biologically active. Synthetic
AL.
analogs
of la,25-(OH)2-D3
Once lct,25-(OH)2-D3 was recognized as the biologically active form of vitamin D3, it became feasible to search for structural analogs with the objective of defining the structural requiremnts for biological activity. Our laboratory currently has on hand some 30 analogs, many of which are shown in Figure 3. All have been synthesized in our laboratory with the exception of la,25-(OH)2D3, the R and S epimers of 24,25-(OH)2-D3 and la,24,25-(OH)3-D3 (kindly supplied by M.
D:
mobilization.
with
target
b
tissue
receptors)
IP, intraperitoneal;
IC,
intracardial.
Uskokovic), 25-OH-D2 (generously provided by H. F. DeLuca), and 25-hydroxydihydrotachysterol3 25-OH-DHT3 (a gift from P. Bell). The reader is asked to refer to Figure 3 whenever the structure of these analogs is discussed. One of the earliest vitamin D analogs shown to possess biological activity is dihydrotachysterol2 (63). This compound was first isolated by von Werder (64) as the product following the reduction of the C10-C19 double bond of tachysterol2, an isomer of vitamin D2 produced by the ultraviolet irradiation of ergosterol (21). The corresponding dihydrotachysterol3 (C8H17 side chain) was prepared some years later (11). DHT3 is a member of the 5,6-trans (SE) series of vitamin D analogs, so named because the A-ring is rotated 180 C about the 5,6-double bond, thus orienting the 3fl-hydroxyl in a position geometrically equivalent to the la-hydroxyl of la,25-(OH)2-D3. Members of the 5,6-trans series of analogs are, therefore, often referred to as pseudo-la-hydroxy analogs. In the chick the dose of DHT3 required to achieve a biological response is 20 times that of a physiological dose of vitamin D3 (30). In the body, DHT3 is further metabolized to 25-OH-DHT3 (65, 66), a compound that has also been synthesized (53, 66). Both 25-OHDHT3 and DHT3 retain their biological activity in nephrectomized rats (67, 68). This indicates that the pseudo-la-OH groups can fulfill to a substantial degree the structural requirement for a la-OH group. The synthesis of 5,6-trans-vitamin D3 was first achieved by utilizing an iodine-catalyzed
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TABLE
PROCSAL
VITAMIN A
03 AtC
D, IT
AND
ANALOGS
1275
METABOLITES
5Z (5,6-ca)
0.25-1014)303
24R,
25’(OH)3D3
25C.26-(OH)303
0.24R.25-(0H)303
ANALOGS
,c
H
OH 3-d-ia-0H03
100HD3
H3C
3-d-3aMs-b-0H03
3d-.25(014)203
oc?ov-D3
i0.OH-sPD3
3-d-3-
3d3.3M,2
Ms-Io-0H03
160H03
o14v3-m
9Cp0HV3fl
H0.f
r
HOT
004
02
OHV3-D
110,000 > 10,000 >10,000 90 600 > 10,000 >10,000 >10,000 > 10,000 >10,000 >
10,000
The data shown are the concentrations of analog required to reduce the binding of 3H-la,25-(OH)2-D3 to its intestinal cytosol-chromatin receptor system by 50%. The assay was conducted as described by Procsal et al. (95).
±
Ia,25-dihydroxycholesterol standard deviation.
and
filipin.
Each
point
(OH)2-D3. The dramatically decreased ability of either 25-OH-D3 or la-OH-D3 to compete with la,25-(OH)2-D3 emphasizes the great importance that must be attributed to the presence of both 25 and la hydroxyl groups in order for a compound to attain biological activity. The 100 times greater ability of 3-D-la,25-(OH)2-D3 to compete than either la-OH-D3 or 25-OH-D3 argues strongly that the requirement for the 3j3-OH group is relatively less stringent than for either the 25-OH-D3 or la-OH groups. The importance of the 25-OH group for biological activity in the intestine is brought out by the greatly enhanced ability of 25-OHDHT3, 25-OH-5,6-t-D3, and 3-D-Ia,25(OH)2-D3 to compete as compared to their non-25-hydroxylated counterparts (DHT3 ,5, 6-t-D3 and 3-D-la-OH-D3, respectively). It is interesting to note that 25-OH-DHT3 is a significantly better competitor than 25-OH5,6-t-D3. This suggests that a C-l9 methyl group in these 5,6-trans analogs permits the analog to interact more effectively with the receptor system than a C- 19 methylene group. Our laboratory has recently succeeded (18) in introducing a hydroxyl into the 19 position of the 5,6-cis analogs lOS,19-dihydrovitamin D3 (DHV3-II) and DHV3-III and the 5,6trans analogs DHT3 and DHV3-IV. It was reasoned that the presence of an hydroxyl at
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FIG. 5. Competition of vitamin D3 analogs with la,25-dihydroxyvitamin D3 for the chick intestinal receptor system. Increasing concentrations of nonradioactive analogs were incubated for 45 mm at 25 C with a reconstituted cytosol-chromatin receptor system in the presence of 2.0 x 10’ M tritiated la,25-(OH)2D3. The percentage of maximum radioactivity tightly bound to the chromatin (radioactivity bound in the absence of analog equals 100%) is plotted as a function of the relative concentration of analog and tritiated la,25-(OH)2D3. Non-radioactive compounds are denoted as follows: (.-#{149}) Ia,25-(OH)2-D3; (U.-), 3-D-la,25-(OH)2-D3; (-), 25-OH-DHT; (D-D). 25-OH-5,6-t-D3; (A-A), 25-OH-D3; (O-O), la-OH-D3; (V-V). 24,25-(OH)2-D3 (R or 5)
VITAMIN
D,
ITS
METABOLITES
Conformational A
theory
further refinement of the requirements for biological activity has recently been proposed by Okamura and co-workers (18, 105). Their model also takes into account the chair-like nature of the A-ring and proposes that the chair conformer in which the la-OH is equatorial is the prefered biologically active
ANALOGS
1279
form. The evidence to support this model is largely derived by a comparison ofthe significant biological activity of DHT3 (e/a ratio of 91/2) with the inactivity of its structural congener DHV3-IV (e/a ratio of 50/50); (24-26). If one were to assume (4, 8, 13) that the mode ofaction of la,25-(OH)2-D3 is like that of other classical steroids, then one might expect that the ultimate magnitude of the response produced by the natural steroid and related analogs would be the same, but that different concentrations would be required to produce the maximum response. In the light of the elegant studies of Samuels and Tomkins (106) the situation does not appear to be 8
Unpublished
DeLuca
(personal
studies,
by
communication),
us,
as well indicate
as
by
H.
that
the
F. R
epimer of 24,25-(OH)2-D3 is more active biologically than the S epimer. Recently Tanaka et al. (I 13) have reported that biosynthesized 24,25-(OH)2-D3 comigrates with 24R,25-(OH)2-D3 in a chromatography system which can separate the R and S epimers. 9 Abbreviations: vitamin D3, (D3); 25-hydroxyvitamin D3 (25-OH-D3); lct,25-dihydroxyvitamin D3, (la,25(OH)2-D3); 24R,25-dihydroxyvitamin D3, (24R,25(OH)2-D3); 25,26-dihydroxyvitamin D3 (25,26-(OH )D7); la,24R,25-trihydroxyvitamin D3. (la,24R,25(OH)3-D3); la-hydroxyvitamin D3, ( la-OH-D3); 3-
,
deoxyla-hydroxyvitamin D3, (3-d- la-OH-D3); 3deoxy- la,25-dihydroxyvitamin D3, (3-d- la,25-(OH )2D3 ); 3-deoxy-3a-methylI a-hydroxyvitamin D3 (3-d-3aMe- la-OH-D3); 3-deoxy-3f3-methylla-hydroxyvitamin D3, (3-d-3fl-Mela-OH-D3): 3-deoxy-3,3-dimethylIahydroxyvitamin D3, (3-d-3,3-(Me)2la-OH-D3); Iahydroxy vitamin D3, (3-d-3,3-(Me)2la-OH-D3); la-hydroxy-epi-vitamin D3, (Ia-OH-epi-D3); 20,21, 22,23,24,25,26,27-octanovitamin D3, (octanor-D3); vitamin D2, (D2); 105, 19-dihydrovitamin D3, (DHV3II or the lOS-a isomer); IOR, 19-dihydrovitamin D3, (DHV3-III or the IOR-a isomer); 19-hydroxy-IOS, 19dihydrovitamin D3, (l9-OH-DHV3-II); 19-hydroxybR, 19-dihydrovitamin D3, (19-OH-DHV 3-Ill). 245,25-dihydroxyvitamin D3, (24S,25-(OH)2-D3); 24homo-25-hydroxyvitamin D3, (24-homo-25-OH-D3); 24-nor-25-hydroxyvitamin D3, (24-nor-25-OH-D3); 23,24-dinor-25-hydroxyvitamin D3, (23,24-dinor-25OH-D3); 22,23,24-trinor-25-hydroxyvitamin D3, (22,23,24-trinor-25-OH-D3); 20,21 ,22,23,24-pentanor25-hydroxyvitamin D3, (20,21 ,22,23,24-pentanor-25OH-D3); SE-vitamin D3, (5E-D3 or 5,6-trans-D3); 25hydroxy-5E-vitamin D3, (25-OH-S E-D3); dihydrotachysterol3, (DHT3 or the lOS-b isomer: also IOS,l9-dihydro-SE-D3); 25-hydroxydihydrotachysterol3, (25-OHDHT3); IOR,19-dihydro-5E-vitamin D3, (DHV3-IV, the bR-b isomer or IOR,l9-dihydro-SE-D3); 19-hydroxydihydrotachysterol3, (19-OH-DHT3); 19-hydroxydihydrovitamin D3-IV, (19-OH-DHV3-IV); dihydrotachysterol2, (DHT2); dihydrovitamin D2-IV, (DHV2IV); isovitamin D3, (iso-D3); isotachysterols, (iso-T3); tachysterol3, (T3).
,
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the 19 position might, to some limited degree, satisfy the requirement for a pseudo 3-OH group in the 5,6-trans series, or the I-OH in the 5,6-cis series. However, no significant competition was observed for any of these compounds in our competitive binding assay. It may be that these compounds will first have to be hydroxylated in the 25 position before their relative ability to bind to the receptor can be evaluated. The insertion of an hydroxyl group into the 24 position of 25-OH-D3 to give either the R or S epimer of 24,25-(OH)2-D3 significantly reduces the ability of the compound to compete with lcv,25-(OH)2-D3. Under these assay conditions, it was not possible to discern any significant difference between the two epimers.8 The naturally occurring epimer of 24, 25-(OH)2-D3 has been shown to have its 24OH in the R configuration (113). In general, there is a remarkable correlation between the relative binding of analogs in the chick intestinal receptor system, and their relative ability to stimulate intestinal calcium absorption. The following conclusions may therefore be drawn with respect to the structural features required for vitamin D-like biological activity in the intestinal cell: 1) All biologically active compounds must have a la-hydroxyl or its geometric equivalent and a 25-hydroxyl group. 2) The presence of a hydroxyl at a geometrically equivalent position to the 3fl-hydroxyl of la,25-(OH)2-D3 is not an absolute requirement. 3) A 5,6-cis geometry of the A-ring is preferred to a 5,6-trans geometry. A comparison of the biological and binding activities of 25-OHDHT3 with those of 25-OH-5,6-t-D3 suggests that in compounds of the 5,6-trans series a C-19 methyl is preferred over a C-19 methylene group. 4) Altering the length or modifying the side chain, including the insertion of a hydroxyl group as in 24,25-(OH)2-D3 or 25,26-(OH)2D3, significantly decreases the activity of a compound.
AND
1280
PROCSAL
AL.
la,25-(OH)2-D3 are already being used clinically to treat patients who suffer from vitamm D-related diseases, principally renal osteodystrophy (108-Ill). As our understanding of the structural basis for biological activity increases, it may become possible to synthesize analogs with predetermined activities or that can act as antivitamins (1 12). The availability of such compounds will undoubtedly serve as a further stimulus for this rapidly expanding area of research. References 1. COBURN,
L. HARTENBOWER AND A. W. J. Med. 121: 22, 1974. NORMAN, A. W., AND H. HENRY. Clin. Orthopaed. Related Res. 98: 258, 1974. HOLICK, M. F., AND H. F. DELUCA. Ann. Rev. Med. 25: 349, 1974. NORMAN, A. W., AND H. HENRY. Rec. Prog. Hormone Res. 30: 431, 1974. OMDAHL, J. L., AND H. F. DELUCA. Physiol. Rev. 53: 327, 1973. HAUSSLER, M. R., J. F. MYRTLE AND A. W. NORMAN. J. Biol. Chem. 243: 4055, 1968. LUND, J., AND H. F. DELUCA. J. Lipid Res. 7: 739, 1966. HAU5SLER, M. R. Nutr. Rev. 32: 257, 1974. HOLICK, M. F., AND H. F. DELUCA. Adv. Steroid Biochem. and Pharmacology 4: III, 1974. SCHNOES, H. K., AND H. F. DELUCA. Vit. Hormones 32: 385, 1975. HAVINGA, E. Experientia 29: 1181, 1973. NORMAN, A. W. Biol. Rev. 43: 97, 1968. NORMAN, A. W. Vit. Hormones 32: 325, 1975. NORMAN, A. W., W. H. OKAMURA AND R. M. WING. Proc. 5th Parathyroid Conf., Amsterdam NORMAN.
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quite so simple. These investigators measured the induction of the enzyme, tyrosine aminotransferase, in hepatoma tissue culture (HTC) cells in tissue culture, utilizing analogs of glucocorticoids as inducers. It became apparent that there existed at least three levels of maximum response, each level being related to a structural class of the analogs. The three classes were: optimal inducers, i.e., those that gave the largest magnitude of response: suboptimal inducers, i.e., those that generated only a fraction of the response of the optimal inducers; and a third class of analogs, which were inactive. Samuels and Tomkins proposed that this classification resulted from the fact that the cytoplasmic glucocorticoid receptor in the HTC cells exists as two allosteric species. If, moreover, only one of the two allosteric species was capable of being “activated” and transported to the nucleus to induce the biological response, then the relative magnitude of response produced by the steroids was the result of their relative affinity for the two forms of the cytoplasmic receptor. Conceivably such a situation may exist also for the vitamin D metabolites and their analogs. The problem is even more complicated in the light of the rapid equilibration in solution of the two conformers of the seco-steroids. Each of these two conformers may have a different affinity for the two presumed allosteric forms of the cytoplasmic receptor for la,25-(OH)2-D3. In this connection it is interesting that the natural hormone, la,25-(OH)2-D3 may be a suboptimal inducer, since the analog, 3-D-laOH-D3 can produce a greater maximum response (107). It follows logically from what has been said that the intracellular sequence of receptor events must contain an irreversible step. Otherwise, the “inactive conformer” could be transformed back to the “active conformer.” Experimental support for our model requires further comparisons of the biological activities of a number of structural stereoisomers that differ in the relative proportion of A-ring conformers. Studies of this type are currently under way. The structure-function studies described here have made it possible to design and construct a number of analogs for clinical use. Compounds such as la-OH-D3 and
ET
VITAMIN Proc.
York: 1959. 22.
24. 25.
26.
27.
28.
pp.
AND New
127-130,
D.,
AND
J.
D.
DUNITZ.
SI.
96:
52.
7317, 1974. E. N., N. L. ALLINGER,
G.
MORRISON.
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Interscience
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Lett.
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R. H. J. Am.
M.
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M.
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F. M.
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21.
D,
PROCSAL
1282
80.
81.
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83.
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85. 86. 87. 88. 89. 90. 91. 92. 93.
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AND
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AND
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