68Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors

Article abstract of DOI:10.1016/j.bmcl.2017.08.065

Molecular imaging using radiolabeled Tyrosine Kinase Inhibitors (TKI) is a promising strategy for detection and staging of EGFR-positive cancers. A novel analogue of one such TKI, Erlotinib has been developed for PET imaging by derivatizing the parent Erl

Full text of DOI:10.1016/j.bmcl.2017.08.065

Accepted Manuscript
6
8Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing
tumors
Akanksha Jain, Mythili Kameswaran, Usha Pandey, Kumar Prabhash, Haladhar
Dev Sarma, Ashutosh Dash
PII:
S0960-894X(17)30857-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.065
BMCL 25259
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 June 2017
7 August 2017
25 August 2017
6
8
Please cite this article as: Jain, A., Kameswaran, M., Pandey, U., Prabhash, K., Sarma, H.D., Dash, A., Ga labeled
Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.065
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1
6
8
Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors
a,d
Akanksha Jain , Mythili Kameswaran , Usha Pandey , Kumar Prabhash ,
a*
a,d
b
c
Haladhar Dev Sarma , Ashutosh Dash
a,d
a
Radiopharmaceuticals Division
Radiation Biology & Health Sciences Division,
c
Bhabha Atomic Research Centre (BARC), Trombay, Mumbai 400 085, INDIA.
b
Tata Memorial Hospital, Parel, Mumbai 400 012, INDIA
Homi Bhabha National Institute, Anushaktinagar, Mumbai 400 094, INDIA.
d
Author for correspondence: Dr. Mythili Kameswaran
Telephone: 91-22-25593740
Fax: 91-22-25519613
E-mail: kmythili@barc.gov.in
6
Keywords: Erlotinib, tyrosine kinase inhibitors, EGFR, Ga, PET imaging, p-NCS-Bn-
8
68
NOTA, Ga-NOTA-Erlotinib
1

2
Abstract
Molecular imaging using radiolabeled Tyrosine Kinase Inhibitors (TKI) is a promising
strategy for detection and staging of EGFR-positive cancers. A novel analogue of one such
TKI, Erlotinib has been developed for PET imaging by derivatizing the parent Erlotinib
molecule for conjugation with the bifunctional chelator p-SCN-Bn-NOTA towards
6
radiolabeling with Ga. NOTA-Erlotinib conjugate was synthesized and characterized by
8
6
NMR and ESI-MS techniques. The conjugate was radiolabeled with Ga in 95±2 % yield,
8
6
as evidenced by HPLC characterization. The log P value of Ga-NOTA-Erlotinib was –
8
6
0.6±0.1). The Ga-NOTA-Erlotinib conjugate was characterized using its Ga-NOTA-
8
nat
(
Erlotinib surrogate. Cell viability studies showed that the NOTA-Erlotinib conjugate retained
6
8
the biological efficacy of the parent Erlotinib molecule. Further, Ga-NOTA-Erlotinib
exhibited an uptake of 9.8±0.4% in A431 cells which was inhibited by 55.1±0.2% on addition
of cold Erlotinib (10 µg) confirming the specificity of the radioconjugate for EGFR
6
8
expressing cells. In the biodistribution studies carried out in tumor bearing SCID mice, Ga-
NOTA-Erlotinib conjugate showed moderate tumor accumulation (1.5±0.1 % ID/g at 30 min
p.i.; 0.7 ±0.2 % ID/g at 1 h p.i.). Hepatobiliary clearance of the radioconjugate was observed.
6
The Ga-NOTA-Erlotinib conjugate was found to have high in vivo stability as determined
8
by the metabolite analysis study using urine sample of the Swiss mice injected with the
6
8
preparation. The overall properties of Ga-NOTA-Erlotinib are promising and merit further
6
exploration. To the best of our knowledge, this is the first report on the design of a Ga
8
labeled Erlotinib for PET imaging of EGFR and opens avenues for the successful
6
development of Ga labeled TKI for imaging of EGFR over-expressing tumors.
8
2

3
In the recent years, small molecule Tyrosine Kinase Inhibitors (TKI) have emerged as
attractive tools for targeting receptors such as Epidermal Growth Factor Receptors (EGFR),
Vascular Endothelial Growth Factor Receptors (VEGFR), Platelet Derived Growth Factor
Receptors (PDGFR) etc. which are involved in the growth, proliferation, differentiation and
1
-3
metastasis of tumor cells.
The small molecule TKIs bind to the intracellular tyrosine
kinase domain of the receptors thus inhibiting the phosphorylation of TK proteins and further
disrupting the signal transduction pathways in cancer cells. In the past decade, more than 10
small molecule TKIs have been approved by the Food and Drug Administration (FDA), USA
4,5
and many TKIs are under development and evaluation. One of the earliest and the most
®
potent TKI is the drug Erlotinib (Tarceva , OSI Pharmaceuticals) which is approved for
treatment of EGFR over-expressing cancers, mainly non small cell lung cancer and
6
,7
pancreatic cancer. Although Erlotinib has shown success in increasing the survival rate of
cancer patients in the clinical trials, only 10-20 % of the patients benefit from it as the
patients acquire resistance to the drug upon continuous treatment, due to mutation of the
8
target receptors.
Molecular imaging using radiolabeled small molecule TKIs such as Erlotinib can be
beneficial in the evaluation of EGFR mutational status before and after treatment to
8
-11
determine the best possible treatment strategy for cancer patients.
There are numerous
literature reports on the development of radiolabeled small molecule TKIs for early detection
and staging of cancers using SPECT (Single Photon Emission Computed Tomography) and
1
2-20
PET (Positron Emission Tomography) techniques.
As compared to SPECT, the higher
sensitivity and superior resolution of PET renders it more beneficial in the visualization and
9,21 11
quantification of the in vivo distribution of TKIs.
C-Erlotinib is one such agent which has
shown promising results in pre-clinical studies for in vivo tumor targeting. In the clinical
3

4
1
studies involving cancer patients, C-Erlotinib has exhibited its utility in patient selection.
1
22-
2
4
11
However, the short half life of C (t1/2 = 20.33 min) and its cyclotron-dependant
1
availability pose severe impediments in the clinical use of C radiotracers as they can only
1
be used at medical centres with an in-house/nearby cyclotron facility. Besides, radiolabeling
1
1
procedures with C are complex and time consuming with low radiochemical yields
1
rendering the production of C radiotracers very expensive. Gallium-68, another PET
1
1
radioisotope, is an attractive alternative to C owing to its cyclotron-independent availability
1
6
from a Ge/ Ga generator. Gallium-68 also has a well developed coordination chemistry
8
68
which allows fast and reproducible radiolabeling in clinical settings. Further, its nuclear
+
25,26
characteristics (t1/2 = 67.71 min; 89% β decay) are suitable for PET imaging.
Thus, in the
6
8
present study, we report the design, synthesis and biological evaluation of a novel Ga
labeled Erlotinib analogue towards PET imaging of EGFR over-expressing cancers.
Erlotinib molecule was suitably functionalized at its terminal alkyne position and
conjugated with the bifunctional chelator p-SCN-Bn-NOTA (S-2-(4-isothiocyanatobenzyl)-
6
,4,7-triazacyclononane-1,4,7-triacetic acid) for stable complexation with Ga. The
8
1
macrocyclic chelator NOTA was chosen for the present study as it is known to complex with
Ga(III) with high thermodynamic stability (log KGa-NOTA=31.0) along with high plasma and in
2
5
vivo stability.
After synthesis of the NOTA-Erlotinib conjugate, it was characterized and then
6
radiolabeled with Ga. The radiolabeled Erlotinib conjugate was characterized using its
8
nat
Ga-NOTA-Erlotinib surrogate. Biological efficacy of NOTA-Erlotinib was determined in
6
A431 cells. Further, in order to assess the specificity of Ga-NOTA-Erlotinib for EGFR, in
8
vitro cell uptake studies were carried in A431 cells (over-expression of EGFR) and A549
2
cells (ten fold lower EGFR expression compared to A431cells). The in vivo biodistribution
7
4

5
and stability analysis was carried out in NOD/Severe Combined Immuno Deficiency (SCID)
mice bearing EGFR positive tumors and Swiss mice, respectively.
The synthesis scheme for preparation of NOTA-Erlotinib conjugate is given in Figure
28
. The terminal alkyne group of Erlotinib was reacted with the azide group of Compound 1
1
using copper(I) catalysed azide-alkyne cycloaddition (CuAAC) ‘click’ reaction to give
Compound 2. The rationale behind the modification of the terminal alkyne group of Erlotinib
for the conjugation with the chelator originated from some literature reports wherein a similar
2
9-31
modification of Erlotinib was carried out.
In one such report by Zhang et al., Zn(II)-
phthalocyanine-Erlotinib conjugates were prepared for photodynamic therapy and the
conjugates reportedly exhibited high uptake in HepG2 and A431 tumor cells, both of which
over-express EGFR, showing that the synthetic modification of terminal alkyne group did not
2
affect the biological activity of the parent compound. Further, various patents which discuss
9
the derivatization of the alkyne group of Erlotinib have reported high uptake of such
3
0,31
conjugates in A431 cells.
Figure 1. Synthesis scheme for preparation of NOTA-Erlotinib conjugate
5

6
The formation of Compound 2 by CuAAC reaction was ascertained using TLC where
a new UV active spot was observed at R
f f
0.3 along with that of Erlotinib at R 0.4 in 5%
MeOH/CHCl solvent system. In addition, a shift in the parent Erlotinib peak was observed in
3
3
the analytical HPLC from 19.0±0.1 min to 20.1±0.2 min. Compound 2 was obtained in ~ 90
2
1
yield. A singlet at 8.12 corresponding to the proton of triazole ring in H-NMR (500 MHz
%
Varian spectrophotometer) and a peak at 139.5 corresponding to quaternary C of triazole ring
1
3
in C-NMR (500 MHz Varian spectrophotometer) confirmed the structure of Compound 2.
The ESI-MS (Varian Pro Star mass spectrometer) data further corroborated its structure. In
the next step, the BOC protected amine group of Compound 2 was de-protected in 4 N HCl.
A new peak at 15±0.2 min in analytical HPLC along with the ESI-MS data confirmed the de-
protection and formation of Compound 3. In the final step, the amine group of Compound 3
was reacted with the isothiocyanate group of p-SCN-Bn-NOTA under mild basic conditions.
A new peak observed in the analytical HPLC at 18±0.2 min was then purified (~95 % purity)
3
by semi-preparative HPLC and its structure (NOTA-Erlotinib) confirmed by H-NMR and
2
1
ESI-MS.
6
NOTA-Erlotinib (Compound 4) was then radiolabeled with Ga following the
8
3
scheme given in Figure 2. Radiolabeling yield of 95 ± 2 % was obtained as ascertained by
3
6
8
68
3
HPLC (Figure. 3a). It was observed that free GaCl eluted at 4±0.2 min while Ga-NOTA-
Erlotinib eluted at 17.3±0.1 min (n=3). The optimum amount of NOTA-Erlotinib ligand
required for ≥ 95% radiolabeling yield was determined to be 50-70 µg and the specific
6
activity of Ga-NOTA-Erlotinib was 3.2±0.8 GBq/µmol of ligand. No further purification of
8
6
the Ga-NOTA-Erlotinib conjugate was carried out prior to the bioevaluation experiments.
8
6

7
6
Figure 2. Radiolabeling of NOTA-Erlotinib conjugate with Ga
8
6
Figure 3. HPLC chromatogram of (a) Ga-NOTA-Erlotinib (b) Ga-NOTA-Erlotinib
8
nat
6
8
In order to chemically characterize the Ga-NOTA-Erlotinib conjugate, its Ga-
nat
3
4
NOTA-Erlotinib surrogate was synthesized at macroscopic levels.
In the HPLC
nat
chromatogram of Ga-NOTA-Erlotinib (Figure 3b), two peaks were observed at 3.8±0.1 min
and 17.05±0.1 min (n=3) respectively. The peak at 3.8 min corresponds to free Ga(III), as
excess of gallium nitrate was taken initially to ensure completion of the reaction. The peak at
1
7.05 min was collected and was evaluated using ESI-MS. Its MS data corresponded to the
7

8
nat
68
Ga-NOTA-Erlotinib conjugate further substantiating the successful synthesis of Ga-
NOTA-Erlotinib conjugate according to Figure 2.
6
The log Po/w value of Ga-NOTA-Erlotinib conjugate was determined to be –
8
3
0.6±0.1). It is known from literature that Erlotinib is a highly lipophilic molecule with poor
5
(
solubility in aqueous media. Conjugation of Erlotinib with NOTA with the aim to radiolabel
6
with Ga concurrently resulted in decrease in the lipophilicity of the radioconjugate, evident
8
from its log P value and also improved its solubility in aqueous medium.
6
The kinetic stability of Ga-NOTA-Erlotinib was ascertained by the in vitro stability
8
experiments by incubating 50 µL of the conjugate in 10 mM EDTA solution (450 µL) for 2 h
3
5 68
and in human serum (450 µL) for 2 h at 37°C.
Ga-NOTA-Erlotinib exhibited high
stability (≥ 95%) even after 2 h incubation in EDTA as well as in human serum. High
stability is a primary requirement of a radiopharmaceutical as it ensures no abnormal organ
distribution of activity. In order to ascertain the TKI properties of NOTA-Erlotinib, cell
36
viability studies were performed in A431 cells and the results are given in Table 1. These
studies confirmed that the NOTA-Erlotinib conjugate retained the TKI properties of the
parent Erlotinib molecule as a decrease in % viability of cells was observed after 72 h
incubation with the conjugate.
Table1: Cell viability studies of NOTA-Erlotinib in A431 cells
%
Cell viability
Time post treatment
3 h
24 h 48 h
72 h
1
µM NOTA-Erlotinib
40.0±0.2
37.5±0.1
28.5±0.4 12.5±0.3
6
The specificity of the Ga-NOTA-Erlotinib to EGFR was determined by in vitro cell
8
3
uptake studies using A431 and A549 cells and the results are given in Figure 4. A431 cells
7
8

9
6
8
showed an uptake of 9.8±0.4 % with Ga-NOTA-Erlotinib. Inhibition up to 55.1±0.2 % was
observed using 10 µg of unlabeled Erlotinib. Higher amounts of Erlotinib could not be used
for the study as the DMSO used for solubilising the Erlotinib was probably damaging the
cells and hindered its action. On the other hand, a good inhibition was observed even when
1
00 µg of unlabeled NOTA-Erlotinib was used for inhibition studies indicating the specificity
6
of Ga-NOTA-Erlotinib to EGFR as represented in Figure 4. However, Ga-NOTA-
8
68
Erlotinib did not show any uptake with A549 cells indicating the resistance of the cell line to
3
8
Erlotinib.
6
Figure 4. In vitro cell uptake and inhibition studies of Ga-NOTA-Erlotinib in A431 and
8
A549 cells
6
The biodistribution pattern of Ga-NOTA-Erlotinib was studied in NOD/SCID mice
8
3
9
bearing EGFR positive tumor at 30 min and 1 h post injection (p.i.) in groups of four each.
9

1
0
6
8
Results of the biodistribution studies are given in Table 2. Uptake of Ga-NOTA-Erlotinib in
the tumor was 1.5±0.1 % ID/g at 30 min p.i. which decreased at 1 h (0.7±0.2 % ID/g) p.i. The
tumor/lungs ratios were moderately high at both 30 min and 1 h p.i. Due to the decrease in
6
lipophilicity of NOTA-Erlotinib, the accumulation of activity of Ga-NOTA-Erlotinib
8
observed in liver at 1 h p.i. (6.0±0.7 % ID/g) was much lower in comparison to that reported
1
for [ C]-Erlotinib (10.5 - 14.4 % ID/g at 1 h p.i.). However, it was still observed that the
1
23
6
major excretion of the Ga-NOTA-Erlotinib conjugate was via the hepatobiliary pathway as
8
indicated by the high accumulation of activity in the GI tract (12.3±3.9 % ID/g 30 min;
1
4.3±3.4 % ID/g at 1 h).
6
Table 2: Biodistribution pattern of Ga-NOTA-Erlotinib in NOD/SCID mice bearing
8
EGFR expressing tumor (n=4)
%
Injected Dose/g ± S.D.
Organs
30 min
2.4 ± 0.2
1 h
1.2 ± 0.2
Blood
Lungs
Heart
1.5 ± 0.20
0.4 ± 0.2
4.4 ± 2.2
12.3 ± 3.9
10.2 ± 0.6
1.1 ± 0.4
4.1 ± 0.7
3.3 ± 1.3
Nil
0.8 ± 0.1
0.1 ± 0.0
3.4 ± 2.6
14.3 ± 3.4
6.0 ± 0.7
0.1 ± 0.01
3.3 ± 0.6
2.4 ± 0.6
Nil
Stomach
Intestine
Liver
Spleen
Kidney
Muscle
Bone
Excreta
(% I.D.)
Tumor
6.7 ± 4.6
17.6 ± 4.7
1.5 ± 0.1
0.7 ± 0.2
Tumor/Lungs
(
1.1 ± 0.2
0.9 ± 0.2
Ratio)
1
0

1
1
6
In order to ascertain the in vivo stability of the Ga-NOTA-Erlotinib, the urine sample of the
8
4
0
Swiss mice injected with the radioconjugate was analysed using HPLC.
The
68
radiochromatogram is given in Figure 5 which shows a single peak corresponding to Ga-
6
NOTA-Erlotinib at 17.3±0.1 min thereby indicating the stability of Ga-NOTA-Erlotinib in
8
vivo.
6
Figure 5. HPLC chromatogram of the urine sample from Swiss mice injected with Ga-
8
NOTA-Erlotinib
6
8
In this study, a novel small molecule EGFR imaging probe, Ga-NOTA-Erlotinib
was designed, synthesized and characterized. The biological evaluation of NOTA-Erlotinib
suggests that the attachment of the chelator to the terminal alkyne group of Erlotinib
preserves the TKI property of the parent molecule. Although the biodistribution studies show
6
moderate tumor uptake, the overall properties of the Ga radioconjugate are promising.
8
Further work by altering the chelators attached to the parent Erlotinib molecule might be
6
8
helpful in modulation of the pharmacokinetics of the resultant Ga-Erlotinib conjugate so
that it exhibits high tumor uptake and low non-target uptake like in liver and intestine.
Acknowledgements
Research at BARC is part of the ongoing activities of the Department of Atomic
Energy, India and is fully supported by government funding. The authors are thankful to Dr.
6
8
68
Aruna Korde, RPhD for providing access to Ge/ Ga generator. Thanks are also due to Dr.
B.S. Tomar, Director, Radiochemistry & Isotope Group, BARC for his support and
encouragement.
1
1

1
2
Conflict of Interest
The authors declare no conflict of interests
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8. Synthesis of tert-butyl-3-azidopropylcarbamate (1):
tert-butyl-3-bromopropylcarbamate (100 mg, 0.42 mmoles) and sodium azide (70 mg,
0.84 mmoles) were dissolved in DMF (1 mL). The reaction mixture was refluxed
overnight at 75°C with stirring. Subsequently, the solvent was evaporated and the
reaction mixture was diluted with chloroform (50 mL) and washed with water (3*50
mL). Organic layer was collected and solvent was evaporated, affording the product as
1 13
pale yellow oil (Compound 1). The product was characterized by IR, H-NMR, C-
NMR and ESI-MS.Yield: 70 mg (70%)
-
1
3
IR (νmax, cm ): 3455m (N-H), 3018s (C-H), 2099s (N ), 1707s (C=O), 1508s, 1367m,
1216s, 1169s, 757m, 667m.
1
H-NMR (500 MHz, CDCl
m, 2H, -CH CH NH-), 1.78-1.76 (m, 2H, -CH
C(CH ).
C-NMR (125 MHz, CDCl
9.1 (N CH CH -), 38.0 (-CH
3
) δ ppm: 3.37-3.35 (t, 2H, N
3
CH
2 2
CH -, J=6.7 Hz), 3.23-3.20
(
2
2
2
CH CH
2
2
-, J=6.7 Hz), 1.44 (s, 9H, -
3 3
)
1
3
3
) δ ppm: 155.9 (-C(O)OC(CH
CH NH-), 29.6 (-CH CH CH
] 200.24; m/z (observed)223
3
)
3
), 79.4 (-C(O)OC(CH
3 3
) ),
4
3
2
2
2
2
2
2
2
-), 28.3 (-(CH ).
3 3
)
8 16 4 2
MS (ESI, +ve mode): Mass (calculated) [C H N O
+
M+Na );
(
Synthesis
of
tert-butyl-3-(4-(3-(6,7-bis(2-methoxyethoxy)quinazolin-4-
ylamino)phenyl)-1H-1,2,3-triazol-1-yl)propylcarbamate (2)
1
5

1
6
To a solution of Erlotinib (50 mg, 0.13 mmoles) and Compound 1 (25 mg, 0.13 mmoles)
in a 1:1 mixture of water and tert-BuOH (6 mL), sodium ascorbate (12 mg, 0.06 mmoles)
and copper sulphate pentahydrate (3 mg, 0.01 mmoles) were added. The reaction mixture
was stirred at 60°C for 4-5 h under nitrogen atmosphere. Progress of the reaction was
3
monitored by TLC (5% MeOH: CHCl ). After completion of the reaction, solvent was
evaporated and the reaction mixture was diluted with chloroform (50 mL) and washed
with water (3*50 mL). Organic layer was collected and purified by column
chromatography affording a yellow solid: R
f
=0.3 (in 5% MeOH in CHCl
3
). The purified
1 13
product was characterized by IR, H-NMR, C-NMR and ESI-MS. Yield: 68 mg (90%)
-1
IR (νmax, cm ): 3403m (N-H), 2928s (C-H), 1692s (C=O amide), 1623s, 1582s, 1511m,
1449m, 1367s, 1244s, 1166s, 1126s, 930s, 865s
1
H-NMR (500 MHz, CDCl ) δ ppm: 8.64 (s,1H, quinazoline-NC(H)NC-), 8.12 (s, 1H,
3
triazole-CH), 7.92-7.90 (m, 3H, quinazoline-CC(H)C(N)C-, -CC(H)C(C)C-, Aryl-
C(H)C(H)C(H)CC(H)-), 7.54-7.53 (d, 1H, Aryl-C(H)C(H)C(H)CC(H)-), 7.46-7.43 (t,
1
H, Aryl-C(H)C(H)C(H)CC(H)-), 7.37 (s, 1H, Aryl-C(H)C(H)C(H)CC(H)-), 4.49-4.46
t, 4H, CH OCH CH O-), 4.32-4.29 (m, 6H, CH OCH CH O-, NCH CH CH NH-), 3.49
s, 6H, CH OCH CH O-), 3.2-3.19 (m, 2H, NCH CH CH NH-), 2.16-2.13 (m, 2H,
CH CH NH-), 1.28 (s, 9H, -C(CH
C-NMR (125 MHz, CDCl ) δ ppm: 156.4 (quinazoline–N(H)C(=N)C-), 156.2(-
C(O)OC(CH ), 154.3, 153.2, 148.8, 147.4, 146.8 (quinazoline-C -), 139.3
triazole quarternary C-), 131.0, 129.4, 121.5, 121.1, 120.3 (Aryl C -), 118.7 (triazole
CH), 79.6 (-C(CH ), 70.8,70.3, 68.9, 68.18 (-OCH CH O-), 59.2 (-OCH ), 47.61 (-
NCH CH CH NH-), 37.3 (-NCH CH CH NH-), 30.6 (-NCH CH CH NH-), 28.3 (-
CH ) .
(
(
3
2
2
3
2
2
2
2
2
3
2
2
2
2
2
NCH
2
2
2
3 3
) )
1
3
3
3
)
3
8 2 3
N H
(
6 4
H
–
3
)
3
2
2
3
2
2
2
2
2
2
2
2
2
(
3 3
)
1
6

1
7
39 7 6
MS (ESI, +ve mode): Mass (calculated) [C30H N O ] 593.3; m/z (observed) 594
+
M+H ), 616 (M+Na )
+
(
Synthesis of 6,7-bis(2-methoxyethoxy)-N-3-(1-(3-aminopropyl)-1H-1,2,3-triazol-4-
yl)phenyl)quinazoline-4-amine (3)
Compound 2 (68 mg, 0.11 mmoles) was dissolved in chloroform (1 mL) to which 4 N
HCl (1 mL) was added. The reaction mixture was left for overnight stirring. Progress of
the reaction was monitored by analytical HPLC (monitoring the UV peak at 254 nm). On
completion of the reaction, solvent was evaporated and the product was washed twice
with methanol. The dried product was characterized by ESI-MS and used directly in the
next step without further purification. Yield: 56 mg (100%).
31 7 4
MS (ESI, +ve mode): Mass (calculated) [C25H N O ] 493.2; m/z (observed) 494.6
+
M+H ).
(
Synthesis of NOTA-Erlotinib (4) conjugate
To a solution of Compound 3 (10 mg, 0.02 mmoles) and p-SCN-Bn-NOTA (8 mg, 0.018
mmoles) in methanol (1 mL), diisopropylethylamine (10 µL, 57 µmoles) was added. The
reaction mixture was stirred overnight at room temperature. Progress of the reaction was
monitored using analytical HPLC monitoring the UV peak at 254 nm using the gradient
elution method described in the previous paragraph. The product was purified using
1
semi-preparative HPLC and was characterized by H-NMR and ESI-MS. Yield: 16 mg
(
96%)
1
H-NMR (500 MHz, MeOH-d ) δ ppm: 8.71 (s,1H, quinazoline-NCHNC-), 8.52(s, 1H,
4
triazole CH-), 8.26 (s, 1H, quinazoline-CC(H)C(N)C-), 8.11 (s, 1H, quinazoline-
CC(H)C(C)C-), 7.76 (m, 2H, Aryl-C(H)C(H)C(H)CC(H)-,Aryl-C(H)C(H)C(H)CC(H)-),
7.55 (m, 1H, Aryl-C(H)C(H)C(H)CC(H)-), 7.31 (m, 5H, Aryl-C(H)C(H)C(H)CC(H)-,
NOTA Aryl-C -CH ), 4.42 (m, 4H, CH OCH CH O-), 3.91 (m, 6H, CH OCH CH O-,
3
6
H
4
2
3
2
2
2
2
1
7

1
8
NCH
NOTA ring -NCH
NCH CHN-, NOTA C
MS (ESI, +ve mode): Mass (calculated) [C45
2
CH
2
CH
2
NH-), 3.72-3.69 (m, 6H, -OCH
COOH), 2.7-2.68 (m, 15H, NOTA ring -NCH
CH -, -NCH CH CH NH-)
3
), 3.5-3.49 (m, 8H, -NCH
2
CH
2 2
CH NH-,
2
2
CH
2
N- and -
2
6
H
4
2
2
2
2
57 11
H N O10S] 943.4; m/z (observed): 943.4
+
M ), 472.4 (M/2+ H )
+
(
2
9. Zhang FL, Huang Q, Zheng K, Li J, Liu JY, Xue JP. A novel strategy for targeting
photodynamic therapy. Molecular combo of photodynamic agent zinc(II) phthalocyanine
and small molecule target-based anticancer drug erlotinib. Chem Commun. 2013; 49:
9570-9572.
3
3
3
0. Shen C, Yuan M, Wu Q, Zhang P. Erlotinib derivative and preparation method and
application thereof. Patent CN10369427 (A), 2014.
1. Huang S, Wang Q, Zheng X et al. EGFR positron tracer, a preparation method and
application thereof. Patent CN104817542 (A), 2015.
2. HPLC characterization was carried out on a JASCO PU 2080 Plus dual pump HPLC
system (JASCO, Japan) equipped with a JASCO 2075 Plus tunable absorption detector
and Gina Star radiometric detector system (Raytest, Germany), using a C18 reversed
phase HiQSil (5 µm, 4 × 250 mm) column. Water (A) and acetonitrile (B), each
containing 0.1% TFA, were used as the mobile phase at a flow rate of 1 mL/min. The
following gradient elution method was adopted for monitoring the progress of the
reaction at 254 nm: 0 min 90% A, 0-27 min 5% A, 30 min 90% A. Purification of the
synthesized NOTA-Erlotinib derivative was performed using a JASCO-PU-2086 PLUS
Intelligent Prep Pump semi preparative HPLC system (JASCO, Japan) having a Mega
Pak SIL C18-10 column (10 × 250 mm) connected with a JASCO UV-2075 Plus
absorption detector. The same solvent system and method was used in both analytical
HPLC as well as semi-preparative HPLC.
1
8

1
9
6
3. Radiolabeling of NOTA-Erlotinib conjugate (4) with Ga
8
3
6
Towards Ga labeling, NOTA-Erlotinib conjugate (100 µL, 0.5 mM in ethanol) was
8
6
8
mixed with 2 M sodium acetate solution (500 µL, pH ~7.5). GaCl
3
(1 mL, 185 MBq) in
0.6 N HCl was added and the reaction mixture (pH ̴ 4) was incubated for 10 min at
6
8
ambient temperature. Radiochemical yield of Ga-NOTA-Erlotinib conjugate (4a) was
determined by HPLC by gradient elution of water (A) and acetonitrile (B), each
containing 0.1% trifluoroacetic acid as mobile phase at 1 mL/min using the following
gradient elution technique: 0-4 min 95% A, 4-15 min 5% A, 15-20 min 5% A, 20-25 min
6
5% A. The amount of NOTA-Erlotinib conjugate used for Ga labeling was optimized
8
9
so as to obtain ≥ 95% yield.
nat
4. Preparation of Ga-NOTA-Erlotinib conjugate
3
To the NOTA-Erlotinib conjugate (100 µL, 0.1 M in ethanol), excess gallium nitrate
solution (1 mL, 0.1 M) was added and the pH was adjusted to 4 with dilute NaOH. The
reaction mixture was incubated at 40°C for 30 min. The product was characterized by
ESI-MS and HPLC following the UV profile at 254 min using the gradient elution
6
technique as described for Ga-NOTA-Erlotinib.
8
MS (ESI, + ve mode): Mass (calculated) [C45H54GaN11O10S] 1009.3; m/z (observed)
+
042.7 (M+MeOH+H )
1
3
5. Partition coefficient (log Po/w) and stability studies
6
8
For determining log Po/w
,
Ga-NOTA-Erlotinib conjugate (100 µL, 11.1 MBq) was
mixed with water (0.9 mL) and octanol (1 mL) and centrifuged to effect separation of the
two layers. Equal aliquots of both layers were counted in a NaI (Tl) well type counter.
log Po/w was expressed as the logarithm of ratio of counts in n-octanol to that in the
aqueous layer. The n-octanol layer was further repartitioned until consistent value of
partition coefficient was obtained.
1
9

2
0
6
8
In order to assess the extent of transchelation in presence of EDTA, 50 µL of Ga-
NOTA-Erlotinib conjugate (6 MBq) was incubated with 10 mM EDTA solution (450
µL) for 2 h. Thereafter stability was determined by HPLC using the method which was
standardised for determining the radiochemical purity of the complex. Likewise, stability
in human serum was determined by incubating 50 µL of the radioconjugate (6 MBq)
with human serum (450 µL) for 2 h at 37°C. The serum proteins were then precipitated
using acetonitrile (500 µL), the solution was centrifuged and the supernatant was
analyzed by the standardised HPLC method to determine the stability in serum.
6. Cell Viability Assay: Trypan Blue Exclusion Assay
3
A431 cells over-expressing EGFR were grown to confluence in DMEM containing 10%
FBS. After harvesting, cells at a density of 5,000 per well were plated in 12-well plates
and left overnight at 37º C in a humidified CO2 incubator. Two groups of cells in
triplicates were taken for this study. The first group was treated with 1 µmol/L of
NOTA-Erlotinib while second group was the untreated group. At intervals of 3, 24, 48
and 72 h post-incubation with NOTA-Erlotinib, viable cells were counted by trypan blue
exclusion assay wherein the adherent and floating cells were collected at each time point,
centrifuged and re-suspended in medium. Cells were then diluted at 1:1 ratio with 0.4%
trypan blue and checked under a light microscope. Viable (unstained) and nonviable
(
blue-stained) cells were counted and % viable cells after treatment with NOTA-
Erlotinib was calculated as (number of viable cells after treatment / total number of cells)
100.
7. In vitro cell uptake and inhibition assays
×
3
6
In order to determine the specificity of Ga-NOTA-Erlotinib to EGFR, in vitro cell
8
uptake studies were carried out in A431 and A549 cells. Cells were grown to confluence
6
in DMEM containing 10% FBS. The cells were harvested and 1 x 10 cells per well
2
0

2
1
were seeded into 12 well tissue culture plates and incubated overnight at 37ºC in a
6
8
2
humidified CO incubator. Subsequently, the cells were incubated with 1 µg of Ga-
NOTA-Erlotinib (~ 4 kBq per well) in 0.05 M sodium acetate (pH 7.0) for 1 h at 37°C.
Cells were washed twice with 1 mL of 0.05 M sodium acetate buffer (pH 7.0),
solubilized with 1 mL of 1 N NaOH and the solution measured for radioactivity
associated with the cells. For inhibition studies, same numbers of cells were incubated
with the parent Erlotinib and/or the NOTA-Erlotinib at various concentrations (viz. 1, 5,
6
0, 50 and 100 µg) and the same amount of Ga-NOTA-Erlotinib (1 µg and 4 kBq per
8
1
well) under identical experimental conditions, in order to confirm the specificity of
68
uptake of Ga-NOTA-Erlotinib to EGFR.
3
3
8. Ikeda R, Vermeulen LC, Lau E, Jiang Z, Kavanaugh SM, Yamada K, Kolesar J M.
Isolation and characterization of Erlotinib-resistant human non-small cell lung cancer
A549 cells. Oncol Lett. 2011; 2: 91-94.
+
9. Biodistribution studies in EGFR tumor bearing NOD/SCID mice
68
Biodistribution pattern of Ga-NOTA-Erlotinib was studied in NOD/SCID mice bearing
EGFR positive tumor at 30 min and 1 h post injection (p.i.) in groups of four each. For
6
raising the tumors, mice weighing 18-20 g were injected with 3×10 A431 cells
subcutaneously in the right flank. Palpable tumors (0.5-0.8 g) developed within two
6
8
weeks of injection. Subsequently, the animals were injected with Ga-NOTA-Erlotinib
in saline (100 µL, 370 KBq) via the lateral tail vein. A carbon dioxide saturated chamber
was used to euthanize the animals. Major organs/tissues and tumor were dissected,
weighed and their radioactivity measured using a flat-bed NaI (Tl) gamma counter.
Radioactivity associated with the organs was corrected for decay and the percentage of
radioactivity dose per gram (% ID/g) was calculated.
2
1

2
2
4
0. Analysis of metabolites
6
8
For metabolite analysis, Ga-NOTA-Erlotinib (100 µL, 370 KBq) was injected in
normal Swiss mice (n=3) intravenously through the lateral tail vein of each animal. Urine
samples from the animals were collected 1 h post injection and acetonitrile was added to
precipitate the proteins. The samples were then centrifuged and the supernatant was
68
analyzed using HPLC using the gradient elution method standardized for Ga-NOTA-
Erlotinib.
2
2

2
3
Table1: Cell viability studies of NOTA-erlotinib in A431 cells
%
Cell viability
Time post treatment
3 h
24 h 48 h
72 h
1
µM NOTA-erlotinib
40.0±0.2
37.5±0.1
28.5±0.4 12.5±0.3
2
3

2
4
6
Table 2: Biodistribution pattern of Ga-NOTA-erlotinib in NOD/SCID mice bearing
8
EGFR expressing tumor (n=4)
%
Injected Dose/g ± S.D.
Organs
30 min
1 h
Blood
2.4 ± 0.2
1.5 ± 0.20
0.4 ± 0.2
4.4 ± 2.2
12.3 ± 3.9
10.2 ± 0.6
1.1 ± 0.4
4.1 ± 0.7
3.3 ± 1.3
Nil
1.2 ± 0.2
Lungs
Heart
0.8 ± 0.1
0.1 ± 0.0
3.4 ± 2.6
14.3 ± 3.4
6.0 ± 0.7
0.1 ± 0.01
3.3 ± 0.6
2.4 ± 0.6
Nil
Stomach
Intestine
Liver
Spleen
Kidney
Muscle
Bone
Excreta
(
6.7 ± 4.6
1.5 ± 0.1
1.1 ± 0.2
17.6 ± 4.7
0.7 ± 0.2
0.9 ± 0.2
% I.D.)
Tumor
Tumor/Lungs
(
Ratio)
2
4

2
5
Highlights
•
•
•
•
•
Alkyne group of Erlotinib modified by CuAAC reaction for conjugation with NOTA
6
NOTA-Erlotinib radiolabeled with Ga in 95±2 % yields
8
6
8
Ga-NOTA-Erlotinib showed 9.8± 0.4% uptake in A431 cells
Inhibition of up to 55.1 ±0.2% on addition of 10 µg erlotinib in A431 cells
+
Moderate tumor accumulation in EGFR SCID mice (1.53±0.11 % ID/g at 30 min p.i)
2
5