Synthesis of D- and L-tyrosine-chlorambucil analogs active against breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2010.10.039

A series of d- and l-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of d- and l-tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent (ER+ and ER-) breast cancer cell lines. The novel analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry of the tyrosine moiety. Interestingly, the d- and l-tyrosinol-chlorambucil derivatives with 10 carbon atoms spacer are selective towards MCF-7 (ER+) breast cancer cell line.

Full text of DOI:10.1016/j.bmcl.2010.10.039

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7388–7392
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of D- and L-tyrosine-chlorambucil analogs active against breast
cancer cell lines
⇑
Caroline Descôteaux, Valérie Leblanc, Kevin Brasseur, Atul Gupta, Éric Asselin, Gervais Bérubé
Département de Chimie-Biologie, Groupe de Recherche en Oncologie et Endocrinologie Moléculaires, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières,
Québec, Canada G9A 5H7
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of
of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of
- and -tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in dif-
ferent hormone-dependent and hormone-independent (ER+ and ERÀ) breast cancer cell lines. The novel
analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure–activ-
ity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry
D- and L-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy
Received 30 August 2010
Revised 6 October 2010
Accepted 8 October 2010
Available online 21 October 2010
D
L
Keywords:
of the tyrosine moiety. Interestingly, the
D- and L-tyrosinol-chlorambucil derivatives with 10 carbon
Tyrosine-chlorambucil hybrids
Anticancer agents
Estrogen receptor
Breast cancer
atoms spacer are selective towards MCF-7 (ER+) breast cancer cell line.
Ó 2010 Elsevier Ltd. All rights reserved.
Over the years much research has been done on breast cancer. It
has been established that tumors may differ according to the pro-
tein they express.1 Breast cancer is no more considered as a single
homogeneous disease and treatment may differ from a patient to
another. An extensive field of investigation is now to target drugs
to specific molecules within the tumors and this is done by using
molecular-targeted cancer therapy.
was used as a tool to improve the delivery and cytotoxicity of the
parent drug.
Natural amino acids have been widely used in production of
pharmaceuticals and food.11
L-Tyrosine (2-amino-3-(4-hydroxy-
phenyl)-propanoic acid) (2) is a natural amino acid, which plays
a crucial role in human development (Fig. 1). It is the precursor
for the synthesis of thyroid hormones and some neurotransmitters
such as dopamine and noradrenaline.12 Recently, it has been
The estrogen receptor alpha (ER
a) has already been used as a
target to deliver a cytotoxic moiety to breast cancer cells.2–4 ER
a
discovered that L-tyrosine and some of its metal complex deriva-
is a cytosolic protein which become overexpressed in several can-
cerous cell types. Estradiol (E2) (1), the female sex hormone, binds
to its receptor and then activates transcription factors and triggers
proliferation (Fig. 1). Recently, our group has synthesized several
E2-linked-drugs in order to carry an anticancer agent, known for
tives show antibacterial activity against Bacillus subtilis, Serratia,
Pseudomonas aeruginosa and Escherichia coli.13 It also acts as an
antifungal agent.
Chlorambucil (CHL) (3) is a bis-alkylating agent which belongs
to the nitrogen mustard family (Fig. 1). It is mainly used to treat
chronic lymphocytic leukemia and malignant lymphomas.14 It is
also useful for the treatment of solid tumors such as advanced
ovarian and breast carcinomas.15 Like other type of anticancer
treatment, administration of CHL causes non negligible toxic side
effects including bone marrow suppression, anemia and reduced
immune system function.16,17
its efficacy, to cells expressing the ERa 5–7
These molecules have
.
shown promising in vitro biological activity and in vivo studies
of one of these revealed selectivity for hormone-dependent breast
cancer.8
Also, non-steroidal cytotoxic hybrid molecules have already
been synthesized in attempt to target the ER
sign was to destroy cancerous cells without activating the ER
a
.4 The aim of such de-
a.
In our search for anticancer agents with better therapeutic in-
dex and selectivity towards breast cancer, we have designed and
synthesized a new class of tyrosine-linked nitrogen mustard hy-
brids (4, 5) (Fig. 1). Many unusual molecular structures can bind
Conjugates comprising a chemotherapeutic agent and an amino
acid (or a derivative thereof) have also shown promising in vivo re-
sults.9 Furthermore, chlorambucil conjugates with lipidic amino
acids were developed as anticancer agent.10 This particular system
to the ERa 18
Thus, we hypothesized that tyrosine could be used
.
as a building block to target the estrogen receptor as its structure
could mimic, albeit slightly, the steroid backbone of 17b-estradiol
(1) (Fig. 2). Thus, the phenolic group of tyrosine is used as a poten-
tial bioisosteric function imitating the phenol group of estradiol.
⇑
Corresponding author. Tel.: +1 819 376 5011x3353.
E-mail address: Gervais.Berube@uqtr.ca (G. Bérubé).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.039

C. Descôteaux et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7388–7392
7389
OH
O
Cl
N
O
OH
Cl
NH2
HO
HO
HO
HO
1
2
3
3
Cl
R
O
R
N
O
Cl
HN
HN
HO
N
n
3
H
Cl
O
N
Cl
4, R = CO2CH3, CH2OH
5, n = 5, 10; R = CO2CH3, CH2OH
Figure 1. Structures of estradiol (1), L-tyrosine (2), chlorambucil (3) and tyrosine derivatives 4 and 5.
For this purpose, tyrosine was used as the starting material to
which chlorambucil was added directly (4) or via a spacer (5) to
construct several tyrosine-chlorambucil hybrids. Chlorambucil
was linked to the tyrosine framework at the a-amine function. This
position seems suitable as it should not affect functional groups
possibly involved in the receptor recognition. The final compounds
are either tyrosine methyl ester or tyrosinol analogs. In each case,
both, natural (L-tyrosine) and non-natural (D-tyrosine) amino acids
were used.
This study presents the design, synthesis and biological activi-
ties of these new tyrosine-chlorambucil conjugates (4, 5). The hy-
brids were made using two distinct synthetic methodologies
(linear and convergent synthesis) which will be described and
compared in terms of their efficiency.
With the linear synthesis (Scheme 1), tyrosine was initially
transformed into the corresponding methyl ester hydrochloride
salt (6) with thionyl chloride in methyl alcohol (MeOH) at reflux
in 96% yield. Condensation of compound 6 with chlorambucil in
presence of 1-hydroxybenzotriazole (HOBt), dicyclohexylcarbodi-
imide (DCC) and triethylamine (Et3N) in N,N-dimethylformamide
(DMF) at room temperature (22 °C) yielded compound 4a in 81%
Figure 2. Superimposition of 17b-estradiol (1) and tyrosine (2).
Moreover, molecular modeling displays the possible interactions of
the novel hybrids with the estrogen receptor alpha (ongoing
study). This encouraged us to perform this exploratory work. Be-
sides, the cytotoxic activity of chlorambucil and the known anti-
bacterial activity of tyrosine could together (1) generate powerful
compounds interacting with the ERa, (2) being more specific to
female cancer cells, and (3) more potent than each of the indepen-
dent components.
O
O
OH
(i)
OCH3
NH3Cl
NH2
HO2C
m
NH2
m = 5 or 10
HO
HO
HO
(ii)
6, (96%)
2, D- or L-Tyrosine
(iv)
R
NH
O
t-Boc
HO2C
N
3
m
H
Cl
N
7, (97%)
4a, R = CO2CH3 (81%)
4b, R = CH2OH (78%)
(iii)
Cl
N
(v)
O
Cl
R
OCH3
O
Cl
HN
HN
t-Boc
HO
HO
N
m
N
3
(vi) ( ii)
m
H
H
O
O
8, (96%)
5a, R = CO2CH3 (60%)
5b, R = CH2OH (57%)
(iii)
Scheme 1. Reagents and conditions (i) SOCl2, MeOH, reflux, 3.5 h; (ii) Chlorambucil, HOBt, DCC, Et3N, dry DMF, 22 °C; (iii) LiBH4 excess, dry Et2O, 22 °C; (iv) Boc-ON, 1,4-
dioxane, water, Et3N, 22 °C; (v) 6, HOBt, DCC, Et3N, dry DMF, 22 °C; (vi) excess TFA, DCM, 22 °C, 10 min.

7390
C. Descôteaux et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7388–7392
yield. Borohydride reduction of compound 4a with excess of lith-
ium borohydride (LiBH4) in dry diethyl ether (Et2O) at room tem-
perature (22 °C) gave the desired tyrosinol compounds 4b in 78%
yield. Chlorambucil was also linked via a 5 or a 10 carbon atoms
spacer. The amino acid-spacer was first protected using 2-(t-but-
oxycarbonyloxyimino)-2-phenylacetonitrile (Boc-ON) in 1,4-dioxane,
water and Et3N at room temperature (22 °C) yielding N-Boc-amino
acid (7) in 97% yield. Then, the corresponding N-Boc protected
amino acid-alkyl chain (7) was linked to tyrosine methyl ester (6)
via an amide function. Activation and condensation were performed
using DCC and HOBt in DMF linking 7 to 6 to give compound 8
in 96% yield. Deprotection was achieved with treatment of the
tBoc-compound (8) with trifluoroacetic acid in dichloromethane
(DCM). The resulting oil was then coupled to chlorambucil (3)
using DCC and HOBt in DMF. The tyrosine methyl ester chlorambu-
cil derivatives 5a were obtained in 60% yield. The pure compounds
were then reduced in the presence of LiBH4 in Et2O and provide the
corresponding tyrosinol derivatives 5b in 57% yield.
With the convergent synthesis (Scheme 2), chlorambucil was
first linked to the amino acid-spacer. The acid function of the chlor-
ambucil was activated with isobutylchloroformate (ClCO2 Bu) and
i
then coupled to the amino acid function using hexamethyldisilaz-
ane (HMDS), trimethylsilyl chloride (TMSCl), and
a catalytic
amount of sulfuric acid.19 The resulting amide (9) was isolated in
98% yield. The amino acid-CHL derivative (9) was then coupled,
in presence of DCC and HOBt, to the tyrosine methyl ester, ob-
tained as described above with the linear synthesis. The tyrosine
methyl ester-CHL analogs (5a) were obtained in 70% yield. The fi-
nal chemical step, reduction with LiBH4 in Et2O, provides the de-
sired tyrosinol derivatives in 57% yield. All the new compounds
were fully characterized by their respective IR, 1H NMR and 13C
NMR spectroscopy and by HRMS.20
Briefly, compounds 5a and 5b were synthesized using two dif-
ferent approaches of synthesis. With the linear path (Scheme 1),
D- or L-tyrosine were transformed into 5a and 5b through four
and five chemical steps, respectively. By the convergent path
(Scheme 2), chlorambucil was first linked to the spacer and then,
the resulting moiety was conjugated to the tyrosine methyl ester
hydrochloride. The same derivatives (5a and 5b) were obtained
in two and three chemical steps. The convergent path is the most
effective with fewer chemical steps and better global yields. Com-
pounds 5a and 5b were obtained in 54% and 31%, respectively, with
the linear path and in 67% and 38% with the convergent path.
In order to verify if some epimerization occurred during the for-
mation of the S- and R-tyrosine methyl ester hydrochlorides (S-6
and R-6), the corresponding Mosher’s amides were synthesized
(Scheme 3). Thus, independent treatment of S-6 and R-6 with com-
Cl
N
O
Cl
HO2C
NH2
(i)
m
HO2C
(ii)
N
3
m = 5 or 10
m
9, (98%)
H
O
O
OH
OCH3
NH3Cl
NH2
HO
HO
mercially available S-(+)-a-methoxyphenyl acetic acid, HOBt, DCC,
2, D- or L-Tyrosine
6, (96%)
Et3N in DMF gave the corresponding amides S,S-10 and R,S-10 with
good yields. Proton nuclear magnetic resonance (1H NMR) spectro-
scopic analysis of the two diastereoisomers showed no trace of epi-
merization in the final products. Further confirmation of this was
done by the preparation of a 1:2 mixture of S,S-10 and R,S-10 mol-
ecules which shows by 1H NMR many distinct signals assigned to
each specific diastereoisomers. For example, the methyl ester and
methyl ether signals are more deshielded with the S,S-10 deriva-
tive compared to the R,S-10 derivative. This confirms that no epi-
merization occurred during the first step of the synthesis of the
tyrosine-chlorambucil analogs described above.
(iii)
Cl
R
N
Cl
O
HN
HO
N
3
m
H
O
5a, R = CO2CH3 (70%)
5b, R = CH2OH (57%)
(iv)
The newly synthesized compounds (Table 1) were evaluated for
their in vitro cytotoxic activity using estrogen receptor positive
(MCF-7) and negative (MDA-MB-231) breast cancer cell lines.
Chlorambucil (3) was used as a positive control. MTT assays21
Scheme 2. Reagents and conditions (i) (1) HMDS, H2SO4 cat., DCM, Et3N, TMSCl; (2)
CHL, HOBt, DCC, Et3N, DMF; (ii) SOCl2, MeOH, reflux, 3.5 h; (iii) Chlorambucil–
NH(CH2)mCO2H, m = 5 or 10, HOBt, DCC, Et3N, dry DMF, 22 °C; (iv) LiBH4 excess, dry
Et2O, 22 °C.
O
O
O
OH
(i)
(ii)
OCH3
O
OCH3
NH2
HN
H
NH3Cl
HO
HO
HO
H3CO
S-2, L-Tyrosine (S-1)
S,S-10 (54%)
S-6 (96%)
O
O
O
OH
(ii)
OCH3
(i)
OCH3
NH2
HN
H
NH3Cl
O
HO
HO
HO
H3CO
R-2, D-Tyrosine (R-1)
R,S-10 (82%)
a-methoxyphenyl acetic acid, HOBt, DCC, Et3N, dry DMF, 22 °C.
R-6 (96%)
Scheme 3. Reagents and conditions (i) SOCl2, MeOH, reflux, 3.5 h; (ii) S-(+)-

C. Descôteaux et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7388–7392
7391
Table 1
on MCF-7 cells. Its enantiomer, compound
L
-5b, is also very active,
Compound numbers and descriptors
nearly 7 times more active than chlorambucil (3). Interestingly,
these two compounds contain a 10 carbon atoms spacer in their
structure. They are also both tyrosinol analogs, while their methyl
ester derivatives are much less active on the same hormone-
dependent cancer cell line.
Compound
#
Chain length
(n (or m))
R
CHL
or
3
—
—
4a
4b
5a
5b
5a
5b
—
—
5
5
10
10
CO2CH3
CH2OH
CO2CH3
CH2OH
CO2CH3
CH2OH
D
L Tyrosine
The observed biological activity of these two hybrids (
D-5b and
L-5b) could be explained by an increase in solubility due to the
hydroxymethyl (CH2OH) function, which can create hydrogen
bond with water and biological residues. Even if a longer alkyl
chain should create a diminution in solubility, the CH2OH residue
could help to counteract this effect. Furthermore, other derivatives
bearing a CH2OH residue were synthesized and it was found to be
beneficial for biological activity.7 Of note, the best cytotoxic activ-
ity was obtained with the longer alkyl chain spacer which could
enhance cellular penetration. In earlier studies, it was observed
that, on several non-steroidal estrogen cytotoxic molecules, the
length of the side chain bearing the cytotoxic portion should be
about 11 carbon atoms long for optimal biological activity.22
In order to further confirm the observed selectivity of the mol-
ecules bearing a 10 carbon atoms spacer for hormone-dependent
showed that the newly synthesized tyrosine-chlorambucil hybrids
of type 4 and 5 (Table 2) possess moderate to significant cytotoxic
activity. Some selected compounds (3, L-4 and L-5) were tested on
additional ER- (MDA-MB-436, MDA-MB-468) breast cancer cell
lines to further assess selectivity on ER+ MCF-7 cancer cells
(Table 3).
All compounds were first tested on hormone-dependent (MCF-
7) and hormone-independent (MDA-MB-231) cancer cell lines.
Generally, biological activity results of all compounds tested re-
vealed a greater cytotoxic activity, compared to chlorambucil (3;
cancer cells, the complete L-tyrosine-chlorambucil (L-4 and L-5)
series was selected for additional biological assays on two ER-
cancer cell lines (MDA-MB-436 and MDA-MB-468). The results
IC50 >130
l
M for all cell lines) itself. The
L
-tyrosine-chlorambucil
-analogs (Table 2).
D-5b is eight times more active than the parent drug
analogs are generally more active than the
Compound
D
showed that compounds L-5a and L-5b, with n = 10, are active
against hormone-dependent cancer cells whereas they do not
reach IC50 with all hormone-independent cancer cells tested. In
light of these results, we can confirm that tyrosine-chlorambucil
derivatives bearing a 10 carbon atom spacers are specific to
hormone-dependent MCF-7 cancer cells.
Table 2
Inhibitory concentration of chlorambucil (3) and compounds 4 and 5 on both ER+ and
ERÀ breast cancer cell lines
a
a
In conclusion, tyrosine-based chlorambucil hybrids have been
synthesized in good yields starting from D- and L-tyrosine through
two different methodologies. The convergent synthesis was more
effective compared to the linear synthesis. The former was per-
formed with fewer chemical steps and with about 24% increased
in overall yield. Most of the tyrosine-chlorambucil analogs are
Compound
MCF-7 (ER+) IC50
(lM)
MDA-MB-231 (ERÀ) IC50
(lM)
3
130.36 2.92
31.92 0.89
136.85 6.79
37.83 1.52
L
L
L
L
L
L
-4a
34.11 2.04
25.43 2.06
62.16 5.50
67.90 8.68
19.39 2.66
50.01 5.51
31.41 3.22
152.37 11.28
66.98 3.28
NR
39.57 2.40
-4b
36.81 3.97
-5a, n = 5
-5b, n = 5
-5a, n = 10
-5b, n = 10
55.09 5.33
more cytotoxic than chlorambucil itself. Compounds
D-5b and
NR
L
-5b, bearing a 10 carbon atoms spacer between the chlorambucil
NR
and the tyrosinol moiety, were the most active compounds and
were selective toward hormone-dependent breast cancer cells
(MCF-7). Further evaluation of these compounds is in progress in
our laboratory.
47.23 5.81
D
D
D
D
D
D
-4a
29.78 3.86
-4b
—
-5a, n = 5
-5b, n = 5
-5a, n = 10
-5b, n = 10
—
NR
NR
16.27 2.00
Acknowledgments
NR: not reached.
The authors thank the Fonds de Recherche sur la Nature et les
Technologies du Québec (FQRNT) for valuable financial support.
The Natural Sciences and Engineering Research Council of Canada
(NSERC) student fellowship to C. Descôteaux is gratefully acknowl-
edged. We also thank É. Froehlich B.Sc. students who participated
to the synthesis of these molecules.
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 3
Inhibitory concentration of chlorambucil (3) and compounds L-4 and L-5 on ERÀ
breast cancer cell lines
References and notes
Compound MDA-MB-468 (ERÀ) IC50
(l
M) MDA-MB-436 (ERÀ) IC50
M)
a
a
(l
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131.83 7.87
15.42 1.21
146.88 10.69
62.51 2.49
L
L
L
L
L
L
-4a
24.38 1.65
14.96 1.10
28.92 0.71
NR
55.08 2.90
59.04 3.31
77.28 7.07
NR
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-5a, n = 5
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20. Spectral data for N-chlorambucil-L-tyrosine methyl ester (L-4a): IR (KBr, mmax,
25.4, (1C hidden). ESI+ HRMS [M+H]+ calculated for C30H42Cl2N3O5 = 594.2496;
found = 594.2490.
Spectral data for N-((6-N-chlorambucilamino) hexanoyl)- -tyrosinol
(L-5b,
n = 5): IR (NaCl,
m
max, cmÀ1) 3100–3400 (2Â O–H and 2ÂLN–H), 1639 (2Â
C@O, 2Â NHCO), 1523 and 1246 (C–N–H). 1H NMR (Acetone-d6, d ppm) 8.33
(1H, s, OH), 7.12 (1H, br t, hidden, CH2NHCO), 7.07 and 7.06 (4H, 2d, J = 8.6 Hz
and J = 8.2 Hz, 3-CH tyr and 3-CH CHL), 6.87 (1H, d, J = 8.2 Hz, CHNHCO), 6.74
(2H, d, J = 8.2 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 4.06 (1H, m,
CHNH), 3.74 (8H, m, 2Â CH2Cl and 2Â NCH2), 3.51 (2H, d, J = 5.1 Hz, CH2OH),
3.14 (2H, m, CH2NHCO), 2.88 (1H, s, CH2OH), 2.58–2.87 (2H, m, CH2CHNH),
2.52 (2H, t, J = 7.6 Hz, CH2CH2Ph), 2.17 (2H, t, J = 7.4 Hz, CH2NHCOCH2), 2.10
(2H, t, J = 6.3 Hz, CHNHCOCH2), 1.78–1.93 (2H, m, CH2CH2CH2Ph), 1.38–1.55
(4H, m, CH2CH2NHCO and CHNHCOCH2CH2), 1.22 (2H, m, CH2CH2CH2CH2CH2).
13C NMR (Acetone-d6, d ppm) 172.54 (CONH), 172.48 (CONH), 156.1 (1-C tyr),
144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.9 (4-C tyr), 129.7 (2C,
3-C CHL), 115.3 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.8 (CH2OH), 53.3 (2C, 2Â
NCH2CH2Cl), 53.2 (CHNH), 41.0 (2Â C, 2Â NCH2CH2Cl), 39.0 (CH2NHCO), 36.2
(CH2CHNH), 36.1 (CHNHCOCH2), 35.6 (CH2CH2Ph), 34.3 (CH2NHCOCH2), 27.9
(COCH2CH2CH2Ph), 26.4 (CH2CH2CH2CH2CH2), 25.6 (NHCOCH2CH2CH2CH2CH2),
(1Â CH2 hidden). ESI+ HRMS [M+H]+ calculated for C29H42Cl2N3O4 = 566.2547;
found = 566.2541.
cmÀ1) 3100–3450 (O–H and N–H), 1741 (C@O, COOCH3), 1650 (C@O, NHCO),
1517 and 1221 (C–N–H). 1H NMR (Acetone-d6, d ppm) 8.27 (1H, s, OH), 7.25
(1H, d, J = 8.2 Hz, CHNHCO), 7.05 and 7.03 (4H, 2d, J = 8.6 Hz and J = 9.0 Hz, 3-
CH tyr and 3-CH CHL), 6.75 (2H, d, J = 9.0 Hz, 2-CH tyr), 6.70 (2H, d, J = 9.0 Hz, 2-
CH CHL), 4.63 (1H, m, CHNH), 3.74 (8H, dt, J = 3.5 Hz and J = 1.8 Hz, CH2Cl and
NCH2), 3.65 (3H, s, OCH3), 2.82–3.08 (2H, m, CH2CHNH), 2.46 (2H, t, J = 7.0 Hz,
CH2CH2Ph), 2.18 (2H, d, J = 7.0 Hz, NHCOCH2), 1.76–1.87 (2H, m, CH2CH2CH2).
13C NMR (Acetone-d6, d ppm) 172.43 (NHCOCH2), 172.38 (COOCH3), 156.5 (1-C
tyr), 144.8 (1-C CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL),
127.9 (4-C tyr), 115.4 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 54.0 (CHNH), 53.3 (2C,
2Â NCH2CH2Cl), 51.5 (OCH3), 41.0 (2Â C, 2Â NCH2CH2Cl), 36.9 (CH2CHNH),
35.0 (CH2CH2Ph), 34.0 (NHCOCH2), 27.7 (CH2CH2CH2). ESI+ HRMS [M+H]+
calculated for C24H31Cl2N2O4 = 481.1655; found = 481.1650.
Spectral data for N-((11-N-chlorambucilamino)undecanoyl)-L-tyrosine methyl
ester. (
L
-5a, n = 10): IR (NaCl,
m
max, cmÀ1) 3250–3450 (O–H and 2Â N–H), 1745
(C@O, COOCH3), 1656 and 1614 (2Â C@O, 2Â NHCO), 1516 and 1216 (C–N–H).
1H NMR (Acetone-d6, d ppm) 8.49 (1H, s, OH), 7.23 (1H, d, J = 7.8 Hz, CHNHCO),
7.12 (1H, br t apparent, partly hidden, CH2NHCO), 7.06 and 7.03 (4H, 2d,
J = 8.6 Hz and J = 8.2 Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 8.2 Hz, 2-CH
tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.65 (1H, m, CHNH), 3.74 (8H, m, 2Â
CH2Cl and 2Â NCH2), 3.65 (3H, s, OCH3), 3.19 (2H, q, J = 6.3 Hz, CH2NHCO),
2.81–3.07 (2H, m, CH2CHNH), 2.52 (2H, t, J = 7.6 Hz, CH2CH2Ph), 2.16 (4H, 2d
overlapped, J = 7.4 Hz, CH2NHCOCH2 and CHNHCOCH2), 1.78–1.93 (2H, m,
CH2CH2CH2Ph), 1.26–1.52 (16H, #m and s, 8Â CH2). 13C NMR (Acetone-d6, d
ppm) 172.5 (2C, 2Â CONH), 172.4 (COOCH3), 156.6 (1-C tyr), 144.9 (1-C CHL),
130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 127.8 (4-C tyr), 115.4
(2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.9 (CHNH), 53.3 (2C, 2Â NCH2CH2Cl), 51.5
(OCH3), 41.0 (2C, 2Â NCH2CH2Cl), 39.0, 36.9, 35.7, 35.6, 34.3, 27.9, 26.4, 25.4,
(6C hidden). ESI+ HRMS [M+H]+ calculated for C35H52Cl2N3O5 = 664.3279;
found = 664.3273.
Spectral data for N-chlorambucil-L-tyrosinol(L-4b): IR (NaCl, m
max, cmÀ1) 3100–
3400 (O–H and N–H), 1646 (C@O, NHCO), 1516 and 1250 (C–N–H). 1H NMR
(Acetone-d6, d ppm) 8.16 (1H, s, OH), 7.08 and 7.04 (4H, 2d, J = 9.0 Hz and
J = 9.0 Hz, 3-CH tyr and 3-CH CHL), 6.87 (1H, d, J = 8.2 Hz, CHNHCO), 6.75 (2H,
d, J = 7.0 Hz, 2-CH tyr), 6.70 (2H, d, J = 7.4 Hz, 2-CH CHL), 4.08 (1H, m, CHNH),
3.74 (8H, dt, J = 1.8 Hz and J = 3.5 Hz, 2Â CH2Cl and 2Â NCH2), 3.51 (2H, t,
J = 5.3 Hz, CH2OH), 2.62–2.88 (2H, m, CH2CHNH), 2.46 (2H, t, J = 7.6 Hz,
CH2CH2Ph), 2.14 (2H, d, J = 7.4 Hz, NHCOCH2), 1.76–1.87 (2H, m, CH2CH2CH2).
13C NMR (Acetone-d6, d ppm) 172.4 (NHCOCH2), 156.0 (1-C tyr), 144.8 (1-C
CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.9 (4-C tyr), 129.7 (2C, 3-C CHL),
115.2 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.5 (CH2OH), 53.3 (3C, CHNH and 2Â
NCH2CH2Cl), 41.0 (2C, 2Â NCH2CH2Cl), 36.2 (CH2CHNH), 35.5 (CH2CH2Ph), 34.1
(NHCOCH2), 27.8 (CH2CH2CH2). ESI+ HRMS [M+H]+ calculated for
Spectral data for N-((11-N-chlorambucilamino) undecanoyl)-L-tyrosinol (L-5b,
n = 10): IR (KBr,
m
max, cmÀ1) 3200–3500 (2Â O–H and 2Â N–H), 1649 (2Â C@O,
NHCO), 1540 and 1212 (C–N–H). 1H NMR (Acetone-d6, d ppm) 8.28 (1H, s, OH),
7.15 (1H, br t apparent, partly hidden, CH2NHCO), 7.07 (4H, d, J = 8.6 Hz, 3-CH
tyr and 3-CH CHL), 6.85 (1H, d, J = 8.2 Hz, CHNHCO), 6.74 (2H, d, J = 8.2 Hz, 2-
CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.10 (1H, m, CHNH), 3.75 (8H, m, 2Â
CH2Cl and 2Â NCH2), 3.50 (2H, t, J = 4.9 Hz, CH2OH), 3.18 (2H, m, CH2NHCO),
2.81 (1H, s, CH2OH), 2.64–2.84 (2H, m, CH2CHNH), 2.51 (2H, t, J = 7.6 Hz,
CH2CH2Ph), 2.16 (2H, t, J = 7.4 Hz, CH2NHCOCH2), 2.10 (2H, t, J = 7.4 Hz,
CHNHCOCH2), 1.81–1.92 (2H, m, CH2CH2CH2Ph), 1.25–1.54 (16H, #m and s,
8Â CH2). 13C NMR (Acetone-d6, d ppm) 172.5 (CONH), 172.3 (CONH), 156.1 (1-C
tyr), 144.9 (1-C CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.8 (4-C tyr), 129.7
(2C, 3-C CHL), 115.2 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.7 (CH2OH), 53.3 (3C,
2Â NCH2CH2Cl and CHNH), 41.0 (2Â C, 2Â NCH2CH2Cl), 38.9, 36.2, 35.6, 34.2,
26.8, 25.8, (8Â CH2 hidden). ESI+ HRMS [M+H]+ calculated for
C23H31Cl2N2O3 = 453.1706; found = 453.1703.
Spectral data for N-((6-N-chlorambucilamino)hexanoyl)-L-tyrosine methyl
ester (
L
-5a, n = 5): IR (NaCl,
m
max, cmÀ1) 3150–3500 (O–H and 2Â N–H), 1745
(C@O, COOCH3), 1646 (2Â C@O, 2Â NHCO), 1523 and 1253 (C–N–H). 1H NMR
(Acetone-d6, d ppm) 8.48 (1H, br s, OH), 7.27 (1H, d, J = 8.2 Hz, CHNHCO), 7.16
(1H, br t, J = 5.9 Hz, CH2NHCO), 7.07 and 7.04 (4H, 2d, J = 8.6 Hz and J = 8.6 Hz,
3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz,
2-CH CHL), 4.65 (1H, m, CHNH), 3.74 (8H, m, 2Â CH2Cl and 2Â NCH2), 3.65 (3H,
s, OCH3), 2.85–3.17 (4H, #m, CH2CHNH and CH2NHCO), 2.52 (2H, t, J = 7.6 Hz,
CH2CH2Ph), 2.16 (4H, m apparent, CH2NHCOCH2 and CHNHCOCH2), 1.82–1.89
(2H, m, CH2CH2CH2Ph), 1.19–1.54 (6H, #m, 3Â CH2). 13C NMR (Acetone-d6, d
ppm) 172.5 (CONH), 172.4 (CONH), 172.1 (COOCH3), 156.5 (1-C tyr), 144.9 (1-C
CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.0 (4-C tyr),
115.4 (2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.8 (CHNH), 53.3 (2C, 2Â NCH2CH2Cl),
51.5 (OCH3), 41.0 (2C, 2Â NCH2CH2Cl), 39.0, 36.9, 35.7, 35.5, 34.2, 27.9, 26.4,
C
34H52Cl2N3O4 = 636.3329; found = 636.3327.
21. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
22. He, Y.; Groleau, S.; Gaudreault, R. C.; Caron, M.; Thérien, H.-M.; Bérubé, G.
Bioorg. Med. Chem. 1995, 5, 2217.