Process development and scale-up of T3-Sulfate, a new prodrug alternative to the conventional hormone therapy of hypothyroidism

Article abstract of DOI:10.1021/op500222p

An efficient and scalable preparation of the thyroid hormone analogue O-[3-iodo-4-(sulfooxy)phenyl]-3,5-diiodo-L-tyrosine sodium salt (T3-sulfate, 1) is reported. The synthesis involved monoiodination of O-(4-hydroxyphenyl)-3,5-diiodo- L-tyrosineto give liothyronine (2) which was sulfated with chlorosulfonic acid in N,N -dimethylacetamide. Crude T3-sulfate was initially purified by chromatography on polystyrene resin Amberlite XAD 1600 and then crystallized with ethanol. This strategy was scaled-up to give a process suitable for the production of kilogram quantities of API, needed to support preclinical and clinical studies.

Full text of DOI:10.1021/op500222p

Article
pubs.acs.org/OPRD
Process Development and Scale-up of T3-Sulfate, A New Prodrug
Alternative to the Conventional Hormone Therapy of
Hypothyroidism
Luciano Lattuada,* Maria Argese, Valeria Boi, Laura Galimberti, and Sonia Gazzetto
Bracco Imaging SpA, Bracco Research Centre, Via Ribes 5, Colleretto Giacosa (TO) 10010, Italy
ABSTRACT: An efficient and scalable preparation of the thyroid hormone analogue O-[3-iodo-4-(sulfooxy)phenyl]-3,5-diiodo-
L-tyrosine sodium salt (T3-sulfate, 1) is reported. The synthesis involved monoiodination of O-(4-hydroxyphenyl)-3,5-diiodo-L-
tyrosine to give liothyronine (2) which was sulfated with chlorosulfonic acid in N,N-dimethylacetamide. Crude T3-sulfate was
initially purified by chromatography on polystyrene resin Amberlite XAD 1600 and then crystallized with ethanol. This strategy
was scaled-up to give a process suitable for the production of kilogram quantities of API, needed to support preclinical and
clinical studies.
vivo half-life of a week1b and undergoes enzymatic deiodination
INTRODUCTION
Hypothyroidism is a common and well-known endocrine
■
in peripheral tissues to liothyronine,1,2 which is the most active
biological hormone. Sodium levothyroxine is usually taken daily
disease, occurring in about 4−5% of the adult population in
in a single dose (e.g., 125 μg per day in a 70 kg adult)1c
reducing the symptoms of hypothyroidism.
Western countries.1 Hypothyroidism is related to an insufficient
blood concentration of thyroid hormones,2 precisely liothyr-
onine3 (2) and thyroxine4 (3) (Figure 1), due to several causes
The administration of both thyroxine and liothyronine has
been also studied since the addition of the more active
liothyronine could help in reaching the optimal plasma level of
both hormones, consequently attaining their physiological
concentrations in target tissues. A controversial debate is still
ongoing since the results of several studies performed following
this approach did not always show a real improvement in
patients’ quality of life. Moreover, the much shorter half-life of
liothyronine (1 day) does not allow a single daily dosage, which
would make therapeutical treatment more awkward for
patients.5
A possible pharmacological alternative to liothyronine could
be its derivative T3-sulfate 1 (Figure 1).6 In fact, one of the
ways the organism disposes of lyothyronine is by an in vivo
sulfation, mainly performed in the liver.7 On the other hand, it
has been found that in some tissues there is a desulfation
process capable of restoring liothyronine from T3-sulfate.8
Preclinical studies demonstrated that T3-sulfate could be
considered as a pro-drug of liothyronine, and its in vivo
enzymatic conversion is able to ensure a stable level of
liothyronine for more than 48 h.9 Therefore, the thyroxine/T3-
sulfate combination therapy could represent a reliable
alternative to the conventional sodium levothyroxine treatment.
Clinical studies are currently ongoing in this respect.10
This manuscript describes the development of a scalable
process for the preparation of T3-sulfate on a kilogram scale
and the synthesis of the related impurities.
Figure 1. Structures of thyroid hormones and analogues: T3-sulfate 1,
liothyronine 2, thyroxine 3, and sodium levothyroxine 4.
such as autoimmune thyroiditis, thyroidectomy, radioiodine
therapy, or to iodine deficiency in the diet, especially in poor
countries. Symptoms associated with hypothyroidism are quite
severe and include tiredness, depression, bradycardia, weight
gain, poor concentration, muscle pain, dry skin, hair loss, and
cold intolerance.
The treatment of choice for hypothyroidism is the life-long
oral administration of synthetic sodium levothyroxine (4),
which is the sodium salt of thyroxine (Figure 1). Sodium
levothyroxine is absorbed in the small intestine,4c it has an in
RESULTS AND DISCUSSION
■
Process Development. The syntheses of T3-sulfate (1)
reported in literature were performed only with 125I or 131I-
Received: July 8, 2014
© XXXX American Chemical Society
A
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labelled liothyronine in a microgram scale or less, employing
sulfuric acid, chlorosulfonic acid, and trimethylamine or
pyridine complexes of sulfur trioxide as sulfating agents.6
Among these and other sulfating agents we have chosen
chlorosulfonic acid11 because it is widely available from
different commercial sources, economical and more effective
in terms of yields.6a The following parameters were studied for
the process optimization: (i) solvent, (ii) stoichiometric excess
of chlorosulfonic acid, (iii) time and temperature reaction, (iv)
quenching conditions, (v) purification.
Solvent. Our preliminary experiments were carried out on
gram scale using chlorosulfonic acid and N,N-dimethylforma-
mide (DMF) as solvent, as reported.6a The results were
disappointing since the yield of T3-sulfate was lower than
expected, and several byproducts were formed, in particular
compounds 5 and 6 (Figure 2), whose structures were assigned
cetamide (DMAC). In this case the reaction between
chlorosulfonic acid and DMAC to give the adduct 8 (Figure
3) cannot be excluded, but the formation of a byproduct such
as 5 was not observed. This is owing to the fact that probably
path b is suppressed due to the greater steric hindrance of the
iminium carbon atom in 8.
Chlorosulfonic Acid. The excess of chlorosulfonic acid
employed in the reaction was found to be a critical issue.
According to relevant literature,6a a huge ratio of 100 mol of
chlorosulfonic acid per mol of liothyronine should be used, but
these conditions are unlikely for economic reasons and
temperature control, since the addition of chlorosulfonic acid
to DMAC is exothermic. Moreover, we found that the amount
of chlorosulfonic acid is directly related to the formation of the
byproduct 12 (Scheme 1). This byproduct cannot be
eliminated during the purification step, and because of this it
must be kept as low as possible in the crude. In order to ensure
good conversion of starting material and at the same time a
lower amount of byproduct 12, an optimized ratio of 8 mol of
chlorosulfonic acid per mol of liothyronine was found to be the
best compromise. It must be pointed out that small traces of
sulfuryl chloride (SO2Cl2) and/or pyrosulfuryl chloride
(ClSO2OSO2Cl) in commercial chlorosulfonic acid might
contribute to the formation of 12. Indeed, an increased amount
of 12 was found when chlorosulfonic acid doped with sulfuryl
chloride (up to 8%) was employed. This byproduct was
synthesized following the strategy shown in Scheme 1 in order
to confirm the structure and to get a sufficient amount as a
reference standard. The synthesis involved chlorination of O-
(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine (9)13 with a solution
of chlorine in acetic acid then iodination with iodine, potassium
iodide, and ethylamine, to give compound 11. Then the final
reaction with chlorosulfonic acid in DMAC gave 12 with an
overall yield of 40%. This strategy was preferred over the direct
chlorination of liothyronine (2) which resulted in incomplete
conversion and formation of a dichlorinated byproduct which
was difficult to separate.
Figure 2. Byproducts formed when chlorosulfonic acid is used in
DMF.
on the basis of HPLC-MS analysis. The explanation could be
the formation of a chlorosulfonic acid−DMF adduct12 such as 7
(Figure 3). Liothyronine can react with 7 following two
Time and Temperature Reaction. A calorimetric evaluation
showed that the reaction between chlorosulfonic acid and
liothyronine is exothermic with an adiabatic increasing of
temperature of 27 °C and a heat release of 176 kcal mol−1,
based on liothyronine. Moreover, it was found that the heat
evolution rate is dependent on chlorosulfonic acid addition
rate, and no accumulation occurred at the end of the addition.
In all the initial laboratory trials the temperature of the reaction
mixture was set at 0 °C. The optimization work revealed that a
temperature range of 5−15 °C was easier to control during
Figure 3. Possible adducts chlorosulfonic acid−DMF 7 and
chlorosulfonic acid−DMAC 8.
different pathways: (a) nucleophilic attack of phenol group at
the sulfur atom giving the desired product 1, and (b)
nucleophilic attack of amino group at the carbon atom
generating byproduct 5. After screening several different
solvents, the best results were obtained with N,N-dimethyla-
Scheme 1. Synthesis of chlorinated byproduct 12
B
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scale-up without increasing the amount of byproducts. With all
the chlorosulfonic acid added the reaction mixture should be
left for completion at 20 °C for at least 2 h. Longer reaction
times should be avoided as they displayed an increase in
byproducts. This is especially true for compound 13 (Figure 4)
deriving from sulfation of both phenol and amino groups of
liothyronine. Also 13 was independently synthesized to confirm
the structure.
after being reconditioned with water, was ready to be employed
for another trial of purification.
Scale-up. The scale-up of T3-sulfate required the availability
of significant amounts of the costly starting material
liothyronine (2). We decided to synthesize this key
intermediate in house. For its preparation, among the several
strategies reported in literature,14 we chose the monoiodination
of O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine (9) (Scheme
3).14d The choice of this strategy was also based on the
expertise that we had previously acquired in the manufacturing
plant synthesis of sodium levothyroxine (4) on a multikilogram
scale.15 The intermediate 9 was synthesized in kilogram
quantities starting from L-tyrosine and following the procedure
reported by Chalmers and co-workers.13
Monoiodination of 9 was achieved with iodine and sodium
iodide in an aqueous solution of ethylamine.16 This method
was preferred over the classical route employing aqueous
ammonia, since it avoids the risk of formation of explosive
nitrogen iodides. Moreover, 9 is much more soluble in aqueous
solution of primary or secondary amines than aqueous
ammonia. Amines can also increase the reactivity of iodine by
the formation of a charge transfer complex, which has also been
documented in the case of morpholine.17
Liothyronine (2), obtained as sodium salt, was converted
into the zwitterionic form by treatment with acetic acid then
reacted with chlorosulfonic acid in DMAC (Scheme 3). The
reaction mixture was quenched with sodium carbonate and
purified by elution through a column of Amberlite XAD 1600.
The fractions with suitable purity were concentrated, and T3-
sulfate was crystallized by addition of ethanol and cooling to 0
°C. After filtration and drying, T3-sulfate was isolated 68% yield
and 99.0% purity (HPLC area %). The scale-up of this process
enabled us to obtain 0.98 kg of pure T3-sulfate needed for
preclinical and clinical evaluation as API.
Figure 4. Structure of disulfated byproduct 13.
Quenching Conditions. Stability tests showed that aqueous
solutions of T3-sulfate are not stable in acidic conditions,
regenerating liothyronine by hydrolysis of the sulfate moiety.
For this reason, the reaction mixture cannot be simply
quenched by addition of water but needs to be slowly poured
into an aqueous solution of sodium bicarbonate or sodium
carbonate in excess, maintaining the temperature at 30 °C.
Similarly, when the reaction is ongoing, the same quenching
step must be always carried out on each sample for HPLC
analysis, in order to obtain realistic information on liothyronine
conversion.
Purification. The aqueous solution obtained from the
quenching step is a complex mixture containing T3-sulfate,
several byproducts, DMAC, and a large quantity of inorganic
salts. We found an easy and convenient purification was the
elution of this mixture through a column of Amberlite XAD
1600, a polystyrene, nonionic, macroreticular resin. Elution
with water allowed to dispose of all inorganic salts and the
majority of DMAC, while T3-sulfate and the byproducts
remained fixed on the resin. All of the last traces of DMAC and
hydrophilic byproducts, such as 13 and 14, were removed
carrying on the elution with water/acetone 95:5 (v/v).
Compound 14, as well as 15 (Scheme 2), derive from the
sulfation of O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine (9) and
thyroxine (3). These two impurities are always present in small
amounts in liothyronine starting material. Byproducts 14 and
15 were also synthesized as reported in Scheme 2. T3-sulfate
was recovered from the column simply by increasing the
acetone amount in the eluent. For the synthesis of laboratory
lots, the fractions with a content of product greater than 99%
(HPLC area) were pooled, concentrated, and treated with
acetone to give, after filtration and drying, T3-sulfate as a white
powder. The column was finally washed with pure acetone to
elute all of the more lipophilic compounds such as unreacted
liothyronine (2) and byproduct 15. At this point the column,
CONCLUSION
■
In conclusion, we have developed a practical and scalable
process for the synthesis of T3-sulfate, a thyroid hormone
analogue. The synthesis has been optimized and scaled-up to a
kilogram scale, with an overall yield of 68% starting from O-(4-
hydroxyphenyl)-3,5-diiodo-L-tyrosine (9). In addition, the
syntheses of the main impurities have been reported.
EXPERIMENTAL SECTION
■
Instrumentation and Materials. 1H- NMR and 13C NMR
spectra were recorded on Bruker DRX400 NMR spectrometer.
The mass spectra were recorded on a ThermoFinnigan mass
spectrometer TSQ700, electrospray ionization. FT-IR spectra
were determined in KBr matrix on a PerkinElmer SPEC-
TRUM2000 spectrophotometer. HPLC analyses were collected
on Agilent 1100 or 1200 liquid chromatographs using a diode
array detector, and the results are reported in area percentage
(area %). Elemental analyses and calorimetry (RC1 Mettler
Scheme 2. Synthesis of sulfated byproducts 14 and 15
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Scheme 3. Synthesis of T3-sulfate
calorimeter) were performed by Redox laboratories, Monza,
Italy. O-(4-Hydroxyphenyl)-3,5-diiodo-L-tyrosine (9)13 and O-
(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-L-tyrosine (thyroxine,
3)15 were synthesized following the procedures reported in
literature. Chlorosulfonic acid was purchased from CABB,
Merck, Sigma-Aldrich, or Acros. Amberlite XAD 1600 and XE
750 were purchased from Rohm&Haas. All other materials
were purchased from commercial suppliers and used without
any further purification. Caution must be taken in handling
chlorine, chlorosulfonic acid, and all the compounds here
reported, which should be considered as highly potent
substances.
HPLC conditions. Stationary phase: Gemini C18, 5 μm, 150
mm × 4.6 mm i.d. (Phenomenex); mobile phase: eluent A =
0.05 M KH2PO4, eluent B = MeCN, gradient elution: t = 0 min
(10% B), t = 45 min (70% B), flow rate: 1.5 mL min−1; column
temperature: 50 °C; UV detector at 225 nm.
(1H, m), 6.74 (1H, d, J = 9 Hz), 7.09 (1H, s), 7.44 (1H, d, J = 9
Hz), 7.86 (2H, s); 13C NMR (DMSO-d6) δ 35.9, 55.9, 91.3,
92.8, 116.4, 121.8, 125.1, 140.0, 141.7, 149.5, 152.6, 170.5; MS
m/z: calcd. for [C15H11I3NNaO7S−Na]− 729.7, found 729.8;
IR (υmax, cm−1) 3595, 3300, 1629, 1246, 1182, 1054, 1027;
Elem. Anal.: Calcd for C15H11I3NNaO7S: C 23.93, H 1.47, N
1.86, Na 3.05, I 50.56, S 4.26. Found: C 23.62, H 1.88, N 1.80,
Na 2.91, I 51.30, S 4.28.
O-[3-Chloro-4-hydroxyphenyl]-3,5-diiodo-L-tyrosine
(10).18 To a vigorously stirred mixture of O-(4-hydroxyphen-
yl)-3,5-diiodo-L-tyrosine (9) (10.0 g; 19 mmol) in AcOH (500
mL), 37% HCl (4.7 g; 48 mmol) was slowly added obtaining a
clear solution. A solution of chlorine (CAUTION) in AcOH
(1.5% w/w; 90 g; 19 mmol) was added in one portion, at room
temperature. After 30 min a second amount of chlorine in
acetic acid (1.5% w/w; 18 g; 4 mmol) was added, and after
further 30 min nitrogen was bubbled into the solution to
remove any trace of chlorine. The solution was evaporated, and
the residue was crystallized from 2 M HCl (160 mL) and
ethanol (30 mL). The solid obtained was filtered then dissolved
in EtOH (50 mL) and water (80 mL), and a 30% (w/w)
aqueous solution of sodium acetate was added until pH 5. The
resulting suspension was filtered and the solid washed with
water (10 mL) then dried at 40 °C for 20 h under vacuum to
give 10 (10.5 g; 99%) as white powder. HPLC purity area
O-[3-Iodo-4-(sulfooxy)phenyl]-3,5-diiodo-L-tyrosine
Sodium Salt (1). A solution of iodine (0.48 kg, 1.90 mol) and
sodium iodide (0.65 kg, 4.34 mol) in water (3.5 L) was added
to a solution of O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine (9)
(1 kg, 1.90 mol), sodium iodide (0.32 kg, 2.13 mol), and 70%
aq ethylamine (2.8 kg, 43.5 mol) in water (3 L). The reaction
mixture was stirred for 6 h at room temperature then cooled to
0 °C, stirred for 4 h, and filtered. The solid was washed with
water (2.4 L), then suspended in water (15 L), and acetic acid
(0.62 L) was added. The suspension was stirred for 2 h and the
solid filtered and washed with water (3.5 L). The solid was
resuspended in water (15 L), stirred, filtered, and washed with
water (3.5 L) to give crude liothyronine (2). The solid was
suspended in DMAC (13 L) and the remaining water was
eliminated by distillation under vacuum. The suspension was
cooled to 5 °C, and under nitrogen atmosphere, chlorosulfonic
acid (1.54 kg, 13.21 mol) was slowly added, keeping the
temperature below 10 °C. The temperature was raised to 20
°C, and the mixture stirred for 1 h then added to a solution of
sodium carbonate (2.27 kg, 21.45 mol) in water (30 L). The
solution was eluted through an Amberlite XAD 1600 (12.5 L)
resin column. The resin was initially eluted with water (87.5 L)
then with water/acetone solutions starting with 95:5 (v/v) and
up to 70:30 (v/v). Fractions with the suitable purity were
pooled and concentrated by distillation under vacuum. The
suspension was cooled to 40 °C, and ethanol (6.6 L) was added
obtaining a clear solution which was cooled to 0 °C causing
precipitation. The solid was filtered, washed with 96% ethanol
(4 L), and dried at 40 °C under vacuum to give 1 (0.98 kg;
1
95.1%; H NMR (DMSO-d6) δ 2.92 (1H, m), 3.16 (1H, m),
3.45 (2H, bm), 3.62 (1H, m), 6.61 (1H, d, J = 6 Hz), 6.71 (1H,
s), 7.01 (1H, d, J = 6 Hz), 7.86 (2H, s); 13C NMR (DMSO-d6)
δ 35.2, 55.2, 92.6, 115.4, 116.6, 117.7, 120.3, 139.1, 141.4,
149.0, 149.2, 152.4, 170.1; MS m/z: calcd. for [C15H12ClI2NO4
+ H]+ 559.9, found 559.8.
O-[3-Chloro-5-iodo-4-hydroxyphenyl]-3,5-diiodo-L-ty-
rosine (11). A 70% aqueous solution of ethylamine (69 g;
1070 mmol) was slowly added in 30 min to a suspension of 10
(10 g; 17.9 mmol) in water (85 mL) keeping the temperature
below 20 °C. A solution of KI (9.38 g; 57 mmol) and iodine
(5.62 g; 22.2 mmol) in water (27 mL) was added in 2 h,
keeping the temperature below 20 °C. The solution was stirred
for 20 h; then a 5% (w/w) solution of Na2S2O5 was added until
complete reaction of excess iodine. The solution was heated to
50 °C; acetic acid (75.2 g; 1250 mmol) was added until final
pH 5. The resulting suspension was heated to 60 °C and
filtered. The solid was washed with hot water (485 g. 65 °C)
then dried at 40 °C for 20 h under vacuum to give 11 (10.9 g;
1
89%) as a white powder. HPLC purity area 88.8%; H NMR
(DMSO-d6) δ 2.85 (1H, m), 3.17 (1H, m), 3.40 (2H, bm), 3.57
(1H, m), 6.81 (1H, s), 7.10 (1H, s), 7.86 (2H, s); 13C NMR
(DMSO-d6) δ 35.5, 55.4, 89.8, 92.4, 116.9, 121.4, 124.4, 139.6,
1
68%) as a white powder. HPLC purity 99.0 area %; H NMR
(DMSO-d6) δ 2.87 (1H, m), 3.17 (1H, m), 3.40 (2H, bm), 3.55
D
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141.4, 148.5, 150.0, 151.9, 169.9; MS m/z: calcd. for
[C15H11ClI3NO4−H]− 683.7, found 683.9.
gradient. The fractions containing the product were evaporated
under vacuum and the solid residue stirred for 2 h with CH2Cl2
(15 mL). The solid was filtered and treated again with CH2Cl2
(15 mL), as described above, in order to eliminate the last
traces of DMAC. The solid was filtered, dried, and dissolved in
water, and 1 M HCl was added until pH 6.2. The suspension
was evaporated, and the solid was dried at 40 °C for 20 h under
vacuum to give 14 (3.28 g; 54.5%) as a white powder. HPLC
purity 98.8 area %; 1H NMR (DMSO-d6) δ 2.87 (1H, m), 3.19
(1H, m), 3.46 (2H, bm), 3.55 (1H, m), 6.67 (2H, d, J = 9 Hz),
7.10 (2H, d, J = 9 Hz), 7.86 (2H, s); 13C NMR (DMSO-d6) δ
35.8, 55.9, 93.0, 116.0, 122.8, 139.7, 141.7, 149.0, 152.8, 153.0,
170.9; MS m/z: calcd for [C15H12I2NNaO7S−Na]− 603.8,
found 604.0; Elem. Anal.: Calcd for C15H12I2NNaO7S: C 28.73,
H 1.93, N 2.23, Na 3.67, I 40.47, S 5.11. Found: C 28.69, H
1.36, N 2.23, Na 4.28, I 39.97, S 4.91.
O-[3,5-Diiodo-4-(sulfooxy)phenyl]-3,5-diiodo-L-tyro-
sine Sodium Salt (15). In nitrogen atmosphere chlorosulfonic
acid (14.0 g; 120 mmol) was slowly added over 1 h to a
vigorously stirred suspension of O-(4-hydroxy-3,5-diiodophen-
yl)-3,5-diiodo-L-tyrosine (3) (10 g; 13 mmol) in DMAC (200
mL), maintaining the temperature at 0 °C. The reaction
mixture was then allowed to warm to room temperature and
reacted for 4 h. The mixture was slowly poured in 1.5 h into a
stirred solution of NaHCO3 (32.8 g; 390 mmol) in water (450
mL). The gelatinous solid precipitated was completely
dissolved by addition of Na2CO3 (4.65 g; 44 mmol). The
resulting solution was eluted through an Amberlite XAD 1600
(250 mL) resin column. The resin was initially eluted with
water to eliminate salts and DMAC; then an acetone/water
gradient was applied. The fractions containing the product were
collected and evaporated, and the residue was treated with
acetone (8 mL). The suspension was filtered and the solid
dried at 40 °C for 48 h under vacuum to give 15 (2.06 g; 18%)
as a white powder. HPLC purity 97.0 area %; 1H NMR
(DMSO-d6) δ 2.80 (1H, m), 3.14 (1H, m), 3.51 (1H, m), 7.14
(2H, s), 7.87 (2H, s); 13C NMR (DMSO-d6) δ 37.4, 56.5, 92.5,
93.5, 126.5, 141.5, 141.7, 150.1, 151.9, 153.0, 173.6; MS m/z:
calcd. for [C15H10I4NNaO7S−Na]− 855.6, found 855.7; Elem.
Anal.: Calcd for: C15H10I4NNaO7S: C 20.50, H 1.15, N 1.59,
Na 2.62, I 57.76, S 3.65. Found: C 20.70, H 0.91, N 1.63, Na
4.26, I 57.04, S 3.44.
O-[3-Chloro-5-iodo-4-(sulfooxy)phenyl]-3,5-diiodo-L-
tyrosine Sodium Salt (12). In a nitrogen atmosphere
chlorosulfonic acid (63.4 g; 544 mmol) was slowly added
over 1.5 h to a vigorously stirred suspension of 11 (15.55 g;
22.7 mmol) in DMAC (300 mL), maintaining the temperature
at 0 °C. The reaction mixture was allowed to warm to room
temperature and reacted for 4 h. The mixture was slowly
poured into a stirred 8% (w/w) aq solution of NaHCO3 (684
g; 651 mmol); then a 15% (w/w) aq solution of Na2CO3 was
added until pH 7. The resulting solution was loaded onto an
Amberlite XE 750 (650 mL) resin column. The resin was
initially eluted with water then with an acetone/water gradient
up to acetone/water 1:1 (v/v). The fractions containing the
product were concentrated, and 1 M HCl was added until pH
6.2. The solution was evaporated, and the solid was dried at 40
°C for 20 h under vacuum to give 12 (8.0 g; 45%) as a white
1
powder. HPLC purity area 99%; H NMR (DMSO-d6) δ 2.86
(1H, m), 3.18 (1H, m), 3.39 (2H, bm), 3.57 (1H, m), 6.81
(1H, d, J = 1.5 Hz), 7.12 (1H, d, J = 1.5 Hz), 7.86 (2H, s); 13C
NMR (DMSO-d6) δ 35.8, 55.5, 92.7, 96.0, 117.5, 125.0, 129.2,
139.9, 141.8, 146.6, 152.2, 153.0, 170.5; MS m/z: calcd. for
[C15H10ClI3NNaO7S−Na]− 763.7, found 763.8; Elem. Anal.:
Calcd for C15H10ClI3NNaO7S: C 22.88, H 1.28, N 1.78, Cl
4.50, Na 2.92, I 48.35, S 4.07. Found: C 22.68, H 0.89, N 1.76,
Cl 4.61, Na 3.48, I 48.69, S 3.76.
O-[3-Iodo-4-(sulfooxy)phenyl]-3,5-diiodo-N-sulfo-L-ty-
rosine (13). In nitrogen atmosphere chlorosulfonic acid (6.15
g; 53 mmol) was slowly added over 15 min to a vigorously
stirred suspension of O-(4-hydroxy-3-iodophenyl)-3,5-diiodo-L-
tyrosine (2) (5 g; 7.7 mmol) in pyridine (100 mL), maintaining
the temperature at 15−20 °C. The reaction mixture was stirred
for 1 h then slowly poured into a stirred solution of NaHCO3
(2.77 g; 34 mmol) in water (150 mL), and 1 M NaOH was
added until pH 8.2. The resulting solution was concentrated to
about 50 mL then loaded onto an Amberlite XAD 1600 (130
mL) resin column. The resin was initially eluted with water
then with an acetone/water gradient. The fractions containing
the product were evaporated, and the glassy residue obtained
was treated twice with acetone (20 mL). The suspension was
filtered and the solid dried at 40 °C for 48 h under vacuum to
give 13 (1.73 g; 27%) as a white powder. HPLC purity 99.4
area %; 1H NMR (D2O) δ 2.91 (2H, d, J = 6 Hz), 3.86 (1H, t, J
= 6 Hz), 6.90 (1H, dd, J1 = 3 Hz, J2 = 9 Hz), 7.28 (1H, d, J = 3
Hz), 7.35 (1H, d, J = 9 Hz), 7.81 (1H, s); 13C NMR (D2O) δ
37.7, 59.7, 90.2, 91.3, 117.2, 122.8, 126.2, 140.0, 141.8, 147.1,
151.8, 154.2, 178.8; MS m/z: calcd. for [C15H10I3NNa2O10S2−
H−Na]−2 415.3, found 415.7; Elem. Anal.: Calcd for:
C15H10I3NNa2O10S2: C 21.07, H 1.18, N 1.64, Na 5.38, I
44.52, S 7.50. Found: C 20.34, H 1.31, N 1.62, Na 7.86, I 39.42,
S 7.94.
AUTHOR INFORMATION
Corresponding Author
*E-mail: luciano.lattuada@bracco.com.
■
Present Address
M.A.: Euticals SpA, Via Volturno 41/43, Rozzano (MI) 20089,
Italy.
Notes
The authors declare no competing financial interest.
O-[4-(Sulfooxy)phenyl]-3,5-diiodo-L-tyrosine Sodium
Salt (14). In nitrogen atmosphere chlorosulfonic acid (8.39
g; 72 mmol) was slowly added in 45 min to a vigorously stirred
suspension of O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine (9)
(5.04 g; 9.6 mmol) in DMAC (100 mL), maintaining the
temperature at 0 °C. The reaction mixture was allowed to warm
to room temperature and reacted for 4 h. The mixture was
slowly poured into a stirred solution of NaHCO3 (19.6 g; 233
mmol) in water (225 mL). The resulting solution was eluted
through an Amberlite XAD 1600 (125 mL) resin column. The
resin was initially eluted with water then with an acetone/water
ACKNOWLEDGMENTS
■
We would like to thank Dr. Marino Brocchetta for helpful
discussions and support of this work.
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