METHODS OF SYNTHESIS AND TECHNOLOGY OF DRUG PRODUCTION

METHODS FOR THE SYNTHESIS OF THYROXINE AND TRIIODOTHYEONINE

P. M. Kochergin, R. M. Palei, UDC 615.357:577.175.443.444].012.1[048.8]

A. N. Kravchenko, and E. V. Popova

Thyroxine (I, T~, tetraiodothyronine, L-~-amine-~-[3,5-diiodo-4-(3',5'-diiodo-4'-hydroxy-

phenoxy)phenyl]propionic acid) and triiodothyronine (II, T 3, L-~-amino-$-[3,5-diiodo-4-(3'-
iodo-4'-hydroxyphenoxy)phenyl]propionic acid)* are hormones produced by the thyroid glands
of humans and animals. They are responsible for vitally important functions in the human
body: they regulate energy processes, tissue oxygen utilization, growth, and tissue differentia-
tion; they affect the states of the nervous and cardiovascular systems. Deficient or excess
secretion of I and II result in a number of severe diseases, the thyroid hormones I and II
have consequently found wide medical use [7, 23, 27, 28, 40, 59, 69, 70]. The similarity
in structure results in similarities in biological actions, with the difference that II is
3-5 times more active than I [7, 13, 63].
Apart from their therapeutic uses, I and II have recently become useful as starting mate-
rial for the preparation of diagnostic reagents, in radioimmunoassay kits [44, 80], and in
immunoenzyme assay kits [3, 4, 8, 61], which allow easy and rapid assessment of the functional
state of the thyroid gland in patients.
The biological actions of I and II have been reviewed in detail [2, 7, 23, 27, 28, 40, 59,.
69, 70], though the methods of their synthesis have not yet been considered systematically.
The only review available describes the chemistry of I, covering data up to 1950 [51].
The aim of the present review is to attempt to generalize the various experimental and
clinical data on the synthesis of I and II, as methods for their synthesis is important for
the industrial production of thyroid hormones.

I I

i~ i ,r-" NH z i~ -~
I Z
3' 2' 3 Z

5' 6 5 6 .N]"[Z
'~ I I

JqHz I ~Z
l~
I I

I~" N"

NHz HttZ

I 1

~ NH7
1~IJl ]

~I]le alternative names for these hormones, L-~-[3,5-diiodo-4-(3',5'-diiodo-4'-hydroxyphenoxy)-

phenyl]alanine (I) and L-6-[3,5-diiodo-A-(3'-iodo-4'-hydroxyphenoxy)phenyl]alanine (II), are
often found in the literature.
S. Ordzhonikidze Science Research Chemicopharmaceutical Institute, Moscow. Translated
from Khimiko-farmatsevticheskii Zhurnal, Vol. 24, No. 6, pp. 43-49, June, 1990. Original arti-
cle submitted June 29, 1989.

430 0091-150X/90/2406-0430512.50 9 1991 Plenum Publishing Corporation

 Scheme 1

2I' [o] Iz
/F Iz X Iz 2Z

Scheme 2

+
I
~r .~r
I I

~ 0 NO Z ~ ~ O NHz H+
I I
XlZ 2~

IO ~ O ~ M eJ T = N I C [ I Ou--q~ Meo O GN Sr;CI~

5Z7

-~ PhCONHCHzCOOH
I
2Z~ 0

_~CH]'-/>'- Ph H20
o =

I I
MeO~ o~CH= "-7;'~"+~HCOPh
'CCOOH n MeO-</~\~-O~ ~3H=CCOOEt
f I)-~J ~woPh
I
HI m H O - ~ O~CIi2CI HCOOH
I NHZ

D-7 J%eparation~ ~-I + D-I

into enatiomers

The importance of the thyroid hormones and their major role in the etiology of the en-
docrine disease hypothyroidism were already known at the end of the last century. English
and German investigators exctracted a substance (iodothyrine), with a high (up to 75%) content
of iodine, using acid and alkaline hydrolysis of the thyroid gland [59]. In 1914, Kendall
extracted a pure compound from the thyroid gland, and designated it thyroxine, though he
mistakenly suggested it to be an iodinated indole derivative [59, 69].
The chemical structure of I was determined in 1926-1927 by Harington and Barger [47,
~8, 59, 69].

431
 II was extracted from human blood and bull thyroid gland in 1951-1953 by Gross and Pitt-
Rivers [45, 46, 69], and by Roche et al. [71-77]. These two groups independently determined
its structure, and carried out the first syntheses [45, 46, 60, 69, 72, 76].
In structural terms, I and II are tetra- and tri-iodo derivatives of the amino acid thyro-
nine (III, Y), which does not occur in nature: thyronine is in turn a derivative of the amino
acid tyrosine (IV), which is widely distributed in the animal and plant worlds.
I and II are optically active substances, as each contains an asymmetric carbon atom.
They can therefore exist as two enantiomers: the levorotatory and the dextrorotatory, and
as a racemate (L-, D-, and DL-forms, respectively). The natural compounds I and II are L-
enantiomers, and have the same configuration as L-IV [41, 69].
I and II are stable, solid, crystalline substances. As a-amino acids, they are amphoteric,
and form salts with both alkalis and with mineral acids. I and II themselves are used for
clinical purposes, as thyroid hormones, as are their sodium salts and hydrochlorides, in the
L-forms. The latter is because the L enantiomers have significantly greater activity in com-
parison with the D enantiomers and the racemates. Thus, according to [13], L-II is 14 times
more active than D-II, and 1.7 times more active than DL-II.
D-I (dextrothyroxine) and its sodium salt also have clinical uses, but in other diseases;
for example, in atherosclerosis, as D-I has anticholesterolemic properties [63].
Apart from I and II, h ~ a n and animal blood and thyroid glands are now known to contain
seven other iodinated thyronine (III) and tyrosine (IV) derivatives: 3,3',5'-triodothyronine
(V), 3,5-diiodothyronine (VI), 2',6'-diiodothyronine (VII), 3,3'-diiodothyronine (VIII), 3-
iodothyronine (IX), 3-iodotyrosine (X), and 3,5-diiodotyrosine (XI).
Compounds V-XI are generally not considered to be hormones [40, 69, 74, 75], and biologi-
cal roles have not yet been discovered for most of them. It is known only that X and XI are
intermediates in the biosynthesis of I and II. It should, however, be noted that XI is used
as a therapeutic agent for the treatment of endocrine diseases [7], but is thus far excluded
from general use because of its low activity.
Scheme 3
I I

MOz
NOz 2XZZ ~ R=Me, PhCO
I O=r-O,
RCONHCH~H~EO O CHi~ ~

xx~ ~ M e . Ph
I I
H~
--------RO 0 CH=Q- OOOMe------RO-<~ ~}-O-~' "2-CH=C-COOMe
t

- - ~OR ~{Z NHCOR

NO7.
XX V// XXVI//
1

Ro o -CO?Me SO - m=
I-I*
L ooR j
XX//
I 1
o_ o_ o-oooMo
I I
.Z~
I
HCfX~; ~--~ "~_ C Separationinto
----- HO O CH~ HCOOH ~ L - ~ E 7 "r D-~
A.C~O -- ~ enantiomers I(. i(~)
9' ~l-lCHO HIO H) H20

DL- xxx, L-2X~ D-~

L-I D-I

432
 The biosynthesis of I and II takes place in the thyroid gland, and consists of a number
of steps [40, 69] (Scheme i).
The first step is the concentration of inorganic iodides from the blood into the thyroid
gland. This process is regulated by the hormone thyrotropin. Iodides are oxidized by perox-
idase to free iodine, which iodinates tyrosine (IV) bound to the protein thyroglobulin, to
form 3-iodo- and 3,5-diiodotyrosines (X and XI). X and XI residues react together to form
I and II, which remain bound to thyroglobulin. It has been suggested that this oxidative
reaction has a radical-mediated mechanism, with the removal of an a-alanine fragment, in the
form of a-aminoacrylic acid, which is then transformed to pyruvic acid and ammonia. Proteol-
ysis of thyroglobulin-bound I and II results in the formation of the free hormones, which
enter the blood.
I has also been synthesized from XI in vitro: by the oxidation of XI with air oxygen
in weakly alkaline conditions in the presence of an Mn30 . catalyst [9, ii, 65-69, 79]. How-
ever, this simple one-step synthesis of I from the readily available XI has not found use for
therapeutic preparations, because of its low yield and the loss of optical activity.
Other possible mechanisms of the biosynthesis of I and II have also been considered [40,
69]: partial oxidation of XI produces diiodoarylpyruvic acid [XII], which reacts with the
initial substrate XI to form I, II being produced by deiodination of I (see Scheme i).
This route for biosynthesis has also been demonstrated chemically: by oxidation with
air oxygen of a mixture of XI and XIII in aqueous buffer (pH 7.6), producing I with a yield
of 23-36% [53, 62] (see Scheme I).
A hormonal preparation of natural origin, thyroidin, has been used for several decades
in the therapy of endocrine diseases with altered thyroid function. This is a defatted and
dried thyroid gland extract from slaughter animals, containing I and II in the ratio 4-5:1
[6, 7, 23, 25-28, 50, 59, 69].
The use of this preparation is now declining because of the synthetic production of I
and II.
The pharmaceutical industry has great experience in the extraction of natural hormones
from animal materials [25, 26]. However, this is not a good route for the preparation I and
II, becuase of their low concentrations in the thyroid and the complexity of their extraction
and purification [50, 59, 69]. As a result, investigators have turned their attention to
the development of synthetic methods for the production of I and II. Such methods have been
developed, and have been scaled up for industrial production in several countries (USA, Switz-
erland, FRG, Belgium, Hungary, GDR, and others).
The availability of synthetic I and II resulted in the possibility of developing state
of the art therapeutic preparations with strictly controlled dosing levels, including a num-
ber of preparations for pediatric use. The synthetics also allow I and II to be used together
in different combinations, and with potassium iodide [7].
Preparative methods for the synthesis of I and II are described below.
The first synthesis of I, which confirmed its structure, was carried out in 1927 by
Harington and Barger [48]. These workers started with 4-methoxyphenol (XIII) and 1,2,3-tri-
iodo-5-nitrobenzene (XIV), from which, via a series of reactions through compounds XV-XXIII,
I was produced in the form of a racemate (Scheme 2). This material appeared to be biological-
ly identical to the natural hormone extracted from the thyroid gland. DL-I was resolved to
its optical enantiomers using a-phenylethylamine [49]. Reduction of L-I to thyronine (III)
showed that this hormone has the same configuration as the natural configuration as the amino
acid tyrosine (IV) [41].
This syntesis of I was improved by Nahm and Siedel [64], by the replacement of XIV with
3-iodo-4-hydroxy-5-nitrobenzaldehyde (XXIV) using Scheme 3 (via compounds XXV-XXXI). These
authors noted that the separation of DL-I into optical enantiomers was difficult. They there-
fore suggested that this operation should be carried out at the stage of N-formyl-3,5-diiodo-
thyronine (DL-XDL\I) using brucine (dimethoxystrychnine) [22, 41, 49, 64]. L-I and D-I were
obtained by hydrolysis of the formyl group and subsequent iodination of the resulting compounds
L-XXIII and D-XXIII ~see Scheme 3).
These methods have not been used industrially, since they are based on the use of starting
compounds of low availability, the large number of stages, and the production of racemic mix-
tures (I, XXIII), requiring rather complex separation into enantiomers.

433
 Scheme 4

z ~ J~Iz /-- ~OMe

.NO?. J~rOz
XXXIII

J~O~ r NO 2 3
~ci I ~ In-~OC~OH
------"=- HO-(' ")-CT'I"LQHCOOEt----Z-"~ITs"O-~,
" x/~"CH~"~COOEtl ~

J~Oz ~'~ L ~Oz -J

xxx/v x~Y~'

.N'Oz
= lv~O_~~CHzOI~COEt HZ ~.
_ _ PIC
XXXI//

-----,,-- MeO ~ /_
CH,2~HCOOEt
~ H~SO4
------
%~-Iz
~'XXVII

XXXI/I.7

I
~/-C~I~HC HIor. H B r
. -- / - - ~ ~OH
I
xxx/x I I,

I I
I

In 1949 Hems et al. [42] described a new stereospecific synthesis of I. The widely avail-
able L-tyrosine (IV) was used as starting material; this was prepared by hydrolysis of kera-
tin-containing waste products of meat factories, or by enzymatic synthesis [29-31].
3,5-dinitrotyrosine (XXXII)was prepared by nitration of IV, using a mixture of nitric
and sulfuric acids. Subsequent acetylation of XXXII and esterification of XXXIII produced
the ethyl ester of N-acetyl-3,5-dinitrotyrosine (XXXIV). Treatment of EXD(IV with p-toluol-
sulfochloride and p-methoxyphenol in anhydrous pyridine without extraction of the intermed-
iate tosylate (XXXV) resulted in the synthesis of the corresponding diphenyl ester (XXXVI),
from which the diamine (XXXVII) was produced by hydrogenation. Diazotization of XXXVII, fol-
lowed by replacement of the diazo group of YXKXVIII with iodine, using the Sandmeier reaction
produced the ethyl eser of N-acetyl-3,5-diiodo-4'-methoxythyronine (XXXIX). All the protecting
groups were removed by heating this compound with concentrated HI or HBr, resulting in the
formation of L-diiodothryonine (XXIII). The Harington-Barger scheme was then followed, with
iodination of XXIII with iodine (2 moles) in the presence of potassium iodide and ethylamine,
producing L-I with a yield of 26% in terms of IV (Scheme 4).

After the discovery of II, scheme 4 was successful]y used for its synthesis, by iodina-
tion of L-XXIII with an equimolar quantity of iodine in the presence of potassium iodide in
aqueous ammonia or primary or secondary amines [i0, 12, 13, 18, 24]. A patented procedure
I15] uses N-iodoacetamide as iodinating agent, and the reaction takes place in anhydrous meth-
anol in the presence of triethylamine. The yield of L-II ranged from 50-95% in terms of L-
XXIII, or 8-15% in terms of the initial L-IV {Scheme 5) [i0, 12, 13, 15, 18, 24].

434
 Scheme 5

L-XXIll I.~ L-II

K1

MeCONHI
l
Scheme 6

~o -• m
"NaI04
or mffr o ~ o x"

2Z 2X/ X=l,13p
I
_+_
I

D-II was synthesized by a route analogous to that used for L-II, by the iodination of
D-XXIII with iodine, and DL-II was synthesized by iodination of D-XXIII by N-potassium-iodo-p-
toluolsulfonamide [16, 64].
These syntheses of I and II have a number of disadvantages, the main ones being the nitra-
tion of IV to XXXII and the substitution of the two amino groups with iodine by the Sandmeier
reaction, the use of anhydrous pyridine and concentrated HI or HBr, and the low yields of
I and II (8-26% in terms of the initial compound IV).
It should also be noted that the iodination of XXIII produces heterogeneous products.
During preparation of I, compound II is formed as an admixture, while I is always present in
preparations of it. A proportion of XXIII also remains unreacted. As a result, purification
of I and II must be carried out carefully, with methods including preparative chromatography
[12, 13, 69].
Despite these disadvantages, the synthesis of I and II using schemes 4 and 5 has industri-
al value. Attention should be brought to a number of later papers, e.g., [24], and patents,
e.g., [i0, 19], describing a number of modifications both of individual stages, and of methods
of extraction and purification of the final products I and II.
A number of authors [32, 37, 38] have tried to prepare XXIII from 3,5-diiodotyrosine
(XI), though these attempts were unsuccessful, which is probably explained by the difficulty
in forming diphenyl esters using 2,6-diiodophenols [32, 37, 38, 41, 51].
Although slowly, 2,6-diiodophenols do react with diaryliodonium iodides (or bromides)
with the formation of the respective diphenyl esters. This resulted in the proposal of a sec-
ond stereospecific synthesis of L-I and L-II from L-IV through L-XI [15, 17, 20, 52] (Scheme
6).
The entire synthesis of I and II consists of seven steps. Treatment of anisole (XL)
with sodium iodate and potassium iodide or a mixture of iodine pentoxide, followed by addition
of sodium bromide resulted in the synthesis of 4,4'-dimethoxydiphenyliodonium iodide and the
respective bromide (XLI) [14, 33]. The second component, the ethyl ester of N-acetyl-3,5-di-
iodotyrosine (XLIII) was obgtained by iodination of IV with iodine or with iodine-chlorine
[i, 35], with subsequent acylation with XI and esterification with XLII. The reaction of
XLIII with XLI produces the ester XXXIX. Heating XXX IX with HI or HBr produces 3,5-diiodo-
thyronine (XXIII). This was iodinated to produce L-I and L-II with yields of 25% in terms
of the starting compound IV II0, 12-15, 18, 21] (Scheme 6).
Although this synthesis of I and II is two steps shorter compared with the synthesis
via 3,5-dinitrotyrosine (XXXII) bv Scheme 4, it has a significant disadvantage, namely the
preparation of iodonium iodide {or bromide) (XLI). This substance may be explosive, as are

435
 Scheme 7
z

I IzI I

Scheme 8
I

Iz

I ~ I 1

other iodinated compounds [5]. The low yields of XLI (12-47%) and XXXIX (50-60%), and the
long reaction time required (24-80 h) should also be noted.
Iodination of thryonine (III) may be an attractive route for the synthesis of I and II.
The authors who first synthesized this amino acid [48] attempted to iodinate it, but did not
obtain compound I.
The iodination of III was later studied in more detail by Roche et al. [72, 73, 76, 77].
They also failed to obtain I and II, since iodination occurred on the more distal benzene
ring B, with the formation of 3'-iodo- and 3',5'-diiodothyronine (XLIV, XLV) (Scheme 7). When
3-iodothyronine (IX) was iodinated, the reaction also took place in ring B, with the formation
of 3,3'-diiodo- and 3,3',5'-triiodothyronines VIII and V {73, 75] (Scheme 8).
Further iodination of V and VIII was not discussed.
Finally, after the structures of I and II were established and methods for their synthesis
were developed, and their great medical value for the treatment of a number of diseases were
recorded, a number of papers appeared in the literature discussing the synthesis of different
derivatives of these hormones, and of compounds of similar structure [40, 69]. The basic
aim of these studies was to determine the relationship between the structure and the biologi-
cal actions of the compounds, and to attempt to produce compounds more active than I and II.
A number of authors [40, 69] synthesized derivatives of I and II containing OH-, NH2,
and COOH- groups, even adding a third iodinated benzene ring (compounds XLVI and XLVII) [39].

I. I. I

I/~ ]/ I/ ~Z
~E

I I

Other studies included the substitution of iodine atoms in compounds I and II for atoms
of other halogens, the introduction of alkyl radicals at the 5' position, the replacement of
the bridging oxygen atom with sulfur, the positioning of iodine atoms in other positions of
benzene rings A and B, the substitution of the ~-alanine side chain with fragments of other
amino acids, and the synthesis of analogs of I and II based on o- and m-tyrosines and other
compounds [34, 36, 40, 43, 54-58, 69, 78].
The biological investigation of these compounds showed that they were either inactive
er had weak thyroid activity. Since attempts to synthesize different derivatives of 1 and
7I have not produced useful therapeutic preparations, their methods of synthesis are not re-
viewed here,

436
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