SAR study of tyrosine-chlorambucil hybrid regioisomers; Synthesis and biological evaluation against breast cancer cell lines

Article abstract of DOI:10.1007/s00726-011-1152-3

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta-and para-tyrosine-chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine-chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER and ER-) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure L-ortho, L-meta-and L-paratyrosine phenolic regioisomers. The synthesis of all tyrosine-chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERa)] are discussed in relation to the biological activity. Springer-Verlag 2011.

Full text of DOI:10.1007/s00726-011-1152-3

Amino Acids (2012) 43:923–935
DOI 10.1007/s00726-011-1152-3
ORIGINAL ARTICLE
SAR study of tyrosine–chlorambucil hybrid regioisomers;
synthesis and biological evaluation against breast cancer cell lines
•
Caroline Descoteaux Kevin Brasseur
•
ˆ
Valerie Leblanc Sophie Parent
•
•
´
Eric Asselin Gervais Berube
´
•
´
´
Received: 27 June 2011 / Accepted: 8 November 2011 / Published online: 20 November 2011
Ó Springer-Verlag 2011
Abstract Amino acids were transformed and coupled to
chlorambucil, a well-known chemotherapeutic agent, in an
attempt to create new anticancer drugs with selectivity for
breast cancer cells. Among the amino acids available,
tyrosine was selected to act as an estrogenic ligand. It is
hypothesized that tyrosine, which shows some structural
similitude with estradiol, could possibly mimic the natural
hormone and, subsequently, bind to the estrogen receptor.
In this exploratory study, several tyrosine-drug conjugates
have been designed. Thus, ortho-, meta- and para-tyro-
sine–chlorambucil analogs were synthesized in order to
generate new anticancer drugs with structural diversity,
more specifically in regards to the phenol group location.
These new analogs were produced in good yield following
efficient synthetic methodology. All the tyrosine–chlor-
ambucil hybrids were more effective than the parent drug,
chlorambucil. In vitro biological evaluation on estrogen
receptor positive and estrogen receptor negative (ER? and
ER-) breast cancer cell lines revealed an enhanced cyto-
toxic activity for compounds with the phenol function
located at position meta. Molecular docking calculations
were performed for the pure L-ortho, L-meta- and L-para-
tyrosine phenolic regioisomers. The synthesis of all tyro-
sine–chlorambucil hybrid regioisomers and their biological
activity are reported herein. Possible orientations within the
targeted protein [estrogen receptor alpha (ERa)] are dis-
cussed in relation to the biological activity.
Keywords Ortho-tyrosine–chlorambucil hybrid Á Meta-
tyrosine–chlorambucil hybrid Á Para-tyrosine–chlorambucil
hybrid Á Tyrosine–chlorambucil hybrid Á Breast cancer
Introduction
Cancer is a devastating disease, which continues to grow in
size worldwide. In recent years, much effort has been
invested in developing new treatment for cancer. Usually,
the most effective treatments are nonspecific to cancer site
and produce undesirable side effects (Magrath 1994; Pal-
mer and Wallace 2010). Hence, in order to overcome this
lack of specificity, drug targeting is still a current and
important research subject.
The estrogen receptor alpha (ERa) is a nuclear protein,
which is present in breast cancer cells and becomes over-
expressed in cancerous cells (Shanle and Xu 2010). Some
research groups have already looked at the possibility of
targeting this protein with known cytotoxic compounds
(Gust et al. 2009). It has been discovered that the toxic
moiety can concentrate only to female organs and thereby
kill cancerous cells. Recently, the anticancer property of
chlorambucil (CLL) (1) has been exploited by linking it to
estradiol (2), the sex female hormone (Gupta et al. 2010)
(Fig. 1). The new estradiol–chlorambucil hybrids (3) were
made to target cells expressing the ERa. It is important to
indicate that an estrogenic drug could, not only target the
cells, but also induce some transcriptional activity as it was
observed for other type of estrogen-cytotoxic derivatives
(Van Themsche et al. 2009). Hence, non-steroidal drugs
with structural similarities with the natural ligand could
possibly avoid such estrogenic activity.
ˆ
C. Descoteaux Á K. Brasseur Á V. Leblanc Á S. Parent Á
´
´
´
E. Asselin Á G. Berube (&)
Groupe de Recherche en Oncologie et Endocrinologie
´
´
Moleculaires, Departement de Chimie-Biologie, Universite du
Quebec a Trois-Rivieres, C.P. 500, Trois-Rivieres,
´
´
`
`
`
Natural products such as monoclonal antibodies,
enzymes, proteins, peptides and amino acids have already
´
Quebec G9A 5H7, Canada
e-mail: Gervais.Berube@uqtr.ca
123

ˆ
C. Descoteaux et al.
924
Fig. 1 Chemical structures of
chlorambucil (1), 17b-estradiol
(2), 17b-estradiol–chlorambucil
hybrids (3), tyrosine–
chlorambucil hybrids (4a, 4b)
and L-tyrosine (5)
OH
Cl
N
O
Cl
HO
HO
3
1
2
OH
OH
H
N
n
O
Cl
Cl
HO
N
3, n = 2, 4, 6
R
O
HN
3
HO
Cl
4, m = 5, 10; R = CO2CH3 or CH2OH
N
4a
Cl
Cl
O
N
R
O
O
Cl
OH
HN
m
N
NH2
HO
3
H
HO
4b
5
been transformed and used to improve anticancer treatment
(Sachdeva 1998). Drug delivery systems were conceived
and developed based on these natural entities, making the
new compounds well tolerated by the human body.
modeling study predicted possible interactions with the
ERa. However, despite the theoretical binding predictions
and despite the interesting cytotoxicity observed, these
molecules had very little in vitro affinity for the ERa when
tested experimentally.
Amino acid-drug conjugates have also been designed.
Several amino acids (proline, asparagine and glutamine)
were linked to nitrogen mustard analogs (Chrzanowski
et al. 2003; Gengrinnovitch and Izakovich 2005). These
amino-acid drug conjugates were then used as prodrugs to
treat cancer cells. Furthermore, the development of amino
acid-antioxidant conjugates and amino acid-anti-angio-
genic conjugates were also recently reported in the litera-
The study of interactions between the ERa and its
potential ligands is a time consuming and difficult task.
Structure activity relationship (SAR) studies have revealed
that various compounds with diverse and unrelated struc-
tures can bind to the ERa (Demyttenaere-Kovatcheva et al.
2005). This can be explained by the motility and plasticity
of the ERa ligand-binding cavity. Nevertheless, other
studies have found some structural requirements for spe-
cific binding to the ERa (Muthyala et al. 2003). First of all,
a phenol group, acting like the A-ring of estradiol, must be
a part of the ligand. In addition, a second hydroxyl group or
´
ture (Ortial et al. 2006; Durand et al. 2009; Contino-Pepin
et al. 2009). Hence, amino-acid derivatives are a promising
avenue, which must be further exploited in the search for
new drug candidates.
˚
a phenol function must be placed approximately 11 A from
Lately, we have reported the synthesis of D- and
L-tyrosine–chlorambucil molecules (4) (Descoteaux et al.
2010). L-Tyrosine (5) was selected as ligand to reach the
ERa as its structure shows likeness with the steroid back-
bone. These new tyrosine analogs showed potent activity
against hormone-dependent breast cancer cells. Molecular
the A-ring phenol. These two polar entities act as contact
points within the ERa binding cavity via hydrogen bonding
network.
Keeping in mind the importance of the phenol group,
and being aware that subtle structure discrepancies can
123

SAR study of tyrosine–chlorambucil hybrid regioisomers
925
change the binding affinity to the ERa, we looked into the
possibility of varying the position of the –OH group on
the phenol portion of the tyrosine–chlorambucil hybrids.
The aim of this study was to investigate how the phenol
location could affect the cytotoxic activity of these mole-
cules as well as their binding affinity.
Materials and methods
Chemistry
All reactions were performed with ACS Fisher solvents. In
some cases, solvent, as well as starting materials and
reactants, were first purified and dried by standard means
(Perrin and Armarego 1988). Anhydrous reactions required
an inert atmosphere of dry nitrogen. DL-o-tyrosine was
purchased from ABCR GmbH & Co.KG, Karlsruhe, Ger-
many, L-m-tyrosine was purchased from PepTech Corpo-
ration, Burlington, MA, USA and L-p-tyrosine was
purchased from Laboratoire Mat, Quebec, Canada. Of note,
the pure L-o-tyrosine was not commercially available. The
6-aminohexanoic acid and 11-aminoundecanoic acid were
purchased from Sigma-Aldrich Canada Ltd., Oakville,
Ontario, Canada. All reactions were monitored by UV
fluorescence or staining with iodine on Sigma T 6145
In an attempt to study this, several tyrosine-based
scaffold, DL-ortho- (DL-o-), L-meta- (L-m-) and L-para- (L-p-)
tyrosine, were used as starting materials. It is noteworthy
that the pure L-o-tyrosine was not commercially available.
However, the mixture of isomers is adequate for this
investigation, which was mainly aimed at studying the
influence of the phenol location on the cytocidal activity of
the tyrosine–chlorambucil hybrids. The chlorambucil
moiety was either directly linked to the amino acid ligand
or via an alkyl amide spacer. All of the new DL-o- (6, 7),
L-m- (8, 9) and L-p-tyrosine–chlorambucil (10, 11) (Fig. 2)
hybrid molecules synthesized were evaluated for their in
vitro cytotoxic activity on breast cancer cell lines (MCF-7,
ER? and MDA-MB-231, ER-). This manuscript gives a
detailed description of the synthesis and cytotoxic activities
of these novel tyrosine–chlorambucil hybrids. Finally,
following molecular docking calculations, the orientation
of L-o- (12, ortho-), L-m- (12, meta-) and L-p- (12, para-)
tyrosine derivatives into the targeted protein, the ERa, was
also investigated and the results are reported herein.
˚
commercial TLC plates (polyester silica gel 60 A,
0.25 mm). Purifications were done using flash column
chromatography according to the method of Still et al.
(1978) on Silicycle UltraPure Flash Silica Gel, 40–63 lm
mesh. Hexanes and acetone were distilled before their use
as chromatography eluent.
The infrared spectra were taken on a Nicolet Impact 420
FT-IR and a Thermo Nicolet IS10 FT-IR with ATR.
OH
OH
Cl
R
N
R
O
O
Cl
HN
HN
3
m
N
H
3
Cl
Cl
O
R
N
7
Cl
6
Cl
HO
HO
N
R
O
Cl
HN
HN
3
m
N
3
H
O
R
O
R
N
9
8
Cl
Cl
N
O
Cl
HN
HN
3
m
HO
HO
N
H
HO
3
Cl
O
R
O
N
11
10
Cl
a: R = CO2CH3
b: R = CH2OH
6a, R = CO2CH3; 6b, R = CH2OH
8a, R = CO2CH3; 8b, R = CH2OH
10a, R = CO2CH3; 10b, R = CH2OH
7a, R = CO2CH3, m = 5 or 10; 7b, R = CH2OH, m = 5 or 10
9a, R = CO2CH3, m = 5 or 10; 9b, R = CH2OH, m = 5 or 10
11a, R = CO2CH3, m = 5 or 10; 11b, R = CH2OH, m = 5 or 10
HN
O
12, R = CH2OH, ortho-, meta- or para
-
12
Fig. 2 DL-o- (6–7), L-m- (8–9), L-p-tyrosine–chlorambucil (10–11) hybrids and N-acetyl tyrosinols (12) regioisomers used as model for docking
calculations
123

ˆ
C. Descoteaux et al.
926
Sodium chloride, potassium bromide pellets or ATR dia-
mond accessory were used for analysis. Mass spectral
assays were obtained using a MS model 6210, Agilent
technology instrument. The high-resolution mass spectra
(HRMS) were obtained by TOF (time of flight) using ESI
(electrospray ionization) using the positive mode (ESI?)
CH2NHCO), 2.51 (2H, t, J = 7.4 Hz, CH2CH2Ph), 2.27
(2H, t, J = 7.2 Hz, CH2COOH), 2.18 (2H, t, J = 7.4 Hz,
CH2NHCOCH2), 1.78–1.96 (2H, m, CH2CH2CH2Ph),
1.28–1.67 (6H, m, 39 CH2). 13C-NMR (Acetone-d6, d
ppm): 174.3 (COOH), 173.0 (CONH), 144.8 (1-C CLL),
130.9(4-C CLL), 129.7(2C, 3-CCLL), 112.5 (2C, 2-CCLL),
53.3 (2C, 29 NCH2CH2Cl), 41.0 (29 C, 29 NCH2CH2Cl),
39.1, 35.6, 34.3, 33.6, 29.4, 27.9, 26.5, 24.7. ESI ? HRMS:
[M ? H]? calculated for C20H31Cl2N2O3 = 417.1706;
found = 417.1707.
´ ´ ` ´
(analysis at Universite du Quebec a Montreal).
Nuclear magnetic resonance (NMR) spectra were
recorded on a Varian 200 MHz NMR apparatus. Samples
were dissolved in deuteroacetone (acetone-d6) or deutero-
dimethylsulfoxide (DMSO-d6) for data acquisition using
tetramethylsilane as internal standard (TMS, d 0.0 ppm for
1H-NMR and 13C-NMR). Chemical shifts (d) are expressed
in parts per million (ppm), the coupling constants (J) are
expressed in hertz (Hz). Multiplicities are described by the
following abbreviations: s for singlet, d for doublet, dd for
doublet of doublets, dt for doublet of triplets, t for triplet, q
for quartet, m for multiplet, #m for several multiplets, br s
for broad singlet, br d for broad doublet and br t for broad
triplet.
Spectral data for N-(11-chlorambucilamino)-undecanoic
acid (14, m = 10). IR (NaCl, mmax, cm-1): 3300 (N–H),
3200–2600 (O–H), 1714 (C=O, COOH), 1618 (C=O,
NHCOO), 1526 and 1250 (C–N–H). 1H-NMR (Acetone-d6,
dppm): 7.07 (2H, d, J = 8.6 Hz, 3-CH CLL), 7.03 (1H, br s,
NH), 6.72 (2H, d, J = 9.0 Hz, 2-CH CLL), 3.75 (8H, m, 29
CH2Cl and 29 NCH2), 3.17 (2H, m, CH2NHCO), 2.90 (1H,
brs, COOH), 2.51(2H, t, J = 7.4 Hz, CH2CH2Ph), 2.27(2H,
t, J = 7.4 Hz, CH2COOH), 2.14 (2H, t, J = 7.2 Hz,
CH2NHCOCH2), 1.80–1.92 (2H, m, CH2CH2CH2Ph),
1.41–1.62 (4H, m, 29 CH2), 1.30 (12H, 69 CH2). 13C-NMR
(Acetone-d6, dppm): 174.0 (COOH), 172.1 (CONH), 144.9
(1-C CLL), 131.0 (4-C CLL), 129.7 (2C, 3-C CLL), 112.5
(2C, 2-C CLL), 53.3 (2C, 29 NCH2CH2Cl), 41.0 (2C, 29
NCH2CH2Cl), 39.0, 35.5, 34.2, 33.6, 29.8, 29.5 (2C), 29.3
(3C), 27.9, 27.0, 25.0. ESI ? HRMS: [M ? H]? calculated
for C25H41Cl2N2O3 = 487.2489; found = 487.2485.
General procedure for the preparation
of the N-chlorambucil-aminoalkyl carboxylic
acid (14, m = 5 or 10)
The amino acid (13) (6.57 mmol) was dissolved in
dichloromethane. Hexamethyldisilazane (21.69 mmol) and
concentrated sulfuric acid (catalytic) were added. The
mixture was stirred and heated to reflux under nitrogen
atmosphere until complete dissolution. The solution was
kept to reflux for 0.5 h. After cooling down, benzene, tri-
ethylamine (4.91 mmol) and chlorotrimethylsilane
(4.91 mmol) were added. The resulting mixture was stirred
at room temperature for 12 h. In another flask, chlorambucil
(1) (3.29 mmol) was dissolved in dichloromethane at 0°C.
Triethylamine (3.62 mmol) and isobutylchloroformate
(3.62 mol) were added and the mixture was kept at 0°C for
1 h. Then, the chlorambucil solution was added to the
activated amino acid solution and the mixture was stirred at
room temperature for 4 h. Work-up was done by diluting
with ethyl acetate and by washing the organic phase with
chlorhydric acid 10% solution (29) and with a saturated
sodium chloride solution. The organic phase was dried with
anhydrous magnesium sulfate, filtered and evaporated. The
product was purified by flash chromatography (hex-
anes:acetone, 7:3) to give the desired material in 98% yield.
Spectral data for N-(6-chlorambucilamino)-hexanoic
acid (14, m = 5). IR (NaCl, mmax, cm-1): 3300 (N–H),
3200–2600 (O–H), 1716 (C=O, COOH), 1622 (C=O,
NHCOO), 1522 and 1255 (C–N–H). 1H-NMR (Acetone-d6,
d ppm): 7.26 (1H, br s, NH), 7.06 (2H, d, J = 8.6 Hz,
3-CH CLL), 6.70 (2H, d, J = 8.6 Hz, 2-CH CLL), 3.73
(8H, m, 29 CH2Cl and 29 NCH2), 3.19 (2H, m,
General procedure for the preparation of tyrosine-methyl
ester hydrochloride regioisomers (16)
(DL-o-tyr, L-m-tyr, L-p-tyr)
A solution of tyrosine 15 (DL-o-, L-m- or L-p-, 1.67 mmol)
in anhydrous methanol was stirred at 0°C. Then, thionyl
chloride (5.00 mmol) was slowly added to the solution.
The mixture was heated to reflux for 12 h. Then, the sol-
vent was evaporated and the resulting crude compound was
recristalized in diethyl ether. The final material was fil-
tered, washed with cold ether and dried in a desiccator for
one day. The crude hydrochloric acid salt, obtained in a
100% yield, was pure enough to be used as such in the next
step.
Spectral data for DL-o-tyrosine methyl ester hydrochloride
(16, DL-o-). IR (ATR, mmax, cm-1): 3258 (N–H), 3076 (O–H),
1734 (C=O ester), 1246 (CO–O–C). H-NMR (DMSO-d6,
1
dppm): 8.74 (3H, br s, NH3), 7.05 (1H, t, J = 10.9 Hz,
5-CH tyr), 7.03 (1H, d J = 6.6 Hz, 3-CH tyr), 6.84 (1H, d,
J = 7.4 Hz, 6-CH tyr), 6.71 (1H, t, J = 6.9 Hz, 4-CH tyr),
4.08 (1H, t, J = 6.7 Hz, CHNH3), 3.58 (3H, s, OCH3), 3.02
(2H, d, J = 6.7 Hz, CH2CH), 2.48 (1H, s, OH). 13C-NMR
(DMSO-d6, d ppm): 170.0 (COOCH3), 156.2 (1-C tyr),
131.5 (3-C tyr), 129.1 (2-C tyr), 121.2 (5-C tyr), 119.4 (4-C
tyr), 115.4 (6-C tyr), 52.9 (CHNH3), 52.4 (OCH3), 32.0
123

SAR study of tyrosine–chlorambucil hybrid regioisomers
927
(CH2CH). ESI ? HRMS: [M ? H]? calculated for
C10H14NO3 = 196.0968; found = 196.0966.
CHNH), 3.74 (8H, dt, J = 4.7 Hz and J = 1.2 Hz,
CH2Cl and NCH2), 3.63 (3H, s, OCH3), 2.92–3.20 (2H,
m, CH2CHNH), 2.46 (2H, t, J = 7.7 Hz, CH2CH2Ph),
2.17 (2H, t, J = 7.4 Hz, NHCOCH2), 1.76–1.87 (2H, m,
CH2CH2CH2). 13C-NMR (Acetone-d6, d ppm): 172.48
(NHCOCH2), 172.3 (COOCH3), 155.4 (1-C tyr), 144.6 (1-C
CLL), 131.1 (3-C tyr), 130.6 (C, 4-C CLL), 129.5 (2C, 3-C
CLL), 128.0 (2-C tyr), 123.5 (5-C tyr), 119.5 (4-C tyr), 115.1
(C, 6-C tyr), 112.2 (2C, 2-C CLL), 53.0 (2C, 29
NCH2CH2Cl), 52.8 (CHNH), 51.2 (OCH3), 41.8 (29 C, 29
NCH2CH2Cl), 34.9 (CH2CHNH), 33.7 (CH2CH2Ph), 32.4
(NHCOCH2), 27.4 (CH2CH2CH2). ESI ? HRMS:
[M ? H]? calculated for C24H31Cl2N2O4 = 481.1655;
found = 481.1652.
Spectral data for L-m-tyrosine methyl ester hydrochloride
(16, L-m-). IR (ATR, mmax, cm-1): 3243 (N–H), 3194 (O–H),
1745 (C=O ester), 1231 (CO–O–C). H-NMR (DMSO-d6,
1
dppm): 9.53 (1H, br s, OH), 8.66 (3H, br s, NH3), 7.08 (1H, t,
J = 7.7 Hz, 5-CH tyr), 6.69–6.59 (2H, m, 4-CH tyr and
6-CH tyr), 6.62 (1H, s, 2-CH tyr), 4.16 (1H, t, J = 6.25 Hz,
CHNH3), 3.65 (3H, s, OCH3), 3.14–2.92 (2H, m, CH2CH).
13C-NMR (DMSO-d6, dppm): 170.0 (COOCH3), 158.0 (1-C
tyr), 136.2(3-C tyr), 130.0(5-Ctyr), 120.3(4-Ctyr), 116.7(2-
C tyr), 114.8 (6-C tyr), 53.7 (CHNH3), 53.0 (OCH3), 36.3
(CH2CH). ESI ? HRMS: [M ? H]? calculated for
C10H14NO3 = 196.0968; found = 196.0964.
Spectral data for L-p-tyrosine methyl ester hydrochloride
(16, L-p-). IR (KBr, mmax, cm-1): 3354 (N–H), 3078 (O–H),
1742 (C=O ester), 1224 (CO–O–C). H-NMR (DMSO-d6,
Spectral data for N-chlorambucil-L-m-tyrosine methyl
ester (8a). IR (ATR, mmax, cm-1): 3100–3450 (O–H and
N–H), 1748 (C=O, COOCH3), 1643 (C=O, NHCO), 1519
(C–N–H). 1H-NMR(Acetone-d6,dppm): 8.31 (1H, s, OH), 7.28
(1H, d, J = 8.2 Hz, CHNHCO), 7.06 (1H, t, J = 8.4 Hz,
5-CH tyr), 7.03 (2H, d, J = 9.0 Hz, 3-CH CLL), 6.72 (1H,
s, 2-CH tyr), 6.70 (4H, 3d, J = 9.0 Hz, 4-CH tyr, 6-CH tyr
and 2-CH CLL), 4.72 (1H, m, CHNH), 3.74 (8H, dt,
J = 4.7 Hz and J = 1.2 Hz, CH2Cl and NCH2), 3.66 (3H,
s, OCH3), 2.85–3.12 (2H, m, CH2CHNH), 2.46 (2H, t,
J = 7.7 Hz, CH2CH2Ph), 2.18 (2H, t, J = 7.4 Hz,
NHCOCH2), 1.76–1.88 (2H, m, CH2CH2CH2). 13C-NMR
1
d ppm): 9.46 (1H, br s, OH), 8.60 (3H, br s, NH3), 7.01
(2H, d, J = 8.6 Hz, 3-CH tyr), 6.73 (2H, d, J = 8.60 Hz,
2-CH tyr), 4.16 (1H, m, CHNH3), 3.67 (3H, s, OCH3), 3.03
(2H, m, CH2CH). 13C-NMR (DMSO-d6, d ppm): 170.1
(COOCH3), 157.4 (1-C tyr), 131.0 (2C, 3-C tyr), 125.0
(4-C tyr), 116.1 (2C, 2-C tyr), 54.1 (CHNH3), 53.2
(OCH3), 35.7 (CH2CH). ESI ? HRMS: [M ? H]? calcu-
lated for C10H14NO3 = 196.0968; found = 196.0968.
General procedure for the preparation of N-chlorambucil-
DL-o-, N-chlorambucil-L-m- and N-chlorambucil-L-p-
tyrosine methyl ester (6a, 8a and 10a):
(Acetone-d6, d ppm): 172.18 (NHCOCH2), 172.10
(COOCH3), 157.4 (1-C tyr), 144.6 (1-C CLL), 138.7 (3-C
tyr), 130.6 (4-C CLL), 129.5 (2C, 3-C CLL), 129.3 (5-C
tyr), 120.2 (4-C tyr), 116.0 (2-C tyr), 113.7 (6-C tyr), 112.2
(2C, 2-C CLL), 53.4 (CHNH), 53.0 (2C, 29 NCH2CH2Cl),
51.3 (OCH3), 40.8 (29 C, 29 NCH2CH2Cl), 37.3
(CH2CHNH), 34.8 (CH2CH2Ph), 33.7 (NHCOCH2), 27.4
(CH2CH2CH2). ESI ? HRMS: [M ? H]? calculated for
C24H31Cl2N2O4 = 481.1655; found = 481.1650.
The appropriate tyrosine methyl ester 16 (0.32 mmol) was
dissolved in dimethylformamide and triethylamine
(0.32 mmol) was added. At the same time, a solution of
chlorambucil (1) (0.49 mmol) in dimethylformamide was
activated
using
dicyclohexylcarbodiimide
(DCC)
(0.52 mmol) followed by N-hydroxybenzotriazole (HOBt)
(0.52 mmol). The tyrosine solution was then added to the
activated acid chlorambucil solution. The mixture was
stirred at room temperature under nitrogen at room tem-
perature for 24 h. The solution was diluted with ethyl
acetate and water, and then washed with water (49). The
organic phase was dried with sodium sulfate, filtered and
evaporated. The product was further purified by flash
chromatography (hexanes: acetone, 4:1) to give a pure
compound in 81% yield.
Spectral data for N-chlorambucil-L-p-tyrosine methyl
ester (10a). The spectral data for this particular compound
were reported elsewhere and are in accordance with the
literature (Descoteaux et al. 2010).
General procedure for the preparation of N-chlorambucil-
DL-o-, N-chlorambucil-L-m- and N-chlorambucil-L-p-
tyrosinol (6b, 8b and 10b)
The N-chlorambuciL-tyrosine methyl ester (6a, 8a or 10a)
(0.15 mmol) was dissolved in diethyl ether and dichloro-
methane and was stirred under nitrogen atmosphere. The
resulting solution was cooled down with an ice and water
bath, afterwards lithium borohydride (0.90 mmol) was
added. The mixture was kept at 0°C for 3 h. Afterwards
sodium sulfate decahydrate (0.3 g) was added. Work-up
was done by diluting with diethyl ether and the organic
phase was washed with saturated ammonium chloride
Spectral data for N-chlorambucil-DL-o-tyrosine methyl
ester (6a). IR (ATR, mmax, cm-1): 3150–3400 (O–H and
N–H), 1745 (C=O, COOCH3), 1652 (C=O, NHCO), 1524
(C–N–H). 1H-NMR (Acetone-d6, d ppm): 8.74 (1H, s,
OH), 7.39 (1H, d, J = 7.4 Hz, CHNHCO), 7.01–7.13
(2H, m, 3-CH and 5-CH tyr), 7.03 (2H, d, J = 8.6 Hz,
3-CH CLL), 6.67–6.88 (2H, m, 4-CH and 6-CH tyr),
6.69 (2H, d, J = 9.0 Hz, CH CLL), 4.75 (1H, m,
123

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C. Descoteaux et al.
928
General procedure for the preparation of N-[(N-
chlorambucilamino)alcanoyl]- DL-o-, L-m-
and L-p-tyrosine methyl ester (7a, 9a and 11a)
solution (29) and with water (49). The organic phase was
dried with anhydrous sodium sulfate, filtered and evapo-
rated. The product was purified by flash chromatography
(hexanes: acetone, 7:3) to give a pure compound in 88%
yield.
The appropriate tyrosine methyl ester hydrochloride 16
(0.41 mmol) was dissolved in dimethylformamide and
triethylamine (0.41 mmol). The latter was added in order to
neutralize the hydrochloride salt. At the same time, a
solution of N-chlorambucil-aminoalkyl carboxylic acid 14
(m = 5 or 10) (0.62 mmol) in dimethylformamide was
activated using DCC (0.66 mmol) followed by HOBt
(0.66 mmol). The tyrosine solution was then added to the
activated N-chlorambucil-aminoalkyl carboxylic acid
solution. The mixture was stirred at room temperature for
24 h. The solution was diluted with ethyl acetate and water,
and then washed with water (49). The organic phase was
dried with sodium sulfate, filtered, and evaporated. The
product was further purified by flash chromatography
(hexanes: acetone, 7:3) to give a pure compound in 71%
yield.
Spectral data for N-chlorambucil-DL-o-tyrosinol (6b).
IR (ATR, mmax, cm-1): 3100–3450 (O–H and N–H), 1638
(C=O, NHCO), 1517 and 1242 (C–N–H). 1H-NMR
(Acetone-d6, d ppm): 9.03 (1H, s, OH), 7.35 (1H, d,
J = 6.3 Hz, CHNHCO), 7.02–7.11 (2H, m, 3-CH tyr and
5-CH tyr), 7.07 (2H, d, J = 8.6 Hz, 3-CH CLL), 6.71
(2H, d, J = 9.0 Hz, 2-CH CLL), 6.69–6.85 (2H, m,
4-CH tyr and 6-CH tyr), 4.18 (1H, t, J = 5.7 Hz,
CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 29
CH2Cl and 29 NCH2), 3.58 (2H, dt, J = 1.2 Hz and
J = 5.5 Hz, CH2OH), 2.92 (1H, s, CH2OH), 2.78–2.91
(2H, m, CH2CHNH), 2.52 (2H, t, J = 7.4 Hz,
CH2CH2Ph), 2.25 (2H, t, J = 7.3 Hz, NHCOCH2),
1.83–1.94 (2H, m, CH2CH2CH2). 13C-NMR (Acetone-d6,
d ppm): 173.8 (NHCOCH2), 156.0 (1-C tyr), 144.7 (1-C
CLL), 130.9 (4-C CLL), 130.6 (3-C tyr), 129.5 (2C, 3-C
CLL), 127.7 (5-C tyr), 124.6 (2-C tyr), 119.3 (4-C tyr),
115.8 (6-C tyr), 112.2 (2C, 2-C CLL), 62.3 (CH2OH),
54.1 (CHNH), 53.0 (2C, 29 NCH2CH2Cl), 40.7 (2C, 29
NCH2CH2Cl), 35.1 (CH2CHNH), 33.9 (CH2CH2Ph),
31.9 (NHCOCH2), 27.5 (CH2CH2CH2). ESI ? HRMS:
[M ? H]? calculated for C23H31Cl2N2O3 = 453.1706;
found = 453.1702.
Spectral data for N-[(6-N-chlorambucilamino)hexa-
noyl]-DL-o-tyrosine methyl ester (7a, m = 5). IR (ATR,
m
max, cm-1): 3100–3400 (O–H and 29 N–H), 1745 (C=O,
COOCH3), 1634 (29 C=O, 29 NHCO), 1520 and 1253
(C–N–H). 1H-NMR (Acetone-d6, d ppm : 9.04 (1H, s, OH),
7.36–7.20 (2H, 2d apparent, CHNHCO and CH2NHCO),
7.06–7.10 (2H, m, 3-CH and 5-CH tyr), 7.08 (2H, d,
J = 7.0 Hz, 3-CH CLL), 6.75–6.92 (2H, 2d apparent,
4-CH and 6-CH tyr), 6.72 (2H, d, J = 6.6 Hz, 2-CH CLL),
4.67 (1H, m, CHNH), 3.75 (8H, s, 29 CH2Cl and 29
NCH2), 3.63 (3H, s, OCH3), 3.17 (2H, br m, CH2NHCO),
2.92–3.17 (2H, m, CH2CHNH), 2.53 (2H, t, J = 7.0 Hz,
CH2CH2Ph), 2.15 (4H, m apparent, CH2NHCOCH2 and
CHNHCOCH2), 1.84–1.95 (2H, m, CH2CH2CH2Ph),
1.28–1.58 (6H, #m, 39 CH2). 13C-NMR (Acetone-d6, d
ppm): 172.4 (CONH), 172.2 (2C, CONH and COOCH3),
155.5 (1-C tyr), 144.7 (1-C CLL), 131.1 (3-C tyr), 130.7 (4-
C CLL), 129.5 (2C, 3-C CLL), 128.0 (2-C tyr), 123.6 (5-C
tyr), 119.5 (4-C tyr), 115.1 (6-C tyr), 112.2 (2C, 2-C CLL),
53.2 (CHNH), 53.0 (2C, 29 NCH2CH2Cl), 51.2 (OCH3),
40.7 (2C, 29 NCH2CH2Cl), 38.6, 35.4, 35.3, 34.0, 32.2,
29.0, 27.7, 25.8, 24.9. ESI ? HRMS: [M ? H]? calculated
for C30H42Cl2N3O5 = 594.2496; found = 594.2488.
Spectral data for N-chlorambucil-L-m-tyrosinol (8b). IR
(ATR, mmax, cm-1): 3100–3450 (O–H and N–H), 1619
(C=O, NHCO), 1526 and 1254 (C–N–H). H-NMR (Ace-
1
tone-d6, d ppm): 8.34 (1H, s, OH), 7.08 (1H, t, J = 7.4 Hz,
5-CH tyr), 7.03 (2H, d, J = 8.6 Hz, 3-CH CLL), 6.97–7.01
(1H, m, CHNHCO), 6.70 (2H, d, J = 8.6 Hz, 2-CH CLL),
6.64–6.79 (3H, #m, 2-CH tyr, 4-CH tyr and 6-CH tyr), 4.14
(1H, m, CHNH), 3.74 (8H, dt, J = 1.2 Hz and J = 4.7 Hz,
29 CH2Cl and 29 NCH2), 3.53 (2H, d, J = 4.7 Hz,
CH2OH), 2.67–2.91 (2H, m, CH2CHNH), 2.46 (2H, t,
J = 7.5 Hz, CH2CH2Ph), 2.16 (2H, t, J = 7.4 Hz,
NHCOCH2), 1.77–1.88 (2H, m, CH2CH2CH2). 13C-NMR
(Acetone-d6, d ppm): 172.4 (NHCOCH2), 157.4 (1-C tyr),
144.6 (1-C CLL), 140.6 (3-C tyr), 130.7 (4-C CLL), 129.5
(2C, 3-C CLL), 129.1 (5-C tyr), 120.3 (4-C tyr), 116.1 (2-C
tyr), 113.1 (6-C tyr), 112.2 (2C, 2-C CLL), 63.2 (CH2OH),
53.0 (2C, 29 NCH2CH2Cl), 52.8 (CHNH), 40.8 (2C, 29
NCH2CH2Cl), 36.8 (CH2CHNH), 35.3 (CH2CH2Ph), 33.9
(NHCOCH2), 27.6 (CH2CH2CH2). ESI ? HRMS:
[M ? H]? calculated for C23H31Cl2N2O3 = 453.1706;
found = 453.1707.
Spectral data for N-[(6-N-chlorambucilamino)hexa-
noyl]-L-m-tyrosine methyl ester (9a, m = 5). IR (ATR,
m
max, cm-1): 3150–3450 (O–H and 29 N–H), 1745 (C=O,
COOCH3), 1638 (29 C=O, 29 NHCO), 1517 and 1217
(C–N–H). 1H-NMR (Acetone-d6, d ppm): 8.66 (1H, s,
OH), 7.24 (1H, d, J = 7.8 Hz, CHNHCO), 7.16 (1H, m,
CH2NHCO), 7.09 (1H, t apparent, J = 8.2 Hz, 5-CH tyr),
7.07 (2H, d, J = 8.6 Hz, 3-CH CLL), 6.71 (2H, d,
J = 8.6 Hz, 2-CH CLL), 6.64–6.75 (3H, m apparent,
2-CH tyr, 4-CH tyr and 6-CH tyr), 4.71 (1H, m, CHNH),
Spectral data for N-chlorambucil-L-p-tyrosinol (10b).
The spectral data for this particular compound were
reported elsewhere and are in accordance with the literature
(Descoteaux et al. 2010).
123

SAR study of tyrosine–chlorambucil hybrid regioisomers
929
3.75 (8H, dt, J = 1.2 Hz and J = 5.1 Hz, 29 CH2Cl and
29 NCH2), 3.66 (3H, s, OCH3), 3.17 (2H, m, CH2NHCO),
2.84–3.10 (2H, m, CH2CHNH), 2.52 (2H, t, J = 7.7 Hz,
CH2CH2Ph), 2.18 (4H, m apparent, CH2NHCOCH2 and
CHNHCOCH2), 1.82–1.94 (2H, m, CH2CH2CH2Ph),
1.20–1.59 (6H, #m, 39 CH2). 13C-NMR (Acetone-d6, d
ppm): 172.5 (CONH), 172.1 (CONH), 172.0 (COOCH3),
157.6 (1-C tyr), 144.6 (1-C CLL), 138.6 (3-C tyr), 130.6
(4-C CLL), 129.5 (2C, 3-C CLL), 129.2 (5-C tyr), 120.1 (4-
C tyr), 116.3 (2-C tyr), 113.8 (6-C tyr), 112.2 (2C, 2-C
CLL), 53.3 (CHNH), 53.0 (2C, 29 NCH2CH2Cl), 51.3
(OCH3), 40.7 (2C, 29 NCH2CH2Cl), 38.7, 37.4, 35.4 (2C),
34.0, 27.7, 26.1, 25.1. ESI ? HRMS: [M ? H]? calculated
for C30H42Cl2N3O5 = 594.2496; found = 594.2491.
Spectral data for N-[(6-N-chlorambucilamino)hexa-
noyl]-L-p-tyrosine methyl ester (11a, m = 5). The spectral
data for this particular compound were reported elsewhere
and are in accordance with the literature (Descoteaux et al.
2010).
6-CH tyr), 4.69 (1H, m, CHNH), 3.74 (8H, dt, J = 1.6 Hz
and J = 5.1 Hz, 29 CH2Cl and 29 NCH2), 3.65 (3H, s,
OCH3), 3.19 (2H, q, J = 5.9 Hz, CH2NHCO), 2.83–3.10
(2H, m, CH2CHNH), 2.51 (2H, t, J = 7.6 Hz,
CH2CH2Ph), 2.16 (4H, 2t overlapped, J = 7.2 Hz,
CH2NHCOCH2 and CHNHCOCH2), 1.81–1.92 (2H, m,
CH2CH2CH2Ph), 1.25–1.55 (16H, #m and s, 89 CH2).
13C-NMR (Acetone-d6, d ppm): 172.2 (CONH), 172.1 (2C,
COOCH3 and CONH), 157.5 (1-C tyr), 144.6 (1-C CLL),
138.6 (3-C tyr), 130.7 (4-C CLL), 129.5 (2C, 3-C CLL),
129.2 (5-C tyr), 120.1 (4-C tyr), 116.1 (2-C tyr), 113.6 (6-C
tyr), 112.2 (2C, 3-C CLL), 53.4 (CHNH), 53.0 (2C, 29
NCH2CH2Cl), 51.3 (OCH3), 40.7 (2C, 29 NCH2CH2Cl),
38.8, 37.4, 35.5, 35.3, 34.0, 29.6, 29.2, 29.1, 28.9, 27.7,
26.7, 25.4. ESI ? HRMS: [M ? H]? calculated for
C35H52Cl2N3O5 = 664.3279; found = 664.3270.
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-L-p-tyrosine methyl ester (11a, m = 10). The spec-
tral data for this particular compound were reported
elsewhere and are in accordance with the literature
(Descoteaux et al. 2010).
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-DL-o-tyrosine methyl ester (7a, m = 10). IR (ATR,
m
COOCH3), 1645 and 1602 (29 C=O and 29 NHCO), 1520
max, cm-1): 3200–3400 (O–H and 29 N–H), 1749 (C=O,
General procedure for the preparation
of N-[(N-chlorambucilamino)alcanoyl]- DL-o-,
L-m- and L-p-tyrosinol (7b, 9b and 11b)
1
and 1213 (C–N–H). H-NMR (Acetone-d6, d ppm): 8.83
(1H, br s, OH), 7.31 (1H, d, J = 7.4 Hz, CHNHCO),
7.03–7.12 (3H, m, CH2NHCO, 3-CH tyr and 5-CH tyr),
7.07 (2H, d, J = 8.6 Hz, 3-CH CLL), 6.69–6.89 (2H, m,
4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 9.0 Hz,
2-CH CLL), 4.69 (1H, m, CHNH), 3.74 (8H, dt,
J = 1.6 Hz and J = 5.1 Hz, 29 CH2Cl and 29 NCH2),
3.62 (3H, s, OCH3), 3.19 (2H, q, J = 5.9 Hz, CH2NHCO),
2.90–3.16 (2H, m, CH2CHNH), 2.52 (2H, t, J = 7.5 Hz,
CH2CH2Ph), 2.17 (4H, m, CH2NHCOCH2 and CHNH-
COCH2), 1.82–1.90 (2H, m, CH2CH2CH2Ph), 1.25–1.54
(16H, #m and s, 89 CH2). 13C-NMR (Acetone-d6, d ppm):
172.3 (2C, 29 CONH), 172.1 (COOCH3), 155.4 (1-C tyr),
144.7 (1-C CLL), 131.1 (3-C tyr), 130.7 (4-C CLL), 129.4
(2C, 3-C CLL), 128.0 (2-C tyr), 123.6 (5-C tyr), 119.4 (4-C
tyr), 115.1 (6-C tyr), 112.2 (2C, 3-C CLL), 53.0 (2C, 29
NCH2CH2Cl), 52.8 (CHNH), 51.1 (OCH3), 40.7 (2C, 29
NCH2CH2Cl), 38.8, 35.5, 35.3, 34.0, 33.7, 32.4, 29.5,
29.2, 29.1, 29.0, 28.8, 27.7, 26.6. ESI ? HRMS:
[M ? H]? calculated for C35H52Cl2N3O5 = 664.3279;
found = 664.3271.
The N-[(N-chlorambucilamino)alcanoyl)-tyrosine methyl
ester (7a, 9a or 11a) (0.12 mmol) was dissolved in diethyl
ether and dichloromethane and was stirred under nitrogen
atmosphere. The resulting solution was cooled down with
an ice and water bath; afterwards lithium borohydride
(0.73 mmol) was added. The mixture was kept at 0°C for
3 h. Afterwards, sodium sulfate decahydrate (0.3 g) was
added. Work-up was done by diluting with diethyl ether
and washing the organic phase with saturated ammonium
chloride solution (29) and with water (49). The organic
phase was dried with anhydrous sodium sulfate, filtered
and evaporated. The product was purified by flash chro-
matography (hexanes: acetone, 3:2) to give a pure com-
pound in 82% yield.
Spectral data for N-[(6-N-chlorambucilamino)hexa-
noyl]-DL-o-tyrosinol (7b, m = 10). IR (ATR, mmax, cm-1):
3100–3400 (29 O–H and 29 N–H), 1638 (29 C=O, 29
NHCO), 1520 and 1242 (C–N–H). 1H-NMR (Acetone-d6, d
ppm): 9.16 (1H, br s, OH), 7.37 (1H, d, J = 6.6 Hz,
CHNHCO), 7.17 (1H, br t, J = 5.1 Hz, CH2NHCO),
7.02–7.11 (2H, m apparent, 3-CH tyr and 5-CH tyr), 7.06
(2H, d, J = 8.6 Hz, 3-CH CLL), 6.70–6.88 (2H, m
apparent, 4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 8.5 Hz,
2-CH CLL), 4.01 (1H, m, CHNH), 3.89 (1H, m, CH2OH),
3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 29 CH2Cl and
29 NCH2), 3.51 (2H, d apparent, J = 5.8 Hz, CH2OH),
3.19 (2H, dq, J = 1.8 Hz and J = 6.8 Hz, CH2NHCO),
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-L-m-tyrosine methyl ester (9a, m = 10). IR (ATR,
m
COOCH3), 1642 and 1613 (29 C=O and 29 NHCO), 1517
max, cm-1): 3150–3400 (O–H and 29 N–H), 1745 (C=O,
1
and 1253 (C–N–H). H-NMR (Acetone-d6, d ppm): 8.44
(1H, br s, OH), 7.26 (1H, d, J = 7.8 Hz, CHNHCO), 7.10
(1H, m, CH2NHCO), 7.04–7.13 (1H, m5-CH tyr), 7.06
(2H, d, J = 8.2 Hz, 3-CH CLL), 6.71 (2H, d, J = 9.0 Hz,
2-CH tyr), 6.65–6.75 (3H, m,2-CH tyr, 4-CH tyr and
123

ˆ
C. Descoteaux et al.
930
2.71–2.93 (2H, m, CH2CHNH), 2.51 (2H, t, J = 7.4 Hz,
CH2CH2Ph), 2.19 (4H, m apparent, CH2NHCOCH2 and
CHNHCOCH2), 1.78–1.93 (2H, m, CH2CH2CH2Ph),
1.27–1.67 (6H, #m, 39 CH2). 13C-NMR (Acetone-d6, d
ppm): 173.8 (CONH), 172.4 (CONH), 156.0 (1-C tyr),
144.7 (1-C CLL), 131.0 (4-C CLL), 130.7 (3-C tyr), 129.5
(2C, 3-C CLL), 127.7 (5-C tyr), 124.7 (2-C tyr), 119.3 (4-C
tyr), 115.7 (6-C tyr), 112.2 (2C, 2-C CLL), 62.6 (CH2OH),
53.2 (CHNH), 53.0 (2C, 29 NCH2CH2Cl), 40.8 (29 C, 29
NCH2CH2Cl), 38.6, 35.7, 35.4, 34.0, 31.8, 29.2, 27.7,
26.1, 25.1. ESI ? HRMS: [M ? H]? calculated for
C29H42Cl2N3O4 = 566.2547; found = 566.2540.
CH2OH), 2.52 (2H, t, J = 7.6 Hz, CH2CH2Ph), 2.22 (2H, t,
J = 7.2 Hz, CH2NHCOCH2), 2.16 (2H, t, J = 7.2 Hz,
CHNHCOCH2), 1.81–1.93 (2H, m, CH2CH2CH2Ph),
1.29–1.59 (16H, #m and s, 8x CH2). 13C-NMR (Acetone-d6,
d ppm): 173.9 (CONH), 172.0 (CONH), 156.0 (1-C tyr),
144.7 (1-C CLL), 130.9 (4-C CLL), 130.7 (3-C tyr), 129.4
(2C, 3-C CLL), 127.7 (5-C tyr), 124.6 (2-C tyr), 119.2 (4-C
tyr), 115.8 (6-C tyr), 112.2 (2C, 2-C CLL), 62.3 (CH2OH),
53.1 (CHNH), 53.0 (2C, 29 NCH2CH2Cl), 40.7 (29 C, 29
NCH2CH2Cl), 38.8, 35.7, 35.3, 34.0, 31.9, 29.6, 29.2 (3C),
29.0 (2C), 26.7. ESI ? HRMS: [M ? H]? calculated for
C34H52Cl2N3O4 = 636.3329; found = 636.3318.
Spectral data for N-[(6-N-chlorambucilamino)hexanoyl)-
L-m-tyrosinol (9b, m = 10). IR (ATR, mmax, cm-1):
3100–3400 (29 O–H and 29 N–H), 1683 (29 C=O, 29
NHCO), 1520 and 1253 (C–N–H). 1H-NMR (Acetone-d6, d
ppm): 8.59 (1H, s, OH), 7.20 (1H, br t, J = 5.3 Hz,
CH2NHCO), 7.07 (1H, t, J = 7.4 Hz, 5-CH tyr), 7.06 (2H, d,
J = 8.6 Hz, 3-CH CLL), 6.98 (1H, d, J = 8.5 Hz,
CHNHCO), 6.64–6.76 (3H, m, 2-CH tyr, 4-CH tyr and
6-CH tyr), 6.71 (2H, d, J = 8.5 Hz, 2-CH CLL), 4.13 (2H, m,
CHNH and CH2OH), 3.74 (8H, dt, J = 1.2 Hz and
J = 5.1 Hz, 29 CH2Cl and 29 NCH2), 3.52 (2H, t,
J = 5.1 Hz, CH2OH), 3.16 (2H, m, CH2NHCO), 2.64–2.91
(2H, m, CH2CHNH), 2.51 (2H, t, J = 7.6 Hz, CH2CH2Ph),
2.14 (4H, m apparent, CH2NHCOCH2 and CHNHCOCH2),
1.82–1.89 (2H, m, CH2CH2CH2Ph), 1.20–1.58 (6H, #m, 39
CH2). 13C-NMR (Acetone-d6, d ppm): 172.51 (CONH),
172.46 (CONH), 157.5 (1-C tyr), 144.6 (1-C CLL), 140.5 (3-
C tyr), 130.6 (4-C CLL), 129.5 (2C, 3-C CLL), 129.0 (5-C
tyr), 120.3 (4-C tyr), 116.3 (2-C tyr), 113.1 (6-C tyr), 112.2
(2C, 2-C CLL), 63.4 (CH2OH), 53.0 (2C, 29 NCH2CH2Cl),
52.8 (CHNH), 40.8 (29 C, 29 NCH2CH2Cl), 38.7
(CH2NHCO), 36.8(CH2CHNH), 35.9(CHNHCOCH2), 35.6
(CH2CH2Ph), 35.4 (CH2NHCOCH2), 34.0, 27.7, 26.2, 25.3.
ESI ? HRMS: [M ? H]? calculated for C29H42Cl2N3O4 =
566.2547; found = 566.2541.
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-L-m-tyrosinol (9b, m = 10). IR (ATR, mmax, cm-1):
3100–3400 (29 O–H and 29 N–H), 1634 (29 C=O,
NHCO), 1520 and 1246 (C–N–H). 1H-NMR (Acetone-d6, d
ppm): 8.45 (1H, s, OH), 7.14 (1H, br t apparent,
CH2NHCO), 7.07 (1H, t, J = 7.6 Hz, 5-CH tyr), 7.06 (2H,
d, J = 8.6 Hz, 3-CH CLL), 6.97 (1H, d, J = 7.8 Hz,
CHNHCO), 6.65–6.77 (3H, m, 2-CH tyr, 4-CH tyr and
6-CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH CLL), 4.08 (1H,
m, CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 29
CH2Cl and 29 NCH2), 3.52 (2H, t, J = 5.1 Hz, CH2OH),
3.19 (2H, q, J = 6.5 Hz, CH2NHCO), 2.92 (1H, s,
CH2OH), 2.65–2.91 (2H, m, CH2CHNH), 2.52 (2H, t,
J = 7.4 Hz, CH2CH2Ph), 2.13 (4H,
m
apparent,
CH2NHCOCH2 and CHNHCOCH2), 1.82–1.93 (2H, m,
CH2CH2CH2Ph), 1.25–1.56 (16H, #m and s, 89 CH2).
13C-NMR (Acetone-d6, d ppm): 172.5 (CONH), 172.2
(CONH), 157.5 (1-C tyr), 144.6 (1-C CLL), 140.6 (3-C
tyr), 130.7 (4-C CLL), 129.5 (2C, 3-C CLL), 129.0 (5-C
tyr), 120.3 (4-C tyr), 116.2 (2-C tyr), 113.1 (6-C tyr), 112.2
(2C, 2-C CLL), 63.3 (CH2OH), 53.0 (2C, 29
NCH2CH2Cl), 52.8 (CHNH), 40.7 (2C, 29 NCH2CH2Cl),
38.8, 36.8, 36.0, 35.4, 34.0, 29.6, 29.3, 29.2, 29.1, 29.0,
28.9, 27.7, 26.7, 25.6. ESI ? HRMS: [M ? H]? calculated
for C34H52Cl2N3O4 = 636.3329; found = 636.3326.
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-L-p-tyrosinol (11b, m = 10). The spectral data for
this particular compound were reported elsewhere and are
in accordance with the literature (Descoteaux et al. 2010).
Spectral data for N-[(6-N-chlorambucilamino)hexa-
noyl]-L-p-tyrosinol (11b, m = 5). The spectral data for this
particular compound were reported elsewhere and are in
accordance with the literature (Descoteaux et al. 2010).
Spectral data for N-[(11-N-chlorambucilamino)undeca-
noyl]-DL-o-tyrosinol (7b, m = 10). IR (ATR, mmax, cm-1):
3100–3400 (29 O–H and 29 N–H), 1631 (29 C=O,
NHCO), 1524 and 1246 (C–N–H). 1H-NMR (Acetone-d6, d
ppm): 9.11 (1H, s, OH), 7.36 (1H, d, J = 5.9 Hz,
CHNHCO), 7.02–7.10 (3H, m, 3-CH tyr, 5-CH tyr and
CH2NHCO), 7.07 (2H, d, J = 8.6 Hz, 3-CH CLL),
6.68–6.86 (2H, m, 4-CH tyr and 6-CH tyr), 6.71 (2H, d,
J = 8.6 Hz, 2-CH CLL), 4.11 (1H, m, CHNH), 3.74 (8H, dt,
J = 1.6 Hz and J = 5.1 Hz, 29 CH2Cl and 29 NCH2), 3.58
(2H,br d, J = 5.1 Hz, CH2OH), 3.18 (2H, q, J = 5.8 Hz,
CH2NHCO), 2.70–2.93 (3H, m and br s, CH2CHNH and
Biology
In vitro cytotoxic activity
The cytotoxicity of the tyrosine–chlorambucil regioisomers
(6a–11a and 6b–11b, m = 5 or 10) was evaluated on
MCF-7 (ER?) and MDA-MD-231 (ER-) breast cancer cell
lines. MTT (3-(4,5-dimethylthiazol-2-yl)-phenyl-tetrazo-
lium bromide) assay, a standard colorimetric test, was used
for measuring cellular proliferation (Carmichael et al.
1987). Briefly, tumor cell lines were added into 96-well
123

SAR study of tyrosine–chlorambucil hybrid regioisomers
931
tissue culture plates in culture medium and incubated at
37°C in a 5% CO2 atmosphere. Dilutions were done using
cremophore:ethanol (1:1) solution. Cells were incubated
with or without drugs for 72 h. Culture plates were pro-
cessed using MTT for 3.5 h afterwards SDS solubilisation
solution (HCl 0.010 M, sodium dodecyl sulfate solution
10%) was added. The absorbance was read using a scan-
ning multiwell spectrophotometer (FLUOStar OPTIMA) at
565 nm. All measurements were carried in triplicates. The
results were compared with those of a control reference
plate fixed on the treatment day, and the growth inhibition
percentage was calculated for each drug contact period.
optimization was performed according to molecular
mechanics (MM2) calculations with ChemBio3D Ultra
11.0 program. Even if the synthesis was achieved with a
mixture of DL-ortho-tyrosine precursor, the pure L-o-ty-
rosinol enantiomer was chosen for docking calculations.
The resulting ligand file was saved as a pdb file. Next, in
ArgusLab, the binding site was defined. ArgusDock search
˚
docking engine was used with a grid resolution of 0.40 A.
Then, docking calculations were performed with a flexible
ligand mode.
Results and discussion
Molecular modelling
Synthesis of tyrosine–chlorambucil hybrid regioisomers
17b-estradiol (2), L-o-N-acetyltyrosinol (12, ortho-), L-m–
N-acetyltyrosinol (12, meta-) and L-p-N-acetyltyrosinol
(12, para-), our model compounds, were docked into the
ERa. This was done using ArgusDock docking engine,
implemented in ArgusLab 4.0. This software is freely
distributed for Windows platforms by Planaria Software
(http://www.ArgusLab.com) (Thompson 2004). Although
lagging behind in accuracy when compared to other
docking programs, previous research proved that results
from ArgusLab are biologically meaningful (Joy et al.
2006). The crystal structure of ERa in complexation with
17b-estradiol (1A52) was extracted from the Research
Collaboratory for Structural Bioinformatics Protein Data
Bank (RCSB PDB) (Tanenbaum et al. 1998; Berman et al.
2000). Ions, ligands and subunits not involved in the ligand
binding were removed from the original structure file. The
receptor input pdb file was then generated. Separatly,
ligands files were prepared. The N-acetyl-tyrosinols (12)
(Fig. 3) were used as a model for two main reasons: (1)
smaller ligand involve less calculation time and more
accurate results and (2) the N-acetyl-tyrosinol derivatives
show similarities with our final tyrosine–chlorambucil
hybrid molecules. For each ligand [17b-estradiol (2),
L-o-, L-m- and L-p-N-acetyltyrosinol (12)], structural
The synthesis of regioisomers 6–11 was accomplished
using the convergent synthesis previously reported for the
para-tyrosine–chlorambucil series (Descoteaux et al. 2010)
(Scheme 1). The ease of synthesis and the excellent global
yields obtained in our previous study encourage us to use
this methodology for the two other commercially available
regioisomers of tyrosine (ortho- and meta-). Moreover, as
proved in our earlier study, the initial stereochemistry of
the amino acids (with D- or L-para-tyrosine) was main-
tained all along the synthesis, giving optically pure final
compounds (Descoteaux et al. 2010). For this study,
DL-ortho-tyr and L-meta-tyr are used as the starting mate-
rial. So, the stereochemistry of the final hybrids made from
L-meta-tyrosine was maintained during the course of the
synthesis.
6-Aminohexanoic acid (13, m = 5) and 11-aminoun-
decanoic acid (13, m = 10) were first coupled to chlor-
ambucil (1). Thus, chlorambucil (1) was activated [with
isobutylchloroformate (ClCOi2Bu)] and then coupled to
the amino acid function using hexamethyldisilazane and
trimethylsilyl chloride in acidic conditions. Separately,
DL-o-tyrosine (15, ortho-), L-m-tyrosine (15, meta-) and
L-p-tyrosine (15, para-) were transformed into the
(a)
(b)
(c)
Ar-OH
Arg394
Ar-OH
Arg394
Ar-OH
Arg394
His524
His524
His524
Glu353
Glu353
Glu353
Fig. 3 17b-estradiol (2), L-m- (12, meta-) and L-p- (12, para-) N-acetyltyrosinol within the active site of the ERa. The main interactions are
occurring on ring A of the steroid nucleus (a) or on the aromatic hydroxy group (Ar–OH) of the tyrosinol derivatives (b, c)
123

ˆ
C. Descoteaux et al.
932
Scheme 1 Synthesis of
tyrosine–chlorambucil hybrid
regioisomers (6a–11a and 6b–
11b)
Cl
N
Cl
O
HO2C
NH2
(i)
m
HO2C
N
3
13, m = 5 or 10
m
14, m = 5 or 10, (98%)
H
O
O
R
OH
OCH3
(ii)
(iii)
HO
HO
HO
NH2
NH3Cl
HN
3
15, o-, m- or p-Tyrosine
16, (96%)
O
Cl
N
6a, 8a, 10a, R = CO2CH3 (70%)
6b, 8b, 10b, R = CH2OH (57%)
Cl
(iv)
(v)
Cl
Cl
R
O
N
O
HO
HN
N
m
3
H
7a, 9a, 11a, R = CO2CH3 (70%)
7b, 9b, 11b, R = CH2OH (57%)
m = 5 or 10
(iv)
Reagents: i) 1. HMDS, H2SO4 cat., DCM, triethylamine, TMSCl; 2. CLL, HOBt, DCC, triethylamine, DMF;
(ii) MeOH, SOCl2,; (iii) CLL, HOBt, DCC, triethylamine, DMF; (iv) LiBH4, Et2O, 0oC to 22oC; (v) 14, m = 5 or
10, HOBt, DCC, triethylamine, DMF.
corresponding methyl ester hydrochloride salt (16) with
thionyl chloride in methanol. The corresponding tyrosine
methyl ester hydrochloride salts were obtained in 96 to
100% yield. Then, under standard reaction conditions
(HOBt and DCC), the resulting tyrosine esters were cou-
pled to chlorambucil (1) via an amide function giving the
tyrosine–chlorambucil hybrids without spacer. Purification
gave compounds 6a, 8a and 10a in 68%, 81% and 81%
yield, respectively. The tyrosine methyl esters (16) were
also linked to chlorambucil (1) via an alkyl chain spacer.
The six carbon atoms and the eleven carbon atoms deriv-
atives prepared earlier (14, m = 5 or 10) were coupled to
the tyrosine ester (16) moiety. Again, standard reaction
conditions (HOBt and DCC) were used for the coupling
reaction. Subsequently, purification gave compounds 7a
(m = 5, 10), 9a (m = 5, 10) and 11a (m = 5, 10) in good
yields (about 72%). Finally, all tyrosine–chlorambucil
methyl ester derivatives (6a, 7a, 8a, 9a, 10a, and 11a) were
submitted to selective reduction of the ester function with
lithium borohydride in dry diethyl ether. The correspond-
ing tyrosinol-chlorambucil hybrids (6b, 7b, 8b, 9b, 10b
and 11b) were obtained in good to excellent yields
(36–88%). All the new compounds were fully character-
estrogen-receptor negative (MDA-MB-231, ER-) tumor
cell lines using the MTT colorimetric assay (Carmichael
et al. 1987). Chlorambucil (1) was used as the control on
both human mammary carcinomas.
As shown by the MTT assays, all the derivatives tested
were more cytotoxic than the parent drug, chlorambucil
(from 1.7 to 7.4 times), on both breast cancer cell lines
(Tables 1, 2). The methyl ester (6a–11a) (m = 5, 10) as
well as the tyrosinol (6b–11b) (m = 5, 10) derivatives
presented comparable biological activity. Chlorambucil is
a known bis-alkylation agent which forms covalent adducts
with DNA (Armitage 1993). It can initiate apoptosis
regardless of the presence or absence of the ER protein.
However, the new tyrosine–chlorambucil hybrids show
greater activity than chlorambucil (1) itself, which is
already used for solid breast cancer treatments. This could
reveal a possible beneficial role of the tyrosine ligand to
target the drug where it is needed. Thus, the objective to
increase the activity of chlorambucil by using an estrogen-
like ligand was achieved. This particular molecular com-
bination could possibly diversify the use for this type of
nitrogen-mustard anticancer agent in the future.
The new tyrosine–chlorambucil hybrids were active on
both breast cancer cell lines (ER? and ER-). This can be
explained by the complexity of the protein contents of
these cell types. The hormone-independent MDA-MB-231
cancer cell line does not express the ERa protein, unlike
the MCF-7 cell line. However, other similar proteins,
which can be ER subtypes, are also present in ER- cell
lines. A possible interaction of the tyrosine with one (or
more) of these proteins could explain the activity observed
1
ized by their respective IR, H-NMR, 13C-NMR and mass
spectra.
In vitro biological activity
All the DL-o-, L-m- and L-p-tyrosine–chlorambucil hybrid
regioisomers (6–11) were evaluated for their cytotoxic
activity on estrogen-receptor positive (MCF-7, ER?) and
123

SAR study of tyrosine–chlorambucil hybrid regioisomers
933
Table 1 Cytocidal activity of tyrosine–chlorambucil regioisomers (6a–11a and 6b–11b, m = 5, 10) on estrogen receptor positive (ER?) MCF-7
breast cancer cell lines
Compound
DL-o
Compound
L-m
Compound
L-p
IC50, lMa
CLL ratio
IC50, lMa
CLL ratio
IC50, lMa
CLL ratio
6a
52.63 4.68
20.54 1.97
44.97 3.93
43.71 5.37
NR
2.48
6.35
2.90
2.98
–
8a
17.72 1.27
23.42 2.94
18.63 2.32
48.31 1.36
41.00 3.97
21.68 2.63
7.36
5.57
7.00
2.70
3.18
6.01
10a
31.92 0.89
34.11 2.04
25.43 2.06
62.16 5.50
67.90 8.68
19.39 2.66
4.08
3.82
5.13
2.10
1.92
6.72
6b
8b
10b
7a, m = 5
7b, m = 5
7a, m = 10
7b, m = 10
1
9a, m = 5
9b, m = 5
9a, m = 10
9b, m = 10
11a, m = 5
11b, m = 5
11a, m = 10
11b, m = 10
47.73 3.61
130.36 2.92
2.73
NR not reached
a
Inhibitory concentration (IC50, lM) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent the
mean SEM of three independent experiments. The cells were incubated for a period of 72 h
Table 2 Cytocidal activity of tyrosine–chlorambucil regioisomers (6a–11a and 6b–11b, m = 5, 10) on estrogen receptor negative (ER-) MDA-
MB-231 breast cancer cell lines
Compound
DL-o
Compound
L-m
Compound
L-p
IC50, lMa
CLL ratio
IC50, lMa
CLL ratio
IC50, lMa
CLL ratio
6a
57.45 5.38
26.47 2.09
38.18 2.77
33.97 3.19
NR
2.38
5.17
3.58
4.03
–
8a
32.24 2.85
28.71 3.51
34.01 3.37
45.07 5.04
63.03 4.00
71.83 5.65
4.24
4.77
4.02
3.04
2.17
1.91
10a
37.83 1.52
39.57 2.40
36.81 3.97
55.09 5.33
NR
3.62
3.46
3.72
2.48
–
6b
8b
10b
7a, m = 5
7b, m = 5
7a, m = 10
7b, m = 10
1
9a, m = 5
9b, m = 5
9a, m = 10
9b, m = 10
11a, m = 5
11b, m = 5
11a, m = 10
11b, m = 10
79.37 6.54
136.85 6.79
1.72
NR
–
NR not reached
a
Inhibitory concentration (IC50, lM) as obtained by the MTT assay. Experiments were performed in duplicates and the results represent the
mean SEM of three independent experiments. The cells were incubated for a period of 72 h
on ER- cells with the tyrosine derivatives. Moreover, the
new tyrosine–chlorambucil molecules, being bis-alkylating
agents, show the same pattern of activity in vitro than
chlorambucil. They are active on both cell types. It is
important to indicate that the desired selectivity towards
ER? cancer cells might be expressed more clearly in vivo
as it was previously reported for other anticancer agents
(Otto et al. 1991; Karl et al. 1988).
cells. This specificity could be explained by the phenol
group in meta position, which could possibly adopt a
structure which favorises better orientation and stronger
interactions within the ERa compared to the other two
regioisomers (ortho and para). Otherwise, the location of
the phenol group itself (ortho-, meta- or para-) might play
a role in the overall cytocidal activity observed with these
various hybrids. Indeed, it is reported that m-tyrosine
is significantly more phytotoxic than its structural isomers
o- and p-tyrosine (Bertin et al. 2007). Furthermore, it is
reported that phenolic compounds exhibiting antioxidant
properties can contribute to the destruction of cancer cells
(Durand et al. 2009). The slightly greater activity observed
for the m-tyrosine regioisomer might be explained by these
factors being more important for the meta isomer compared
to the ortho and para isomers.
Aforementioned, this study was undertaken in order to
verify the influence of the position of the phenol hydroxyl
group on the biological activity. Interestingly, among all
the regioisomers tested, compound 8a, with the phenol
group located in meta position, showed the most significant
cytocidal activity [7.36 times more active than chloram-
bucil (1)]. Generally, all the L-m-tyrosine methyl ester
derivatives are more active than their corresponding DL-o-
and their L-p- analogs. Furthermore, in most cases, the L-m-
tyrosine–chlorambucil hybrids reveals to be slightly more
specific (more active) on hormone-dependent breast cancer
cells compared to hormone-independent breast cancer
Some molecules (9a, 9b, 11a and 11b) (m = 5) have
been submitted to estrogen receptor alpha affinity evalua-
tion. Even if these specific molecules presented non neg-
ligible cytotoxic activity, only very little affinity was
123

ˆ
C. Descoteaux et al.
934
observed experimentally. Consequently, further investiga-
tions will be done to elucidate the exact mode of action of
these novel tyrosine–chlorambucil hybrids.
ester analogs or between Ar–OH and –CH2OH for the ty-
rosinol analogs) are not sufficiently distant to allow H
bonding interactions being formed between the ligand and
all the known ERa binding sites (Arg394, Glu353 and
His524). So, molecules with polar regions more distant
from each other would possibly fit much better into the
ERa binding cavity.
Docking
To the best of our knowledge, to date, no study have been
performed in order to verify if tyrosine can theorically bind
into the ERa pocket. In attempt to explain the mode of
binding, preliminary molecular docking calculations have
been done using Arguslab program. This was done in order
to verify if the amino acid squeletton could assume an
orientation close to specific residue (Arg394, Glu353 and
His524) within the ERa and, by doing so, establish H
bonding interactions as estradiol. Such interactions could
allow the tyrosine moiety reach cells expressing ERa and
then allow chlorambucil to act on cancer cells more
specifically.
Conclusion
Regiosomers of tyrosine were coupled to chlorambucil (1),
directly or via a 5 carbon or a 10 carbon atoms spacer.
Eighteen compounds were synthesized in 38 to 67%
overall yield using an efficient synthetic methodology. The
association of tyrosine to chlorambucil gave compounds
with higher cytotoxicity than chlorambucil itself when
tested on human breast cancer cell lines. One goal of this
study was to verify the influence of the phenol group
location on the biological activity of the new compounds.
The m-tyrosine–chlorambucil hybrids (6 and 8, m = 5, 10)
showed greater cytotoxicity compared to the other regioi-
somers. Moreover, the m-tyrosine molecules were slightly
more specific for hormone-dependent cancer cells. Dock-
ing calculations gave us insight to the mode of binding of
the m-tyrosine derivatives. The m-tyrosine regioisomer
seems to mimic closely the 3-OH group of estradiol into
the ERa pocket when compared to the two other regioi-
somers. This can explain, in part, the greater biological
activity observed for the meta regioisomer. Also, the
antioxidant properties of the meta phenolic compounds
could contribute to the overall cytotoxicity. This explor-
atory study also suggests that larger ligands could fit more
adequately into the ERa binding cavity. Hence, a second
series of bulkier tyrosine–chlorambucil hybrids are pres-
ently designed in our laboratory in order to obtain ligand
with enhanced affinity for the ERa.
In light of our molecular modeling results, the three
regioisomers [L-o-, L-m- and L-p-N-acetyltyrosinol, (12)]
seem to be oriented in different ways into the ERa binding
pocket (Fig. 3). Figure 3a shows the native ligand,
17b-estradiol (2), within the active site of the ERa. The
m-tyrosinol derivative (12, meta-) (Fig. 3b) adopts a con-
formation similar to that of estradiol (Fig. 3a). The docking
calculations demonstrate that the phenol group of the
m-tyrosinol is located in the A-ring region of estradiol, near
residues Arg394 and Glu353. Beside, the same docking
calculations were performed with the para regioisomer (12,
para-). This time, the same phenol group appears to be
close to the His524 residue (Fig. 3c) an orientation
completely opposite to that observed with m-tyrosinol. The
L-o-N-acetyltyrosinol (12, ortho-) behaves as its para reg-
ioisomer (figure not shown). Hence, these results demon-
strate that the meta-tyrosine derivative mimics the estradiol
ligand much better than its ortho and para analogs. Also,
all N-acetyltyrosine methyl esters were docked into the
ERa. Each molecule behaves as its corresponding ortho,
meta or para tyrosinol analog (12) where, in each cases, the
phenol group is oriented similarly into the ERa pocket
(figure not shown). The m-tyrosine ester derivative still
positions itself into the ERa like the natural ligand. Of note,
the docking of the various tyrosinol and tyrosine ester
derivatives led repeatedly to the same results.
Acknowledgments The authors wish to thank the Fonds de
´
Recherche sur la Nature et les Technologies du Quebec (FQRNT) for
essential financial support. The NSERC student fellowship for Car-
ˆ
oline Descoteaux is gratefully acknowledged.
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