Synthesis and characterization of well-defined l-lactic acid-caprolactone co-oligomers and their rhenium (I) and technetium(I) conjugates

Article abstract of DOI:10.1016/j.jorganchem.2012.06.001

Staring from l-lactide and ε-caprolactone, the corresponding lactic-caprolactone cooligomer with hydroxyl and carboxylic acid groups were synthesized. These oligomers were connected with chelating groups through a long chain tether, ready for transition metal binding. Coordination of organometallic rhenium(I) and technetium(I) complexes generated the conjugates in high yield and short time, satisfying the requirements for short-lived radiopharmaceuticals in clinical applications. A reasonable pharmacophore model has been established to guide the design of well-defined lactic acid oligomer for nuclear medicine.

Full text of DOI:10.1016/j.jorganchem.2012.06.001

Journal of Organometallic Chemistry 716 (2012) 95e102
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and characterization of well-defined
L
-lactic acid-caprolactone
co-oligomers and their rhenium (I) and technetium(I) conjugates
*
Hua Zhu, Zhi Yang , Nan Li, Xue-Juan Wang, Feng Wang, Hua Su, Qing Xie, Yan Zhang, Yun-Xia Ma,
Bao-He Lin
Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute,
Beijing 100142, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Staring from L-lactide and ε-caprolactone, the corresponding lactic-caprolactone cooligomer with
Received 21 April 2012
Received in revised form
1 June 2012
hydroxyl and carboxylic acid groups were synthesized. These oligomers were connected with chelating
groups through a long chain tether, ready for transition metal binding. Coordination of organometallic
rhenium(I) and technetium(I) complexes generated the conjugates in high yield and short time, satis-
fying the requirements for short-lived radiopharmaceuticals in clinical applications. A reasonable
pharmacophore model has been established to guide the design of well-defined lactic acid oligomer for
nuclear medicine.
Accepted 7 June 2012
Keywords:
Lactic acid
ε-Caprolactone
Tricarbonyl rhenium
Technetium-99m radiolabeling
Oligomer
Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction
structural studies. A series of blends of biodegradable polymers
using PCL and PLA were prepared by the variation of the mass
Biodegradable polymers are degraded in the environment by
the action of naturally occurring microorganisms. The conjugates of
biodegradable polymers with radiopharmaceutical agents would
allow new insight into their bio-distribution and controlled release.
The coordination and characterization of fac-[M(CO)3]þ conjugate
complexes would benefit their applications in SPECT image or
radio-therapy. Biodegradable nature of these conjugates would also
alleviate environmental contamination for un-used radiopharma-
ceuticals [1].
Aliphatic polyesters are the most known and studied biodegrad-
able polymers, including poly(lactide) (PLA) and poly(caprolactone)
(PCL). PLA is prepared from renewable resources and commercially
available from a variety of manufacturers [2]. PCL is a FDA-approved
biodegradable polymer and has been used in various medical and
pharmaceutical applications, including controlled drug delivery,
implants, and surgical dressings [3].
fraction across the composition range [4e7]. Traditionally, PLA and
PCL have been prepared via the ring opening polymerization of
L-lactide and ε-caprolactone using a catalyst, such as stannous
octanoate. Significant effort has been devoted to the development
of length-controlled polymerization process in recent years [8,9].
Although there is growing academic and industrial interest in PLA,
PCL, or their copolymers (PLA-PCL), the synthesis of well-defined
PLA or PCL oligomers have been scarcely reported [10,11].
Technetium-99m (140 keV (89%), T1/2 ¼ 6.02 h) complexes
present a major synthetic challenge with respect to designing
radiopharmaceuticals that have suitable in vivo stabilites. Alberto
et al developed
a particularly stable and kinetically inert
[M(H2O)3(CO)3]þ core (M ¼ 99mTc, 186/188Re) reagent [12,13], which
can be used to form complexes with various ligands, including the
bis(picolyl)amine moiety [14e16]. Saatchi have synthesized length-
controlled PLA by ring-opening polymerization of L-lactide and
Preparation of monodisperse co-oligomers of poly(lactide)-
poly(caprolactone) (PLA-PCL) would enable a wide range of
connected with rhenium(I) for medicinal applications [17,18].
Previously, we reported the synthesis of oligomeric (monomer to
heptamer) lactic acids, and their conjugates with a chelating agent
through a short oligoethylene oxide spacer, which can bind with
a radioactive transition metal ion [19,20]. Here, we report the
synthesis of molecularly defined LA-CL co-oligomers, their conju-
gates with rhenium(I) and 99mTc(I). We intended to develop these
* Corresponding author. Department of Nuclear Medicine, Peking University
School of Oncology, Beijing Cancer Hospital & Institute. Beijing 100142, China. Tel./
fax: þ86 10 88196196.
E-mail address: pekyz@163.com (Z. Yang).
0022-328X/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.06.001

96
H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
complexes as new agents for applications in SPECT image or
chemotherapy.
colorless oil. 1H NMR (CDCl3, 400 MHz)
d: 7.30e7.38 (m, Ar, 5H),
5.10 (s, CO2CH2Ph, 2H), 3.61 (t, J ¼ 6.5 Hz, CH2OH, 2H), 2.35
(t, J ¼ 6.5 Hz, CH2CO2Bn, 2H),1.45e1.65 (m, CH2(CH2)3CH2, 6H), 0.88
2. Experimental
(s, (CH3)3CSi, 9H), 0.09 (s, (CH3)2Si, 6H). IR (KBr) n: 2991, 2802,1555,
1326, 1107, 618 cmꢀ1. ESI-HRMS: calcd for C19H33O3Si [M þ H]þ
2.1. Materials and methods
337.2199, found 337.2107.
All of the starting materials and reagents were commercially
available and used directly without further purification. High
resolution mass spectra were obtained on a Thermo-MAT95XP
mass spectrometer using electron impact ionization. Matrix assis-
ted laser desorption/ionization (MALDI TOF-HRMS) was carried out
on a Thermo Finnigan Bioanalysis DYNAMO TOF Mass Spec Spec-
trometer. NMR spectra were recorded on a Bruker Avance 400 or
500 MHz spectrometer. IR spectra were recorded on an Avataar 370
FT-IR spectrometer (250e4000 cmꢀ1). The elemental analyses were
conducted using Elementar Analysen-System GmbH (Germany)
vario EL III. High performance liquid chromatography (HPLC)
analyses of the Re and 99mTc complexes were performed on
2.2.1.4. Synthesis of 6-(tert-Butyldimethyl)siloxyhexanoic acid
(H5). PdeC (10 wt %, 0.88 g) was added into a solution of H4
(3.36 g, 10 mmol) in ethyl acetate (50 mL), and the reaction
mixture was stirred for
2 h at room temperature under
dihydrogen. The resulting mixture was filtered through celite, and
the cake was washed with 100 mL of ethyl acetate. The combined
filtrate was concentrated under reduced pressure to give H5
2.34 g (yield 95%) as clear and colorless oil. 1H NMR (CDCl3,
500 MHz)
d
: 3.56 (t, J ¼ 6.5 Hz, CH2OSi, 2H), 2.31 (t, J ¼ 7.5 Hz,
CH2CO2H, 2H), 1.30e1.55 (m, SiOCH2(CH2)3CH2CO2H, 6H), 0.84
(s, (CH3)3CSi, 9H), 0.05 (s, (CH3)2Si, 6H). IR (KBr) n: 3201, 2908,
1675, 1542, 1126, 1102, 713 cmꢀ1. ESI-HRMS: calcd for C12H26O3Si
a hypersil BDS C-18 reversed-phase column (5
m
m, 60% CH3CN/40%
[M þ H]þ, 246.1651; found, 246.1649.
H2O, flow rate 1.0 mL/min), using a dionex P680 system equipped
with a tunable absorption detector and a PDA-100 photodiode-
array detector.
2.2.1.5. Synthesis of 6-[(S)-2-((S)-2-(tertbutyldimethylsilyloxy)prop-
anoyloxy)propanoate] hexanoic benzyl ester (L5). L4 (5.52 g,
20 mmol), EDC (4.20 g, 22 mol, 1.1 eq), and DMAP (2.68 g, 22 mmol,
1.1 eq) were dissolved in CH2Cl2 (50 mL). H3 (4.44 g, 20 mmol) was
added into the mixture, and the solution was stirred at room
temperature for 10 h. The reaction mixture was then added into
H2O (50 mL), and the product was extracted with additional CH2Cl2
(2 ꢁ 50 mL). The combined organic layer was dried over Na2SO4,
filtered, and the crude product was purified via silica gel flash
column chromatography, to give L5 7.20 g (yield 75%) as colorless
2.2. Chemical synthesis
2.2.1. Synthesis of L-lactic acid-caprolactone co-oligomers
2.2.1.1. Synthesis of 6-hydroxyhexanoic acid (H2). ε-Caprolactone
(22.8 g, 0.20 mol), H2O (300 mL), and sodium hydroxy (NaOH)
(8.4 g, 0.21 mol) were added in a round bottom flask. This reaction
mixture was stirred at room temperature overnight. The clear
reaction mixture was then extracted with ethyl acetate (EtOAc)
(4 ꢁ 50 mL), and the organic phase was, dried by anhydrous sodium
sulphate (Na2SO4), to give the product H2 26.0 g (yield 98%) as
oil. 1H NMR (CDCl3, 400 MHz)
d: 7.25 (b, Ar, 5H), 5.12e5.30
(m, CO2CH2Ph and CO2CH(CH3)CO2, 3H), 4.38 (q, J ¼ 6.8 Hz,
SiOCH(CH3)CO, 1H), 4.10 (t, J ¼ 6.6 Hz, COOCH2(CH2)3, 2H), 2.35
(t, J ¼ 8.6 Hz, CH2CO2Bn, 2H), 1.56e1.65 (m, OCH2(CH2)3CH2CO2Bn,
6H), 1.42e1.50 (m, OCH(CH3)CO2CH(CH3)COO, 6H), 0.93
(s, (CH3)3CSi, 9H), 0.11 (s, (CH3)2Si, 3H), 0.09 (s, (CH3)2Si, 3H). IR
a clear white solid. 1H NMR (CDCl3, 300 MHz)
3.55 (t, J ¼ 6.2 Hz, CH2OH, 2H), 2.3 (t, J ¼ 7.2 Hz, CH2CO2H, 2H), 1.6
(m, HOCH2(CH2)3CH2CO2H, 6H). IR (KBr) : 3260, 2912, 2849, 1600,
1256, 583 cmꢀ1
d: 3.7 (b, CH2OH, 1H),
n
.
(KBr) n
: 2986, 1765, 1231, 1103, 1055, 743 cmꢀ1. ESI-HRMS: calcd for
C25H41O7Si [M þ H]þ 481.2622, found 481.2496.
2.2.1.2. Synthesis of benzyl 6-hydroxyhexanoate (H3). H2 (13.20 g,
0.10 mol), triethylamine (30.3 g, 0.30 mol), and dichloromethane
(CH2Cl2, 60 mL) were added in a round bottom flask. Benzyl
bromide (BnBr, 12.2 g, 0.12 mol) in 40 mL CH2Cl2 was dropped into
the mixture, and the solution was stirred overnight at room
temperature. Water (100 mL) was added to the reaction mixture,
and the solution was extracted with CH2Cl2 (2 ꢁ 100 mL). The
organic layer was dried over Na2SO4, filtered, and the crude product
was purified via silica gel flash column chromatography to give H3
2.2.1.6. Synthesis of 6-[(S)-2-((S)-2-(tertbutyldimethylsilyloxy)prop-
anoyloxy)propanoate] hexanoic acid (L6). The mono-protected,
acid-functionalized LA-CL-COOH, L6, was prepared using the
general procedure described above for deprotection of benzyl ester
L5 by hydrogenolysis (PdeC/H2). The combined filtrates were
concentrated under reduced pressure to give L6 (95% yield) as
colorless oil. 1H NMR (CDCl3, 500 MHz)
d
: 5.10 (q, J ¼ 6.8 Hz,
CO2CH(CH3)CO2, 1H), 4.40 (q, J ¼ 6.8 Hz, SiOCH(CH3)CO, 1H), 4.11
(t, J ¼ 6.5 Hz, COOCH2(CH2)3, 2H), 2.35 (t, J ¼ 7 Hz, CH2CO2H, 2H),
1.58e1.65 (m, OCH2(CH2)3CH2CO2H, 6H), 1.38e1.50 (m, OCH(CH3)
CO2CH(CH3)COO, 6H), 0.93 (s, (CH3)3CSi, 9H), 0.11 (s, (CH3)2Si, 3H),
18.89 g (yield 85%) as colorless oil. 1H NMR (CDCl3, 400 MHz)
d:
7.30e7.38 (m, Ar, 5H), 5.12 (s, CO2CH2Ph, 2H), 3.63 (t, J ¼ 6.5 Hz,
CH2OH, 2H), 2.38 (t, J ¼ 6.5 Hz, CH2CO2Bn, 2H), 1.45e1.65 (m,
HOCH2(CH2)3CH2CO2Bn, 6H). IR (KBr)
n
: 3180, 2992, 2840, 1650,
0.09 (s, (CH3)2Si, 3H). IR (KBr) n: 2916, 1815, 1301, 1213, 1156,
1126, 638 cmꢀ1
.
723 cmꢀ1. ESI-HRMS: calcd for C18H34O7SiNa [M þ Na]þ 413.1971,
found 413.1690.
2.2.1.3. Synthesis of 6-(tert-Butyldimethyl)siloxyhexanoic benzyl
ester (H4). H3 (31.62 g, 0.12 mol), imidazole (10.30 g, 0.15 mol),
and dimethylformamide (DMF, 20 mL) were added in a round
bottom flask. tert-Butyldimethylsilyl chloride (TBDMSCl, 22.65 g,
0.15 mmol) was added into the mixture, which was stirred over-
night at room temperature under dinitrogen atmosphere. The
resulting mixture was poured into a separation funnel containing
150 mL of saturated aqueous NaHCO3, and the product was
extracted with hexane (3 ꢁ 150 mL). The organic phase was dried
over Na2SO4, filtered, and the crude product was purified via silica
gel flash column chromatography, to give H4 26.88 g (yield 80%) as
2.2.1.7. Synthesis of 6-[(S)-2-((S)-2- hydroxypropanoyloxy) prop-
anoate] hexanoic benzyl ester (L7). The mono-protected, hydroxyl-
functionalized LA-CL-OH, L7, was prepared using the general
procedure described above for deprotection of TBDMS group of the
protected Monomer, L5 (TBAF/THF). The crude product was purified
via silica gel flash column chromatography, to give L7 (yield 75%) as
a colorless oil.1H NMR (CDCl3, 400 MHz)
d: 7.25 (b, Ar, 5H), 5.12e5.30
(m, CO2CH2Ph and CO2CH(CH3)CO2, 3H), 4.38 (q, J ¼ 6.8 Hz,
SiOCH(CH3)CO, 1H), 4.10 (t, J ¼ 6.6 Hz, COOCH2(CH2)3, 2H), 2.35
(t, J ¼ 8.6 Hz, CH2CO2Bn, 2H), 1.56e1.65 (m, OCH2(CH2)3CH2CO2Bn,

H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
97
6H), 1.42e1.50 (m, OCH(CH3)CO2CH(CH3)COO, 6H), 0.93
(s, (CH3)3CSi, 9H), 0.11 (s, (CH3)2Si, 3H), 0.09 (s, (CH3)2Si, 3H). IR (KBr)
ESI-HRMS: calcd for C42H65N4O12Si [M þ H]þ 845.4368, found
845.3624.
n
: 3206, 3012, 1745, 1531, 1130, 1089, 813 cmꢀ1. ESI-HRMS: calcd for
C19H27O7 [M þ H]þ 367.1757, found 367.1901.
2.2.3.2. Synthesis of HO-hexamer-alkyl-tri (C2). Acetic acid (0.5 g,
8.3 mmol) was added into a solution of C1 (0.34 g, 0.4 mmol) in THF
(2 mL). To this solution was added TBAF (1.0 M solution in THF,
0.25 g, 0.8 mmol), and the solution was stirred for 48 h at room
temperature. The reaction mixture was added into aqueous NaHCO3
(40 mL), and the crude product was extracted with additional EtOAc
(2 ꢁ 40 mL). The combined organic layer was washed with H2O and
saline, and dried over Na2SO4, filtered, and the crude product was
purified via silica gel flash column chromatography, to give C2
2.2.1.8. Synthesis of LA-CL dimer (L8). The doubly protected LA-CL
dimer, L8, was prepared using the general procedure described
above for esterification of LA-CL-COOH (L6) and LA-CL-OH (L7). The
crude product was purified via silica gel flash column chromatog-
raphy, toyieldL8(50%yield)ascolorlessoil.1HNMR(CDCl3, 400 MHz)
d: 7.25 (b, Ar, 5H), 5.12e5.30 (m, CO2CH2Ph and CO2CH(CH3)CO2, 5H),
4.38 (q, J ¼ 6.8 Hz, SiOCH(CH3)CO, 1H), 4.10 (m, COOCH2(CH2)3, 4H),
2.35 (m, CH2CO2Bn, 4H), 1.56e1.65 (m, OCH2(CH2)3CH2CO2Bn, 12H),
1.42e1.50 (m, OCH(CH3)CO2CH(CH3)COO, 12H), 0.93 (s, (CH3)3CSi,
0.24 g (yield 82%) as a light yellow oil. IR (KBr)
1238, 833, 764 cmꢀ1 1H NMR (CDCl3, 400 MHz)
Py- H, 2H), 7.55 (d, J ¼ 7.8 Hz, Py-
H, 2H), 7.68 (t, J ¼ 7.6 Hz, Py-
2H), 7.16 (t, J ¼ 6.1 Hz, Py- H, 2H), 6.22 (t, J ¼ 5.5 Hz, CONHCH2, 1H),
n
: 2921, 1605, 1503,
: 8.52 (d, J ¼ 4.7 Hz,
H,
d
9H), 0.11 (s, (CH3)2Si, 3H), 0.09 (s, (CH3)2Si, 3H). IR (KBr)
n: 2896,1695,
s
g
b
1230,1112,1046, 734 cmꢀ1. ESI-HRMS: calcd for C37H59O13Si [M þ H]þ
d
739.3725, found 739.3710.
5.12e5.23 (m, CO[OCH(CH3)CO]5N, 5H), 4.38 (q, J ¼ 6.8 Hz,
SiOCH(CH3)CO, 1H), 3.82 (s, eN(CH2)2, 4H), 3.14e3.24
(m, NHCH2CH2, 2H), 2.54 (t, J ¼ 7.1 Hz, CH2N(CH2)2, 2H), 1.44e1.58
(m, Si[OCH(CH3)CO]6N and NHCH2CH2(CH2)2CH2CH2N, 22H), 1.25
2.2.2. Synthesis of 6-(N-dipicoylaminoethyl)hexylamine (N4)
Compound N2eN3 were synthesized according to our previously
published procedure with slight modification [20,21]. PdeC (10 wt
%, 0.40 g) was added to a solution of N3 (1.99 g, 5 mmol) in absolutely
methanol (20 mL), and the reaction mixture was stirred for 2 h at
room temperature under dihydrogen. The resulting mixture was
filtered and the cake was washed with 20 mL of ethyl acetate. The
combined filtrate was concentrated under reduced pressure to give
(b, NH(CH2)2(CH2)2(CH2)2N, 4H); 13C NMR (CDCl3):
d
: 175.20 (CO2N,
1C), 170.63 (CO2C, 1C), 169.73 (CO2C, 1C), 169.68 (CO2C, 1C), 169.64
(CO2C, 1C), 168.62 (CO2C, 1C), 159.23 (Py- C, 2C), 148.71 (Py- C, 2C),
136.75 (Py- C, 2C), 123.17 (Py- C, 2C), 122.14 (Py- C, 2C), 71.79
a
s
g
b
d
(OCH2CH2N, 1C), 69.80 (CO2CH(CH3)CO2, 1C), 69.19 (CO2CH(CH3)
CO2, 1C), 68.96 (CO2CH(CH3)CO2, 1C), 68.88 (CO2CH(CH3)CO2, 1C),
66.68 (CO2CH(CH3)CON, 1C), 60.00 (Py(CH2)2, 2C), 54.28 (CH2CH2N,
1C), 39.29 (CONHCH2CH2O, 1C), 29.20 (CH2CH2N, 1C), 26.85
(CH2CH2CH2N, 1C), 26.73 (CONCH2CH2CH2, 1C), 26.56 (CONCH2CH2,
1C), 20.50 (HOCH(CH3)CO, 1C), 17.79 (CO2CH(CH3)CO2, 1C), 16.72
(CO2CH(CH3)CO2, 1C), 16.69 (CO2CH(CH3)CO2, 1C), 16.65
(CO2CH(CH3)CO2, 1C), 16.64 (CO2CH(CH3)CO2, 1C). ESI-HRMS: calcd
for C36H51N4O12 [M þ H]þ 731.3498, found 731.3499.
N41.41 g (yield 98%) as pale red oil.1H NMR (CDCl3, 500 MHz)
(d, J ¼ 4.1 Hz, Py- H, 2H), 7.63 (dt, J ¼ 7.7 Hz, J ¼ 1.7 Hz, Py- H, 2H),
7.52 (d, J ¼ 7.8 Hz, Py- H, 2H), 7.12 (t, J ¼ 5.2 Hz, Py- H, 2H), 3.79
(s, eN(CH2)2, 4H), 2.62 (d, J ¼ 7.1 Hz, CH2NCH2, 2 H), 2.52 (t, J ¼ 7.3 Hz,
CONCH2CH2, H), 1.75 (s, CH2CH2CH2NH2, 2H), 1.50e1.56
d: 8.50
s
g
b
d
2
(m, CH2NH2, 4H), 1.20e1.29 (m, (CH2)2(CH2)2NH2, 4 H); IR (KBr):
2924, 2028, 1917 cmꢀ1; EI-HRMS: calcd for C18H26N4 298.2157,
found 298.2157.
2.2.3.3. Synthesis of HO-hexamer-alkyl-tri-Re(CO)3 (C3). C2 (73.0
mg, 0.10 mmol) was dissolved in 2 mL methanol. (Et4N)2[Re(CO)3Br3]
(85 mg, 0.11 mmol) was added into the mixture, and the solution was
stirredfor30minatroomtemperatureunderdinitrogen. Thereaction
mixture was evaporated to dryness, and the residue was purified via
silica gel flash column chromatography, to yield C3 90 mg (90% yield)
as a light yellow solid. The sample for element analysis was recrys-
2.2.3. Synthesis of the conjugates between lactic acid and
tricarbonyl rhenium complex
2.2.3.1. Synthesis of hexamer-alkyl-tri (C1). A mixture of lactic acid
hexamer (0.56 g, 1.0 mmol), EDC (0.21 g, 1.1 mmol, 1.1 eq), and HOBt
(0.147g,1.1mmol,1.1eq), weredissolvedinCH2Cl2 (50mL). N4(0.30g,
1.0 mmol) was added into the mixture, which was stirred at room
temperature for 10 h. The crude product was added into H2O (50 mL),
the product was extracted with additional CH2Cl2 (2 ꢁ 50 mL). The
organic layer was dried over Na2SO4, filtered, and the crude product
was purified via silica gel flash column chromatography to give C1
tallized from CHCl3 and hexane. IR (KBr)
1238, 772 cmꢀ1 1H NMR (CD3OD, 400 MHz)
Py-
H, 2H), 7.94 (td J1 ¼ 8.0 Hz, J2 ¼ 1.1 Hz, Py-
(d, J ¼ 7.8 Hz, Py- H, 2H), 5.13e5.22
n
: 2921, 2030, 1920, 1511,
: 8.85 (d, J ¼ 5.4 Hz,
H, 2H), 7.56
d
s
g
b
H, 2H), 7.36 (t, J ¼ 6.6 Hz, Py-
d
0.63 g (yield 75%) as colorless oil.1H NMR (CDCl3, 500 MHz)
J ¼ 4.0 Hz, Py- H, 2H), 7.65 (t, J ¼ 7.5 Hz, Py- H, 2H), 7.52 (d, J ¼ 8.0 Hz,
Py- H, 2H), 6.18 (t, J ¼ 5.5 Hz, CONHCH2,
H, 2H), 7.15 (t, J ¼ 6.0 Hz, Py-
d: 8.51 (d,
(m, CO[OCH(CH3)CO]4OCH(CH3), 4H), 5.06 (q, OCH(CH3)CON,
J ¼ 5.0 Hz, 1H), 4.86 (s, eN(CH2), 2H), 4.32 (q, J ¼ 5.0 Hz, HOCH(CH3)
CO, 1H), 3.80 (s, eN(CH2), 2H), 3.20e3.30 (m, NHCH2CH2, 2H), 1.97
s
g
b
d
1H), 5.13e5.23 (m, CO[OCH(CH3)CO]5N, 5H), 4.38 (q, J ¼ 6.0 Hz,
SiOCH(CH3)CO,1H), 3.80 (s, eN(CH2)2, 4H), 3.11e3.25 (m, NHCH2CH2,
2H), 2.53 (t, J ¼ 7.3 Hz, CH2N(CH2)2, 2H), 1.43e1.57 (m, Si[OCH(CH3)
CO]6N and NHCH2CH2(CH2)2CH2CH2N, 22H), 1.23e1.28 (m,
NH(CH2)2(CH2)2(CH2)2N, 4H), 0.90 (s, (CH3)3CSi, 9H), 0.11 (s, (CH3)2Si,
(t,
CO]6N and NHCH2CH2(CH2)2CH2CH2N, 22H), 0.82e0.91 (m,
NH(CH2)2(CH2)2(CH2)2N, 4H); 13C NMR (CDCl3):
: 197.56 (fac-
Re(CO)3, 3C), 176.04 (CO2N, 1C), 172.94 (CO2C, 1C), 171.71 (CO2C, 1C),
171.68 (CO2C, 1C), 171.47 (CO2C, 1C), 171.11 (CO2C, 1C), 162.51 (Py- C,
2C), 153.43 (Py- C, 2C), 141.92 (Py- C, 2C), 127.17 (Py- C, 2C), 124.93
(Py- C, 2C), 72.90(OCH2CH2N, 1C), 72.13 (CO2CH(CH3)CO2, 1C),
J
¼
7.1 Hz, CH2N(CH2)2, 2H), 1.42e1.62 (m, Si[OCH(CH3)
d
a
3H), 0.083 (s, (CH3)2Si, 3H). 13C NMR (CDCl3):
d
: 173.53 (CO2N, 1C),
170.68 (CO2C,1C), 169.98 (CO2C, 1C), 169.85 (CO2C, 1C), 169.58 (CO2C,
1C), 168.62 (CO2C, 1C), 159.96 (Py- C, 2C), 148.92 (Py- C, 2C), 136.41
(Py- C, 2C), 122.90 (Py- C, 2C), 121.91 (Py- C, 2C), 71.80 (OCH2CH2N,
s
g
b
d
a
s
71.24 (CO2CH(CH3)CO2, 1C), 70.89 (CO2CH(CH3)CO2, 1C), 70.60
(CO2CH(CH3)CO2, 1C), 70.18 (CO2CH(CH3)CON, 1C), 69.10 (Py(CH2)2,
2C), 67.83 (CH2CH2N, 1C), 40.25 (CONHCH2CH2O, 1C), 30.37
(CH2CH2N,1C), 27.75 (CH2CH2CH2N,1C), 27.64 (CONCH2CH2CH2, 1C),
26.49 (CONCH2CH2, 1C), 20.91 (HOCH(CH3)CO, 1C), 18.42
(CO2CH(CH3)CO2, 1C),17.38 (CO2CH(CH3)CO2, 1C), 17.35 (CO2CH(CH3)
CO2, 1C), 17.32 (CO2CH(CH3)CO2, 1C), 17.25 (CO2CH(CH3)CO2, 1C).
Elemental Analysis: Calcd for [C39H50N4O15Re]Brꢂ2CHCl3: C 37.31, H
3.97, N 4.24; found: C 37.61, H 3.83, N 3.80. MALDI/DHB-HRMS: calcd
for C39H50O15N1485Re, 999.28425.
g
b
d
1C), 69.76 (CO2CH(CH3)CO2, 1C), 69.13 (CO2CH(CH3)CO2, 1C), 68.65
(CO2CH(CH3)CO2,1C),68.46(CO2CH(CH3)CO2,1C), 67.97(CO2CH(CH3)
CON, 1C), 60.43 (Py(CH2)2, 2C), 54.31 (CH2CH2N, 1C), 39.30 (CON-
HCH2CH2O,1C), 29.25 (CH2CH2N,1C), 26.98 (CH2CH2CH2N,1C), 26.92
(CONCH2CH2CH2, 1C), 26.63 (CONCH2CH2, 1C), 25.68 ((CH3)3CSi, 3C),
21.22 (SiOCH(CH3)CO, 1C), 18.27 ((CH3)3CSi, 1C), 17.79 (CO2CH(CH3)
CO2, 1C), 16.72 (CO2CH(CH3)CO2, 1C), 16.70 (CO2CH(CH3)CO2, 1C),
16.63 (CO2CH(CH3)CO2, 2C), e4.92 ((CH3)2Si,1C), e5.30 ((CH3)2Si,1C).

98
H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
O
O
OTBDMS
BnO
OTBDMS
H 4
HO
H 5
O
O
O
H2O
BnBr/NEt3
O
OH
OH
H 3
HO
BnO
H 2
H 1
EDC/DMAP
DCM
TBDMSCl/
imidazole
OTBDMS O
O
OH
O
O
O
O
OTBDMS O
O
H2/Pd-C
EtOAc
BnOH/CSA
PhCH3
O
OH
L 4
O
DMF
O
O
O
O
Bn
O
Bn
L 2
L 1
L 3
OTBDMS O
O
H2/Pd-C
EtOAc
OH
OTBDMS O
O
O
O
O
L 6
EDC/DMAP
DCM
O
O
OBn
O
O
OH
O
O
L 5
TBAF
THF
O
OBn
L 7
O
OTBDMS O
O
O
O
O
O
OBn
_
O
Bn: PhCH2
TBDMS:
H3C CH3
O
O
O
O
Si
CH3
H3C
H3C
L 8 : PLA-PCL oligomers
Scheme 1. Synthetic scheme for well-defined L-lactic acid-caprolactone co-oligomers.
2.3. Radio-labeling of 99mTc for SPECT imaging
100 mL) were added into a 5-mL serum vial. The reaction mixture
was heated at 42 ꢃC for 30 min, and the progress of the reaction was
monitored by radio-TLC. After the mixture was cooled to room
temperature, the radiotracer was purified by HPLC using the same
conditions as those for the analysis of C2 and C3. The radioactivity
of the eluate was counted and calibrated against the standard
solutions.
2.3.1. Radio-synthesis of the conjugate 99mTc(CO)3-lactic acid
For radio-synthesis of the conjugate 99mTc(CO)3-lactic acid:
[
99mTc(CO)3(H2O)3]þ was prepared as reported before [22,23], with
radiochemical purity of <95%, as determined by radio-HPLC. An
aqueous [99mTc(CO)3(H2O)3]þ solution (0.2 mL) and C2 (10ꢀ4 M,
PyCHO/AcOH
Py
CbzCl
N
N
Pd-C
H2
NaBH(OAc)3
N
CbzHN
N
H2N
NH2
CbzHN
NH2
H2N
DCM
DCM
6
N 3
6
N 1
6
N 2
Py
N 4
Py
N
NH2
N 4
OTBDMS O
O
OH
O
O
Py
Py
Py
Py
6
Py
TBAF
THF
O
EDC/HOBt
DCM
N
6N
N
H
6 N
OTBDMS O
O
H
O
5
5
C 1
C 2
OH
O
5
γ
LA-Hexamer
δ
β
α
σ
N
N
OH
O
O
O
CO
[(Et4N)2][Re(CO)3Br3]
MeOH
O
O
O
O
N
Re CO
CO
O
N
H
O
O
O
C 3
Scheme 2. Synthetic scheme for Re(CO)3-coordinated lactide conjugate.

H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
99
Fig. 1. IR spectra of C2 (left) and its corresponding [Re(CO)3]þ-conjugate C3 (right).
2.3.2. In vitro stability of the conjugate 99mTc(CO)3-lactic acid
The stability of the conjugate 99mTc(CO)3-lactic acid was studied
by measuring the radiochemical purity using radio-HPLC at
different time intervals after preparation. The complex was added
to a test tube with PBS. The mixture was incubated by shaking the
test tube at 37 ꢃC in an incubation apparatus. The radiochemical
purity was measured at 30 min and 1, 2, 4, and 8 h by radio-HPLC.
catalyzed ring opening reactions. Initial studies using benzyl
alcohol (BnOH) as protect group of H2 gave low chemical yield [10].
The carboxylic acid-protected H3 was prepared instead from H2 by
treatment with BnBr/NEt3. Reaction of H3 with TBDMSCl led to H4
in high yield, with both hydroxyl and carboxylic acid group pro-
tected at this stage. Hydrogenolysis of H4 gave the carboxylic acid
CL H5 almost quantitatively.
The coupling of the orthogonally protected LA dimers L4 and CL
monomer H3 in the presence of EDC and DMAP proved to be a high
yielding procedure affording the LA-CL oligomer, L5, with minimal
side products (Scheme 1). The prowess of orthogonal protecting
groups is shown in the synthesis of L6 and L7 from the same L5. The
comboxylic acid group was released by hydrogenolysis to generate
L6, and the hydroxy group was activated by treatment with tetra-n-
butyl ammonium fluoride (TBAF) in the presence of acetic acid to
give L7. The coupling of carboxylic acid and alcohol group using the
same procedure to synthesize L5 gave the LA-CL dimer L8.
Following the protocols established in Scheme 1, each of the
molecularly defined LA-CL oligomers could be synthesized.
3. Result and discussion
3.1. Synthesis of L-lactic acid-caprolactone co-oligomers
The synthesis of LA, CL and LA-CL oligomers relies on the
availability of orthogonal protecting groups for carboxylic acid and
hydroxyl group (Scheme 1). Benzyl (Bn) ester and t-butyldime-
thylsily (TBDMS) ether were selected to protect carboxylic and
hydroxyl group [20]. Starting from -lactide (L1) and ε-caprolactone
(H1), the corresponding dimer L2 and monomer H2 were obtained
L
in quantitative yields by continuous extraction of the base-
Fig. 2. (a) ESI-HRMS analysis of C2, and (b) MALDI/DHB-HRMS analysis of C3.

100
H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
Fig. 3. 1H NMR analysis of compound (a) C2, and (b) C3.
3.2. Synthesis and characterization of conjugates between lactic
acid and Re(CO)3 complexes
achieved by hydrogenolysis (Pd/C, H2), leading to compound N4.
The coupling of the free amine in N4 and the free carboxylic acid in
lactic acid hexamer [19,20] by EDC/HOBt gave intermediate C1.
Deprotection of TBDMS groups generated C2 in good yield. The
rhenium(I) species was introduced using tricarbonyl rhenium
complex ([Et4N]2[Re(CO)3Br3]) prepared from [Bu4N][ReO4] [24].
To illustrate the stability of the conjugate, the crude product C3
was purified via silica gel flash chromatography without any sign
of decomposition.
3.2.1. Synthesis of the conjugates
The chemical structure of the chelate, and the structure and
length of the spacer, greatly affect the radiolabeling efficiency in
the complex formation step. There are a number of reports on the
preparation of tridentate chelating systems with variable spacer
groups comprising terminal primary amine [20]. Here, we present
a new procedure to synthesize tridentate chelating compound N4
(Scheme 2). Starting from mono-Cbz-protected diaminohexane N2,
the two py groups in N3 were introduced by in situ imine
formation with 2-pyridinecarbaldehyde and reduction with
sodium borohydride triacetate. Cbz-deprotection of N3 was
3.2.2. Characterization of conjugate
A number of unique feature of the conjugates in spectroscopy
have been observed.1H NMR spectra of the hydroxyl-protected
oligomers showed unique resonances for three methyl groups
OH
O
N
Py
Py
CO
OH
O
[
99mTc(CO)3(H2O)3]+
O
O
N
99mTc CO
N
N
H
6 N
N
H
O
5
CO
O
5
C 2
C 4
Scheme 3. Radio-synthesis of 99mTc-lactic acid conjugate.

H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
101
Fig. 4. HPLC analyses of compounds C2 (a. UV detector) C3 (b. UV detector) and C4 (c.
g detector).
around TBDMS group region with chemical shifts at 0.90, 0.11, and
0.08 ppm. The small quartet (J ¼ 6.0 Hz) at 4.38 ppm, due to the
unique CH group next to TBDMS group (or HO group in the end of
the chain), also functioned as a quantitative reference peak to
monitor the number of repeating units. The analytical data
(1H NMR, 13C NMR, and ESI-HRMS) for C1 and C2 are given in the
electronic supporting information Figs. S3eS6.
lactic acid conjugate C4 (Fig. 4) exceeded 90%. The conjugate and its
precursor were well separated, and the conjugate was purified by
semi preparative HPLC.
3.4. In vitro stability of 99mTc-lactic acid conjugate
The radio-conjugate, C4, showed no degradation in PBS solution
over 8 h at room temperature (Fig. 5). Decomposition or dissocia-
tion of the complexes to either [99mTc(CO)3]þ or other side products
was not observed for all of the complexes under the conditions
used.
Infrared spectroscopy of the conjugates showed unique features
of [Re(CO)3]þ with strong band and intense absorption at 1920 and
2030 cmꢀ1, which were attributed to (CeO) of the fac-Re(CO)3þ
n
units (Fig. 1). These absorption bands are significantly blue shifted
compared with the starting material ([Et4N]2[Re(CO)3Br3]) (1871,
1998 cmꢀ1) [25].
MALDI/DHB mass spectrometry of the conjugates and their
precursors shows the fingerprint of oligomer chain (Fig. 2) [26].
Inductively coupled plasma-mass spectrometry (ICP-MS) measured
the actual amount of rhenium in the conjugates (Fig. S8). The
isotope pattern of rhenium complex matches the simulation signal.
1H and 13C NMR spectra of the rhenium complex C3 and its
precursor C2 show that the rhenium core is an electron-
withdrawing group, making the chemical shift moving down-field
(Fig. 3). The 13C NMR spectra of the rhenium complexes C3 also
showed three carbonyl peaks in the range 197.0e198.0 ppm (Fig. S7).
3.3. HPLC analysis of the conjugate 99mTc-lactic acid
The radiochemical conjugate of technetium(I) complex was
synthesized following the same procedure as that for Rhenium (I)
complex (Scheme 3). The radiochemical purity of the crude 99mTc-
Fig. 5. In vitro PBS stability of 99mTc-lactic acid conjugate.

102
H. Zhu et al. / Journal of Organometallic Chemistry 716 (2012) 95e102
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We have prepared molecularly defined LA-CL oligomers with
terminal functional groups of hydroxyl and carboxylic acid. These
oligomers were connected with chelating groups through a long
chain tether, ready for transition metal binding. Coordination of
organometallic rhenium(I) and technetium(I) complexes generated
the conjugates in high yield and short time, satisfying the require-
ments for short-lived radiopharmaceuticals in clinical applications.
These conjugates are stable in PBS at RT, ready for SPECT image or
radio-therapy study. The availability of these well-defined oligo-
mers of LA-CL radiochemical conjugates allows new insight into the
drug distribution and controlled release in future studies.
[15] L.E. Jennings, N.J. Long, Chem. Commun. 24 (2009) 3511e3524.
[16] H. Zhu, L.L. Huang, Y.Q. Zhang, X.P. Xu, Y.H. Sun, Y.M. Shen, J. Biol. Inorg. Chem.
15 (2010) 591e599.
Acknowledgment
[17] K. Saatchi, U.O. Hafeli, Dalton Trans. 39 (2007) 4439e4445.
[18] K. Saatchi, U.O. Hafeli, Biocojug. Chem. 20 (2009) 1209e1217.
[19] H. Zhu, L.L. Huang, Y.Q. Zhang, Y.M. Shen, Chin. J. Org. Chem. 31 (2011)
87e95.
This work was supported by Grant No. 81071198 and No.
81172082 from the National Natural Science Foundation of China.
[20] H. Zhu, X.P. Xu, W. Cui, Y.Q. Zhang, H.P. Mo, Y.M. Shen, J. Poly. Sci. Part A 49
(2011) 1745e1752.
Appendix A. Supplementary material
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Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jorganchem.2012.06.001.
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