Design and synthesis of peptide conjugates of phosphoramide mustard as prodrugs activated by prostate-specific antigen

Article abstract of DOI:10.1016/j.bmc.2016.04.035

A series of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl phosphoramide mustard conjugates (1a-e) was designed and synthesized as potential prodrugs for site-specific activation by PSA in prostate cancer cells. All conjugates were found to be substrates of PSA with cleavage occurring between Gln and the para-aminobenzyl (PAB) linker. Structure-activity relationship studies on these conjugates indicated that introduction of electron-withdrawing fluorine(s) on the phenyl ring in the PAB linker uniformly improved the chemical stability of the conjugates while the position of substitution affected differently the self-immolative process of conjugates upon proteolysis. Introduction of a fluorine at ortho position to benzylic phosphoramide as in 1b results in better stability of the conjugate prior to activation while maintaining its antiproliferative activity upon activation by PSA. The conjugate 1b with 2-fluoro substitution was identified as a promising lead for further evaluation and optimization in the development of prostate cancer-targeted prodrugs.

Full text of DOI:10.1016/j.bmc.2016.04.035

Bioorganic & Medicinal Chemistry 24 (2016) 2697–2706
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of peptide conjugates of phosphoramide
mustard as prodrugs activated by prostate-specific antigen
Xinghua Wu a, Longqin Hu a,b,
⇑
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 February 2016
Revised 8 April 2016
Accepted 17 April 2016
Available online 19 April 2016
A series of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl phosphoramide mustard conjugates (1a–e)
was designed and synthesized as potential prodrugs for site-specific activation by PSA in prostate cancer
cells. All conjugates were found to be substrates of PSA with cleavage occurring between Gln and the
para-aminobenzyl (PAB) linker. Structure–activity relationship studies on these conjugates indicated that
introduction of electron-withdrawing fluorine(s) on the phenyl ring in the PAB linker uniformly improved
the chemical stability of the conjugates while the position of substitution affected differently the self-
immolative process of conjugates upon proteolysis. Introduction of a fluorine at ortho position to benzylic
phosphoramide as in 1b results in better stability of the conjugate prior to activation while maintaining
its antiproliferative activity upon activation by PSA. The conjugate 1b with 2-fluoro substitution was
identified as a promising lead for further evaluation and optimization in the development of prostate can-
cer-targeted prodrugs.
Keywords:
Phosphoramide mustard prodrug
Prostate-specific antigen
Site-specific activation
Self-immolative linker
Structure–activity relationship
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
antigen feature: A PSA-specific antibody conjugated to a cytotoxic
drug can be developed to improve cytotoxic selectivity that will be
According to American Cancer Society, prostate cancer is the
second most common cancer found in American men after skin
cancer.1 Current treatment options include surgery, radiation, hor-
mone deprivation, and chemotherapy. After initial treatment,
many patients may still progress to metastatic prostate cancer
for which there is no effective therapy. Several recently approved
drugs, such as abiraterone, enzalutamide, sipuleucel-T, docetaxel,
cabazitaxel and Radium-223, are only providing an extended sur-
vival benefit of several months.2–7 Thus, there is still an urgent
need for new drugs that provide better antitumor selectivity and
effectiveness.8
Prostate-specific drug design is an attractive approach to local-
ize the cytotoxicity at the prostate tumor site.8 Prostate-specific
antigen (PSA) is among the most studied targets for the design of
next generation of drugs against prostate cancer. PSA is an antigen
with a serine protease activity and is over-expressed in the pros-
tate tissue and prostate carcinoma.9 The PSA in systemic circula-
tion is enzymatically inactive due to formation of complexes
with the protease inhibitors present in the blood, while its protease
activity is confined to the prostate or prostate-derived cancer
cells.10,11 One approach to obtain PSA-targeted drugs is to use PSA’s
localized in the prostate cancer tissues.12 Another approach is to
use PSA’s protease activity: A prodrug can be designed and acti-
vated only upon exposure to the enzymatically active PSA in the
prostate cancer tissues, while the prodrug remains inactive in sys-
temic circulation. Incorporation of PSA-specific peptides with var-
ious anticancer cytotoxic agents has been reported to exhibit
selective cytotoxicity against PSA-expressing cancer cells.8,13–22
Some dual-action prodrugs were also designed to increase uptake
of drugs by tumor cells, like albumin-binding PSA-specific peptide
drug conjugates23,24 and PSA-specific peptide drug conjugates
linked with an HER2-specific peptide.25 In this paper, we describe
our PSA-targeted prodrug approach to release cytotoxic phospho-
ramide mustard site-specifically in prostate tumor tissues by the
proteolytic action of PSA.
2. Design and synthesis
By design, the PSA-specific peptide-drug conjugate remains
inactive until it reaches the prostate-derived tumor tissues. Upon
proteolytic cleavage by PSA, the cytotoxic drug is released locally
in the microenvironment that surrounds prostate cancer cells.
The anticancer selectivity is, therefore, achieved through this
site-specific activation mechanism. PSA as an enzyme is sensitive
to structural bulkiness at the cleavage site: Coupling the peptide
⇑
Corresponding author. Tel.: +1 848 445 5291; fax: +1 732 445 6312.
E-mail address: LongHu@rutgers.edu (L. Hu).
http://dx.doi.org/10.1016/j.bmc.2016.04.035
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

2698
X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
substrate directly with a bulky anticancer drug molecule, such as
doxorubicin and paclitaxel, could lead to slow cleavage and thus
insufficient activation by PSA. For better cleavage and activation
by PSA, it is essential to insert a spacer between the cleavage site
and the bulky anticancer drug. Thus, upon proteolytic cleavage
by PSA, a spacer-drug conjugate is released in the prostate tumor
tissue instead of the free drug.26 The spacer-drug conjugate may
still be in the form of a prodrug with no or low cytotoxicity, requir-
ing a post PSA-cleavage process to restore the cytotoxicity of the
parent drug. However, the potential problem is that the spacer-
drug conjugate may enter into systemic circulation, and thus could
be distributed to other tissues. The subsequent proteolysis of the
spacer-drug conjugate by other proteases, however, can be non-
specific, and the release of the active drug in the blood or other tis-
sues could lead to undesired effects as a result of decreased
selectivity.
Our approach was to select a small molecule cytotoxic agent
that could be coupled to a PSA-specific peptide with or preferably
without a spacer. We previously reported several PSA-specific pep-
tide conjugates of 4-aminocyclophosphamide without a spacer,
which showed good substrate activity toward PSA.27 These conju-
gates, however, did not present satisfactory cytotoxicity toward
PSA-secreting LNCaP prostate cancer cells. We rationalized that,
after cleavage of the peptide by PSA, the immediately released 4-
aminocyclophosphamide was less cytotoxic and needed to decom-
pose to the phosphoramide mustard as the ultimate anti-prolifer-
mustard 3 before it could be conjugated to an amino acid or pep-
tide. We selected the conjugation of the first amino acid 7 to the
azide 6 through selenocarboxylate/azide amidation, which gives
the amino acid–PAB–phosphoramide mustard 8 upon deprotec-
tion. The last step in the synthesis is the fragment condensation
of the remaining peptide with the amino acid–PAB–phospho-
ramide mustard 8 followed by any necessary deprotection to give
the desired peptide–PAB–phosphoramide mustard conjugate 1.
3.2. Synthesis of 4-azidobenzyl phosphoramide mustards 6a–e
The synthesis started from preparing various 4-azidaobenzyl
alcohols 12a–e as shown in Scheme 3. Briefly, the nitro group of
5b–d was converted to the amino group by catalytic hydrogena-
tion, affording nearly quantitative yields of the corresponding
4-aminobenzoic acids 10b–d. 4-Aminobenzoic acid (10a) and
4-amino-2,3,5,6-tetrafluorobenzoic acid (10e) were commercially
available. The amino group was then replaced by the azido group
to afford 4-azidobenzoic acids 10a–e in yields between 80% and
98%, following the standard diazotization/azido-displacement pro-
tocol. The benzoic acid group was converted to the HOBt activated
ester and then selectively reduced by NaBH4 in THF to benzyl alco-
hol without affecting the azido group, providing the corresponding
alcohols 12a–e in yields between 70% and 77%. The desired 4-
azido-benzyl phosphoramides 6a–e were synthesized via a 3-step
one-pot procedure by first deprotonation of the 4-azidobenzyl
alcohol with 1.0 equiv of n-BuLi at À78 °C followed by phosphory-
lation with bis-(2-chloroethyl)phosphoramidic dichloride and then
ammonolysis with ammonia. The desired products were obtained
in yields between 48% and 55%.
ative agent. Our new strategy was then to insert
a self-
immolative linker, para-aminobenzyl (PAB), between a PSA-speci-
fic peptide and the phosphoramide mustard 3. After proteolytic
cleavage of the PSA-specific peptide followed by a spontaneous
[1,6]-elimination process, the cytotoxic phosphoramide mustard
3 is released tracelessly in the prostate tumor tissues producing
the desired alkylating activity as shown in Scheme 1. The substitu-
tion on the phenyl ring in the PAB linker could be used to modulate
the stability of the prodrug, the rate of cleavage by PSA, and the
rate of phosphoramide mustard release.
3.3. Synthesis of H-glutaminyl-4-aminobenzyl phosphoramide
mustards 8a–e
We previously reported a one-pot selenocarboxylate/azide ami-
dation procedure for direct coupling of a N-protected amino acid
with an azide to form an amide.28–31 Herein, the same selenocar-
boxylate/azide amidation methodology was used to synthesize
3. Results and discussion
3.1. Chemistry
a
N -protected glutaminyl-4-aminobenzyl phosphoramides. We first
a
synthesized the Boc and Cbz protected analogs, namely, N -Boc-
a
glutaminyl-4-aminobenzyl phosphoramide mustards and N -Cbz-
glutaminyl-4-aminobenzyl phosphoramide mustards. However,
the removal of Boc- and Cbz-protecting groups could not be done
without affecting the phosphoryl functionality under the deprotec-
In our previous work, we used three different PSA-specific pep-
tides to couple with 4-aminocyclophosphamide.27 The PSA prote-
olysis study showed that Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-
aminocyclophosphamide conjugate had the best PSA substrate
activity. Therefore, we used this same peptide for our new
approach for the traceless release of the phosphoramide mustard
3. The structure–activity relationship (SAR) study was performed
on the self-immolative PAB linker by introducing fluorine(s) to
the aromatic ring. A novel synthetic methodology, selenocar-
boxylic acid/azide amidation,28 was developed for the convergent
synthesis of the peptide-Linker-phosphoramide mustard conju-
gates as shown in Scheme 2. This is because we needed to use a
more stable intermediate like 4-azidobenzyl phosphoramide mus-
tard 6 instead of the 4-aminobenzyl analog 2, as the latter would
spontaneously decompose and release the phosphoramide
a
tion conditions tried. N -Fmoc-glutamine was not used because of
a
its poor solubility in THF. N -Trifluoroacetyl glutamine (7) was
soluble in THF and was readily prepared by the treatment of
L
-glutamine with S-ethyl trifluorothioacetate in 84% yield. Thus,
a
N -TFA-glutamine was used to prepare H-glutaminyl-4-aminoben-
zyl phosphoramide mustards 8a–e (Scheme 4).
a
The synthesis of N -TFA-glutaminyl-4-aminobenzyl phospho-
ramides 13a–e started from the selenocarboxylation of
a
N -TFA-glutamine via the treatment of the mixed anhydride of
a
N -TFA-glutamine with freshly prepared LiAlHSeH. The in situ gen-
a
erated N -TFA-glutaminyl selenocarboxylate was used directly to
react with 4-azidobenzyl phosphoramide mustards 6a–e, affording
Cl
Cl
Cl
Spontaneous
decomposition
O
P
NH2
O
P
NH2
O
P
NH2
PSA
O
O
N
O
N
O
N
Peptide
N
H
H2N
Cl
Cl
X
X
Cl
1
2
3
HN
X
4
Scheme 1. Proposed activation of peptide–PAB–phosphoramide mustard conjugates by PSA proteolysis.

X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
2699
Cl
Cl
Selenocarboxlic acid/Azide amidation
followed by deprotection
O
P
NH2
COOH
Cl
Cl
O
N
O
P N
O2N
N3
O
O
X
X
Fragment condensation
5
6
H2N
NH2
N
H
X
H
R1
N
COOH
8
PG
R1
7
O
R2
O
R4
H
H
N
N
R
R
Peptide
synthesis
N
COOH
N
H
H
O
9
R3
O
R5
Cl
O
P
O
O
N
O
R2
O
R4
H
N
H
N
H
N
NH2
N
H
1
N
H
N
H
Cl
X
R1
O
O
R3
O
R5
Peptide
PAB Linker
Drug
Scheme 2. Convergent synthesis of peptide-Linker-drug conjugates through selenocarboxylate/azide amidation methodology.
R1
R1
R1
R2
COOH
R3
R2
COOH
R3
1. NaNO2
2. NaN3
R2
N3
COOH
R3
1.HOBt/DCC
2.NaBH4
THF, r.t.
H2/Pd-C
MeOH, r.t.
O2N
H2N
6 N H2SO4
R4
R4
R4
5b-d
10a-e
11a-e
Cl
1. BuLi
R1
R1
R4
O
2.Cl2PON(CH2CH2Cl)2
3. NH3
R2
N3
R2
N3
a
b
c
d
e
R1, R2, R3, R4 = H
OH
R3
O P N
NH2
R1 = F; R2, R3, R4 = H
R1, R3, R4 = H; R2 = F
R1, R3 = F; R2, R4 =H
R1, R2, R3, R4 = F
THF, -78 °C
R3
6a-e
Cl
R4
12a-e
Scheme 3. Synthesis of 4-azido-benzyl phosphoramide mustards 6a–e.
Cl
Cl
Cl
O
R1
R1
R4
O
P
NH2
O
P
NH2
1.
, TEA
R2
R2
Cl
O
O
O
O
N
O
O
N
H
N
H
N
2. LiAlHSeH
3. 6a-e
F3C
F3C
H2N
O
N
H
R3
OH
N
H
R3
K2CO3
Cl
ACN/H2O (3/1)
O
O
O
O
R4
a
b
c
d
e
R1, R2, R3, R4 = H
THF
R
1 = F; R2, R3, R4 = H
R1, R3, R4 = H; R2 = F
R1, R3 = F; R2, R4 =H
R1, R2, R3, R4 = F
NH2
NH2
NH2
7
13a-e
8a-
e
Scheme 4. Synthesis of H-glutaminyl-4-aminobenzyl phosphoramide mustards 8a–e.
the desired products 13a–e in yields between 20% and 87%. The
O
O
TFA protecting group was smoothly removed by the treatment of
O
O
H
a
N
N
H
OH
N -TFA-glutamine conjugates 13a–e with 4 equiv of K2CO3 in
O
N
N
H
aqueous acetonitrile at room temperature. The resulting
H-glutaminyl-4-aminobenzyl phosphoramide mustards 8a–e were
purified by flash column chromatography and their structures
were confirmed by LC/MS and NMR.
O
O
OH
HO
9
1. HBTU/DIPEA, 8a-e
2. 10 eq DEA
Cl
Cl
R1
O
3.4. Synthesis of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl
phosphoramide mustards 1a–e
R2
HO
O
O
O
O
O P N
NH2
H
N
H
N
N
O
N
N
N
R3
H
H
H
O
O
R4
The protected tetrapeptide, Fm-glutaryl-Hyp-Ala-Ser-Chg-OH
(9), was synthesized on solid phase using Fmoc chemistry and
Wang resin.32 The tetrapeptide 9 was first activated by formation
of the HOBt-activated ester using HBTU/DIPEA protocol, followed
by amidation with the H-glutaminyl-4-aminobenzyl phospho-
ramide mustards 8a–e in NMP (Scheme 5). The conjugated prod-
ucts were collected as white solids by precipitation from 5%
aqueous NaHCO3. The obtained conjugates were immediately dis-
solved in acetonitrile/methanol (50:50, v/v) and treated with 10
equiv of diethylamine at room temperature to remove the
OH
a
b
c
d
e
R1, R2, R3, R4 = H
HO
R1 = F; R2, R3, R4 = H
R1, R3, R4 = H; R2 = F
R1, R3 = F; R2, R4 =H
R1, R2, R3, R4 = F
H2N
1a-e
O
Scheme 5. Synthesis of Glutaryl-Hyp-Ala-Ser-Chg-Gln-aminobenzyl phospho-
ramide mustards 1a–e.
fluorenylmethyl protecting group of N-terminal glutarate. The
desired peptide–PAB–phosphoramide mustard conjugates 1a–e
were purified in 28–40% yields by preparative HPLC.

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X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
3.5. PSA substrate activity of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-
aminobenzyl phosphoramide mustards 1a–e
1.0
0.8
0.6
0.4
0.2
0.0
1a
1b
1c
1d
1e
The peptidyl-4-aminobenzyl phosphoramide mustards 1a–e
were evaluated as substrates of PSA at an enzyme/substrate molar
ratio of 1:100, while L-377,202, N-glutaryl-Hyp-Ala-Ser-Chg-Gln-
Ser-Leu-Doxorubicin, was used as a control. The stock solution of
each substrate was prepared as a 10 mM solution in DMSO. The
substrates were then incubated with 1% mol of PSA at 37 °C in
10 mM Tris/HCl buffer containing 0.01% Tween-20Ò at pH 8.0.
Aliquots were withdrawn at various time intervals, quenched by
acetonitrile (20%), and stored frozen prior to HPLC analysis.
All peptide-Linker-drug conjugates 1a–e were cleaved by PSA in
a time-dependent manner as observed by HPLC analysis (Fig. 1).
The pentapeptide segment, Glutaryl-Hyp-Ala-Ser-Chg-Gln-OH,
was confirmed by LC/MS, indicating that the proteolytic cleavage
occurred after the glutamine residue. For the control substrate,
L-377,202, the complete disappearance of the substrate was
observed in about 2 h with a half-life of 30 min. For peptide-4-
aminobenzyl phosphoramide mustards 1a–e, the substrate con-
centration did not reach zero after 5-h incubation. This may be
due to the inactivation of PSA by the released alkylating phospho-
ramide mustard 3 or the electrophilic quinonimine methides 4.
0
50
100
150
200
250
300
350
400
450
500
Time (h)
Figure 2. Stability of peptide-4-aminobenzyl phosphoramide conjugates 1a–e in
50 mM Tris/HCl buffer, pH 8.0 containing 10 mM CaCl2 and 0.1% Tween-20 at 37 °C.
Table 1
Stability, PSA cleavage, and antiproliferative activity of the peptide-conjugates
a
b
Prodrugs
t1/2 (h)
IC50 (lM)
PSAÀ
PSA+
DU145
LNCaP
3.6. Stability of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl
phosphoramide mustards 1a–e
PSAÀ
PSA+
L-377,202
1a
1b (2-F)
1c (3-F)
1d (2,6-diF)
1e (2,3,5,6-tetraF)
NA
46
105
465
131
178
0.6
2.1
0.93
0.65
1.7
>100
35
>100
>100
>100
>100
1.1
11.0
5.3
29.0
20.0
>100
Conjugates 1a–e were incubated at 37 °C in 10 mM Tris/HCl
buffer containing 0.01% TWEEN-20Ò at pH 8.0. Aliquots were with-
drawn at various time intervals and stored frozen prior to HPLC
analysis (Fig. 2). The half-lives were calculated based on the disap-
pearance of the conjugates and summarized in Table 1: The conju-
gate 1a had the shortest half-life of 46 h; Introduction of fluorine at
the 2-, 2,6-, and 2,3,5,6-position of phenyl ring increased the stabil-
ity of the peptide-Linker-drug conjugates (1b, 1d, 1e) by about 2, 3
and 4 folds, respectively. Introduction of fluorine at the 3-position
of phenyl ring increased the stability of 1c by about 10 folds. The
degradation presumably goes through the hydrolysis of the P–O
bond or the 1,6-elimination process, shown in Scheme 6. To under-
stand the degradation pathway, 4-nitrobenzyl phosphoramide
mustard as the control was incubated in the same buffer at
37 °C. After 3 days of incubation, there was no detectable degrada-
tion of 4-nitrobenzyl phosphoramide mustard, indicating that the
phosphoryloxy functionality was stable toward hydrolysis under
1.1
a
Stability and substrate activity of conjugates in 50 mM Tris Buffer, pH 8, and
37 °C. Each conjugate (100 M) was incubated in 50 mM Tris/HCl buffer, pH 8.0
containing 10 mM CaCl2 and 0.1% Tween-20 at 37 °C.
PSA-producing LNCaP and non-PSA-producing DU145 cell lines were treated
with conjugates at concentrations ranging from 0.01 to 100
viability determined using the MTT assay.
l
b
lM for 72 h and cell
the same conditions. Therefore, the degradation of conjugates
was most likely due to the 1,6-elimination. Introduction of elec-
tron-withdrawing fluorine atom(s) decreased the electron density
of the aromatic ring and thus slowed down the 1,6-elimination
process, resulting in enhanced stability of the fluoro-substituted
peptide-Linker-drug conjugates. It is clear that the 1,6-elimination
process is facilitated by the electron density of the aromatic ring:
The rate of 1,6-elimination increases with increasing electron den-
sity of aromatic ring in the PAB linker. This is consistent with our
observation that reduction of 4-nitrobenzyl phosphoramide mus-
tard leads to elimination and release of phosphoramide
mustard.33,34
L-377,202
1a
1.0
1b
1c
1d
1e
0.8
3.7. Antiproliferative activity of Glutaryl-Hyp-Ala-Ser-Chg-Gln-
4-aminobenzyl phosphoramide mustards 1a–e
0.6
0.4
0.2
0.0
The antiproliferative activities of peptidyl-4-aminobenzyl phos-
phoramide mustards (1a–e) were evaluated using the standard
MTT assay in LNCaP cells that express PSA and DU145 cells that
do not express PSA with L-377,202 used as a positive control.
The IC50 values were calculated and summarized in Table 1.
DU145 cell line was not sensitive to L-377,202 and conjugates
1b–e. The conjugate 1a, however, was cytotoxic towards DU145
0
50
100
150
200
250
300
350
400
450
500
Time (min)
cells with an IC50 of 35 lM, presumably due to its chemical insta-
bility leading to the release of phosphoramide mustard: this is con-
sistent with its relatively shorter half-life of 46 h in the buffer; and
after 72 h of incubation under cell culture conditions prior to the
Figure 1. The disappearance of peptide-4-aminobenzyl phosphoramide conjugates
1a–e (0.16 mM) in the presence of PSA in 50 mM Tris/HCl buffer, pH 8.0 containing
10 mM CaCl2 and 0.1% Tween-20 at 37 °C.

X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
2701
H2O
Cl
Cl
Cl
Cl
O
P
O
P
hydrolysis
O
N
O
N
OH
Peptidyl
NH2
Peptidyl
O
NH2
Peptidyl
N
H
N
H
N
H
1e
14e
1,6-elimination
Cl
Cl
H2O
O
P
NH2
N
Peptidyl
H2O
N
H
Peptidyl
N
Scheme 6. Proposed degradation pathways of peptide-Linker-drug conjugate 1e.
MTT assay, over 60% of 1a had already decomposed and released
the cytotoxic phosphoramide mustard. All peptidyl-4-aminobenzyl
phosphoramide mustard conjugates 1a–e were cytotoxic towards
the PSA-secreting LNCaP cell line with the 2-fluoro substituted
conjugate 1b showing the highest cytotoxicity against LNCaP cells
cytotoxicity. Introduction of a fluorine atom at the meta position
led to significant decrease in cytotoxicity. Decreasing electron
density of the PAB linker would improve the stability of the con-
jugate but adversely affect the release of cytotoxic phospho-
ramide mustard from the conjugates upon proteolysis. The
conjugate 1b was identified as a promising lead for further eval-
uation and optimization, representing a balance of the desired
chemical stability, PSA substrate activity, antiproliferative activ-
ity, and selectivity.
(IC50 = 5.3
M), and 1c (IC50 = 29
Compared to 1a, the 3-fluoro substituted conjugate 1c showed
l
M), followed by 1a (IC50 = 11.0
lM), 1d (IC50 = 20.0 -
l
lM).
about 3-fold decreased cytotoxicity towards PSA-expressing LNCaP
cells; the 2,6-difluoro substituted conjugate 1d showed about 2-
fold decreased cytotoxicity; and the 2,3,5,6-tetrafluoro substituted
conjugate 1e did not show cytotoxicity at the highest assay con-
5. Experimental section
5.1. General methods
centration of 100 lM. These results suggest that the rate of self-
immolative 1,6-elimination process decreased because of the
decreased electron density of the PAB linker caused by the induc-
tive effects of electron-withdrawing fluorine(s). The lack of cyto-
toxicity of 1e was because the 1,6-elimination process was
completely halted by the introduction of the four electron-with-
drawing fluorine atoms, and thus the release of phosphoramide
mustard was blocked. To demonstrate our hypothesis, 4-azido-
2,3,5,6-tetrafluorobenzyl phosphoramide mustard (6e) was
reduced to the corresponding amine by catalytic hydrogenation.
As expected, there was no 1,6-elimination occurring after reduced
to 4-amino-2,3,5,6-tetrafluorobenzyl phosphoramide mustard that
was isolated as a stable product. However, the cytotoxicity of 2-flu-
oro substituted conjugate 1b was improved by 2 folds. We postu-
lated that the ortho-fluorine of 1b facilitated the cleavage of
benzylic C–O bond through a 1,6-elimination process via the reso-
nance effect of fluorine. Both the chemical stability of the conju-
gates and the release of phosphoramide mustard from the
conjugates upon proteolysis were impacted by the self-immolative
PAB linker through the electron-withdrawing inductive effect as
well as the electron-donating resonance effect of fluorine
substitutions.
Moisture-sensitive reactions were performed in flame-dried
glassware under a positive pressure of nitrogen or argon. Air and
moisture-sensitive materials were transferred by syringe or can-
nula under an argon atmosphere. Except for redistillation prior to
use, solvents were either ACS reagent grade or HPLC grade. Unless
otherwise stated, all reactions were magnetically stirred and mon-
itored by thin-layer chromatography (TLC) using 0.25 mm What-
man precoated silica gel plates and/or LC–MS. Flash column
chromatography (FCC) was conducted on a Teledyne ISCO Combi-
Flash Companion Automated Flash Chromatographic System (Tele-
dyne Technologies, Thousand Oaks, CA) using pre-packed silica gel
columns. TLC plates were visualized using either 7% (w/w) ethano-
lic phosphomolybdic acid or 1% (w/w) aqueous potassium perman-
ganate containing 1% (w/w) NaHCO3. Yield refers to
chromatographically and spectroscopically (1H NMR) homoge-
neous material, unless otherwise noted.
Infrared spectra were recorded with Perkin–Elmer model 1600
series FTIR spectrometer using polystyrene as an external standard.
Infrared absorbance is reported in reciprocal centimeters (cmÀ1
)
with broad signals denoted by br. 1H NMR spectra were recorded
on Varian Gemini 200, 300, or 400 MHz spectrometers as indicated
at ambient temperature and calibrated using residual undeuter-
ated solvents as the internal reference. 13C NMR spectra were
recorded at 50 MHz on a Varian Gemini 200 MHz spectrometer
or 75 MHz on a Varian Gemini 300 MHz spectrometer. Chemical
shifts are reported in parts per million (d) relative to CDCl3 (d
7.27 ppm for 1H and 77.2 ppm for 13C) or CD3OD (d 3.31 ppm for
1H and 49.2 for 13C). Coupling constants (J values) are given in
hertz (Hz). The following abbreviations were used to explain the
multiplicities: s = singlet; d = doublet; t = triplet; q = quartet;
m = multiplet; br = broad. High resolution mass spectral (HRMS)
data were obtained from the University of Kansas Mass Spectrom-
etry Laboratory (Lawrence, KS).
4. Conclusions
We designed, synthesized and evaluated a series of peptidyl-4-
aminobenzyl phosphoramide mustard conjugates as prodrugs for
site-specific activation by PSA in prostate cancer cells. The design
principle is based on proteolytic cleavage of the PSA-specific pep-
tide to activate the self-immolative 4-aminobenzyl (PAB) linker
that releases cytotoxic phosphoramide mustard via a 1,6-elimina-
tion process. All conjugates were found to be the substrates of
PSA. The proteolytic cleavage by PSA occurred between Gln and
the PAB linker. In vitro antiproliferative activity assay of these
conjugates showed promising cytotoxicity and selectivity. Struc-
ture–activity relationship studies on these conjugates indicated
that introducing electron-withdrawing fluorine(s) on the phenyl
ring in the PAB linker improved the stabilities of the conjugates.
However, introducing a fluorine atom at the ortho position to
benzylic phosphoramide was crucial to maintain the conjugate’s
Purified PSA (99%, 1.37 mg protein/ml) was purchased from
Fitzgerald (North Acton, MA). Cell growth medium was prepared
by adding L-glutamine (2 mM), fetal bovine serum (10%), penicillin
G (100 units/mL) and streptomycin sulfate (100 units/mL) to phe-
nol red-containing RPMI 1640.

2702
X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
Moisture-sensitive reactions were performed in oven-dried
the desired product were pooled and lyophilized to afford the pep-
tide as a white powder. The purity and identity were confirmed by
HPLC and LC/MS. The purity of all intermediates was >90% and the
purity of final target prodrug conjugates was >95%.
glassware under a positive pressure of argon or nitrogen. Air and
moisture sensitive materials were transferred via syringe or can-
nula under argon or nitrogen atmosphere. Solvents were either
ACS reagent grade or HPLC grade and used directly without further
purification unless otherwise stated: THF and dichloromethane
were dried by pressure filtration under nitrogen through activated
alumina; N,N-dimethylformamide was dried and redistilled over
calcium hydride. Reagents purchased were ACS grade or better
and used without further purification unless otherwise stated:
the concentration of n-BuLi in hexane was measured by using
diphenylacetic acid protocol before use. All reactions were magnet-
ically stirred and monitored by thin-layer chromatography (TLC)
using Whatman polymer-backed F254 silica gel plates and/or Shi-
madzu 2010 LC–MS system. Flash column chromatography was
performed using silica gel (Merck 230–400 mesh) or using a Tele-
dyne ISCO CombiFlash Companion Automated Flash Chromato-
graphic System with prepacked silica gel columns. Yields were
based on the chromatographically pure compounds. Melting points
were determined on a Mel-Temp capillary apparatus and are
uncorrected. Infrared spectra were recorded with Thermo-Nicolet
Avatar 360 FTR spectrometer and the absorbance is reported in
reciprocal centimeters (cmÀ1). All 1H and 13C NMR spectra were
recorded on a Varian Gemini 200 MHz spectrometer at ambient
temperature and calibrated using residual undeuterated solvents
as the internal reference. The following abbreviations were used
to indicate the multiplicities: s = singlet; d = doublet; dd = doublets
of doublet; dt = doublets of triplet; t = triplet; td = triplets of dou-
blet; q = quartet; m = multiplet; br = broad.
5.3. Synthesis of fluorinated 4-aminobenzoic acids 10b–d
To a solution of 5b–d (1.0 mmol) in methanol (20 mL) was
added 10% Pd–C (10 mg, 0.01 mmol). The resulting mixture was
stirred at room temperature under a hydrogen balloon for 16 h
and filtered through a Celite 545 pad. The filtrate was concentrated
to dryness to afford the corresponding aminobenzoic acid 10b–d
that was used directly for the next step without purification.
5.4. Synthesis of fluorinated 4-azidobenzoic acids 11a–e
To an ice-cold suspension of 10a–e (1.0 mmol) in 6 N H2SO4
(20 mL) was added dropwise an aqueous solution of sodium nitrite
(83 mg, 1.2 mmol) over 10 min. The resulting mixture was stirred
at 0 °C for additional 30 min. An aqueous solution of sodium azide
(98 mg, 1.5 mmol) was added dropwise to the above mixture. The
reaction was then carried out at room temperature for 1 h. The
reaction mixture was extracted with EtOAc (3 Â 30 mL). The com-
bined EtOAc phase was washed with water and brine, and dried
over Na2SO4. After removal of Na2SO4 via filtration, the filtrate
was concentrated to dryness. The crude product was purified by
flash column chromatography (hexane to 50% EtOAc/hexane) to
afford the desired azidobenzoic acid 11a–e.
The reactions were monitored using Schimadzu LCMS-2010
system equipped with a Chromolith SpeedROD RP-18e column
(50 Â 4.6 mm). Solvent A was 0.1% HCOOH/H2O and solvent B
was 0.1% HCOOH/CH3CN. The gradient was 10–90% of solvent B
over 10 min with a flow rate at 1 mL/min. The kinetic studies were
5.4.1. 4-Azido-benzoic acid (11a)
A white solid (163 mg, 98%); 1H NMR (200 MHz, DMSO-d6): d
7.96 (d, 2H, J = 8.4 Hz), 7.17 (d, 2H, J = 8.4 Hz); 13C NMR (50 MHz,
DMSO-d6): 167.5, 143.4, 131.9, 128.7, 119.7; MS (ESIÀ): m/z (inten-
sity), 162.0 ([MÀH]À, 100%).
performed on
4.6 Â 150 mm) at 1 mL/min with a gradient of 10–80% of acetoni-
trile containing 0.1% trifluoroacetic acid over 15 min on
a
Waters symmetry C18 column (3.5 lm,
5.4.2. 4-Azido-2-fluorobenzoic acid (11b)
a
A white solid (163 mg, 83%); 1H NMR (200 MHz, CDCl3): d 7.78
(dd, 1H, J = 8.6 Hz, J = 8.4 Hz), 6.71 (d, 1H, dd, 1H, J = 8.4 Hz,
J = 2.2 Hz), 6.62 (dd, 1H, J = 11.4 Hz, J = 2.2 Hz); 13C NMR (50 MHz,
CDCl3): 164.0, 162.8 (d, J = 250.0 Hz), 146.2 (d, J = 10.0 Hz), 133.6,
114.2 (d, J = 3.0 Hz), 107.3 (d, J = 26.0 Hz). MS (ESIÀ): m/z (inten-
sity), 180.1 ([MÀH]À, 100%).
HP1090 system. The peptide–aminoarylmethylphosphoramide
mustards were purified using Gilson automatic preparative HPLC
system equipped with C18 column (250 Â 20 mm) at 12 mL/min
with a gradient of 30–90% of acetonitrile containing 0.1% trifluo-
roacetic acid over 10 min.
5.2. Solid-phase synthesis of peptide
5.4.3. 4-Azido-3-fluorobenzoic acid (11c)
A white solid (163 mg, 90%); 1H NMR (200 MHz, CD3OD): d 7.77
(d, 1H, J = 7.8 Hz), 7.69 (d, 1H, J = 11.4 Hz), 7.10 (dd, 1H, J = 7.8 Hz,
J = 7.2 Hz); 13C NMR (50 MHz, CD3OD): 167.5, 154.9 (d,
J = 250.0 Hz), 133.3 (d, J = 10.0 Hz), 129.1 (d, J = 6.0 Hz), 127.2 (d,
J = 3.0 Hz), 121.3, 118.4 (d, J = 20.0 Hz); IR (KBr): 2146, 1691,
1432, 1298 cmÀ1; MS (ESIÀ): m/z (intensity), 180.1 ([MÀH]À,
100%).
The peptide, Fm-glutaryl-Hyp-Ala-Ser-Chg-OH, was synthe-
sized using Fmoc chemistry on 4-hydroxymethylphenoxy
(WANG-type HMP) resin purchased from Advanced Chemtech.
a
N -Fmoc protected
L-amino acids and coupling reagents were pur-
chased from Advanced Chemtech. Side-chain protection was Ser(t-
Bu) and Lys(Cl-Z). First amino acid was attached to the resin via its
a
C-terminal using HOBt/DMAP/DIC protocol. All N -Fmoc protected
L-amino acids were used as 3-fold excess amount for each coupling
5.4.4. 4-Azido-2,6-difluorobenzoic acid (11c)
in NMP. Following completion of the assembly on the resin sup-
port, the Fmoc protecting group was removed via the standard
20% piperidine/NMP protocol followed by washing with NMP 5
times and introduction of N-terminal capping group using acetic
anhydride. Deprotection and cleavage of the peptide from the resin
support were performed using 90% TFA/CH2Cl2. The benzyl ester
and fluorenylmethyl ester (OFm) were not affected under these
conditions. After removal of solvents under reduced pressure, the
peptide was purified by preparative-HPLC on reversed phase C18
column. A linear gradient was used from 10% solvent A to 90% sol-
vent B with a flow rate of 12 mL/min, where solvent A was 0.1%
TFA/H2O and solvent B was 0.1% TFA/CH3CN. The UV detection
wavelength was set at 220 nm. Homogeneous fractions containing
A white solid (163 mg, 83%); 1H NMR (200 MHz, Acetone-d6): d
6.87 (d, 2H, J = 9.2 Hz); 13C NMR (50 MHz, Acetone-d6): 171.9,
162.1 (dd, J = 255.0 Hz, J = 8.4 Hz), 161.4, 146.0 (t, J = 12.9 Hz),
108.2 (t, J = 19.8 Hz); IR (KBr): 3432, 2119, 1738, 1636, 1312,
1280 cmÀ1; MS (ESIÀ): m/z (intensity), 154.0 ([MÀCOOH]À, 100%).
5.4.5. 4-Azido-2,3,5,6-tetrafluorobenzoic acid (11e)
A yellow solid (188 mg, 80%); 1H NMR (200 MHz, CDCl3): d
10.34 (br s, –COOH); 13C NMR (50 MHz, CDCl3): 164.3, 146.0
(dm, J = 265.0 Hz), 140.7 (dm, J = 265.0 Hz), 124.8 (t. J = 13.0 Hz),
106. (t, J = 13.0 Hz), 51.7; IR (film): 3388, 2123, 1652, 1489, 1241,
1002, 945 cmÀ1. MS (ESIÀ): m/z (intensity), 234.0 ([MÀH]À, 100%).

X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
2703
5.5. Synthesis of fluorinated 4-azido-benzyl alcohols 12a–e
(50 mL) followed by extraction with dichloromethane
(3 Â 50 mL). The combined organic phase was washed with water
(50 mL) and saturated brine (50 mL), and dried over Na2SO4. After
filtration off Na2SO4, the filtrate was concentrated to dryness under
reduced pressure. The crude product was purified by flash column
chromatography (dichloromethane to 5% methanol in dichloro-
methane) to afford the desired product 6a–e.
To a solution of 12a–e (1.0 mmol) in THF (10 mL) was added
HOBt (135 mg, 1.0 mmol) and DCC (227 mg, 1.1 mmol). The result-
ing mixture was stirred at room temperature for 1 h. The white
precipitates were filtered off and rinsed with THF (2 Â 5 mL). The
filtrate was added dropwise to a suspension of NaBH4 (38 mg,
1.0 mmol) in THF (10 mL) over 30 min. The reaction was carried
out at room temperature for additional 1.5 h and quenched by add-
ing 10 ml 1.0 N HCl. After evaporation of THF under reduced pres-
sure, the remaining aqueous solution was extracted EtOAc
(3 Â 20 mL). The combined EtOAc phase was washed with water
and brine, and dried over Na2SO4. After removal of Na2SO4 via fil-
tration, the filtrate was concentrated to dryness. The crude product
was purified by flash column chromatography (hexane to 50%
EtOAc/hexane) to afford the desired alcohol 12a–e.
5.6.1. 4-Azido-benzyl phosphoramide mustard (6a)
A yellow semi-solid (0.965 g, 55%); 1H NMR (200 MHz, CD3OD):
d 7.45 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 8.4 Hz), 4.97 (d, 2H,
J = 7.4 Hz), 3.61–3.69 (m, 4H), 3.33–3.48 (m, 4H); 13C NMR
(50 MHz, CD3OD): 139.5, 133.3 (d, J = 8.0 Hz), 128.6, 118.2, 65.7
(d, J = 5.0 Hz), 48.8 (d, J = 5.0 Hz), 41.2; IR (film): 3415, 1655,
1437, 1407, 1315, 1022, 953 cmÀ1; MS (ESI+): m/z (intensity),
352.1 ([M+H]+, 100%), 354.1 ([M+H]+ + 2, 65%), 356.1 ([M+H]+ + 4,
10%), 393.2 ([M+H]+ + CH3CN, 20%), 395.2 ([M+H]+ + 2 + CH3CN,
13%), 397.2 ([M+H]+ + 4 + CH3CN, 2%).
5.5.1. 4-Azido-benzyl alcohol (12a)
A yellow solid (115 mg, 77%); 1H NMR (200 MHz, CDCl3): d 7.31
(d, J = 8.4 Hz), 7.00 (d, J = 8.4 Hz), 4.61 (s, 2H), 2.81 (br s, –OH); 13
C
5.6.2. 4-Azido-2-fluorobenzyl phosphoramide mustard (6b)
A yellow semi-solid (0.959 g, 52%); 1H NMR (200 MHz, CDCl3): d
7.24 (t, 1H, J = 8.0 Hz), 6.63 (dd, 1H, J = 2.2 Hz, J = 8.0 Hz), 6.54 (dd,
1H, J = 2.2 Hz, J = 8.0 Hz), 4.79 (d, 2H, J = 7.2 Hz), 3.28–3.45 (m, 4H),
3.13–3.26 (m, 4H); 13C NMR (50 MHz, CDCl3): 161.2 (d,
J = 248.9 Hz), 142.2 (d, J = 9.9 Hz), 131.5 (d, J = 5.4 Hz), 120.2 (dd,
J = 15.0 Hz, J = 8.0 Hz), 114.8 (d, J = 3.4 Hz), 106.6 (d, J = 25.0 Hz),
60.7 (d, J = 4.1 Hz), 49.1 (d, J = 5.0 Hz), 42.4; IR (film): 3431, 2118,
1621, 1505, 1303, 1214, 1091, 1012, 980 cmÀ1; MS (ESI+): m/z
(intensity), 370.1 ([M+H]+, 100%), 372.1 ([M+H]+ + 2, 65%), 374.1
([M+H]+ + 4, 10%), 411.1 ([M+H]+ + CH3CN, 20%), 413.1 ([M
+H]+ + 2 + CH3CN, 13%), 415.1 ([M+H]+ + 4 + CH3CN, 2%).
NMR (50 MHz, CDCl3): 139.4, 137.7, 128.6, 119.2, 64.6; IR (film):
3332, 2107, 1506, 1287 cmÀ1
.
5.5.2. 4-Azido-2-fluorobenzyl alcohol (12b)
Yellow oil (124 mg, 74%); 1H NMR (200 MHz, CDCl3): d 7.27 (t,
1H, J = 8.0 Hz), 6.73 (dd, 1H, J = 8.0 Hz, J = 2.2 Hz), 6.63 (dd, 1H,
J = 10.6 Hz, J = 2.2 Hz), 4.58 (s, 2H), 3.63 (br s, –OH); 13C NMR
(50 MHz, CDCl3): 160.7 (d, J = 250.0 Hz), 140.9 (d, J = 10.0 Hz),
130.2 (d, J = 6.0 Hz), 124.2 (d, J = 15.0 Hz), 114.5 (d, J = 3.0 Hz),
106.2 (d, J = 25.0 Hz), 58.1 (d, J = 4.0 Hz); IR (film): 3321, 2116,
1621, 1583, 1502, 1301, 1212 cmÀ1
.
5.5.3. 4-Azido-3-fluorobenzyl alcohol (12c)
5.6.3. 4-Azido-3-fluorobenzyl phosphoramide mustard (6c)
A yellow semi-solid (0.959 g, 52%); 1H NMR (200 MHz, CDCl3): d
7.06–7.25 (m, 3H), 5.00 (dd, 2H, J = 7.8 Hz, J = 4.0 Hz), 3.66–3.73
(m, 4H), 3.43–3.55 (m, 4H); 13C NMR (50 MHz, CDCl3): 154.3 (d,
J = 248.5 Hz), 134.7 (t, J = 7.5 Hz), 127.4 (d, J = 10.0 Hz), 123.7 (d,
J = 3.4 Hz), 120.8, 115.7 (d, J = 19.7 Hz), 65.5 (d, J = 3.1 Hz), 48.9
(d, J = 4.6 Hz), 42.3; IR (film): 3424, 2134, 2099, 1643, 1509,
1218 cmÀ1; MS (ESI+): m/z (intensity), 370.10 ([M+H]+, 100%),
372.10 ([M+H]+ + 2, 65%), 374.10 ([M+H]+ + 4, 10%), 411.1 ([M+H]-
+ + CH3CN, 30%), 413.1 ([M+H]+ + 2 + CH3CN, 20%), 415.1 ([M
+H]+ + 4 + CH3CN, 3%).
Yellow oil (117 mg, 70%); 1H NMR (200 MHz, CDCl3): d 6.94–
7.08 (m, 3H), 4.57 (s, 2H), 2.59 (br s, –OH); 13C NMR (50 MHz,
CDCl3): 154.7 (d, J = 250.0 Hz), 139.2 (d, J = 6.0 Hz), 126.8 (d,
J = 12.0 Hz), 122.9 (d, J = 4.0 Hz), 120.9, 115.0 (d, J = 20.0 Hz), 63.8
(d, J = 1.0 Hz); IR (film): 3334, 2134, 2096, 1508, 1315 cmÀ1
.
5.5.4. 4-Azido-2,6-difluorobenzyl alcohol (12d)
A white solid (131 mg, 71%); 1H NMR (200 MHz, CDCl3): d 6.56
(d, 2H, J = 7.6 Hz), 4.66 (s, 2H), 2.39 (br s, –OH); 13C NMR (50 MHz,
CDCl3): 162.1 (dd, J = 250.0 Hz, J = 10.0 Hz), 142.5 (t, J = 13.0 Hz),
113.0 (t, J = 20.0 Hz), 102.6 (d, J = 30.0 Hz), 52.4 (t, J = 4.0 Hz); IR
(film): 3330, 2116, 1641, 1587, 1441, 1237, 1060, 1026 cmÀ1
.
5.6.4. 4-Azido-2,6-difluorobenzyl phosphoramide mustard (6d)
A light yellow solid (0.970 g, 50%); 1H NMR (200 MHz, CD3OD):
d 6.72 (d, 2H, J = 8.4 Hz), 4.95 (d, 2H, J = 7.0 Hz), 3.52–3.60 (m, 4H),
3.26–3.39 (m, 4H); 13C NMR (50 MHz, CD3OD): 163.6 (dd,
J = 250.0 Hz, J = 9.5 Hz), 145.2 (t, J = 13.3 Hz), 110.5 (td,
J = 20.0 Hz, J = 8.4 Hz), 103.9 (dd, J = 29.6 Hz, J = 2.0 Hz), 55.4 (dd,
J = 8.0 Hz, J = 3.8 Hz), 50.6 (d, J = 4.6 Hz), 43.0; MS (ESI+): m/z
(intensity), 388.2 ([M+H]+, 100%), 390.2 ([M+H]+ + 2, 65%), 392.2
([M+H]+ + 4, 10%), 429.3 ([M+H]+ + CH3CN, 20%), 431.3 ([M
+H]+ + 2 + CH3CN, 13%), 433.3 ([M+H]+ + 4 + CH3CN, 2%).
5.5.5. 4-Azido-2,3,5,6-tetrafluorobenzyl alcohol (12e)
A white solid (155 mg, 70%); 1H NMR (200 MHz, CDCl3): d 4.71
(s, 2H), 2.85 (br s, –OH); 13C NMR (50 MHz, CDCl3): 144.6 (dm,
J = 245.0 Hz), 139.7 (dm, J = 245.0 Hz), 119.2 (t. J = 20.0), 113.3 (t,
J = 20.0 Hz), 51.7; IR (film): 3388, 2123, 1652, 1489, 1241, 1002,
945 cmÀ1
.
5.6. Synthesis of fluorinated 4-azido-benzyl phosphoramide
mustards 6a–e
5.6.5. 4-Azido-2,3,5,6-tetrafluorobenzyl phosphoramide
mustard (6e)
To a solution of alcohol 12a–e (5.0 mmol) in anhydrous THF
(25 mL) was added a solution of BuLi in cyclohexane (2.0 M,
2.75 mL) at À78 °C. After 20 min, the above solution was trans-
ferred to a pre-cooled solution of bis(2-chloroethyl)phospho-
ramidic dichloride (1.43 g, 5.5 mmol) in THF (25 mL) at À78 °C
via cannula. The resulting mixture was stirred at À78 °C for 5 h fol-
lowed by bubbling with ammonia for 10 min. The reaction mixture
was allowed to gradually warm up to room temperature over 2 h.
After removal of THF via distillation under reduced pressure, the
residue was suspended in saturated aqueous sodium bicarbonate
A dark green solid (1.02 g, 48%); 1H NMR (200 MHz, CD3OD): d
5.03 (d, 2H, J = 7.8 Hz), 3.55–3.62 (m, 4H), 3.32–3.41 (m, 4H); 13C
NMR (50 MHz, CD3OD): 145.4 (dm, J = 251.2 Hz), 140.4 (dm,
J = 250.5 Hz), 121.0 (tt, J = 12.2 Hz, J = 3.1 Hz), 110.4 (td,
J = 12.4 Hz, J = 8.4 Hz), 54.2, 49.1 (d, J = 5.0 Hz), 42.3; IR (film):
3241, 3113, 2964, 2159, 2124, 1654, 1496, 1238 cmÀ1; MS (ESI+):
m/z (intensity), 424.0 ([M+H]+, 100%), 426.0 ([M+H]+ + 2, 65%),
428.0 ([M+H]+ + 4, 10%), 465.1 ([M+H]+ + CH3CN, 20%), 467.1 ([M
+H]+ + 2 + CH3CN, 13%), 469.1 ([M+H]+ + 4 + CH3CN, 2%).

2704
X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
5.7. Synthesis of TFA–
L
-glutamine (7)
([MÀOP(O)NH2N(CH2CH2Cl)2]+ +H2O, 100%), 407.3 ([MÀOP(O)
NH2N(CH2CH2Cl)2]+ +H2O + CH3CN, 40%), 590.3 ([M+Na]+, 20%),
592.3 ([M+Na]+ + 2, 13%), 594.3 ([M+Na]+ + 4, 2%).
To a solution of
L-glutamine (14.6 g, 0.1 mol) in 1 N NaOH
(100 mL, 0.1 mol) was added S-ethyl thioacetate (17 mL,
0.13 mol). The reaction was carried out at room temperature for
24 h. The reaction solution was acidified with 12 N HCl to pH
ꢀ2.0, saturated with NaCl, and then extracted with EtOAc. The
combined EtOAc phase was washed with water, brine and dried
over Na2SO4. After removal of Na2SO4 via filtration, the filtrate
was concentrated to dryness to afford the desired product 7
(20.4 g) as white powder in a yield of 84%. 1H NMR (200 MHz, CD3-
OD): d 4.47 (dd, 1H, J = 9.0 Hz, J = 4.8 Hz), 2.35–2.42 (m, 4H), 2.21–
2.32 (m, 1H), 1.96–2.18 (m, 1H); 13C NMR (50 MHz, CD3OD): 175.6,
171.4, 157.2 (q, J = 37.0 Hz), 115.5 (q, J = 285.0 Hz), 51.8, 30.5, 25.6;
MS (ESIÀ): m/z (intensity), 241.1 ([MÀH]À, 100%).
a
5.8.3. 4-(N -TFA–
L-glutaminyl)amino-3-fluoro-benzyl
phosphoramide mustard (13c)
A yellow semi-solid (114 mg, 20%); 1H NMR (200 MHz, CD3OD):
d 7.83 (t, 1H, J = 8.0 Hz), 7.15–7.26 (m, 2H), 4.93 (d, 2H, J = 7.2 Hz),
4.61 (dd, 1H, J = 8.6 Hz, J = 5.2 Hz), 3.59–3.66 (m, 4H), 3.33–3.45
(m, 4H), 2.37–2.44 (m, 2H), 2.09–2.27 (m, 2H); MS (ESI+): m/z
(intensity), 348.2 ([MÀOP(O)NH2N(CH2CH2Cl)2]+, 100%), 366.3
([MÀOP(O)NH2N(CH2CH2Cl)2]+ +H2O, 55%), 407.3 ([MÀOP(O)
NH2N(CH2CH2Cl)2]+ +H2O + CH3CN, 15%), 568.3 ([M+H]+, 40%),
570.3 ([M+H]+ + 2, 26%), 572.3 ([M+Na]+ + 4, 4%), 590.3 ([M+Na]+,
30%), 592.3 ([M+Na]+ + 2, 20%), 594.23 ([M+Na]+ + 4, 3%).
a
a
5.8.
Synthesis
of
4-(N -TFA–
L
-glutaminyl)aminobenzyl
5.8.4. 4-(N -TFA–
L-glutaminyl)amino-2,6-difluoro-benzyl
phosphoramide mustards 13a–e
phosphoramide mustard (13d)
A yellow semi-solid (422 mg, 72%); 1H NMR (200 MHz, CD3OD):
d 7.31 (d, 1H, J = 9.6 Hz), 5.00 (d, 2H, J = 7.0 Hz), 4.50 (dd, 1H,
J = 8.6 Hz, J = 5.2 Hz), 3.53–3.64 (m, 4H), 3.33–3.44 (m, 4H), 2.30–
2.41 (m, 2H), 2.04–2.25 (m, 2H); 13C NMR (50 MHz, CD3OD):
177.3, 170.9, 163.0 (dd, J = 246.5 Hz, J = 9.9 Hz), 159.1 (q,
J = 37.5 Hz), 142.4 (t, J = 14.0 Hz), 117.3 (q, J = 285.3 Hz), 109.2
(td, J = 19.8 Hz, J = 8.4 Hz), 103.7 (d, J = 29.6 Hz), 55.4, 50.6 (d,
J = 4.9 Hz), 50.4, 42.9, 32.0, 28.0; IR (film): 3295, 3075, 2966,
1698, 1668, 1615, 1556, 1425, 1215, 1012 cmÀ1; MS (ESIÀ): m/z
(intensity), 584.1 ([MÀH]À, 100%), 586.1 ([MÀH]À + 2, 65%), 588.1
([MÀH]À + 4, 10%), 698.1 ([MÀH]À + TFA, 40%), 700.1 ([MÀH]À + 2
+ TFA, 26%), 702.1 ([MÀH]À + 4 + TFA, 4%).
To a solution of TFA-glutamine (2.0 mmol) and N-methylpiper-
idine (0.244 mL, 2.0 mmol) in THF (10 mL) was added a solution of
isopropylchloroformate in toluene (2.0 mL, 2.0 mmol) at À15 °C.
The resulting mixture was stirred for 30 min at À15 to À10 °C.
Then, the obtained mixed anhydride solution was added into the
freshly prepared solution of LiAlHSeH in THF via cannula over a
period of 5 min. The reaction mixture was stirred for additional
30 min below 5 °C under nitrogen atmosphere. Then, a solution
of azide 6a–e (1.0 mmol) in THF (1 mL) was added into the above
selenocarboxylate solution via syringe. The reaction was carried
out at room temperature for 24 h. After evaporation of THF under
reduced pressure, the residue was suspended in a saturated
sodium bicarbonate aqueous solution (25 mL) followed by extrac-
tion with EtOAc (4 Â 50 mL). The combined organic phase was
washed with 1.0 M HCl (50 mL), water (50 mL) and saturated brine
(50 mL), and dried over Na2SO4. After removal of Na2SO4 through
filtration, the filtrate was concentrated to dryness under reduced
pressure. The crude product was purified by flash column chro-
matography (FCC) (dichloromethane to 10% methanol/dichloro-
methane) on silica gel to provide desired product 13a–e.
a
5.8.5. 4-(N -TFA–
L-glutaminyl)amino-2,3,5,6-tetrafluoro-benzyl
phosphoramide mustard (13e)
A yellow semi-solid (541 mg, 87%); 1H NMR (200 MHz, CD3OD):
d 5.13 (d, 2H, J = 7.6 Hz), 4.66 (dd, 1H, J = 8.0 Hz, J = 5.2 Hz), 3.60–
3.68 (m, 4H), 3.37–3.47 (m, 4H), 2.40–2.47 (m, 2H), 2.07–2.37
(m, 2H); 13C NMR (50 MHz, CD3OD): 177.3, 171.3, 159.2 (q,
J = 37.6 Hz), 147.6 (dm, J = 245.0 Hz), 143.8 (dm, J = 245.0 Hz),
118.5 (t, J = 14.8 Hz), 117.3 (q, J = 285.0 Hz), 114.9 (td, J = 17.7 Hz,
J = 8.0 Hz), 55.5, 54.9, 50.6 (d, J = 4.9 Hz), 43.0, 32.0, 28.1; MS
(ESIÀ): m/z (intensity), 620.1 ([MÀH]À, 100%), 622.1 ([MÀH]À + 2,
65%), 624.1 ([MÀH]À + 2, 10%).
a
5.8.1. 4-(N -TFA–
L
-glutaminyl)amino-benzyl phosphoramide
mustard (13a)
A yellow semi-solid (138 mg, 25%); 1H NMR (200 MHz, CD3OD):
d 7.62 (d, 2H, J = 8.4 Hz), 7.39 (d, 2H, J = 8.4 Hz), 4.97 (d, 2H,
J = 7.2 Hz), 4.56 (dd, 1H, J = 8.0 Hz, J = 5.4 Hz), 3.61–3.68 (m, 4H),
3.32–3.41 (m, 4H), 2.38–2.45 (m, 2H), 2.09–2.27 (m, 2H); 13C
NMR (50 MHz, CD3OD): 175.5, 168.7, 157.2 (q, J = 37.5 Hz), 137.5,
132.5 (d, J = 7.5 Hz), 127.6, 119.5, 115.3 (q, J = 285.0 Hz), 65.9 (d,
J = 5.3 Hz), 53.5, 48.8 (d, J = 5.0 Hz), 41.2, 30.3, 26.4; IR (film):
3261, 3063, 1715, 1608, 1545, 1213, 1185, 1159, 980 cmÀ1; MS
(ESI+): m/z (intensity), 572.1 ([M+Na]+, 100%), 574.1 ([M+Na]+ + 2,
65%), 576.1 ([M+Na]+ + 4, 10%).
5.9. Synthesis of fluorinated and non-fluorinated 4-
glutamylamino-benzyl phosphoramide mustards 8a–e
L-
A solution of 13a–e (1.0 mmol) and potassium carbonate
(552 mg, 4.0 mmol) in 75% aqueous acetonitrile (20 mL) was stir-
red at room temperature for 48 h. The completion of reaction
was confirmed by LC/MS and TLC. The aqueous phase was satu-
rated with sodium chloride followed by extraction with acetoni-
trile (3 Â 10 mL). The combined acetonitrile phase was dried over
Na2SO4 followed by filtration off Na2SO4. The filtrate was concen-
trated to dryness. The crude was purified by flash column chro-
matography (FCC) (dichloromethane to 25% methanol/
dichloromethane containing 1% concd NH4OH) on silica gel to pro-
vide the desired product 8a–e.
a
5.8.2. 4-(N -TFA–
L
-glutaminyl)amino-2-fluoro-benzyl
phosphoramide mustard (13b)
A yellow semi-solid (312 mg, 55%); 1H NMR (200 MHz, CD3OD):
d 7.84 (dd, 1H, J = 12.4 Hz, J = 1.8 Hz), 7.66 (t, 1H, J = 8.2 Hz), 7.54
(dd, 1H, J = 8.4 Hz, J = 2.2 Hz), 5.21 (d, 2H, J = 6.8 Hz), 4.75 (dd,
1H, J = 8.0 Hz, J = 5.2 Hz), 3.82–3.89 (m, 4H), 3.58–3.68 (m, 4H),
2.57–2.67 (m, 2H), 2.22–2.48 (m, 2H); 13C NMR (50 MHz, CD3OD):
177.4, 170.8, 161.9 (d, J = 244.3 Hz), 159.0 (q, J = 36.8 Hz), 141.5 (d,
J = 11.0 Hz), 131.7 (d, J = 5.3 Hz), 120.8 (dd, J = 15.0 Hz, J = 8.4 Hz),
117.3 (q, J = 284.9 Hz), 116.6 (d, J = 3.0 Hz), 108.1 (d, J = 26.6 Hz),
61.9, 53.4, 50.6 (d, J = 5.0 Hz), 43.1, 32.3, 28.2; IR (film): 3404,
2962, 1666, 1627, 1549, 1189, 1161, 998 cmÀ1; MS (ESI+): m/z
(intensity), 348.2 ([MÀOP(O)NH2N(CH2CH2Cl)2]+, 25%), 366.2
5.9.1. 4-L-Glutaminylamino-benzyl phosphoramide mustard
(8a)
A yellow semi-solid (326 mg, 72%); 1H NMR (200 MHz, CD3OD):
d 7.66 (d, 2H, J = 8.4 Hz), 7.40 (d, 2H, J = 8.4 Hz), 4.97 (d, 2H,
J = 7.6 Hz), 3.61–3.69 (m, 4H), 3.38–3.48 (m, 4H), 2.36–2.45 (m,
2H), 2.09–2.27 (m, 2H); IR (film): 3202, 3064, 1671, 1203,
1134 cmÀ1; MS (ESI+): m/z (intensity), 476.1 ([M+Na]+, 100%),

X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
2705
478.1 ([M+Na]+ + 2, 65%), 480.1 ([M+Na]+ + 4, 10%). HRMS (FAB+)
temperature for 30 min followed by addition of a solution of 8a–
e (0.1 mmol) in NMP (0.5 mL). The reaction was carried out at
room temperature for additional 2 h and quenched by adding
ice-cold 5% aqueous sodium bicarbonate (10 mL). The resulting
mixture was stirred at 0–5 °C for 10 min and the resulting white
precipitates were collected via centrifugation followed by succes-
sively washing with distilled water. The obtained solid was then
dissolved in 50% MeOH/CH3CN and treated with 10 equiv of DEA
for 1 h. After removal of solvents under reduced pressure, the
remaining solid was suspended in diethyl ether and collected via
centrifugation. The crude products were purified by preparative-
HPLC on reversed phase C18 column. A linear gradient was used
from 10% solvent A to 90% solvent B with a flow rate of 12 mL/
min, where solvent A was 0.1% TFA/H2O and solvent B was 0.1%
TFA/CH3CN. The UV detection wavelength was set at 245 nm.
Homogeneous fraction containing the desired products were
pooled and lyophilized to afford the desired products as white
powder. The purities and identities were confirmed by HPLC and
LC/MS and the purity of the prodrug conjugates was >95%.
m/z calcd for C16H26Cl2N5O4P, [M+H]+, 454.1178, found 454.1178.
5.9.2. 4-L-Glutaminylamino-2-fluoro-benzyl phosphoramide
mustard (8b)
A yellow semi-solid (358 mg, 76%); 1H NMR (200 MHz, CD3OD):
d 7.65 (dd, 1H, J = 12.4 Hz, J = 1.8 Hz), 7.46 (t, 1H, J = 8.4 Hz), 7.32
(dd, 1H, J = 8.4 Hz, J = 2.2 Hz), 5.00 (d, 2H, J = 7.4 Hz), 4.07 (t, 1H,
J = 6.2 Hz), 3.59–3.66 (m, 4H), 3.36–3.46 (m, 4H), 2.46–2.53 (m,
2H), 2.09–2.25 (m, 2H); 13C NMR (50 MHz, CD3OD): 176.9, 168.6,
162.1 (d, J = 244.7 Hz), 141.0 (d, J = 11.0 Hz), 132.0 (d, J = 5.0 Hz),
121.3 (dd, J = 13.7 Hz, J = 7.6 Hz), 116.5 (d, J = 3.4 Hz), 108.1 (d,
J = 26.6 Hz), 61.8, 54.8, 50.7 (d, J = 5.0 Hz), 43.0, 31.7, 28.2; IR
(film): 3200, 3064, 1670, 1623, 1555, 1203, 1135, 982 cmÀ1; MS
(ESI+): m/z (intensity), 252.1 ([MÀOP(O)NH2N(CH2CH2Cl)2]+,
100%), 494.3 ([M+Na]+, 80%), 496.3 ([M+Na]+ + 2, 52%), 498.3 ([M
+Na]+ + 4, 8%). HRMS (FAB+) m/z calcd for C16H25Cl2FN5O4P, [M
+H]+, 472.1084, found 472.1034.
5.9.3. 4-L-Glutaminylamino-3-fluoro-benzyl phosphoramide
mustard (8c)
5.10.1. Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-benzyl
phosphoramide mustard (1a)
A yellow semi-solid (348 mg, 74%); 1H NMR (200 MHz, CD3OD):
d 8.03 (t, 1H, J = 8.0 Hz), 7.18–7.29 (m, 2H), 4.96 (d, 2H, J = 7.6 Hz),
4.17 (t, 1H, J = 8.0 Hz), 3.60–3.68 (m, 4H), 3.39–3.55 (m, 4H), 2.38–
2.47 (m, 2H), 1.90–2.21 (m, 2H); MS (ESI+): m/z (intensity), 252.1
([M–OP(O)NH2N(CH2CH2Cl)2]+, 100%), 472.2 ([M+H]+, 20%), 474.2
([M+H]+ + 2, 13%), 476.2 ([M+Na]+ + 4, 2%), 494.2 ([M+Na]+, 50%),
496.2 ([M+Na]+ + 2, 33%), 498.2 ([M+Na]+ + 4, 5%). HRMS (FAB+)
m/z calcd for C16H25Cl2FN5O4P, [M+H]+, 472.1084, found 472.1046.
White powder (30 mg, 30%); HRMS (FAB+) m/z calcd for
C
40H62Cl2N9NaO13P, [M+Na]+, 1000.3479, found 1000.3477.
5.10.2. Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-fluorobenzyl
phosphoramide mustard (1b)
White powder (40 mg, 38%); HRMS (FAB+) m/z calcd for
C
40H61Cl2FN9NaO13P, [M+Na]+, 1018.3385, found 1018.3357.
5.9.4. 4-
mustard (8d)
L
-Glutaminylamino-2,6-difluoro-benzyl phosphoramide
5.10.3. Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-3-fluorobenzyl
phosphoramide mustard (1c)
A yellow semi-solid (416 mg, 85%); 1H NMR (200 MHz, CD3OD):
d 7.36 (d, 1H, J = 9.4 Hz), 5.02 (d, 2H, J = 6.8 Hz), 4.61 (t, 1H,
J = 8.6 Hz), 3.52–3.66 (m, 4H), 3.35–3.45 (m, 4H), 2.35–2.43 (m,
2H), 1.91–2.16 (m, 2H); 13C NMR (50 MHz, CD3OD): 177.6, 173.6,
163.1 (dd, J = 246.2 Hz, J = 10.3 Hz), 142.4 (t, J = 14.0 Hz), 109.4
(td, J = 19.8 Hz, J = 8.4 Hz), 103.7 (d, J = 30.4 Hz), 55.7, 55.5 (t,
J = 3.4 Hz), 50.7 (d, J = 4.6 Hz), 43.0, 32.2, 30.6; IR (film): 3269,
3073, 1673, 1638, 1557, 1427, 1203, 1136, 1014 cmÀ1; MS (ESIÀ):
m/z (intensity), 270.1 ([MÀOP(O)NH2N(CH2CH2Cl)2]+, 75%), 512.1
([M+H]+, 100%), 514.1 ([M+H]+ + 2, 65%), 516.1 ([M+Na]+ + 4,
10%). HRMS (FAB+) m/z calcd for C16H24Cl2F2N5O4P, [M+H]+,
490.0989, found 490.0998.
White powder (28 mg, 28%); HRMS (FAB+) m/z calcd for
C
40H61Cl2FN9NaO13P, [M+Na]+, 1018.3385, found 1018.3363.
5.10.4. Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2,6-difluorobenzyl
phosphoramide mustard (1d)
White powder (41 mg, 40%); HRMS (FAB+) m/z calcd for
C
40H60Cl2F2N9NaO13P, [M+Na]+, 1036.3291, found 1036.3181.
5.10.5. Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2,3,5,6-
tetrafluorobenzyl phosphoramide mustard (1e)
White powder (32 mg, 30%); HRMS (FAB+) m/z calcd for
C
40H58Cl2F4N9NaO13P, [M+Na]+, 1072.3103, found 1072.2985.
5.9.5. 4-
L-Glutaminylamino-2,3,5,6-tetrafluoro-benzyl
5.11. Stability study of 1a–e in buffers
phosphoramide mustard (8e)
A yellow semi-solid (410 mg, 78%); 1H NMR (200 MHz, CD3OD):
d 5.14 (d, 2H, J = 8.2 Hz), 4.06 (dd, 1H, J = 7.0 Hz, J = 5.4 Hz), 3.60–
3.68 (m, 4H), 3.35–3.47 (m, 4H), 2.47–2.54 (m, 2H), 1.90–2.28
(m, 2H); 13C NMR (50 MHz, CD3OD): 176.9, 169.6, 147.6 (dm,
J = 245.1 Hz), 143.6 (dm, J = 247.4 Hz), 118.0 (t, J = 14.8 Hz), 115.3
(td, J = 17.7 Hz, J = 8.0 Hz), 55.5, 54.4, 50.6 (d, J = 5.0 Hz), 43.0,
31.5, 28.5; MS (ESIÀ): m/z (intensity), 526.1 ([M+H]+, 100%),
528.1 ([M+H]+ + 2, 65%), 530.1 ([M+Na]+ + 4, 10%), 548.1 ([M
+Na]+, 40%), 550.1 ([M+Na]+ + 2, 26%), 552.1 ([M+Na]+ + 4, 4%).
HRMS (FAB+) m/z calcd for C16H22Cl2F4N5O4P, [M+H]+, 526.0801,
found 526.0796.
The peptide conjugate 1a–e (1 mg) was dissolved in 0.5 mL of
50 mM Na2HPO4/NaH2PO4 buffer (pH 7.4) containing 2% DMSO
or 0.5 mL of 50 mM Tris–HCl/10 mM CaCl2/0.1% Tween-20Ò buffer
(pH 8.0) containing 2% DMSO, respectively. At different time inter-
vals, aliquots (25
lL) were withdrawn and frozen prior to HPLC
analysis (Waters Symmetry C18 column —3.5
l
m, 4.6 Â 150 mm,
gradient elution from 10% to 80% acetonitrile containing 0.1% TFA
at a flow rate at 1 mL/min, detection wavelength at 220 nm and
245 nm). The half-life was determined based on the disappearance
of the peptide conjugate 1a–e.
5.12. Prostate-specific antigen (PSA) assay of 1a–e
5.10. Synthesis of fluorinated and non-fluorinated Glutaryl-
Hyp-Ala-Ser-Chg-Gln-NH-benzyl phosphoramide mustards (1a–
e)
PSA was purchased from CALIBIOCHEM. The stock solution of
peptide conjugate 1a–e (5
PSA buffer (295 L, 50 mM Tris–HCl/10 mM CaCl2/0.1% Tween-
20Ò buffer, pH 8.0), respectively. The prepared solution was
warmed up to 37 °C and then 245 L of solution was transferred
to an eppendorf vial containing PSA (5 L, 2.45 mg/mL), respec-
lL, 10 mM in DMSO) was added to a
l
To
a
solution of Fm-glutaryl-Hyp-Ala-Ser-Chg-OH (9)
(0.1 mmol) and HBTU (0.11 mmol) in NMP (2 mL) was added
DIPEA (0.11 mmol). The resulting mixture was stirred at room
l
l

2706
X. Wu, L. Hu / Bioorg. Med. Chem. 24 (2016) 2697–2706
5. Ryan, C. J.; Smith, M. R.; Bono, J. S. d.; Molina, A.; Logothetis, C. J.; Souza, P. d.;
tively. At different time intervals, aliquots (20 lL) were withdrawn,
quenched with acetonitrile (5
(Waters Symmetry C18 column —3.5
elution from 10% to 80% acetonitrile containing 0.1% TFA at a flow
rate at 1 mL/min, detection wavelength at 220 nm and 245 nm).
The half-life was determined based on the disappearance of pep-
tide conjugate 1a–e.
Fizazi, K.; Mainwaring, P.; Piulats, J.; Ng, S.; Carles, J.; Mulders, P. F. A.; Basch, E.;
Small, E. J.; Saad, F.; Schrijvers, D.; Poppel, H. V.; Mukherjee, S. D.; Suttmann, H.;
Gerritsen, W. R.; Flaig, T. W.; George, D. J.; Yu, E. Y.; Efstathiou, E.; Pantuck, A.;
Winquist, E.; Higano, C. S.; Park, Y.; Kheoh, T.; Griffin, T.; Scher, H. I.; Rathkopf,
D. E. N. Eng. J. Med. 2013, 368, 138.
l
L) and frozen prior to HPLC analysis
l
m, 4.6 Â 150 mm, gradient
6. Scher, H. I.; Fizazi, K.; Saad, F.; Taplin, M. E.; Sternberg, C. N.; Miller, K.; Wit, R.
d.; Mulders, P.; Chi, K. N.; Shore, N. D.; Armstrong, A. J.; Flaig, T. W.; Fléchon, A.;
Mainwaring, P.; Fleming, M.; Hainsworth, J. D.; Hirmand, M.; Selby, B.; Seely, L.;
Bono, J. S. d. N. Eng. J. Med. 2012, 367, 1187.
7. Parker, C.; Nilsson, S.; Heinrich, D.; Helle, S. I.; O’Sullivan, J. M.; Fosså, S. D.;
Chodacki, A.; Wiechno, P.; Logue, J.; Seke, M.; Widmark, A.; Johannessen, D. C.;
Hoskin, P.; Bottomley, D.; James, N. D.; Solberg, A.; Syndikus, I.; Kliment, J.;
Wedel, S.; Boehmer, S.; Dall’Oglio, M.; Franzén, L.; Coleman, R.; Vogelzang, N. J.;
O’Bryan-Tear, C. G.; Staudacher, K.; Garcia-Vargas, J.; Shan, M.; Bruland, Ø. S.;
Sartor, O. N. Eng. J. Med. 2013, 369, 213.
5.13. Cell culture and antiproliferative assays of 1a–e
LNCaP (PSA positive, ATCC) and DU145 (PSA negative, ATCC)
human prostate carcinoma cells were grown as monolayer cultures
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100 units/mL streptomycin sulfate). Cells were cultured in a
humidified atmosphere of 5% CO2 at 37 °C. Media were routinely
changed every 72 h. Cells were split at 80% confluence followed
by trypsinization and subcultured at 1:4. Cells were plated in 96-
well plates (7500 LNCaP cells/well and 500 DU145 cells/well)
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Acknowledgements
We gratefully acknowledge the financial support of grant SNJ-
CCR 700-009 from the State of New Jersey Commission on Cancer
Research, a pilot grant from the Gallo Prostate Cancer Center of the
Cancer Institute of New Jersey, and grant RSG-03-004-01-CDD
from the American Cancer Society.
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