Derivatives of nitrogen mustard anticancer agents with improved cytotoxicity

Article abstract of DOI:10.1002/ardp.202000366

In previous studies, we demonstrated that esters of bendamustine containing a basic moiety are far more cytotoxic anticancer agents than their parent compound and that the substitution of the labile ester moiety by a branched ester or an amide markedly in

Full text of DOI:10.1002/ardp.202000366

Received: 29 September 2020
DOI: 10.1002/ardp.202000366
Revised: 11 November 2020
Accepted: 13 November 2020
|
|
F U L L P A P E R
Derivatives of nitrogen mustard anticancer agents with
improved cytotoxicity
Frauke Antoni
| Günther Bernhardt
Institute of Pharmacy, University of
Regensburg, Regensburg, Germany
Abstract
In previous studies, we demonstrated that esters of bendamustine containing a
basic moiety are far more cytotoxic anticancer agents than their parent compound
and that the substitution of the labile ester moiety by a branched ester or an amide
markedly increases stability in the blood plasma. In the current study, we showed
that this substitution was bioisosteric. Aiming at increased cytotoxicity, we in-
troduced the same modification to related nitrogen mustards: 6‐isobendamustine,
chlorambucil, and melphalan. The synthesis was accomplished using the
coupling reagents N,N′‐dicyclohexylcarbodiimide or 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐
tetramethylaminium tetrafluoroborate. Cytotoxicity against a panel of diverse cancer
cells (carcinoma, sarcoma, and malignant melanoma) was assessed in a kinetic che-
mosensitivity assay. The target compounds showed cytotoxic or cytocidal effects at
concentrations above 1 µM: a striking enhancement over bendamustine and
Correspondence
Frauke Antoni, Institute of Pharmacy,
University of Regensburg, Universitätsstraße
31, 93053 Regensburg, Germany.
Email: frauke.antoni@ur.de
6‐isobendamustine, both ineffective against the selected cancer cells at concentrations
up to 50 µM, and a considerable improvement over chlorambucil, showing some po-
tency only against the sarcoma cells. Melphalan was almost as effective as the target
compounds—derivatization only provided a small improvement. The novel cytostatics
are of interest as model compounds for analyzing a correlation between cytotoxicity
and membrane transport and for the treatment of malignancies.
K E Y W O R D S
bendamustine, chlorambucil, derivatives, melphalan, nitrogen mustards
1
|
INTRODUCTION
contrast, chemotherapy works systemically and is the most effective
[
2,3]
treatment for disseminated tumors.
The era of chemotherapy
[
1]
Cancer is the second leading cause of death worldwide. Surgery
began in 1942 with the discovery of nitrogen mustards—originally
[
2,4]
and radiation are local therapies and cannot eradicate metastatic
produced as chemical warfare agents—as a remedy for cancer.
A
[
2]
cancer, where every organ in the body needs to be reached. By
basic chemical reaction underlies the mechanism of action of
Abbreviations: Boc, tert‐butyloxycarbonyl; CLL, chronic lymphocytic leukemia; DCC, N,N′‐dicyclohexylcarbodiimide; DCM, dichloromethane; DIPEA, N,N‐diisopropylethylamine;
DMF, dimethyformamide; DMSO, dimethylsulfoxide; FCS, fetal calf serum; HL, Hodgkin lymphoma; HPLC, high‐performance liquid chromatography; HRMS, high‐resolution mass spectrometry;
MeCN, acetonitrile; MM, multiple myeloma; NHL, non‐Hodgkin lymphoma; NMR, nuclear magnetic resonance; RP‐HPLC, reversed‐phase high‐performance liquid chromatography;
TBTU, 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminium tetrafluoroborate; TFA, trifluoroacetic acid; TLC, thin‐layer chromatography.
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
©
2020 The Authors. Archiv der Pharmazie published by Wiley‐VCH Verlag GmbH on behalf of Deutsche Pharmazeutische Gesellschaft
Arch Pharm. 2020;e2000366.
wileyonlinelibrary.com/journal/ardp
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https://doi.org/10.1002/ardp.202000366

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ANTONI AND BERNHARDT
|
−
FIGURE 1 The mechanism of the reaction of nitrogen mustards with nucleophiles (Nu ), here shown for mechlorethamine (1)
[
18,19]
nitrogen mustards, namely the intramolecular cyclization in a polar
solvent to form an aziridnium cation, which reacts readily with bio-
nucleophiles (Figure 1), for example nitrogen in the DNA. This re-
action leads to the formation of DNA interstrand crosslinks, which
and was introduced into the market there.
It distinguishes itself
from chlorambucil in the central benzimidazole ring, which is unique
to bendamustine and was intended to include antimetabolite prop-
[
17,18]
erties, which, however, have not yet been confirmed.
Never-
[
4,5]
prevents cell replication and ultimately causes cell death.
theless, bendamustine displays a unique mechanism of action, as it
inhibits mitotic checkpoints, causes inefficient DNA repair, and in-
duces the expression of the protein p53, a tumor suppressor, which
Inspired by this success, further, improved DNA‐alkylating
agents of the nitrogen mustard type were developed as che-
motherapeutics, such as chlorambucil, melphalan, or bendamustine.
Chlorambucil (2) (Figure 2) was first synthesized by Everett
[
20,21]
initiates apoptosis.
Bendamustine was approved in the Federal
Republic of Germany after the iron curtain had fallen, and today it is
sold, among others, under the brand name of Ribomustin for CLL,
[
6]
et al.
in 1953. The nitrogen mustard moiety (i.e., the bis(2‐
[
19,22]
chloroethyl)amino moiety, also called N‐lost moiety) was attached to
a phenyl ring, which withdraws electrons from the nitrogen atom,
thereby disfavoring aziridinium ion formation, which renders the
nitrogen mustard moiety less reactive toward the nucleophilic at-
indolent NHL, and MM.
In the United States of America, the
drug is marketed, among others, under the brand name of Treanda
[
18,19]
and is approved for CLL and indolent NHL.
The assets of
the lack of cross‐
and its superiority
[
18]
bendamustine are a favorable side‐effect profile,
[
7]
[19]
tack. Therefore, chlorambucil and other aromatic analogs are suf-
ficiently deactivated so that they can reach their target DNA sites
before being degraded by reacting with collateral nucleophiles, re-
sulting in reduced toxic side effects and allowing oral administra-
resistances, with many other alkylating agents,
[
22]
to chlorambucil in previously untreated patients with CLL.
Bendamustine is usually dosed intravenously; however, there have
been a couple of patent applications aiming at formulations for oral ad-
[23,24]
[7]
tion.
The butanoic acid side chain makes the compound
ministration.
Esterification of the carboxylic acid moiety may in-
[
8]
hydrophilic. Chlorambucil is sold, among others, under the brand
name of Leukeran and is indicated in the treatment of chronic lym-
phocytic leukemia (CLL), Hodgkin and non‐Hodgkin lymphomas (HL
crease the chances of oral application and, furthermore, has been
reported to enhance the hydrolytic stability of the N‐lost moiety (for
[25–27]
different reasons).
The increasing interest in esters of bendamus-
[
5,9,10]
and NHL), as well as breast and ovarian carcinomas.
tine prompted our working group to perform a study on the pharma-
cological properties of the latter. It was revealed that esters of
bendamustine are by far more potent cytotoxic agents than the parent
compound, especially esters comprising basic moieties, which are charged
under physiological conditions, for example, the 2‐pyrrolidinoethyl ester
Melphalan (3) (Figure 2) differs from chlorambucil in the length of
the alkanoic acid side chain and in the amino group attached to the
latter—the amino acid L‐phenylalanine is the “carrier” of the N‐lost
[11]
moiety. Melphalan was first synthesized in 1954 by Bergel et al.,
aiming at increased tumor selectivity. Indeed, melphalan is imported by
[28]
5
(Figure 3). The basic esters show a pronounced cellular accumula-
[
12]
amino acid transporters
whose expression is upregulated in cancer
Melphalan is used for treating various malignancies including
multiple myeloma (MM), ovarian cancer, breast cancer, and melanoma,
tion for reasons not yet identified, but transport proteins may be in-
[28]
[13]
cells.
volved. However, the basic esters turned out to be especially prone to
hydrolysis at the ester bond in the blood plasma, which was attributed to
[29]
[14,15]
and is sold, among others, under the trade name of Alkeran.
Bendamustine (4) (Figure 3) was initially synthesized in 1963 by
their similarity to substrates of unspecific cholinesterases.
We were
able to overcome this obstacle by substituting the linear ester moiety in 5
by a branched ester (compound 6), which increased the stability to an
[
16,17]
Ozegowski et al.
in the former German Democratic Republic
[29]
acceptable level, and so did the replacement of the ester by an amide
bond (compound 7; unpublished data). Bendamustine, its derivatives 5–7,
and their respective half‐lives in the human blood plasma are depicted in
Figure 3. The decomposition of bendamustine is due to the hydrolysis of
the carbon–chlorine bonds of the N‐lost moiety.
The preparation of 7 and the cytotoxicity analysis of 6 and 7
have not yet been reported, which are a part of the present study.
As other anticancer agents are structurally very similar to bend-
amustine, it is conceivable that these can be improved by the same
FIGURE 2 Structures of the nitrogen mustard anticancer agents
chlorambucil (2) and melphalan (3)

ANTONI AND BERNHARDT
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FIGURE 3 Structures of the nitrogen mustard anticancer agent bendamustine (4) and its derivatives 5, 6, and 7, as well as their respective
half‐lives in human blood plasma, and the rationale of the present study
modification. Aiming at increased cytotoxicity, the present study com-
prises the synthesis and cytotoxicity analysis of basic derivatives of fur-
ther members of the nitrogen mustard family, especially chlorambucil
and melphalan. In addition, we considered an isomer of bendamustine
isobendamustine), which, in contrast to conventional bendamustine,
bears the N‐lost moiety in position‐6 (instead of position‐5) of the
benzimidazole ring, is not commercially available. The latter was
prepared from 4‐(1‐methyl‐6‐bis(2‐hydroxyethyl)aminobenzimid
azol‐2‐yl)butyric acid ethyl ester (8), which we received as a kind
gift from Gemini PharmChem. Compound 8 was first treated with
the chlorinating agent thionyl chloride and then with hydrochloric
acid to hydrolyze the ester bond (Scheme 1).
(
6‐isobendamustine), which we recently described in
a
patent
[30,31]
application.
Cytotoxicity analyses of 6‐isobendamustine are still
pending, and this compound serves as another example of increasing the
cytotoxicity of nitrogen mustards by derivatization. A pyrrolidinoalkyl
chain was introduced into the structure of 6‐isobendamustine,
chlorambucil, and melphalan via amide or branched ester formation
The esters and amides were prepared from their respective
parent compounds and a pyrrolidinoalkyl alcohol or amine. The
bendamustine ester 6 was synthesized with the help of the coupling
reagent N,N′‐dicyclohexylcarbodiimide (DCC), as we described
(Figure 3), as these moieties previously proved superior to a linear ester
[29]
group in terms of stability in the blood plasma.
[
29]
previously ; for the preparation of the novel bendamustine amide
the coupling reagents 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3
‐tetramethylaminium tetrafluoroborate (TBTU) and N,N‐
7
,
2
2
|
RESULTS AND DISCUSSION
|
Synthesis
diisopropylethylamine (DIPEA) were used (Scheme 2). Iso-
bendamustine was converted to the ester 10 and the amide 11 in
the same way as compounds 6 and 7, respectively (Scheme 2). The
chlorambucil ester 12 could not be synthesized with DCC, but
coupling with TBTU and DIPEA was successful, and the same
.1
The anticancer agents bendamustine, chlorambucil, and melphalan
[
30,31]
are marketed
drugs. 6‐Isobendamustine
(9)
(here,

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ANTONI AND BERNHARDT
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SCHEME 1 The synthesis of 6‐
isobendamustine (9). Reagents and conditions:
(
2
I) SOCl , dichloromethane, reflux, 60 min; (II)
6
M HCl aq., 90°C, 2 h
SCHEME 2 The synthesis of the target compounds 6, 7, 10–13, 15, and 16. Reagents and conditions: (a) 1‐(pyrrolidin‐1‐yl)propan‐2‐ol,
N,N′‐dicyclohexylcarbodiimide, dimethylformamide (DMF), 0°C, 15 min → 80°C (microwave), 30 min; (b) respective alcohol or amine,
2
‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminium tetrafluoroborate, N,N‐diisopropylethylamine, DMF, rt, overnight or 60–80°C
(
microwave), 45–120 min; (c) Boc O, NaHCO , tetrahydrofuran/H O 1:1, rt, 30 min; (d) trifluoroacetic acid, dichloromethane, rt, 2 h
2
3
2

ANTONI AND BERNHARDT
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(
a)
(b)
(c)
FIGURE 4 Cytotoxicity of bendamustine (4), its ester 6, and its amide 7 against HT‐29 cells (a), MG‐63 cells (b), and SK‐MEL‐3 cells (c).
Cytotoxic/cytocidal effects correspond to the left y‐axes. The growth curves of untreated control cells (open circles) correspond to the right
y‐axes. Data are mean values ± SEM of two to four independent experiments, each performed in octuplicate
reagents were used to prepare the chlorambucil amide 13 (which is
against the following three cancer cell lines were determined: a
carcinoma (colorectal adenocarcinoma, HT‐29), sarcoma
(osteosarcoma, MG‐63), and a malignant melanoma (SK‐MEL‐3).
[
32]
already known in the literature ) (Scheme 2). Melphalan was first
tert‐butyloxycarbonyl (boc)‐protected (to compound 14), then
treated with the respective alcohol or amide and the established
coupling reagents TBTU and DIPEA, and subsequently deprotected
to the melphalan ester 15 or the amide 16 (Scheme 2).
a
2.2.1
|
Bendamustine and derivatives
[
28]
In accordance with our previous report,
bendamustine proved
2
.2
|
Cytotoxicity
ineffective against HT‐29 carcinoma cells (Figure 4). Also, the che-
mosensitivity of MG‐63 osteosarcoma and SK‐MEL‐3 melanoma cells
was very low with slight cytotoxic effects at concentrations above
30 µM. In contrast, the branched ester 6 and the amide 7 exhibited a
distinct increase in cytotoxicity as compared with the parent
The cytotoxicity of the target compounds was investigated in a
kinetic chemosensitivity assay, which allows the distinction between
[
33]
cytotoxic, cytostatic, and cytocidal drug effects.
The effects

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(
a)
(b)
(c)
FIGURE 5 Cytotoxicity of isobendamustine (9), its ester 10, and its amide 11 against HT‐29 cells (a), MG‐63 cells (b), and SK‐MEL‐3 cells (c).
Cytotoxic/cytocidal effects correspond to the left y‐axes. The growth curves of untreated control cells (open circles) correspond to the right
y‐axes. Data are mean values ± SEM of two to four independent experiments, each performed in octuplicate
compound, showing cytocidal effects against the treated cancer cells
of bendamustine. Hence, the method of increasing the cytotoxicity
by the introduction of a basic moiety was successfully applied to the
6‐isomer of bendamustine, and it became obvious that the change in
the position of the nitrogen mustard moiety at the benzimidazole
ring is well tolerated.
at concentrations of 10–30 µM. This is in good accordance with our
[
28]
previously examined linear esters of bendamustine.
Thus, the
replacement of the linear ester moiety by a branched ester or an
amide group did not only increase the stability in human blood
plasma but also proved to be bioisosteric.
2.2.3 Chlorambucil and derivatives
|
2.2.2
|
Isobendamustine and derivatives
Chlorambucil displays a pronounced structural difference from
bendamustine, as it bears a phenyl ring instead of a benzimidazole
moiety. Its effect against HT‐29 carcinoma cells and SK‐MEL‐3
melanoma cells was rather weak, whereas it showed a cytocidal ef-
fect against MG‐63 osteosarcoma cells at a concentration of 30 µM
(Figure 6). The conversion into the basic ester 12 or amide 13
markedly increased the cytotoxicity as compared with the parent
The comportment of isobendamustine in the chemosensitivity assay
was almost identical to its isomer bendamustine—all three cell lines
were refractory against treatment with isobendamustine (Figure 5).
In contrast, the basic derivatives 10 and 11 exerted a pronounced
effect against the three cell lines (cytocidal at concentrations be-
tween 10 and 30 µM), which is in analogy with the basic derivatives

ANTONI AND BERNHARDT
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(
a)
(b)
(c)
FIGURE 6 Cytotoxicity of chlorambucil (2), its ester 12, and its amide 13 against HT‐29 cells (a), MG‐63 cells (b), and SK‐MEL‐3 cells (c).
Cytotoxic/cytocidal effects correspond to the left y‐axes. The growth curves of untreated control cells (open circles) correspond to the right
y‐axes. Data are mean values ± SEM of two to four independent experiments, each performed in octuplicate
compound, both derivatives showing cytocidal effects against HT‐29
and MG‐63 cells at concentrations of 1–10 µM and against SK‐MEL‐
chlorambucil, which may be due to its property as an amino acid
transporter substrate, causing increased cellular uptake. Melphalan
showed a cytocidal effect on carcinoma HT‐29 and osteosarcoma
MG‐63 cells at concentrations of 30 and 10 µM, respectively
(Figure 7). The chemosensitivity of SK‐MEL‐3 cells was lower, with a
cytostatic effect at a concentration of 30 µM. The basic ester 15 and
amide 16 showed effects similar to the parent compound against
MG‐63 cells. However, HT‐29 and SK‐MEL‐3 cells exhibited a higher
response upon treatment with 15 and 16 than when treated with
melphalan (cytocidal effects at concentrations of 3–30 µM). As in the
case of the chlorambucil derivatives, the amide was somewhat more
potent than the ester. All in all, only a slight improvement in cytotoxicity
of melphalan could be achieved by the introduction of a basic moiety.
Comparing the basic derivatives of bendamustine, isobendamus-
tine, chlorambucil, and melphalan, it becomes apparent that they all
display similar potencies—the growth curves of the individual cell lines
3
cells, which were refractory against treatment with chlor-
ambucil, at concentrations of 10–30 µM. The chlorambucil amide was
more potent than the ester; for instance, the amide exhibited a cy-
tocidal effect against MG‐63 osteosarcoma cells at a concentration
as low as 1 µM, whereas a concentration of 10 µM of the ester was
necessary to achieve the same effect. Taken together, the in-
troduction of a basic moiety via an ester or amide bond turned out an
effective means to increase the potency of chlorambucil.
2.2.4
|
Melphalan and derivatives
Melphalan, an L‐phenylalanine‐nitrogen mustard, was more effective
against the three cell lines than bendamustine, isobendamustine, or

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(
a)
(b)
(c)
FIGURE 7 Cytotoxicity of melphalan (3), its ester 15, and its amide 16 against HT‐29 cells (a), MG‐63 cells (b), and SK‐MEL‐3 cells (c).
Cytotoxic/cytocidal effects correspond to the left y‐axes. The growth curves of untreated control cells (open circles) correspond to the right
y‐axes. Data are mean values ± SEM of two to four independent experiments, each performed in octuplicate
upon treatment with 6, 7, 10–13, 15, and 16 are very much alike. This
can be taken as a hint that the enhancement of cytotoxicity is due to
the same mechanism, for instance, increased cellular uptake. As the
basic pyrrolidine ring is protonated under physiological conditions, the
involvement of cation transporters is conceivable. This would also ex-
plain the smaller potency difference between melphalan and its
derivatives—contrary to bendamustine and chlorambucil, melphalan is
cleaved in the human blood plasma, the labile ester moiety was
substituted by a branched ester, resulting in compound UR‐Ant26
(6) or an amide group, yielding UR‐Ant16 (7), both of which markedly
increased the stability. In the current study, the cytotoxicity of
compounds 6 and 7 was examined. By analogy with the previous
linear esters, they showed cytocidal effects at concentrations be-
tween 10 µM and 30 µM—a striking increase in cytotoxicity as
compared with bendamustine, which was practically ineffective
against the treated cells. These results verify that the replacement of
the labile ester moiety by a branched ester or an amide was
bioisosteric.
[12]
imported into the cell by amino acid transporters,
which is why the
cellular uptake does not leave much room for optimization.
3
|
CONCLUSION
In the hope that this approach of increasing the cytotoxicity
would be applicable to other, related nitrogen mustard anticancer
agents, basic esters and amides of 6‐isobendamustine (compounds
UR‐Ant45 [10] and UR‐Ant48 [11]), chlorambucil (compounds
Previously, we have shown that esters of bendamustine are by far
more cytotoxic anticancer agents than their parent compound,
especially those containing a basic moiety. As the latter was rapidly
UR‐Ant66
[12]
and
UR‐Ant55
[13]),
and
melphalan

ANTONI AND BERNHARDT
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|
1
13
(
compounds UR‐Ant65 [15] and UR‐Ant39 [16]) were synthesized. The
instrument (9.40 T, H: 400 MHz, C: 101 MHz) or an Avance 600
1
13
reaction of the respective parent compound with a pyrrolidinoalkyl al-
cohol or amide and the coupling reagent DCC or TBTU yielded the target
compounds. Cytotoxicity against carcinoma, sarcoma, and melanoma
cells was assessed in a kinetic chemosensitivity assay. The novel deri-
vatives showed cytotoxic or cytocidal effects at concentrations above
instrument with a cryogenic probe (14.1 T, H: 600 MHz,
C:
151 MHz) (Bruker) with TMS as an external standard. The high‐
resolution mass spectrometry (HRMS) analysis (see the Supporting
Information) was performed on an Agilent 6540 UHD Accurate‐Mass
Q‐TOF LC/MS system (Agilent Technologies) using an ESI source.
Preparative HPLC was performed on a system from Knauer, con-
sisting of two K‐1800 pumps and a K‐2001 detector. A Nucleodur 100‐5
C18 (5 µm, 110 Å, 250 × 21 mm; Macherey‐Nagel) (compound 7) or a
Kinetex® XB‐C18 (5 µm, 100 Å, 250 × 21.2 mm; Phenomenex) (all other
compounds) served as RP columns at flow rates of 16 ml/min and 15 ml/
min, respectively. Mixtures of MeCN and 0.1% aq trifluoroacetic acid
(TFA) were used as the mobile phase. The detection wavelength was set
to 220 nm throughout. The solvent mixtures were removed by lyophili-
zation using an Alpha 2‐4 LD lyophilization apparatus (Christ) equipped
with an RZ 6 rotary vane vacuum pump (Vacuubrand).
1
μM. This constitutes a striking enhancement over 6‐isobendamustine,
which was ineffective against the selected cancer cells. These results are
very similar to bendamustine and its derivatives, which shows that the
constitution isomerism, that is, the change in the position of the N‐lost
moiety, is well tolerated. Also, an ample improvement over chlorambucil
was achieved, which only showed a weak potency against the sarcoma
cells. Melphalan was almost as effective as the target compounds—
derivatization provided only a small increase in cytotoxicity. It can be
speculated that the introduction of a basic moiety confers substrate
properties of (cation) transporters, thereby increasing cellular uptake and
ultimately cytotoxicity. This would explain the comparable meager en-
hancement achieved by derivatization of melphalan—the latter already
exploits membrane transport systems, as it is a substrate of amino acid
Analytical HPLC of all compounds, except 12 and 15, was per-
formed on a system from Thermo Separation Products (Dreieich),
composed of an SN400 controller, a P4000 pump, a Degassex
DG‐4400 degasser (Phenomenex), an AS3000 autosampler, and a
Spectra Focus UV–visble detector. A Nucleodur 100‐5 C18 (5 μm,
[12]
transporters.
Taken together, the novel nitrogen mustard chemotherapeutics can
be considered interesting molecular tools for the analysis of a correlation
between cytotoxicity and membrane transport mechanisms. Further-
more, the increased antiproliferative activity suggests higher efficacy in
the treatment of malignancies for which the parent compound is
approved and a possible extension of the scope of indications.
250 × 4.0 mm; Macherey‐Nagel) (compound 7) or
a Kinetex®
XB‐C18 (5 µm, 100 Å, 250 × 4.6 mm; Phenomenex) (all other com-
pounds) served as RP columns at a flow rate of 0.75 ml/min. The oven
temperature was set to 30°C throughout. Mixtures of MeCN (A) and
0.05% aq TFA (B) were used as the mobile phase and degassed with
helium. The detection wavelength was set to 220 nm throughout. So-
lutions for injection (100 µM) were prepared in a mixture of A and B,
corresponding to the mixture at the start of the gradient. The following
linear gradient was applied: 0–30 min: A/B 20:80–95:5, 30–35 min: A/B
95:5. Analytical HPLC of compounds 12 and 15 was performed on a
system from Agilent Technologies (Santa Clara) (Series 1100), com-
prising a G1312A binary pump equipped with a G1379A degasser, a
G1329A ALS autosampler, a G1316A COLCOM thermostated column
compartment, and a G1314A VWD detector. A Kinetex® C18 (2.6 µm,
100 Å, 100 × 3 mm; Phenomenex) served as an RP column at a flow rate
of 0.4 ml/min. The oven temperature was set to 30°C throughout.
Mixtures of MeCN (A) and 0.05% aq TFA (B) were used as the mobile
phase. The detection wavelength was set to 220 nm throughout. Solu-
tions for injection (100 µM) were prepared in a mixture of A and B,
corresponding to the mixture at the start of the gradient. The following
linear gradient was applied: 0–12 min: A/B 20:80–95:5, 12–15 min: A/B
95:5. Retention (capacity) factors were calculated from retention times
4
4
4
|
EXPERIMENTAL
|
Chemistry
.1
.1.1
|
General experimental conditions
Chemicals and solvents were purchased from commercial suppliers
Sigma Aldrich, Merck, VWR, Thermo Fisher Scientific, and TCI) and
(
used without further purification unless stated otherwise. Bend-
amustine was a kind gift from Arevipharma. 4‐(1‐Methyl‐6‐bis(2‐
hydroxyethyl)aminobenzimidazol‐2‐yl)butyric acid ethyl ester was a
kind gift from Gemini PharmChem. Reactions requiring anhydrous
conditions were carried out in dried reaction vessels under an at-
mosphere of argon and anhydrous solvents were used. Millipore
water was used throughout for the preparation of buffers and high‐
performance liquid chromatography (HPLC) eluents. Acetonitrile
(t
R R 0 0 0
) according to k = (t − t )/t (t = dead time of the respective HPLC
(
MeCN) for HPLC (gradient grade) was obtained from Merck.
Microwave reactions were carried out in an Initiator
system).
8
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information.
microwave reactor (Biotage).
Thin‐layer chromatography (TLC) was performed on TLC Silica gel
60 F254 aluminum plates (Merck). Visualization was accomplished by
UV irradiation at wavelengths of 254 and 366 nm or by staining with
ninhydrin (1.5 g ninhydrin, 5 ml acetic acid, and 500 ml 95% ethanol).
The nuclear magnetic resonance (NMR) spectra (see the
Supporting Information) were recorded on an Avance 400
4.1.2 General procedure 1 for ester bond formation
|
The respective carboxylic acid (1.0 eq) was dissolved in anhydrous
DMF (0.2–0.3 M) in a microwave reaction vessel. 1‐(Pyrrolidin‐1‐yl)

10 of 14
ANTONI AND BERNHARDT
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propan‐2‐ol (1.5 eq) was added and the stirred mixture was cooled
down to 0°C using an ice bath. DCC (1.1 eq) was added and stirring
was continued at 0°C for 15 min and at 80°C in a microwave reactor
for 30 min. The mixture was subjected to preparative HPLC (eluent:
MeCN/0.1% aq TFA) and the eluate was lyophilized.
0.307 mmol, 1.1 eq), 2‐(pyrrolidin‐1‐yl)ethan‐1‐amine (35.4 µl,
0.279 mmol, 1.0 eq), and DMF (1 ml). Preparative HPLC (0–30 min:
MeCN/0.1% aq TFA 19:81–55:45, t
R
= 13.0 min) yielded 7 as a yellowish
1
resin (97.5 mg, 0.143 mmol, 51%). H NMR (600 MHz, CDCl
3
): δ
(ppm) = 11.43 (s, 1H), 8.24 (t, J = 4.9 Hz, 1H), 7.39 (d, J = 9.2 Hz, 1H), 6.98
d, J = 2.3 Hz, 1H), 6.95 (dd, J = 9.2 Hz, J = 2.3 Hz, 1H), 3.89 (s, 3H), 3.81 (t,
(
J = 6.6 Hz, 6H), 3.67 (t, J = 6.6 Hz, 4H), 3.49 (q, J = 5.4 Hz, 2H), 3.24 (t,
J = 5.4 Hz, 2H), 3.19 (t, J = 7.4 Hz, 2H), 2.89 (br s, 2H), 2.39 (t, J = 6.7 Hz,
4.1.3
|
General procedure 2 for ester bond formation
1
3
2
H), 2.18 (qi, J = 7.1 Hz, 2H), and 2.08 (br s, 4H). C NMR (150 MHz,
Under dry conditions, the respective carboxylic acid (1.0 eq) was
dissolved in anhydrous DMF (0.2–0.3 M) in a microwave reaction
vessel. DIPEA (2.0 eq) and the coupling reagent TBTU (1.0 eq) were
added, and the solution was stirred at room temperature for 5 min.
CDCl ): δ (ppm) = 173.0, 162.1 (TFA), 161.9 (TFA), 161.6 (TFA), 161.4
3
(TFA), 151.5, 146.1, 132.8, 125.1, 117.5 (TFA), 115.5 (TFA), 112.9, 112.3,
96.4, 55.0, 54.4 (2C), 54.1 (2C), 40.6 (2C), 35.7, 34.3, 31.0, 24.7, 23.2 (2C),
and 22.5. Reversed‐phase high‐performance liquid chromatography (RP‐
1
‐(Pyrrolidin‐1‐yl)propan‐2‐ol (1.5 eq) was added and stirring was
HPLC) (220 nm): 99% (t
R
= 7.8 min, k = 2.3). HRMS (ESI): m/z
+
+
continued for another 45 min at 80°C in a microwave reactor. The
mixture was subjected to preparative HPLC (eluent: MeCN/0.1% aq
TFA) and the eluate was lyophilized. In the case of boc‐protected
compounds, work‐up and deprotection preceded purification of the
final product by preparative HPLC.
[M+H] calcd. for
O·C
C
22
H
34Cl
2
N
5
O : 454.2135, found: 454.2135.
C
22
H
33Cl
2
N
5
4
H
2
F
6
O
4
(454.44 + 228.05).
4‐{6‐[Bis(2‐chloroethyl)amino]‐1‐methyl‐1H‐benzo[d]imidazol‐2‐yl}‐
[30,31]
butanoic acid (6‐isobendamustine) hydrotrifluoroacetate (9)
4.1.4
|
General procedure for amide bond formation
Under dry conditions, the respective carboxylic acid (1.0 eq) was dis-
solved in anhydrous DMF (0.2–0.3 M) in a microwave reaction vessel or a
round‐bottom flask. DIPEA (2.0–3.0 eq) and the coupling reagent TBTU
(1.0 eq) were added and the solution was stirred at room temperature
for 5 min. The appropriate amine (1.0–1.1 eq) was added and stirring was
continued for another 45–120 min at 60–80°C in a microwave reactor or
at room temperature overnight. The mixture was subjected to pre-
parative HPLC (eluent: MeCN/0.1% aq TFA) and the eluate was lyophi-
lized. In the case of boc‐protected compounds, work‐up and deprotection
preceded purification of the final product by preparative HPLC.
4‐(1‐Methyl‐6‐bis(2‐hydroxyethyl)aminobenzimidazol‐2‐yl)‐
butyric acid ethyl ester (800 mg, 2.29 mmol, 1.0 eq) was dissolved in
dichloromethane (DCM) and the chlorinating agent thionyl chloride
(498 µl, 6.87 mmol, 3.0 eq) was added dropwise. The mixture was
refluxed for 60 min and the volatiles were removed under reduced
pressure. The ester bond was hydrolyzed with 6 M HCl aq (4 ml)
90°C for 2 h. The mixture was washed with DCM to remove lipo-
philic impurities and isobendamustine hydrochloride was pre-
cipitated by adding 10 M NaOH aq until a pH of 0–1 was reached.
Recrystallization in isopropanol yielded isobendamustine free base
as a brownish solid (346 mg, 0.966 mmol, 42%). For further pur-
ification, 50 mg of the substance was subjected to preparative HPLC
4
‐{5‐[Bis(2‐chloroethyl)amino]‐1‐methyl‐1H‐benzo[d]imidazol‐2‐yl}‐
N‐[2‐(pyrrolidin‐1‐yl)ethyl]butanamide (isobendamustine 2‐
pyrrolidinoethyl amide)bis(hydrotrifluoroacetate) (7)
R
(gradient: 0–30 min: MeCN/0.1% aq TFA 25:75–55:45, t = 13.0 min)
and the eluate was lyophilized, yielding compound 9 as a brownish
resin (61.8 mg, 0.131 mmol, 94%). Analytical and pharmacological
characterization was performed with the HPLC‐purified substance.
1
6
H NMR (600 MHz, deuterated dimethyl sulfoxide [DMSO‐d ]): δ
(
ppm) = 14.70 (br s, 1H), 12.26 (br s, 1H), 7.58 (d, J = 9.03 Hz, 1H),
.11 (d, J = 2.28 Hz, 1H), 7.08 (dd, J = 9.03 Hz, J = 2.28 Hz, 1H), 3.90
s, 3H), 3.86 (t, J = 6.57 Hz, 2H), 3.80 Hz (t, J = 6.66 Hz, 4H), 3.15 (t,
J = 7.74 Hz, 2H), 2.41 (t, J = 7.1 Hz, 2H), and 2.00 (qi, J = 7.56 Hz, 2H).
7
(
Compound 7 was prepared according to the general procedure for
the amide bond formation (in a round‐bottom flask with stirring over-
night). The reaction was carried out using bendamustine (100 mg,
1
3
6
C NMR (151 MHz, DMSO‐d ): δ (ppm) = 173.7, 158.5 (TFA), 158.3
(TFA), 158.1 (TFA), 157.8 (TFA), 151.8, 145.4, 134.3, 121.9, 117.5
(TFA), 115.6 (TFA), 114.5, 112.7, 94.0, 52.4 (2C), 41.2 (2C), 32.5,
0.279 mmol, 1.0 eq), TBTU (98.6 mg, 0.307 mmol, 1.1 eq), DIPEA (52.2 µl,

ANTONI AND BERNHARDT
11 of 14
|
3
0.8, 24.2, and 21.4. RP‐HPLC (220 nm): 98% (t
R
= 11.7 min, k = 2.9).
Compound 11 was prepared according to the general procedure
for the amide bond formation (in the microwave). The reaction was
carried out using isobendamustine (100 mg, 0.279 mmol, 1.0 eq),
TBTU (89.6 mg, 0.279 mmol, 1.0 eq), DIPEA (97.2 µl, 0.558 mmol, 2.0
eq), 2‐(pyrrolidin‐1‐yl)ethan‐1‐amine (38.9 µl, 0.279 mmol, 1.0 eq),
and DMF (1 ml). Preparative HPLC (0–30 min: MeCN/0.1% aq TFA
+
+
2
HRMS (ESI): m/z [M+H] calcd. for C16
H22Cl
2
N
3
O
: 358.1084,
2 3 2 2 3 2
found: 358.1104. C16H21Cl N O ·C HF O (358.26 + 114.02).
1
‐(Pyrrolidin‐1‐yl)propan‐2‐yl 4‐{6‐[bis(2‐chloroethyl)amino]‐1‐
methyl‐1H‐benzo[d]imidazol‐2‐yl}butanoate (isobendamustine 1‐
methyl‐2‐pyrrolidinoethyl ester) bis(hydrotrifluoroacetate) (10)
15:85–55:45, t
R
= 14.2 min) yielded 11 as a yellow resin (77.2 mg,
1
0
.113 mmol, 41%). H NMR (600 MHz, DMSO‐d
6
): δ (ppm) = 14.98
(
br s, 1H), 10.02 (s, 1H), 8.24 (t, J = 5.69 Hz, 1H), 7.58 (d, J = 9.07 Hz,
H), 7.11 (d, J = 2.25 Hz, 1H), 7.08 (dd, J = 9.07 Hz, J = 2.25, 1H), 3.91
s, 3H), 3.86 (t, J = 6.51 Hz, 4H), 3.80 (t, J = 6.51 Hz, 4H), 3.57 (br s,
1
(
2
H), 3.36 (m, 2H), 3.17 (s, 2H), 3.13 (t, J = 7.71 Hz, 2H), 2.99 (br s,
13
2
H), 2.27 (t, J = 7.24 Hz, 2H), 2.01 (m, 2H), and 1.92 (m, 4H). C NMR
): δ (ppm) = 171.9, 158.8 (TFA), 158.6 (TFA),
(151 MHz, DMSO‐d
6
1
1
3
58.4 (TFA), 158.1 (TFA), 151.9, 145.4, 134.3, 122.0, 117.6 (TFA),
15.6 (TFA), 114.6, 112.7, 94.0, 53.3 (2C), 53.1, 52.4 (2C), 41.2 (2C),
5.0, 33.7, 30.8, 24.3, 22.5 (2C), and 21.8. RP‐HPLC (220 nm): 96%
+
Compound 10 was prepared according to the general procedure 1
for the ester bond formation. The reaction was carried out using iso-
bendamustine (75 mg, 0.209 mmol, 1.0 eq), 1‐(pyrrolidin‐1‐yl)propan‐2‐ol
(t
R
= 9.0 min, k = 2.0). HRMS (ESI): m/z [M+H]
calcd. for
+
C
22
H
34Cl
2
N
5
O : 454.2135, found: 454.2135. C22
H
33Cl
2
N
5
4 2
O·C H -
6 4
F O (454.44 + 228.05).
(40.6 mg, 0.314 mmol, 1.5 eq), DCC (47.5 mg, 0.230 mmol, 1.1 eq), and
DMF (0.5 ml). A yellow coloration and a white precipitate could be ob-
1‐(Pyrrolidin‐1‐yl)propan‐2‐yl 4‐{4‐[bis(2‐chloroethyl)amino]phenyl}‐
butanoate (chlorambucil 1‐methyl‐2‐pyrrolidinoethyl ester)
hydrotrifluoroacetate (12)
served. Preparative HPLC (0–30 min: MeCN/0.1% aq TFA 20:80–55:45,
t
R
= 14.5 min) yielded 10 as a brownish resin (32.8 mg, 0.047 mmol, 22%).
1
More than one diastereoisomer was evident in the NMR spectra.
NMR (600 MHz, DMSO‐d
d, J = 9.13 Hz, 1H), 7.12 (d, J = 2.23 Hz, 1H), 7.08 (dd, J = 9.13 Hz,
J = 2.23 Hz, 1H), 5.13 (m, 1H), 3.91 Hz (s, 3H), 3.86 (t, J = 6.58 Hz, 4H),
H
6
): δ (ppm) = 15.03 (br s, 1H), 10.18 (s, 1H), 7.59
(
3.80 (t, J = 6.58 Hz, 4H), 3.54 (br s, 2H), 3.38 (m, 2H), 3.16 (t, J = 7.56 Hz,
2H), 3.10 (br s, 2H), 2.55 (m, 2H), 2.04 (m, 2H), 1.92 (br s, 4H), and 1.18 (d,
13
6
J = 6.25 Hz, 3H). C NMR (151 MHz, DMSO‐d ): δ (ppm) = 171.7, 158.8
(TFA), 158.5 (TFA), 158.3 (TFA), 158.1 (TFA), 151.7, 145.4, 134.3, 122.0,
117.4 (TFA), 115.5 (TFA), 114.6, 112.7, 94.0, 66.5, 57.4, 54.9 and 53.3
(
the two carbons adjacent to the pyrrolidine nitrogen yielded two sig-
Compound 12 was prepared according to the general procedure
2 for the ester bond formation. The reaction was carried out using
chlorambucil (80 mg, 0.263 mmol, 1.0 eq), TBTU (84.4 mg,
0.263 mmol, 1.0 eq), DIPEA (89.4 µl, 0.526 mmol, 2.0 eq), 1‐
(pyrrolidin‐1‐yl)propan‐2‐ol (51.0 µl, 0.394 mmol, 1.5 eq), and DMF
nals), 52.4 (2C), 41.2 (2C), 32.4, 30.8, 24.1, 22.6 (2C), 21.1, and 17.7. RP‐
HPLC (220 nm): 96% (t
R
= 10.2 min, k = 2.4). HRMS (ESI): m/z
+
+
[M+H] calcd. for
C
23
H
35Cl
2
N
4
O
2
:
469.2132, found: 469.2137.
C
23
H
34Cl
2
N
4
O
2
·C
4
H
2
F
6
O
4
(469.45 + 228.05).
(1 ml). Preparative HPLC (0–30 min: MeCN/0.1% aq TFA
4
‐{6‐[Bis(2‐chloroethyl)amino]‐1‐methyl‐1H‐benzo[d]imidazol‐2‐yl}‐
33:67–69:31, t = 15.5 min) yielded 12 as a brownish resin (49.2 mg,
R
N‐[2‐(pyrrolidin‐1‐yl)ethyl]butanamide (isobendamustine 2‐
pyrrolidinooethyl amide) bis(hydrotrifluoroacetate) (11)
0.093 mmol, 35%). More than one diastereoisomer was evident in
1
the NMR spectra. H NMR (600 MHz, DMSO‐d
6
) δ (ppm) 9.99 (br s,
1
H), 7.02 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 5.13 (m, 1H), 3.69
m, 8H), 3.59–3.45 (m, 2H), 3.44–3.30 (m, 2H), 3.12–3.99 (m, 2H),
.49–2.45 (m, 2H), 2.38–2.27 (m, 2H), 1.99 (m, 2H), 1.86 (m, 2H), 1.77
(
2
13
(
m, 2H), and 1.20 (d, J = 6.3 Hz, 3H). C NMR (151 MHz, DMSO‐d
6
) δ
(ppm) 172.3, 158.6 (TFA), 158.3 (TFA), 158.1 (TFA), 157.9 (TFA),
1
5
44.5, 129.4, 129.3 (2C), 117.5 (TFA), 115.6 (TFA), 111.9 (2C), 66.1,
7.4, 54.9 and 53.3 (the two carbons adjacent to the pyrrolidine
nitrogen yielded two signals), 52.2 (2C), 41.2 (2C), 33.2, 33.0, 26.3,
2.7 and 22.5 (the two pyrrolidine carbons not adjacent to the ni-
trogen yielded two signals), and 17.7. RP‐HPLC (220 nm): 92% (t
.2 min, k = 6.2) (the second peak in the chromatogram is not due to
2
R
=
9

12 of 14
ANTONI AND BERNHARDT
|
impurity but due to decomposition in the aqueous HPLC eluent).
30 min. The suspension was diluted with EtOAc and the organic layer
+
+
HRMS (ESI): m/z [M+H] calcd. for C21
found: 415.1924. C21 32Cl ·C HF
H
33Cl
2
N
2
O
2
: 415.1914,
2 4
was washed with 1 M HCl aq and brine, dried over Na SO , and
H
2
N
2
O
2
2
3
O
2
(414.18 + 114.02).
concentrated under reduced pressure to a brown oil, which was used
1
in subsequent reactions without further purification. H NMR
4
‐{4‐[Bis(2‐chloroethyl)amino]phenyl}‐N‐[2‐(pyrrolidin‐1‐yl)ethyl]‐
3
(400 MHz, CDCl ) δ (ppm) 9.79 (s, 1H), 7.07 (d, J = 7.8 Hz, 2H), 6.62
butanamide (chlorambucil 2‐pyrrolidinooethyl amide)
(d, J = 8.6 Hz, 2H), 4.96 (d, J = 7.4 Hz, 1H), 4.55 (m, 1H), 3.70 (m, 4H),
[32]
hydrotrifluoroacetate (13)
3.61 (m, 4H), 3.13–2.96 (m, 2H), and 1.52 (s, 9H). HRMS (ESI): m/z
+
+
[
M+H] calcd. for C22
2 5
H34Cl N O : 454.2135, found: 454.2135.
C
18
H
2 2 4
26Cl N O (504.32).
1‐(Pyrrolidin‐1‐yl)propan‐2‐yl (2S)‐2‐amino‐3‐{4‐[bis(2‐chloroethyl)‐
amino]phenyl}propanoate (melphalan 1‐methyl‐2‐pyrrolidinoethyl
ester) bis(hydrotrifluoroacetate) (15)
Compound 13 was prepared according to the general procedure for
the amide bond formation (in the microwave). The reaction was carried
out using chlorambucil (100 mg, 0.329 mmol, 1.0 eq), TBTU (105.5 mg,
0
.329 mmol, 1.0 eq), DIPEA (111.8 µl, 0.657 mmol, 2.0 eq), 2‐(pyrrolidin‐
‐yl)ethan‐1‐amine (45.8 µl, 0.362 mmol, 1.1 eq), and DMF (1 ml). Two-
1
fold purification by preparative HPLC (0–30 min: MeCN/0.1% aq TFA
2
0
8
3
2
8:72–68:32, t
R
= 13.5 min) yielded 13 as a yellowish resin (75.5 mg,
1
.147 mmol, 45%). H NMR (600 MHz, DMSO‐d
6
) δ (ppm) 9.80 (br s, 1H),
Compound 15 was prepared according to the general procedure
2 for the ester bond formation. The reaction was carried out using
boc‐melphalan (133 mg, 0.328 mmol, 1.0 eq), TBTU (105 mg,
0.328 mmol, 1.0 eq), DIPEA (111 µl, 0.655 mmol, 2.0 eq), 1‐
(pyrrolidin‐1‐yl)propan‐2‐ol (63.5 µl, 0.491 mmol, 1.5 eq), and DMF
(3 ml). The mixture was diluted with brine (50 ml) and the product
was extracted with EtOAc (3 × 50 ml). The combined organic layers
.07 (m, 1H), 7.01 (d, J = 8.6 Hz, 2H), 6.66 (d, J = 8.6 Hz, 2H), 3.69 (m, 8H),
.57 (m, 2H), 3.36 (q, J = 6.1 Hz, 2H), 3.18 (q, J = 6.0 Hz, 2H), 3.00 (m, 2H),
.44 (t, J = 7.7 Hz, 2H), 2.10 (t, J = 7.4 Hz, 2H), 1.99 (m, 2H), 1.85 (m, 2H),
13
6
and 1.74 (m, 2H). C NMR (151 MHz, DMSO‐d ) δ (ppm) 172.8, 158.6
(TFA), 158.4 (TFA), 158.2 (TFA), 158.0 (TFA), 144.4, 129.8, 129.3 (2C),
117. 6 (TFA), 115.6 (TFA), 111.9 (2C), 53.4 (2C), 53.3, 52.2 (2C), 41.2
(
2C), 35.0, 34.8, 33.6, 27.0, and 22.5 (2C). RP‐HPLC (220 nm): 92%
= 14.3 min, k = 3.7) (the second peak in the chromatogram is not due
to impurity but due to decomposition in the aqueous HPLC eluent).
2 4
were dried over Na SO and concentrated under reduced pressure.
(t
R
For N‐boc deprotection, the residue was dissolved in DCM (2 ml), and
TFA (0.5 ml) was added and the solution was stirred at room tem-
perature for 2 h. Evaporation of the volatiles and purification by
preparative HPLC (0–30 min: MeCN/0.1% aq TFA 21:79–57:43,
+
+
HRMS (ESI): m/z [M+H] calcd. for C20
2 3
H32Cl N O : 400.1917, found:
4
00.1925. C20 31Cl O·C HF (399.18 + 114.02).
H
2
N
3
2
3 2
O
R
t = 13.5 min) yielded 15 as a brown resin (20.2 mg, 0.031 mmol, 10%
(
S)‐3‐{4‐[Bis(2‐chloroethyl)amino]phenyl}‐2‐[(tert‐butoxycarbonyl)‐
for protection, coupling, and deprotection). More than one diaster-
[
34,35]
1
amino]propanoic acid (boc‐melphalan) (14)
eoisomer was evident in the NMR spectra. H NMR (600 MHz,
DMSO‐d
6
) δ (ppm) 10.07 (br s, 1H), 8.57 (m, 3H), 7.08 (m, 2H), 6.72
(m, 2H), 5.16 (m, 1H), 4.33–4.15 (m, 1H), 3.72 (m, 8H), 3.62–3.31 (m,
13
4
H), 3.12–2.91 (m, 4H), 2.05–1.74 (m, 4H), 1.27–1.12 (m, 3H).
) δ (ppm) 168.7, 168.0, 158.8 (TFA), 158.6
TFA), 158.4 (TFA), 158.2 (TFA), 145.7, 145.7, 130.7, 130.6 (2C),
C
NMR (151 MHz, DMSO‐d
6
(
1
5
22.3, 122.0, 117.6 (TFA), 115.6 (TFA), 112.0 (2C), 68.9, 68.6, 57.1,
7.0, 54.8, 53.7, 53.5, 53.3, 53.0, 52.0 (2C), 41.1 (2C), 34.8, 34.8, 22.6
(
2C), 17.3, and 17.2. RP‐HPLC (220 nm): 98% (t
R
= 5.5 min, k = 3.4).
+
+
HRMS (ESI): m/z [M+H] calcd. for C20
found: 416.1871. C20 31Cl ·C
H
32Cl
2
N
3
O
2
: 416.1866,
H
2
N
3
O
2
4
H
2
F
6
O
4
(416.39 + 228.05).
Melphalan (38.0 mg, 0.125 mmol, 1.0 eq) and NaHCO
.374 mmol, 3.0 eq) were dissolved in tetrahydrofuran/H
1 ml and Boc O [di‐tert‐butyl dicarbonate]; 42.9 µl, 0.1787 mmol, 1.5
eq) was added and the mixture stirred at room temperature for
3
(31.4 mg,
0
2
O 1:1
(S)‐2‐Amino‐3‐{4‐[bis(2‐chloroethyl)amino]phenyl}‐N‐[3‐(pyrrolidin‐
1‐yl)propyl]propanamide (melphalan 3‐pyrrolidinopropyl amide)
bis(hydrotrifluoroacetate) (16)
(
2

ANTONI AND BERNHARDT
13 of 14
|
Instruments: Absorbance measurements were carried out with a
GENios Pro microplate reader (equipped with a Xenon arc lamp; Tecan).
Software: All biological data were analyzed with GraphPad Prism
5
(GraphPad Software).
4.2.2 Cell culture
|
Compound 16 was prepared according to the general procedure
for the amide bond formation (in the microwave). The reaction was
carried out using boc‐melphalan (64.2 mg, 0.125 mmol, 1.0 eq), TBTU
All cell lines were cultured in the RPMI‐1640 medium containing
110 mg/l sodium pyruvate, 2.4 g/l HEPES, and 2.0 g/l NaHCO , and
supplemented with 10% (v/v) FCS at 37°C in a water‐saturated at-
mosphere containing 5% CO . Cells were passaged after treatment
3
(
40.0 mg, 0.125 mmol, 1.0 eq), DIPEA (65.1 µl, 0.374 mmol, 3.0 eq), 3‐
pyrrolidin‐1‐yl)propan‐1‐amine (47.3 µl, 0.374 mmol, 3.0 eq), and
2
(
with a solution containing 0.05% trypsin and 0.025% EDTA. All cells
were routinely monitored for mycoplasma contamination by poly-
merase chain reaction using the Venor®GeM mycoplasma detection
kit (Minerva Biolabs), which were found to be negative.
DMF (1 ml). The mixture was diluted with brine (20 ml) and the
product was extracted with EtOAc (3 × 20 ml). The combined organic
layers were dried over Na
pressure. For N‐boc deprotection, the residue was dissolved in DCM
0.5 ml), and TFA (0.5 ml) was added and the solution was stirred at
2 4
SO and concentrated under reduced
(
[
33]
room temperature for 2 h. Evaporation of the volatiles and purification
4.2.3 Chemosensitivity assay
|
by preparative HPLC (0–30 min: MeCN/0.1% aq TFA 15:85–55:45,
t
R
= 14.4 min) yielded 16 as a brownish resin (22.9 mg, 0.036 mmol, 29%
Cells were seeded into 96‐well plates at a density of 1500 cells per well
(MG‐63 and HT‐29 cells) or a density of 3000 cells per well (SK‐MEL‐3
cells) (100 μl/well), and they were allowed to attach to the surface of the
for protection, coupling and deprotection). More than one diastereoi-
1
somer was evident in the NMR spectra. H NMR (600 MHz, DMSO‐d
6
)
δ (ppm) 10.08 (s, 1H), 8.59 (t, J = 5.8 Hz, 1H), 8.20 (br s, 3H), 7.06 (d,
J = 8.7 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 3.86 (m, 1H), 3.71 (s, 8H), 3.51
2
microplates in a water‐saturated atmosphere containing 5% CO at 37°C
overnight. The next day, fresh medium containing the test compounds at
two‐fold final concentrations was added (100 µl/well; giving a final vo-
lume of 200 µl/well). On each plate, vinblastine at a final concentration of
300 nM served as reference cytostatic (positive control); the vehicle
DMSO (0.1%) served as a negative control to monitor cell growth in the
absence of a drug. Each concentration was measured in octuplicate and
the negative control in a 16‐fold replication. Growth of the cells was
stopped after different periods of time by removal of medium and fixa-
tion with 2% (v/v) glutardialdehyde in phosphate‐buffered saline (100 µl/
well). All the plates were stored at 4°C until the end of the experiment
and afterward stained with 0.02% crystal violet in water (100 µl/well) for
(m, 2H), 3.21–3.09 (m, 2H), 3.07–2.98 (m, 2H), 2.95–2.83 (m, 4H),
13
2
.03–1.82 (m, 4H), and 1.79–1.67 (m, 2H). C NMR (151 MHz, DMSO‐
d
1
5
6
) δ (ppm) 168.3, 158.8 (TFA), 158.6 (TFA), 158.4 (TFA), 158.2 (TFA),
45.5, 130.5 (2C), 122.8, 117.8 (TFA), 115.9 (TFA), 112.0 (2C), 53.9,
3.3 and 53.2 (the two carbons adjacent to the pyrrolidine nitrogen
yielded two signals), 52.1 (2C), 51.8, 41.2 (2C), 36.1, 35.9, 25.4, and 22.6
(
2C). RP‐HPLC (220 nm): 97% (t
R
= 8.9 min, k = 1.9). HRMS (ESI): m/z
+
+
[M+H] calcd. for
20 2
C H33Cl N
4
O : 415.2026, found: 415.2034.
C
20
H
32Cl
2
N
4
O·C
4
H
2
F
O
6 4
(415.40 + 228.05).
20 min. Excess dye was removed by rinsing the plates with water three
4
.2
.2.1
|
Biology
General experimental conditions
times. Crystal violet bound by the fixed cells was redissolved in 70%
ethanol (180 µl/well) while shaking the microplates for 2–3 h. The ab-
sorbance (580 nm) as a parameter proportional to the cell mass was
measured using a GENios Pro microplate reader.
4
|
Materials: Commodity chemicals and solvents were purchased from
commercial suppliers (Sigma Aldrich, Merck, VWR, Thermo Fisher
Scientific, Invitrogen, and Serva). Millipore water was used
throughout for the preparation of buffers and aqueous reagent so-
lutions. The pH of buffers was adjusted with NaOH aq or HCl aq. All
cell lines were purchased from the ATCC (American Type Culture
Collection). Tissue culture flasks were procured from Sarstedt. The
RPMI‐1640 medium was purchased from Sigma Aldrich. Fetal calf
serum (FCS) and trypsin/EDTA solution was purchased from
Biochrom. Ninety‐six‐well microplates (PS, clear, F‐bottom, with
lid, sterile) were purchased from Greiner Bio‐One.
Cytotoxic effects were expressed as corrected T/C values
according to
T − C0
C − C0
T/Ccorr [%] =
× 100,
where T is the mean absorbance of the treated cells, C is the mean
absorbance of the negative controls, and C is the mean absorbance of
the cells at the time of compound addition (t ). When the absorbance of
treated cells T was lower than at the beginning of the experiment (C ),
0
0
0
the extent of cell killing was calculated as cytocidal effect according to
T − C0
Stock solutions: The test compounds were dissolved in DMSO at
Cytocidal effect [%] =
× 100.
C0
1
000 times the final concentrations in the chemosensitivity assay.

14 of 14
ANTONI AND BERNHARDT
|
ACKNOWLEDGMENTS
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The authors thank Maria Beer‐Krön for excellent technical assis-
tance, Marlies Antoni for skillful linguistic support, and Christian and
Helmut Schickaneder for helpful discussions. Open Access funding
enabled and organized by ProjektDEAL.
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CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
AUTHOR CONTRIBUTIONS
[24] R. Y. LaBell, P. R. Patel, (Cephalon, Inc.), USA US20120157505A1, 2012.
[
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Frauke Antoni conceived the project with input from Günther Bernhardt.
Frauke Antoni performed the synthesis, the cytotoxicity assays, and the
data analysis with Günther Bernhardt as a supervisor. Frauke Antoni
wrote the manuscript with input from Günther Bernhardt.
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ORCID
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Frauke Antoni
http://orcid.org/0000-0001-8382-6315
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