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462
Pharmaceutical Chemistry of Natural Products
(e) Pommer’s second synthesis: In this method, reaction between β-ionone and esters
of 6-bromo-3-methylhex-2-en-4-ynoic acid under the condition of the Reformatsky
reaction yields a product, which on dehydration with p-toluene sulphonic acid in
benzene followed by partial reduction by hydrogen in the presence of a lead-poisoned
catalyst yields the ethyl esters of retinoic acid. Vitamin A was obtained by reducing the
latter compound with LiAlH
4
.
CH
CH
3
CH C O
-Ionone
BrCH C
2
CCCHCOOR
CH
3
Esters of 6-bromo-3-methyl
hex-2-en-4-enoic acid
Zn/–H O
2
CH
CH
3
CH C CH C
C C CHCOOR
CH
3
(i) Dehydration with -toluene sulphonic acid
(ii) Reduction with H -lead poisoned catalyst
p
2
LiAlH
4
CH
CH
3
CH C CH CH
CH C CHCOOR
CH
3
Ester of retinoic acid
CH
CH
3
CH CH
CH C CH CH OH
2
CH
3
Vitamin A (retinol)
CH C
The ester of 6-bromo-3-methyl-hex-2-en-4-enoic acid used in the above synthesis can be
synthesized as follows:
HOH C
2
C C C
H
OH
CH
3
Pent-2-yne-1,4-diol
(i) (CH CO) O
(ii) CrO
32
3
AcOCH C
2
CCO
CH
3
AcOCH C
2
CCCHCOOC H
25
CH
3
(i) CH BrCOOC H /Zn
(ii) H O
225
2
Hydrolysis/PBr
3
BrCH C
2
CCCHCOOC H
25
CH
3
Esters of 6-bromo-3-methyl
hex-2-en-4-enoic acid
(f) Jansen’s synthesis: Jansen synthesized vitamin A from 2-methyl cyclohexanone.
O
2-Methylcyclohexanone
NaNH
2
CH I
3
O
2,2,6-Trimethyl
cyclohexanone
HC C C
CH CH CH CHOH
CH
3
CH
3
3-Methylocta-3,5-dieno-1-yl-7-ol
OH
C CC
CH
3
CH CH CH CHOH
CH
3
(i) LiAlH
(ii) MnO
4
2
Vitamins
463
CH CH C
CH
3
CH CH CH C
CH
3
O
CH CH C
CH
3
CH CH CH C
CH
3
O
OH
HO
2
CH BrCOOC H /Zn
225
CH CH C
CH
3
CH CH CH C
CH
3
CH COOC H
225
OH
(i) H O
(ii) LiAlH
2
4
CH
3
CH
3
CH CH C CH CH CH C CHCH OH
2
Vitamin A (Retinol)
VITAMIN D
It is a group of fat-soluble secosteroidal vitamin and resembles sterols in structure and functions
like a hormone. About fi ve vitamins (D
1
, D
2
, D
3
, D
4
and D
5
) have been isolated up to now. Vitamin
D
1
has been found to be a molecular compound of D
2
and lumisterol. Vitamin D can be prepared
from irradiation of specifi c sterols called provitamins. Examples of vitamin D are as follows:
1. Vitamin D
2
(ergocalciferol) from ergosterol
2. Vitamin D
3
(cholecalciferol) from 7-dehydro cholesterol
3. Vitamin D
4
from 22,23-dihydroergosterol (22-dihydro ergocalciferol)
4. Vitamin D
5
from 7-dehydro sitosterol (sitocalciferol)
Formation of Vitamin D
2
(Ergocalciferol) from Ergosterol
Ergosterol (plants)
Sunlight
HO
H
HO
Ergocalciferol (D )
2
CH
2
Vitamin D is relatively stable and not destroyed to any appreciable extent by heat or oxidation.
Cooked, dried and stored food retain most of their vitamin D potency.
464
Pharmaceutical Chemistry of Natural Products
Ergocalciferol (vitamin D
2
) is formed from ergosterol present in plants. Cholecalciferol is found
in animals. Both are similar in structure except that ergocalciferol has an additional methyl group
and a double bond.
During cholesterol biosynthesis, 7-dehydro cholesterol is formed as an intermediate, which
on exposure to sunlight is converted to cholecalciferol in the skin (dermis and epidermis). The
synthesis of vitamin D
3
in the skin is proportional to the exposure to sunlight. Dark skin pigment
(melanin) adversely infl uences the synthesis of vitamin D
3
.
History
The symptoms of rickets and the benefi cial effects of sunlight to prevent rickets have been known
for centuries. In 1919, Mellanby discovered that administration of CLO prevents development of
rickets in experimental dog. In 1922, McCollum and his associates established that the antirachitic
principle of CLO is different from vitamin A and named it as vitamin D. In 1924, Hess reported that
irradiation of some foods with UV light induces antirachitic activity. Later in 1931, Augus isolated
vitamin D and named it calciferol.
Dietary Source
Vitamin D is found only in a few foods. The richest natural sources of vitamin D are fi sh liver oils
and saltwater fi sh such as sardines, herring, salmon and mackerel. Eggs, meat, milk and butter
also contain small amounts of the vitamin. Plants are poor sources, while fruits and nuts contain
no vitamin D at all. The amount of vitamin D in human milk is insuffi cient to cover infant needs.
The amount of vitamin D
2
in the liver of fi sh depends on the season and also on the biological
conditions of the environment in which the fi sh is living.
Vitamin D can be provided to the body in the following three ways:
1. Consumption of natural foods.
2. By irradiating foods (like yeast) that contain precursors of vitamin D and fortifi cation of
foods (milk, butter, etc.).
3. Exposure of skin to sunlight for the synthesis of vitamin D.
Deficiency Diseases
Nutrient defi ciencies are usually the result of dietary inadequacy, impaired absorption and
utilization, and increased requirement or increased excretion (loss). Defi ciency of vitamin D occurs
in the following cases:
1. When usual intake is below recommended levels.
2. When there is limited exposure to sunlight.
3. When the kidney cannot convert vitamin D to its active form.
4. When someone cannot adequately absorb vitamin D from the digestive tract.
Vitamin D–defi cient diets are associated with milk allergy, lactose intolerance and strict
vegetarianism. Infants fed with only breast milk receive insuffi cient amounts of vitamin D unless
they also receive appropriate levels of vitamin D supplementation.
Vitamins
465
Defi ciency of vitamin D leads to demineralization of bone. The result is rickets in children and
osteomalacia in adults.
Rickets
The defi ciency of vitamin D
2
causes rickets in children. Rickets is derived from an old English
word wrickken which means twist. It is characterized by bone deformities due to incomplete
mineralization, resulting in soft and pliable bones and delay in teeth formation. These conditions
respond rapidly to vitamin D
2
. Hence, this vitamin is known as antirachitic factor. In rickets, the
plasma level of calcitriol is decreased and alkaline phosphatase activity is elevated. Alkaline
phosphatase is concerned with the process of bone formation. There is an overproduction of
alkaline phosphatase related to more cellular activity of the bone. It is believed to be due to a vain
attempt to induce bone formation.
Osteomalacia
The word osteomalacia is derived from Greek words osteon and malakia (osteon means bone; malakia
means softness). In osteomalacia, demineralization of the bones occurs, which increases their
susceptibility to fractures.
Renal rickets
This is also known as renal osteodystrophy and is seen in patients with chronic renal failure. It is mainly
due to decreased synthesis of calcitriol in kidney. Renal rickets can be treated by administering
calcitriol.
Hypervitaminosis of Vitamin D
Mostly vitamin D is stored in the liver and is slowly metabolized. Vitamin D is the most toxic
vitamin among all the vitamins, in overdoses. Excretion of vitamin D is negligible and hence
excessive administration can lead to toxicity. Weakness, polyurea, intense thirst, diffi culty in
speaking, confusion, weight loss, nausea and vomiting are symptoms of hypervitaminosis D. The
patient may lapse into coma, while cardiac arrhythmias and renal failure may also occur. These
effects are due to hypercalcaemia induced by increased intestinal absorption and mobilization
of calcium from the bones. Hypokalaemia and metabolic alkalosis are also associated with this
condition. Hypervitaminosis of vitamin D may lead to formation of stones in kidney.
RDA
The daily requirement of vitamin D is 400 IU or 10 µg of cholecalciferol. In countries with good
sunlight (like India), the RDA for vitamin D is 200 IU or 5 µg of cholecalciferol.
Isolation
Vitamin D can be isolated from either fi sh liver oil or sterols.
466
Pharmaceutical Chemistry of Natural Products
From fish liver oil
From fi sh liver oil, vitamin D can be separated by two methods.
First method: This method involves four steps.
1. Fish liver oil is saponifi ed with methanolic potassium hydroxide in the presence of an
inert atmosphere using nitrogen. Then the unsaponifi ed portion is separated, whereas the
saponifi ed portion is rejected.
2. To remove vitamin A from unsaponifi ed portion, it is treated with maleic anhydride or
citraconic anhydride.
3. In this step, the vitamin D
2
fraction obtained in the above step is purifi ed and concentrated
by adsorption chromatography or ion-exchange chromatography.
4. Finally, elute precipitation of sterols is done with digitonin, followed by its concentration.
Then this is converted into their ester which upon esterifi cation yields the different vitamin
D.
Second method: In this method, the fi sh liver oil is mixed with constant yield oil. Then the
oil mixture is subjected to molecular distillation where free vitamins get distilled over at 160°C,
whereas the esters get distilled over at 230–235°C. In this method, a new vitamin having lowest
boiling point is discovered and is named as vitamin D
6
.
From sterols
First specifi c sterols are irradiated to convert them into vitamin D. Once 40–60% of the provitamin
is converted into the required vitamin D, the solution is ready for isolation in the absence of
oxygen because the intermediate compound tachysterol absorbs oxygen to form peroxide which is
capable of destroying vitamin D. Tachysterol is removed by condensing the solution with citraconic
anhydride. The crude product is saponifi ed at room temperature and extracted with petroleum
ether and water. Tachysterol–citraconic acid adduct remains in water phase and vitamin D in ether
phase. The ether phase upon crystallization with acetone yields crystals of vitamin D.
Metabolism
Vitamins D
2
and D
3
as such are not biologically active. Hence, they are metabolized identically in
the body and converted to active forms of vitamin D.
Cholecalciferol (D
3
) is fi rst hydroxylated at the 25th position in the presence of a specifi c
hydroxylase present in the liver into 25-hydroxy cholecalciferol (25-OH D
3
). 25-OH D
3
is the
major storage and circulatory form of vitamin D. In kidney, 25-OH D
3
is hydroxylated at the 1st
position in the presence of 25-hydroxy cholecalciferol-1-hydroxylase to produce 1,25-dihydroxy
cholecalciferol (1,25-DHCC). 1,25-DHCC contains three hydroxy groups (1, 3 and 25th carbon),
hence it is referred to as calcitriol. The concentration of 1,25-DHCC is regulated by plasma levels of
calcium and phosphate. They control hydroxylation reaction at position 1. Low plasma phosphate
increases the activity of 25-hydroxy cholecalciferol-1-hydroxylase. Low plasma calcium enhances
the production of parathyroid hormone (PTH) which in turn activates 1-hydroxylase. Thus the
action of phosphate is direct while that of calcium is indirect on kidney 1-hydroxylase.
Vitamins
467
HO
HO
H
Sunlight
Skin
H
HO
HO
H
OH
OH
1
25
7-Dehydrocholesterol
(animals)
CH
2
Cholecalciferol (D )
3
25-Hydroxylase (liver)
25-Hydroxy cholecalciferol (25-OH D )
3
CH
2
H
25
OH
1 -Hydroxylase(kidney)
1,25-Dihydroxy cholecalciferol (1,25-DHCC)
or calcitriol
CH
2
24,25-DHCC is another metabolite of vitamin D. It is also synthesized in the kidney by 24-
hydroxylase. The exact function of 24,25-DHCC is not known. But it is believed that when
calcitriol concentration is adequate, 24-hydroxylase acts leading to the synthesis of less important
24,25-DHCC. In this way, synthesis of 24,25-DHCC is also important to maintain the homeostasis
of calcium.
Qualitative Test for Vitamin D
Dissolve a quantity equivalent to about 1000 units of vitamin D activity in chloroform and add 10
mL of antimony trichloride solution. Pinkish red colour appears at once.
Biochemical Function
Calcitriol is the biologically active form of vitamin D. Calcitriol acts at three different levels
(intestine, kidney and bone) and regulates the plasma levels of calcium and phosphate.
1. On intestine: It increases the intestinal absorption of calcium and phosphate. In the intestinal
cells to form a calcitriol–receptor complex, calcitriol binds with cytosolic receptor. Then this
complex approaches the nucleus and interacts with a specifi c DNA leading to the synthesis
of a specifi c calcium-binding protein. This calcium-binding protein increases the calcium
uptake by the intestine. In intestine, the action of calcitriol is similar to that of the action of
a steroid hormone.
468
Pharmaceutical Chemistry of Natural Products
2. On bone: Calcitriol stimulates the calcium uptake for deposition as calcium phosphate in the
osteoblasts of bone. Thus, calcitriol is essential for bone formation. Along with PTH, calcitriol
increases the mobilization of calcium and phosphate from bone. This causes elevation in the
plasma calcium and phosphate levels.
3. On kidney: In kidney, calcitriol minimizes the excretion of calcium and phosphate by decreasing
their excretion and reabsorption.
The sequence of events that takes place in response to low plasma calcium concentration and
the action of calcitriol on intestine, kidney and bone ultimately leads to increase in plasma calcium
levels.
Structural elucidation of vitamin D
2
or ergocalciferol
6
15
19
21
1
2
3
4
5
7
8
9
10
11
12
13
14
16
17
18
20
22
23
24
25
26
27
CH
2
HO
Ergocalciferol (D )
2
28
The structure of vitamin D
2
is elucidated on the basis of following synthetic and chemical
evidences.
1. Molecular formula: Analytical data reveals that the molecular formula of vitamin D
2
is
C
28
H
44
O. This molecular formula is same as that of ergosterol. This indicates both vitamin
D
2
and ergosterol are isomers of each other. It was also confi rmed by the formation of
vitamin D
2
by irradiation of
ergosterol which is present in plants.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
hv
Vitamin D
2
Ergosterol (plants)
HO
I
II
H
III
IV
2. Presence of secondary alcoholic group: Vitamin D
2
forms monoacetate, monobenzoate,
etc.,
indicating that it contains one hydroxyl group. When vitamin D
2
undergoes oxidation,
it yields a ketone, which indicates that the hydroxyl group present is secondary alcoholic
in nature.
Vitamins
469
CHO
28 44
CH COCl/C H COCl
365
Vitamin D
2
Monoacetate/monobenzoate of
Vitamin D
2
CHO
28 44
Ketone
Vitamin D
2
O
3. Nature of side chain: Ozonolysis of vitamin D
2
yields various products including methyl
isopropyl acetaldehyde. Ergosterol also upon ozonolysis gives the same product. This
suggests that the side chain in vitamin D
2
is the same as that in ergosterol. The formation
of methyl isopropyl acetaldehyde can be explained further if vitamin D
2
has the structure
like this.
Vitamin D
2
IV
ozonolysis
O
3
IV
COOH
CHO
CH CH(CH )
32
CH
3
Methyl isopropyl
acetaldehyde
4. Presence of four double bonds: Vitamin D
2
absorbs four molecules of hydrogen to form
a fully saturated compound called octahydro ergocalciferol (C
28
H
52
O) upon catalytic reduction.
The formation of octahydro ergocalciferol indicates the presence of four double bonds in
vitamin D
2
. From step 3, out of these four double bonds one must be present in side chain
to explain the formation of aldehyde and the remaining three are present in the nucleus of
vitamin D
2
. In addition, the UV spectrum
of vitamin D
2
reveals the presence of a conjugated
triene system which has been further confi rmed by its formation of adduct with maleic
anhydride.
CHO
28 44
Vitamin D
2
4H
2
Reduction
CHO
28 52
Octahydro ergocalciferol
5. Presence of three rings: The molecular formula of parent hydrocarbon of octahydro
ergocalciferol (reduced product of vitamin D
2
) is C
28
H
52
. This corresponds to the general
formula C
n
H
2n–4
for tricyclic compounds (DBE = 28 + 1–(52/2) = 3; three rings are
present).
6. Structure of vitamin D
2
: Vitamin D
2
undergoes reduction with sodium and alcohol to
yield dehydro ergocalciferol. The latter reacts with three moles of perbenzoic acid, which
results in the formation of trioxide. It reveals that the additional unsaturated centre is
introduced during the conversion of ergosterol to vitamin D
2
(see point 1). Furthermore,
the elemental analysis does not show any loss in either carbon or hydrogen during the
conversion. It is assumed that the unsaturated centre is introduced by photochemical
cleavage of one of the rings of ergosterol.
470
Pharmaceutical Chemistry of Natural Products
The above fact is also supported by the following reactions, i.e. vitamin D
2
does not yield
Diels’ hydrocarbon when distilled with selenium. This indicates that vitamin D
2
does not
contain the four-ring system of ergosterol and one of the rings of ergosterol has been
opened in the formation of vitamin D
2
. Now one question will arise: out of four rings
present in ergosterol which of the ring has been opened in the formation of vitamin D
2
?
Explanation of the following reaction satisfactorily answers the above question if the
structure of vitamin D
2
is accepted as 2.
7. In presence of chromic acid or KMnO
4
,
if vitamin D
2
undergoes careful oxidation, it yields
the aldehyde (3), ketone (4) and formic acid (5).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
28
25
27
26
Vitamin D (2)
2
CH
2
H
Cro
3
CH
CHO
CH
917
CH
917
O
HCOOH
Formic acid (5)
Ketone (4)
Aldehyde (3)
HO
I
II
III
IV
Absorption spectrum of semicarbazone of aldehyde (3) reveals that the aldehyde is an
α,β-unsaturated aldehyde and the absence of hydroxyl group, carbon content of (3), i.e.
C
21
H
34
O, indicates the absence of ring (I). The above fact reveals that the ring (II) is opened
between C
9
and C
10
in vitamin D
2
and the compound (3) is formed due to the scission of
molecule at a double bond present between the fi fth and the sixth position. The compound
(3) will be an α,β-unsaturated aldehyde if a double bond is present at seventh and eighth
positions (these double bonds are also present in ergosterol). In addition, isolation of ketone
(4) shows the double bond between the seventh and eighth positions.
8. Vitamin D
2
upon ozonolysis yields formaldehyde (6), keto acid (7) and 2,3-dimethyl bu-
tyraldehyde (8).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
28
25
27
26
CH
2
HO
Vitamin D (2)
2
H
Ozonolysis
O
3
HCHO
Formaldehyde
Keto acid (7)
O
COOH
2,3-Dimethyl
butyraldehyde (8)
CHO
+
Vitamins
471
Isolation of formaldehyde reveals that vitamin D
2
has exocyclic methylene group at C
10
.
This confi rms the opening of ring (II) between ninth and tenth carbons.
Keto acid (C
13
H
20
O
3
) (7) formation reveals that the ring is opened between 9th and 10th
carbon and double bonds are present between 7th, 8th carbon and 22nd, 23rd carbon. The
position of the latter double bond in the side chain has been confi rmed by the isolation of
2,3-dimethyl butyraldehyde (8).
9. The above fact can also be confi rmed by the conversion of acetate of vitamin D
2
to its
corresponding maleic anhydride adduct which in turn is converted to dicarboxylic acid (9).
The structure of this dicarboxylic acid (9) has been further confi rmed by its products (10),
(11), (12) and (13).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
28
25
27
26
HO
CH
2
H
Vitamin D (2)
2
Maleic anhydride
(i) (CH CO) O
32
(ii) CHCO
CHCO
O
(iii) Hydrolysis
HO
III
CHCOOH
CHCOOH
CH
917
Dicarboxylic acid (9)
III IV
Se
Pd–C
(i) H –Pt
(ii) O
2
3
III
III
III
III IV
2,3-Dimethyl naphthalene (10)
CH
3
CH
3
Naphthalene (11) Naphthalene-2-
carboxylic acid (12)
5-Oxo-4a,5,6,7,8,8a-hexahydro
naphthalene-1-carboxylic acid (13)
COOH
COOH
O
The formation of 2,3-dimethyl naphthalene (10) reveals that the carboxyl group may give
rise to methyl groups on selenium dehydrogenation. Similarly, the formation of naphthalene
(11) and naphthalene-2-carboxylic acid (12) reveals that ring (I) and (II) are present in
(9). Furthermore, the formation of (13) from (9) by its reduction followed by ozonolysis
establishes the structure of (9). From the formation of these compounds (10–13) we can
confi rm the structure of (9) and also the position of double bonds between 5th, 6th, 7th,
8th, 10th and 19th carbons.
10. Synthesis: Finally, the structure of vitamin D
2
(2) was confi rmed by its partial synthesis
from the aldehyde (3).