Synthesis and biodistribution studies of 99mTc labeled fatty acid derivatives prepared via “Click approach” for potential use in cardiac imaging

Article abstract of DOI:10.1002/jlcr.3681

¹²³I-Iodophenylpentadecanoic acid (IPPA) is a metabolic agent used in nuclear medicine for diagnosis of myocardial defects. Efforts are underway worldwide to develop a ^99mTc substitute of the above radiopharmaceutical for the aforementioned application. Herein, we report synthesis and biodistribution studies of ^99mTc labeled fatty acids (8, 11, and 15 carbons) obtained via “click chemistry” for its potential use in myocardial imaging. ω-Bromo fatty acids (8C/11C/15C) were synthetically modified at bromo terminal to introduce a heterocyclic triazole with glycine sidearm in a five step procedure. Modified fatty acids were subsequently radiolabeled with preformed [^99mTc(CO)₃]⁺ synthon to yield the desired fatty acid complexes which were evaluated in Swiss mice. All the radiolabeled complexes were obtained with radiochemical purities >80%, as characterized by HPLC. Biodistribution studies of all three complexes in Swiss mice showed myocardial uptake of ~6-9% ID/g at 2?minutes post-injection, close to^*I-IPPA (~9% ID/g). Complexes exhibited significant retention in the myocardium up to 30?minutes (~1% ID/g) but were lower to the standard agent (~7% ID/g). Similar uptake of activity in myocardium for the newly synthesized complexes in comparison to ¹²⁵I-IPPA along with favorable in vivo pharmacokinetics merits potential for the present “click” design of complexes for myocardial imaging.

Full text of DOI:10.1002/jlcr.3681

9
9m
Synthesis and Biodistribution studies of Tc labeled fatty acid derivatives
prepared via ‘Click approach’ for potential use in cardiac imaging
1
,2
1,*
1
2,3
4
Soumen Das, Anupam Mathur, Navin Sakhare, Madhava B. Mallia, H.D. Sarma, S.S.
1
2,3
Sachdev, A. Dash
1
Radiopharmaceuticals Program, Board of Radiation and Isotope Technology (BRIT), Navi Mumbai-400703
INDIA
2
Homi Bhabha National Institute (HBNI), Anushaktinagar, Mumbai-400094 INDIA
3
4
Radiopharmaceutical Division, Radiation Biology and Health Science Division, Bhabha Atomic Research
Centre (BARC), Trombay, Mumbai-400085 INDIA
9
9m
Short title: Evaluation of Tc-fatty acid derivatives for cardiac imaging
*
Author for correspondence: Anupam Mathur
Radiopharmaceuticals Program
Board of Radiation and Isotope Technology
Navi Mumbai - 400 703
India
Telephone
Fax
: 91-22-2788 7250
: 91-22-2788 7218
E-mail
: amathur@britatom.gov.in
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3681
This article is protected by copyright. All rights reserved.

Abstract
1
23
I-Iodophenylpentadecanoic acid (IPPA) is a metabolic agent used in nuclear medicine for
9
9m
diagnosis of myocardial defects. Efforts are underway worldwide to develop a
Tc
substitute of the above radiopharmaceutical for the aforementioned application. Herein, we
9
9m
report synthesis and biodistribution studies of Tc labeled fatty acids (8, 11 and 15 carbons)
obtained via ‘click chemistry’ for its potential use in myocardial imaging.
ω-Bromo fatty acids (8C/11C/15C) were synthetically modified at bromo terminal to
introduce a heterocyclic triazole with glycine sidearm in a five step procedure. Modified fatty
99m
+
acids were subsequently radiolabeled with preformed [ Tc(CO) ] synthon to yield the
3
desired fatty acid complexes which were evaluated in Swiss mice.
All the radiolabeled complexes were obtained with radiochemical purities >80%, as
characterized by HPLC. Biodistribution studies of all three complexes in Swiss mice showed
*
myocardial uptake of ~6-9% ID/g at 2 min post-injection, close to I-IPPA (~9% ID/g).
Complexes exhibited significant retention in the myocardium up to 30 min (~1% ID/g) but
were lower to the standard agent (~7% ID/g).
Similar uptake of activity in myocardium for the newly synthesized complexes in comparison
1
25
to I-IPPA along with favorable in vivo pharmacokinetics merits potential for the present
‘click’ design of complexes for myocardial imaging.
9
9m
Key words: Fatty acid, click chemistry, Tc carbonyl core, myocardial imaging
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Introduction
Mechanistic shift of myocardial metabolism from fats to glucose during ischemic episodes is
a sensitive measure for detecting coronary artery diseases. Radiolabeled fatty acids serve as
1
-3
useful substrates for delineating such abnormalities in a functional myocardium. In this
preview, a number of PET and SPECT labeled fatty acids have been extensively reviewed in
past. PET radiotracers offer advantages in terms of high image resolution and quantitative
4
-7
image analyses, but complex radiosynthesis protocols employing short lived radionuclides
1
23
limits their widespread availability for clinical use. I-15‐(p‐iodophenyl)pentadecanoic acid
1
23
(
IPPA) and
I-15‐(p‐iodophenyl)-3‐methylpentadecanoic acid (BMIPP) are the only
8
-10
SPECT radiotracers that have shown promise for imaging heart metabolism. The relatively
1
23
short half-life of I (~13 h) and its production via high energy beam cyclotron restricts
radiopharmaceutical use around cyclotron facility. To circumvent the above limitations,
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development of
Tc labeled fatty acids will continue to attract attention of scientists
99m
involved in radiopharmaceutical research. Development of
Tc substitute will not only
9
9
99m
ensure continued radioisotope availability via renewable Mo- Tc generator but assure
round the clock availability of the radiotracer via convenient kit labeling strategy.
9
9m
99m
Several Tc labeled fatty acids following different Tc chemistries viz. oxotechnetium (V)
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3+ 11
99m
2+ 12,13 99m
core ([ Tc=O] ),
nitrido core ([ TcN(PNP)] ),
Tc‐4+1 mixed ligand
1
4,15
99m
+ 16,17
approach,
tricarbonyl core ([ Tc(CO) (H O) ] ),
cyclopentadienyl tricarbonyl
3
2
3
9
9m
18-20
99m
21
technetium ( Tc‐CpTT) core
and
Tc-HYNIC , were developed and evaluated in
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past but all lacked favorable characteristics for in vivo imaging.
Tc cores/BFCAs alter in
vivo pharmacokinetic behavior of fatty acids of identical chain lengths. Hence, present work
9
9m
is an evaluation of newly designed
Tc fatty acids of variable carbon chain lengths
2
2
synthesized via ‘click chemistry’ for its potential use in myocardial imaging.
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Copper (I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction (click reaction) between an
azide and an alkyne to give a stable 1,4-disubstituted 1,2,3-triazole has been used extensively
1
8
for preparing F-labeled derivatives and for incorporating metallic radionuclides via
99m
+
23-25
[
Tc(CO) ] core.
Fatty acids of variable chain lengths 8, 11 and 15 carbons were
3
synthetically modified at ω terminal following the same click chemistry approach and labeled
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9m
via
Tc(CO) synthon. The labeled complexes were subsequently evaluated for potential
3
*
towards myocardial imaging in comparison with standard I-IPPA.
Experimental
Materials and Methods
8
-Bromooctanoic
acid,
15-bromopentadecanoic
acid,
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine (DMAP), sodium azide
and palladium on activated carbon (10% wt) were purchased from Aldrich, USA. 11-
bromoundecanoic acid was purchased from TCI Chemicals, Japan. Sodium ascorbate was
obtained from Alfa Aesar (Germany). Benzyl alcohol, copper (II) sulfate, tert-butanol and
trifluoroacetic acid were obtained from SD Fine Chemicals (India). All reagents were of
analytical grade and were used as received without further purification. Sodium pertechnetate
9
9m
99
99m
(
Na TcO4) was eluted with normal saline prior to use from a Mo- Tc column generator,
125
supplied by Board of Radiation and Isotope Technology (BRIT), India.
I-IPPA was
1
3
prepared according to the procedure described previously. Silica gel plates (Silica Gel 60
F ) and 60-120 mesh silica gel used for column chromatography was procured from Merck,
2
54
India. The HPLC of the prepared complexes was carried out on a JASCO PU 2080 Plus dual
pump HPLC system, Japan, with a JASCO 2075 Plus tunable absorption detector and Gina
Star radiometric detector system, using a C18 reversed phase HiQ Sil (5 µm, 4  250 mm)
column. FT-IR spectra were recorded on JASCO FT/IR-460 Spectrophotometer, Japan. The
1
H-NMR spectra were recorded on a 60 MHz Nanalysis NMR spectrometer, Canada. Mass
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L
spectra were recorded on expression CMS Advion Mass Spectrometer, USA using
electrospray ionization (ESI) in both positive and negative modes.
Synthesis
ω-bromo fatty acid benzyl ester (1a-3a)
ω-bromo fatty acid (2.24 mmol) and benzyl alcohol (0.25 mL, 2.5 mmol) in DCM (10 mL)
were stirred overnight at room temperature in presence of EDC (471 mg, 2.46 mmol) and
catalytic amount of DMAP (27 mg, 0.22 mmol). Upon completion of reaction (Cf. TLC), the
reaction mixture was washed with water and DCM layer dried over anhydrous Na SO .
2
4
Thereafter, DCM was removed under vacuum and purified using silica gel column
chromatography to yield the desired compound.
Benzyl 8-bromooctanoate (1a)
Yield: 74% (520 mg) R =0.9 (CHCl )
f
3
-1
IR (neat, ῡ cm ) 1735.6 (C=O ester)
1
H NMR (CDCl , 60 MHz): δ 7.30 (s, 5H, PhCH COO), 5.10 (s, 2H, PhCH COOCH ), 3.30
3
2
2
2
(
t, 2H, J = 8 Hz, BrCH ), 2.31 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.1 (m, 4H, BrCH CH -
2 2 2 2
and BzCOOCH CH -), 1.30 (s, 6H, (CH ) ).
2
2
2 3
Benzyl 11-bromoundecanoate (2a)
Yield: 80% (635 mg)
-1
IR (neat, ῡ cm ) 1735.2 (C=O, ester)
1
H NMR (CDCl , 60 MHz): δ 7.33 (s, 4H, PhCH COO), 5.10 (s, 2H, PhCH COOCH ), 3.36
3
2
2
2
(
t, 2H, J = 8 Hz, BrCH ), 2.34 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.12 (m, 4H,
2
2
BrCH CH - and BzCOOCH CH -), 1.34 (s, 12H, (CH ) ).
2
2
2
2
2 6
Benzyl 15-bromopentadecanoate (3a)
Yield: 70% (644 mg)
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-1
IR (neat, ῡ cm ) 1735.4 (C=O, ester)
1
H NMR (CDCl , 60 MHz): δ 7.37 (s, 4H, PhCH COO), 5.14 (s, 2H, PhCH COOCH ), 3.41
3
2
2
2
(
t, 2H, J = 8 Hz, BrCH ), 2.37 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.2 (m, 4H, BrCH CH -
2 2 2 2
and BzCOOCH CH -), 1.29 (s, 20H, (CH ) ).
2
2
2 10
ω-azido fatty acid benzyl ester (1b-3b)
Sodium azide (130 mg, 2 mmol) was added to a solution of ω-bromo benzyl ester (1a-3a; 0.5
mmol) in DMF (5 mL), and mixture stirred overnight at room temperature. Thereafter DMF
was removed under vacuum and reaction mixture re-dissolved in water (10 mL). The aqueous
mixture was then extracted with CHCl (3 × 10 mL) and pooled extracts were dried over
3
anhydrous Na SO . The chloroform was then removed to give azide derivative which was
2
4
used as such without further purification.
Benzyl 8-azidooctanoate (1b)
Yield: 85% (117 mg)
-1
IR (neat, ῡ cm ) 1735.6 (C=O, ester), 2095.3 (N=N=N azide), 2854.1 (C-H sym), 2926.4 (C-
H asymm)
1
H NMR (CDCl , 60 MHz): δ 7.30 (s, 4H, PhCH COO), 5.10 (s, 2H, PhCH COOCH ), 3.30
3
2
2
2
(
t, 2H, J = 8 Hz, N CH ), 2.31 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.1 (m, 4H, N CH CH -
3 2 2 3 2 2
and BzCOOCH CH -), 1.30 (s, 6H, (CH ) ).
2
2
2 3
Benzyl 11-azidoundecanoate (2b)
Yield: 90% (142 mg)
-1
IR (neat, ῡ cm ) 1735.6 (C=O, ester), 2094.3 (N=N=N, azide), 2855.1 (C-H sym), 2927.4 (C-
H asymm).
1
H NMR (CDCl , 60 MHz): δ 7.33 (s, 4H, PhCH COO), 5.10 (s, 2H, PhCH COOCH ), 3.36
3
2
2
2
(
t, 2H, J = 8 Hz, N CH ), 2.34 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.12 (m, 4H,
3 2 2
N CH CH - and BzCOOCH CH -), 1.34 (s, 12H, (CH ) ).
3
2
2
2
2
2 6
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Benzyl 15-azidopentadecanoate (3b)
Yield: 89% (166 mg)
-1
IR (neat, ῡ cm ) 1735.6 (C=O, ester), 2095.3 (N=N=N, azide), 2854.1 (C-H sym), 2926.4 (C-
H asymm)
1
H NMR (CDCl , 60 MHz): δ 7.37 (s, 4H, PhCH COO), 5.14 (s, 2H, PhCH COOCH ), 3.41
3
2
2
2
(
t, 2H, J = 8 Hz, N CH ), 2.37 (t, 2H, J = 7.2 Hz, BzCOOCH ), 1.60–2.2 (m, 4H, N CH CH -
3 2 2 3 2 2
and BzCOOCH CH -), 1.29 (s, 20H, (CH ) ).
2
2
2 10
ω-(N-Boc-amino propanoic acid) triazole fatty benzyl ester (1c-3c)
N-Boc-propargyl glycine (82 mg, 0.38 mmol) and azido compound (1b-3b, 0.45 mmol) was
refluxed overnight in presence of CuSO .5H O (21 mg, 0.11 mmol) and sodium ascorbate (42
4
2
mg, 0.21 mmol) in tert-butanol: water mixture (1:1 v/v). Reaction mixture was then dried
under vacuum and crude product purified using silica gel column chromatography to yield
compound 1c-3c.
3
-(1-(7-((benzyloxy)carbonyl)heptyl)-1H-1,2,3-triazol-4-yl)-2-N-Boc-aminopropanoic acid
1c)
Yield: 80% (148 mg); R =0.1 (CHCl /MeOH 9:1 v/v )
(
f
3
1
H NMR (CDCl , 60 MHz): δ 7.25-7.5 (m, 6H, PhCH COO and Trz-CH ), 5.09 (s, 2 H,
3
2
2
PhCH COOCH ), 4.0-4.6 (m, 3H, CH -Trz-CH -CH(NHBoc)COOH), 3.1-3.4 (m, 2H, Trz-
2
2
2
2
CH -CH(NHBoc)COOH), 2.1-2.4 (m, 2H, BzCOOCH ), 1.60–1.96 (m, 4H, Trz-CH CH -
2
2
2
2
and BzCOOCH CH -), 1.1-1.6 (m, 15H, C(CH ) and (CH ) ).
2
2
3 3
2 3
+
MS (ESI ): Mass (calculated) C H N O 488.26; m/z (observed) 489.6.
2
5
37
4
6
3
-(1-(10-((benzyloxy)carbonyl)decyl)-1H-1,2,3-triazol-4-yl)-2-N-Boc-aminopropanoic acid
2c)
Yield: 84% (170 mg).
(
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1
H NMR (CDCl , 60 MHz): δ 7.25-7.44 (m, 6H, PhCH COO and Trz-CH ), 5.10 (s, 2H,
3
2
2
PhCH COOCH ), 4.85-5.0 (m, 1H, CH -Trz-CH -CH(NHBoc)COOH), 4.2-4.6 (m, 2H, CH -
2
2
2
2
2
Trz-CH -CH(NHBoc)COOH), 3.1-3.4 (m, 2H, Trz-CH -CH(NHBoc)COOH), 2.34 (t, 2H, J
2
2
=
2.8 Hz, BzCOOCH ), 1.80–2.10 (m, 2H, Trz-CH CH - and BzCOOCH CH -), 1.60–1.75
2 2 2 2 2
(
m, 2H, BzCOOCH CH -), 1.1-1.55 (m, 21H, C(CH ) and (CH ) ).
2 2 3 3 2 6
+
MS (ESI ): Mass (calculated) C H N O 530.66; m/z (observed) 531.8; 441.53 [M-Benzyl].
2
8
43
4
6
3
-(1-(14-((benzyloxy)carbonyl)tetradecyl)-1H-1,2,3-triazol-4-yl)-2-N-Boc-
aminopropanoicacid (3c)
Yield: 82% (182 mg).
1
H NMR (CDCl , 60 MHz): δ 7.25-7.42 (m, 6H, PhCH COO and Trz-CH ), 5.10 (s, 2 H,
3
2
2
PhCH COOCH ), 4.6-5.0 (m, 1H, CH -Trz-CH -CH(NHBoc)COOH), 4.15-4.35 (m, 2H,
2
2
2
2
CH -Trz-CH -CH(NHBoc)COOH), 2.9-3.4 (m, 2H, Trz-CH -CH(NHBoc)COOH), 2.34 (t,
2
2
2
2
1
H, J = 2.8 Hz, BzCOOCH ), 1.75–2.10 (m, 2H, Trz-CH CH - and BzCOOCH CH -), 1.55–
2 2 2 2 2
.70 (m, 2H, BzCOOCH CH -), 1.1-1.55 (m, 29H, C(CH ) and (CH ) ).
2
2
3 3
2 10
+
MS (ESI ): Mass (calculated) C H N O 586.76; m/z (observed) 587.8; 497 [M-Benzyl].
3
2
51
4
6
ω-(amino propanoic acid) triazole fatty acid benzyl ester (1d-3d)
Compound 1c-3c (0.18 mmol) was treated with trifluoroacetic acid (TFA; 2 mL) at room
temperature for 2 h. Thereafter TFA was removed under vacuum to yield the desired
compound and was used as such for next step without further purification.
3
-(1-(7-((benzyloxy)carbonyl)heptyl)-1H-1,2,3-triazol-4-yl)-2-aminopropanoic acid (1d)
Yield: Quantitative (70 mg)
+
MS (ESI ): Mass (calculated) C H N O 388.46; m/z (observed) 389.8.
2
0
29
4
4
3
-(1-(10-((benzyloxy)carbonyl)decyl)-1H-1,2,3-triazol-4-yl)-2-aminopropanoic acid (2d)
Yield: 90% (70 mg)
+
MS (ESI ): Mass (calculated) C H N O 430.54; m/z (observed) 431.9; [M-Benzyl] 341.
2
3
35
4
4
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3
-(1-(14-((benzyloxy)carbonyl)tetradecyl)-1H-1,2,3-triazol-4-yl)-2-aminopropanoic acid (3d)
Yield: 95% (83 mg)
+
MS (ESI ): Mass (calculated) C H N O 486.65; m/z (observed) 487.9; [M-Benzyl] 397.9.
2
7
43
4
4
ω-(amino propanoic acid) triazole fatty acid (1e-3e)
Compound 1d-3d (0.15 mmol) was added to a stirred suspension of 10% Pd-C (20 mg) in
absolute ethanol (10 mL). H gas was bubbled through the above reaction mixture and
2
reducing environment maintained overnight with continued stirring at room temperature.
Upon completion of reaction (Cf. TLC), the reaction mixture was filtered and ethanol
removed under vacuum to yield compound 1e-3e.
8
-(4-(2-amino-2-carboxyethyl)-1h-1,2,3-triazol-1-yl)octanoic acid (1e)
Yield: quantitative (47 mg)
+
MS (ESI ): Mass (calculated) C H N O 298.34; m/z (observed) 320.8 [M+Na-H].
1
3
23
4
4
1
1-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)undecanoic acid (2e)
Yield: quantitative (51 mg)
+
MS (ESI ): Mass (calculated) C H N O 340.42; m/z (observed) 341.5.
1
6
29
4
4
1
5-(4-(2-amino-2-carboxyethyl)-1H-1,2,3-triazol-1-yl)pentadecanoic acid (3e)
Yield: quantitative (60 mg)
+
MS (ESI ): Mass (calculated) C H N O 396.52; m/z (observed) 396.9.
2
0
37
4
4
Radiolabeling
The intermediate [ Tc(CO) ] synthon was produced on addition of freshly eluted sodium
9
9m
+
3
9
9m
pertechnetate (Na TcO ) (1 mL, 740 MBq) to a lyophilized kit containing potassium
4
boranocarbonate (1 mg) and heating the contents in boiling water bath for 30 minutes. The
formation of intermediate was characterized using TLC [MeOH: HCl (95:5 v/v); R ~ 0.4]
f
and HPLC.
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To the freshly prepared intermediate (0.5 mL, 370 MBq), fatty acid derivative (1e/2e/3e) (3-5
mg) in MeOH (0.5 mL) was added and contents heated under boiling water conditions for
another 30 min to yield the respective complexes 1f, 2f and 3f.
Quality control
HPLC
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9m
Formation of Tc-fatty acid complexes (1f-3f) was characterized by reversed phase HPLC.
Gradient elution program was followed using water (solvent A) and acetonitrile (solvent B)
as the mobile phase (0 min 90% A, 30min 10% A, 40 min 0% A) each containing 0.1% TFA.
Flow rate of the solvent was maintained at 1 mL/min. Test solution (20 µL) was injected into
the column using a micro-syringe and elution was monitored by observing the radioactivity
profile. The same C18 reversed phase analytical column was used for the purification of the
radiolabeled complexes 1f, 2f and 3f using the same gradient elution with same mobile phase
but without 0.1%TFA. Peaks corresponding to labeled complexes were collected, dried and
reconstituted in 10% ethanol. Around 7.4 MBq of pure radiolabeled complex was obtained by
this method which was subsequently used for performing in vitro and biodistribution studies.
Partition coefficient (Log Po/w)
The purified complex (1f/2f/3f) (0.1 mL, ~185 kBq) was mixed with double distilled water
(0.9 mL) and n-octanol (1 mL) and vortexed for 3 min. The mixture was then centrifuged at
3
500 g for 3 min to effect clear separation of the two layers. Equal aliquots from the two
layers were withdrawn in triplicates and measured for associated radioactivity. The readings
thus obtained were used to calculate Log Po/w value (octanol-water partition) of the
complexes.
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Serum stability
The purified fatty acid complex (1f/2f/3f) (50 µL, ~ 370 KBq) was incubated in human serum
(450 µL) at 37°C for 30 min. Thereafter, serum proteins were precipitated by addition of
ethanol (500 µL). The solution was centrifuged and the supernatant was analyzed by HPLC
to ascertain the stability of the complex in serum.
Biodistribution studies
All procedures performed herein were in accordance with the national laws pertaining to the
conduct of animal experiments. Normal female Swiss mice (20-25 g body weight) were used
for the in vivo distribution studies. The mice were fasted for 4-5 h prior to the experiment.
9
9m
The HPLC purified
Tc-labelled fatty acid complex (1f/2f/3f) reconstituted in 6% human
serum albumin (HSA) was then administered (100 µL, ~370 KBq) intravenously through the
tail vein of each mouse. Individual sets of animals (n = 3) were utilized for studying
distribution at four different time points (2 min, 5 min, 10 min and 30 min). At the end of
each time point, respective set of animals were sacrificed and the relevant organs and tissue
were excised for the measurement of associated activity. The organs were weighed and the
activity associated with each organ was measured in a flat-bed type NaI(Tl) counter with
9
9m
suitable energy window for
Tc (140 keV ± 10%). The activity associated with each
organ/tissue was expressed as a percent injected dose per gram (% ID per g).
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Results
Three fatty acids of variable chain lengths 8, 11 and 15 carbon were selected for screening
9
9m
the efficacy of new
Tc fatty acid complexes designed via ‘click chemistry’ approach for
myocardial imaging. All the three fatty acids bearing ω-bromo group were modified at bromo
terminal with triazole glycine chelate in a five step synthetic procedure (Scheme 1). The first
step is an esterification reaction where the carboxylic acid group was converted to a benzyl
ester in presence of EDC and DMAP activator. The introduction of benzyl chromophore
favored unambiguous monitoring and product isolation during all the intermediary steps of
reaction scheme. The second step involved direct substitution of bromo group with an azide
-1
functionality via S 2 reaction mechanism. IR stretching frequency at 2095 cm confirmed
N
azide formation, but NMR could not differentiate azide derivative from step 1 bromo
compound. NMR δ values of 1a-3a and 1b-3b were identical probably because of similar
electron pull from both bromine and azide groups. Third step of the reaction is a ‘click
reaction’ where the cyclic triazole ring gets formed via a cycloaddition reaction between
azide group of 1b-3b with the terminal alkyne group of N-Boc-propargyl glycine in presence
of in-situ generated Cu(I) catalyst. Subsequent steps involved deprotection of Boc with TFA,
followed by benzyl group removal with 10% Pd-C catalytic hydrogenation. IR, NMR and
ESI-MS spectroscopic analyses confirmed intermediates and final product formation during
different steps of the reaction.
The radiolabeling procedure (Scheme 2) involved prior generation of intermediate synthon
99m
+
[
Tc(CO) (H2O) ] through a lyophilized kit containing CO donor, potassium
3 3
boranocarbonate (K [H B(CO) ]). The formation yield of intermediate synthon as
2
3
2
99m
characterized using TLC and HPLC was >95%. Subsequently labeled
Tc fatty acid
complexes (1f-3f) were obtained on reaction of intermediate synthon with respective fatty
acid conjugates (1e-3e). HPLC characterization of the three complexes 1f-3f showed
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retention times of 21, 23.5 and 28 min respectively [Figure 1] whereas the intermediate
synthon eluted out as broad peak at 15 min. Radiolabeling yield calculated from the
percentage peak area under the observed complex peak in HPLC spectrum was observed to
be >80% for all three complexes. The increase in HPLC retention times of the respective fatty
acid complexes was consistent with the increasing chain length (lipophilicity). The latter
9
9m
factor was established on ascertaining the Log Po/w of the HPLC purified
Tc labeled fatty
acid complexes (1f-3f) between n-octanol and water (Table 1).The complexes exhibited
serum stability for complete period of study upto 30 minutes.
Table 2 summarizes the results of biodistribution studies carried in normal swiss mice.
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9m
Figure 2 gives a comparison between the myocardial uptake values of the three Tc labeled
1
25
complexes 1f-3f and the reference compound
I-IPPA. All the complexes showed an
increased uptake in myocardium at 2 min post injection (p.i.) with 1f showing the highest
1
25
uptake of 8.70 ±1.41 %ID/g, which was close to uptake shown by I-IPPA [9.51 ± 1.61
%
%
ID/g]. This high initial uptake gradually decreased with time but significant activity (~1
ID/g) was found retained even at 30 min p.i. for all the complexes. The absolute retention
1
25
values observed were much below than that for I-IPPA [7.10 ± 1.79 %ID/g] at 30 min p.i.
Varying the fatty acid carbon backbone from 8 to 15 carbons resulted in no significant
improvement in the myocardial uptake and washout kinetics.
Heart/blood ratio for the complex 3f was initially superior (0.8 vs. 0.3) to the other two
complexes up to 10 min p.i., which decreased to identical ratio (~ 0.4) as observed for 1f and
2
f at 30 min p.i. Significant activity was found associated with the liver owing to the
lipophilic nature of the complexes but rapid movement of activity from liver to GI was
observed for all three complexes with time. Similar washout behavior of the complexes was
observed from other non-target organs viz. blood and lungs, as well. This was a definite
1
25
13
improvement over I-IPPA which had 30.40±6.82 %ID/g and 11.25±1.18 %ID/g activity
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125
associated with liver and lung respectively, at 30 min p.i. Unlike I-IPPA, all the three
complexes showed high activity accumulation in kidneys, followed by rapid decline
indicating renal pathway to be the competing excretion route. The excretion percentage of the
9
9m
Tc complexes via renal decreased with increase in fatty acid chain length (lipophilicity).
Discussion
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9m
There has been prolonged search for a
Tc labeled fatty acid complex that would possess
the advantages of high myocardial uptake, optimum retention and rapid clearance of activity
from blood pool and surrounding non-target organs like liver and lung. Efforts have been put
forward in this direction and several fatty acid analogs have been reported. In all such cases,
as the metal chelate is introduced into the fatty acid chain there is significant alteration in the
properties of the fatty acid molecule, particularly its molar volume and
lipophilicity/hydrophilicity as compared to the parent molecule, which in turn plays an
9
9m
important role in governing the in vivo properties. Hence, a
Tc core with small size but
high stability and lipophilicity is required to counter the effects of introduction of the
99m
+
radiometal. These attributes can be found in the [ Tc(CO) ] core and work with fatty acid
3
analogs having different tridentate donor moiety have been reported before using this core.
9
9m
99m
Also complexes based on [ Tc(CO) -cyclopentadienyl] core, such as
Tc-CpTT-
3
9
9m
18-20
Pentadecanoic acid and
Tc-CpTT-16-oxo-Hexadecanoic acid,
could be found in the
literature but no such literature where the fatty acid chain has been modified through Huisgen
cycloaddition to introduce a tridentate donor set for labeling with the said core is available to
the best of our knowledge.
In the present work, by making use of this reaction, ω-terminal of three fatty acids having 8,
1
1 and 15 carbons respectively has been modified to introduce a tridentate moiety consisting
of an amine nitrogen atom, a carboxylic oxygen atom and the nitrogen atom of the triazole
9
9m
ring to facilitate
Tc labeling through the carbonyl core. Fatty acids with varying chain
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lengths from 8-15 were selected to screen the effect of varying lipophilicity on myocardial
uptake and its in vivo pharmacokinetic behavior. The present ‘click approach’ introduces a
heterocyclic triazole which may mimic the phenyl group present in IPPA. All the ligands
were synthesized with moderate yields and characterized by spectroscopic techniques
confirming their structure. Radiolabelling was carried out following the method described in
the experimental section and the radiolabeled complexes were purified from the excess ligand
mass by HPLC before in vivo studies. The complexes synthesized in the present study are
lipophilic in nature and the longer chain fatty acid complexes of 11 and 15 carbon elute out
broadly in the HPLC spectrum. This may be due to improper elution of the complexes from
the C18 column in the present choice of mobile phase used for characterization. The click
labeling approach do not add radiochemical impurities, as the starting raw materials for
triazole formation viz. fatty acid azide and propargyl glycine, do not independently complex
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9m
with Tc(CO) core and the complexation is feasible only with clicked adducts. The scheme
3
1
followed here for the synthesis of clicked adducts (1e-3e) is via de-protection of clicked
benzyl protected adducts (1d-3d), hence there can be a possibility of benzyl protected
derivative participating in the complexation. But the NMR spectral data confirmed the
absence of such derivatives (2d and 3d) in the final ligands 2e and 3e respectively. However,
to confirm the above proposition a representative complexation of 1d with intermediate
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9m
Tc(CO) core was carried and the complex eluted beyond 30 min (data not shown)
3
indicating significant difference in the HPLC retention times between benzyl protected and
de-protected clicked derivatives. The radiochemical purity observed for the complexes is >
8
0% which is not sufficient for new radiotracer preparation. The lower labeling efficiency
observed may again be due to lipophilic nature of ligands which has affected the solubility of
ligands in water:methanol mixture and hence the final complexation yield. The authors in the
present study have not attempted to optimize the labeling conditions so as to improve the
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labeling efficiency as the focus of the present study was to ascertain the biological efficacy of
1
25
synthesized clicked complexes for myocardial imaging in comparison to I-IPPA. Hence
complexes have been HPLC purified before conducting the biological experiments.
All the complexes exhibited stability in human serum which ensured kinetic inertness of the
99m
complexes in vivo. Biodistribution studies of
Tc fatty acid complexes (1f-3f) in normal
swiss mice showed prominent heart uptake and significant retention up to 30 min p.i. which
is important from SPECT imaging perspective. Another important aspect in myocardial
imaging is the clearance of activity from the blood pool and the surrounding non-target
organs like liver and lungs to obtain high contrast images. Although activity declined rapidly
for this design from non-target organs but heart/blood ratios for all the three complexes were
significantly lower compared to the standard agent. Favorable feature observed for this
‘click’ approach of labeled complexes was their excretion via both hepatobiliary and renal
pathways. Initial uptake in liver was observed to clear rapidly with time. These factors
highlight the significance of the present design, where early improved heart/liver ratios are
expected favoring increased image resolution during metabolic cardiac imaging.
9
9m
Several approaches for labeling long chain fatty acids with Tc have been explored in past.
The results obtained in the present work cannot be directly compared with many of reported
data due to difference in species such as rats utilized for biodistribution experiments. Our
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9m
13,21
group has evaluated before Tc fatty acid utilizing two different approaches.
All three
designs including the present evaluation exhibited high serum stability but suffered from
2
1
reduced myocardial activity retention, albeit HYNIC complex 2e
which showed
moderately high retention values up to 30 min p.i. Among the three labeling approaches, the
1
25
present click design revealed highest myocardial uptake at initial time points close to I-
IPPA. Unlike pharmacokinetics of previous two designs where complexes cleared mainly via
the hepatic route, the clicked complexes excreted via both hepatic and renal pathways.
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Further, the movement of residual activity from liver towards intestines was rapid in
comparison to previous approaches thereby favoring improved resolution for metabolic
cardiac imaging.
Comparing the present in vivo distribution results with most promising [99mTc]MAMA-
1
1
HDA, carrying N S framework and evaluated in the same specie, the present evaluated
2
2
complexes showed improved initial myocardial uptake and heart/liver ratios. Heart/blood
ratio for clicked complex 3f was observed to be similar to [99mTc]MAMA-HDA (~ 0.82) at
5
min p.i. Thus the present design shows reasonable myocardial uptake and favorable in vivo
pharmacokinetics, which merits high potential for metabolic cardiac imaging.
Conclusion
Three fatty acids with 8, 11 and 15 carbon chains were modified at the terminal carbon
through Huisgen 1,3-dipolar cycloaddition reaction to give clicked adducts in high purity and
moderate yields. Radiolabelling of clicked fatty acid ligands with technetium tricarbonyl core
yielded complexes with purity > 80%. Biodistribution studies of these complexes in normal
Swiss mice revealed affinity towards myocardium with significant retention up to 30 min p.i.
The complexes demonstrated improved in vivo pharmacokinetics with rapid washout of
activity from non-target organs via hepatic and renal pathways. All these data provide
conclusive evidence in support of the promising nature of this chemistry but lower retention
125
of tracers in the myocardium in comparison to I-IPPA limit the potential of these tracers as
myocardial metabolic markers.
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Scheme1 Scheme for the synthesis of fatty acid triazole conjugates
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Scheme 2 Radiolabeling of fatty acid triazole conjugates with technetium tricarbonyl core
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9m
Figure 1 HPLC profile of (a) Tc(CO) intermediate synthon (b) Complex 1f (c) Complex
3
2
f and (d) Complex 3f
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9
9m
Figure 2 Myocardial uptake and washout kinetics profile of
complexes (1f-3f) in Swiss mice
Tc(CO) - fatty acid
3
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9
9m
Table1 Partition coefficient (Log P ) of Tc labeled fatty acid complexes 1f-3f
o/w
Complex
Log Po/w
0.10
0.38
1
2
3
f
f
f
0.89
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9
9m
Table 2: Biodistribution studies of Tc fatty acid complexes 1f-3f (values expressed in %ID/g) in
Swiss mice
Complex 1f (n=3)
2
min
5 min
10 min
30 min
Liver
Int + GB
Stomach
Kidney
47.06 (5.59)
3.94 (0.60)
4.92 (1.07)
75.24 (8.41)
8.70 (1.41)
17.22 (3.10)
3.52 (0.58)
23.62 (1.40)
0.36 (0.07)
0.17 (0.02)
0.54 (0.22)
36.20 (3.80)
4.45 (1.95)
5.75 (2.46)
77.22 (13.45)
4.79 (0.59)
9.01 (1.32)
1.98 (0.14)
14.06 (1.27)
0.35 (0.11)
0.13 (0.02)
0.55 (0.12)
20.74 (5.71)
9.58 (2.39)
1.80 (0.43)
39.62 (5.89)
1.87 (0.29)
3.64 (0.58)
0.86 (0.11)
5.65 (0.19)
0.33 (0.4)
13.08 (1.35)
15.34 (0.33)
2.86 (0.14)
19.03 (1.81)
1.21 (0.25)
2.31 (0.90)
0.44 (0.05)
3.18 (0.50)
0.40 (0.18)
0.09 (0.03)
0.60 (0.29)
Heart
Lungs
Muscle
Blood
Heart/Blood
Heart/Liver
Heart/Lung
0.09 (0.01)
0.52 (0.03)
Complex 2f (n=3)
Liver
Int + GB
Stomach
Kidney
46.02 (14.52)
1.94 (1.22)
2.24 (0.93)
18.62 (8.23)
5.82 (1.20)
10.26 (2.39)
0.89 (0.25)
21.41 (5.60)
0.28 (0.06)
0.13 (0.03)
0.58 (0.11)
48.60 (13.86)
2.43 (1.30)
5.39 (2.77)
47.46 (22.60)
3.28 (0.56)
5.82 (2.38)
1.18 (0.35)
10.44 (4.60)
0.35(0.14)
36.95 (10.07)
4.53 (1.39)
6.00 (2.51)
55.27 (15.97)
3.10 (0.31)
5.30 (1.72)
1.40 (0.21)
6.83 (1.35)
0.43 (0.11)
0.08 (0.03)
0.57 (0.15)
12.03 (2.54)
8.77 (2.56)
2.53 (0.24)
17.19 (5.74)
1.00 (0.31)
1.55 (0.49)
0.61 (0.33)
2.38 (0.69)
0.42 (0.01)
0.08 (0.01)
0.65 (0.13)
Heart
Lungs
Muscle
Blood
Heart/Blood
Heart/Liver
Heart/Lung
0.07 (0.02)
0.61(0.19)
Complex 3f (n=3)
Liver
Int + GB
Stomach
Kidney
31.88 (3.94)
0.98 (0.18)
1.51 (0.49)
7.86 (1.62)
5.23 (2.13)
8.41 (1.90)
1.07 (0.80)
19.61 (0.85)
0.28 (0.06)
0.21 (0.11)
0.58 (0.11)
40.10 (10.68)
1.89 (1.09)
1.44 (0.30)
14.17 (8.54)
5.94 (1.04)
6.82 (1.41)
1.19 (0.15)
6.20 (3.55)
0.82 (0.11)
0.14 (0.05)
0.94 (0.55)
21.17(3.07)
5.31 (0.27)
2.35 (0.66)
22.01 (5.86)
3.41 (0.70)
3.24 (0.71)
1.69 (0.68)
4.51 (0.88)
0.76 (0.20)
0.20 (0.05)
1.11 (0.49)
5.29 (0.23)
6.08 (1.17)
2.69 (1.04)
6.73 (0.72)
0.93 (0.13)
1.68 (0.30)
1.24 (0.48)
2.10 (0.2)
Heart
Lungs
Muscle
Blood
Heart/Blood
Heart/Liver
Heart/Lung
0.44 (0.15)
0.18 (0.06)
0.55 (0.29)
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