Synthesis and Evaluation of Radiolabeled Phosphoramide Mustard with Selectivity for Hypoxic Cancer Cells

Article abstract of DOI:10.1021/acsmedchemlett.7b00355

Tumor hypoxia has been widely explored over the years as a diagnostic and therapeutic marker. Herein, we synthesized an alkyne functionalized version of evofosfamide, a hypoxia-selective prodrug. The purpose of this effort was to investigate if this novel 2-nitroimidazole phosphoramide nitrogen mustard (2-NIPAM) retained hypoxia selectivity and could be utilized in radiopharmaceutical development to significantly increase retention of conjugated agents in hypoxic cells. 2-NIPAM demonstrated good hypoxia selectivity with a 62- and 225-fold increase in cytotoxicity toward PC-3 and DU145 human prostate cancer cell lines, respectively, under hypoxic conditions. Radiolabeling of 2-NIPAM with ¹²⁵I was accomplished through a Cu(I)-mediated azide-alkyne cycloaddition reaction. The ¹²⁵I-conjugate demonstrated 13.6 and 17.8% lower efflux rates for DU145 and PC-3 cells, correspondingly, under hypoxic conditions, suggesting that the increased retention is likely due to the known intracellular trapping mechanism. In conclusion, these studies demonstrate the potential of 2-NIPAM in serving as a trapping agent for radiopharmaceutical development.

Full text of DOI:10.1021/acsmedchemlett.7b00355

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Letter
Synthesis and Evaluation of a Radiolabeled Phosphoramide
Mustard with Selectivity for Hypoxic Cancer Cells
Wenting Zhang, Wei Fan, Zhengyuan Zhou, and Jered C Garrison
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00355 • Publication Date (Web): 23 Oct 2017
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Wenting Zhang†, ,‡, Wei Fan†, ,‡, Zhengyuan Zhou₮, Jered Garrison†,§, ,¦, *
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†. Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska
Medical Center, Omaha, NE 68198, United States
§. Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center,
985870 Nebraska Medical Center, Omaha, NE 68198, United States
ǁ. Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, 985830 Nebraska Medical Center,
Omaha, NE 68198, United States
¦. Eppley Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198,
United States
₮. Department of Radiology, Duke University Medical Center, Durham, NC, United States
KEYWORDS: 2-NIPAM, prodrug, radioiodination, prostate cancer.
Macromolecules
ABSTRACT: Tumor hypoxia has been widely explored over the years as a diagnostic and therapeutic marker. Herein, we synthesized
an alkyne functionalized version of evofosfamide, a hypoxia-selective prodrug. The purpose of this effort was to investigate if this
novel 2-nitroimidazole phosphoramide nitrogen mustard (2-NIPAM) retained hypoxia-selectivity and could be utilized in radiophar-
maceutical development to significantly increase retention of conjugated agents in hypoxic cells. 2-NIPAM demonstrated good hy-
poxia-selectivity with a 62- and 225-fold increase in cytotoxicity towards PC-3 and DU145 human prostate cancer cell lines, respec-
tively, under hypoxic conditions. Radiolabeling of 2-NIPAM with 125I was accomplished through a Cu(I)-mediated azide-alkyne
cycloaddition reaction. The 125I-conjugate demonstrated 13.6 and 17.8% lower efflux rates for DU145 and PC-3 cells, correspond-
ingly, under hypoxic conditions suggesting that the increased retention is likely due to the known intracellular trapping mechanism.
In conclusion, these studies demonstrate the potential of 2-NIPAM in serving as a trapping agent for radiopharmaceutical develop-
ment.
Hypoxia is a well-known characteristic of many solid can-
cers. This phenomenon is attributable to the chaotic nature of
tumor vasculature, leading to the inefficient delivery of oxygen
and other nutrients and resulting in a heterogeneous distribution
of these materials in the tumor.1,2 Tumor hypoxia has been im-
plicated in both drug and radiation resistance, which has caused
treatment failure in numerous cancers.3,4 Given this, it is not
surprising that many researchers over the years have focused on
the design of prodrugs that aim to exploit tumor hypoxia for
diagnostic and therapeutic applications.5-8
hypoxia-selectivity of many of these agents is based on biore-
ductive mechanisms, leading to the transformation of these
drugs into reactive species in hypoxic tissues.13 These reactive
species are typically electrophilic metabolites which exhibit in-
creased retention in hypoxic cells by forming intracellular ad-
ducts with opportunistic nucleophilic biomolecules. For hy-
poxia-selective diagnostic agents, the increased formation and
retention of these adducts in hypoxic tissues allows the genera-
tion of target-to-non-target ratios benefiting to in vivo imaging
of hypoxic tissues.14 From a therapeutic perspective, hypoxia
activated chemotherapeutic agents typically generate radicals
(e.g., TPZ) or electrophilic mustard agents (e.g., PR-104 and
evofosfamide) that can induce DNA damage, which ultimately
prompts cancer cell death.15-17 The advantage of these prodrugs
Up to now, various hypoxia based diagnostic (e.g., 18F-
FMISO) and therapeutic (e.g., AQ4N, PR-104 and
evofosfamide) prodrugs have been under development.9-12 The
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is the increased selectivity and cytotoxicity for hypoxic tumors
relative to normoxic non-target tissues.
obtained in three steps with an overall yield of 85%. An Appel
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reaction, utilizing triphenylphosphine and CBr4, was employed
to convert the alcohol into a bromide, giving compound 8. After
work-up, column chromatography was utilized to purify the
compound and remove byproducts (e.g., POPh3) and starting
reagents (e.g., CBr4). Purified 8 was dissolved in Et2O and the
Boc group removed by the introduction of HCl gas yielding in-
termediate 9 in quantitative yield. The structure of 9 was con-
firmed by x-ray crystallography (Figure 2A), but it is notewor-
thy that a molecule of CBr4 also co-crystalized with the desired
compound. After further investigation, it was found that addi-
tional chromatographic purifications of 8 were able to remove
residual CBr4, resulting in the isolation of white crystalline 9.
Overall, the synthetic route in Scheme 1A produced compound
9 in 80% yield. The structures of the compound synthesized in
Our laboratory is interested in the development of diagnostic
and radiotherapeutic agents that can utilize the hypoxic nature
of cancers. Previously, we have found that when 2-nitroim-
dazoles, a class of hypoxia-selective prodrugs, are conjugated
to receptor-targeted peptides, these bioconjugates exhibit in-
creased long-term retention in hypoxic cells, presumably
through adducts formed with intracellular biomolecules.14,18
This initial work has given us the impetus for the continued in-
vestigation and development of other prodrugs that may serve
as hypoxia-selective trapping agents (HSTAs). One of the po-
tential candidates is the 2-nitroimidazole-5-yl methyl based
compound evofosfamide (i.e., TH-302), which is depicted as 2
in Figure 1. Evofosfamide is a hypoxia-activated prodrug that
has undergone several phase I/II/III clinical trials for the treat-
ment of advanced solid cancers, including sarcomas, and oe-
sophageal adenocarcinomas.19-21 This prodrug is activated
through reduction facilitated by cellular reductases (e.g., cyto-
chrome P450) to generate a radical anion. Under hypoxic con-
ditions, the radical anion is exposed to further reduction, lead-
ing to the fragmentation and generation of an active nitrogen
mustard.17 The goal of our work is to develop a derivative of
evosfosfamide that can be easily conjugated to imaging agents
(e.g., fluorophores or radioisotopes) for the in vitro and in vivo
detection of hypoxia or to targeting vectors to enhance the re-
tention of these agents in hypoxic tissues. Herein, we describe
the synthesis of a novel 2-nitroimidazole phosphoramide nitro-
gen mustard (2-NIPAM, 3) that is capable of conjugating to
other moieties through the well-known azide-alkyne cycloaddi-
tion reaction. Utilizing human prostate cancer PC-3 and DU145
cell lines, we have investigated the cytotoxicity and hypoxia se-
lectivity of this analog relative to evosfosfamide (2) and a neg-
ative control (i.e., non-activatable, 1). In addition, we optimized
the azide-alkyne cycloaddition reaction conditions for 2-
NIPAM, radiolabeled the analog with 125I and investigated the
hypoxia trapping capability of this radiolabeled agent under
normoxic and hypoxic conditions. The results obtained from
our investigations have suggested that 2-NIPAM has the poten-
tial to serve as a hypoxia-selective imaging agent and/or trap-
ping agent for receptor-targeted constructs.
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1
this scheme were confirmed by mass spectrometry, H and 13C
NMR spectra. (Figure S2, S3 and S4, correspondingly).
Initial attempts to synthesize the phosphordiamidate mus-
tards centered on utilizing an asymmetric approach using a
phosphoramic dichloride precursor (Scheme S2).26,28 Unfortu-
nately, this approach yielded multiple products with one of the
largest byproducts being a bisphosphoramidate 11. The struc-
ture of 11 was confirmed by X-ray crystallography, Figure 2B.
Attempts to obtain the desired compounds by modifying the re-
action conditions (i.e. substitution of different amines, varying
reaction times and temperatures) were unsuccessful.
With these setbacks, we switched our approach to focus on a
symmetric phosphoramidate mustard synthesis, outlined in
Scheme 1B, to obtain 3 along with our positive (2) and negative
(1) controls. The negative control, 1, is an analog of 2 that uti-
lizes an imidazole instead of a nitroimidazole, thereby eliminat-
ing its ability to fragment and form an active phosphoramidate
mustard. The controls were synthesized by reaction of two
equivalents of bromoethylamine with POCl3 to yield the phos-
phoramidate chloride intermediate. This intermediate was sub-
sequently reacted under basic conditions with (1-methyl-1H-
imidazol-5-yl)methanol or its 2-nitroimidazole derivative to
produce compound 1 in 13.8% yield and 2 in 11.7% yield, re-
spectively. The synthesis of compound 3 was accomplished by
the sequential addition of 2.2 equivalents of 9 with PCl3, fol-
lowed by reaction with 1-methyl-2-nitro-1H-imidazol-5-
yl)methanol and oxidation via tert-butyl hydroperoxide. The
structures of compound 1, 2 and 3 were confirmed by mass
spectrometric, 1H, 13C and/or 31P NMR spectra analysis (Figure
S2, S3, S4, and S5 correspondingly).
Figure 1. Chemical structure of phosphamide mustard nega-
tive control 1, positive control 2 and 2-NIPAM 3.
The synthesis of the phosphoramide mustards 1-3 are out-
lined in Scheme 1. Out first synthetic goal in the synthesis of 2-
NIPAM, 3, was to produce the alkyne containing secondary
amine, 9. The synthesis of compound 9 (N-(2-bromoethyl)prop-
2-yn-1-amine hydrochloride), as outlined in Scheme 1A, was
initiated with the dual protection of 2-aminoethan-1-ol. The
amine and alcohol functional groups of the starting material
were protected with Boc and tert-butyldimethylsilyl groups, re-
spectively.12,22-27 Deprotonation of the protected amine using
sodium hydride and subsequent reaction with propargyl bro-
mide yielded the protected secondary amine 6. Deprotection of
the silyl protecting groups employing a fluoride source (i.e.,
TBAF) provided compound 7. To this point, compound 7 was
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ACS Medicinal Chemistry Letters
Scheme 1. Synthesis of imidazole and its nitro-derivative conjugated phosphoramidate mustard (1-3)
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T
able 1. Cytotoxicity of prostate cancer cell lines with tested
compounds.
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CR
Result (mean±SEM) are from 2 or more independent experiments
carried out in quintuplicate.
HCR: hypoxia cytotoxicity ratio. HCR= IC50(air)/ IC50(N2).
The cytotoxicity of compounds 1, 2 and 3 were evaluated in
PC-3 and DU145 human prostate cancer cell lines under both
normoxic and hypoxic conditions using a viability assay, results
shown in Table 1. As expected, the negative control, 1, demon-
strated an IC50 that was > 0.85 mM for both cell lines. Due to
its inability to form an active phosphoramide mustard under hy-
poxic conditions, the cytotoxicity of 1 was > 100-fold lower
than either 2 or 3. Additionally, the negative control demon-
strated no hypoxia selectivity (i.e., hypoxia cytotoxicity ratio
(HCR) = 1). The positive control, 2, demonstrated increased cy-
totoxicity (IC50 = 4.14 0.66 – 6.49 0.67 µM) in both cell
lines under hypoxic conditions relative to normoxic conditions,
(IC50 = 687 32.6 – 842 52.5 µM). The hypoxia selectivity
for 2 was found to be approximately 2-fold higher in DU145
cells (HCR=203) compared to PC-3 cells (HCR=106). These
results agree with previous reports in which evofosfamide (2)
demonstrated lower hypoxia selectivity in PC-3 cells
(HCR=190) relative to DU145 cells (HCR=240).29 Compound
hypoxia selectivity of 3 was 2-fold lower (HCR = 62) for PC-3
cells, but had similar values (HCR = 225) for DU145 cells.
There is no significant difference in the HCR between com-
pound 2 and compound 3 (P>0.05) in the DU145 cell line, but
a significant difference in PC-3 cells (P<0.05) was observed.
Representative images depicting the cytotoxicity for both cell
lines incubated for 2h with 1ｘ10-5 M 2-NIPAM under hypoxic
and normoxic conditions are shown in Figure 3. At any rate,
these studies demonstrate that incorporation of the alkyne
groups into the phosporamide structure did not substantially im-
pact the cytotoxicity or hypoxia selectivity of 3. This strongly
suggests that compound 3 is able to undergo the same activation
mechanism that has already been established for 2 (i.e.,
evofosfamide ).30
To evaluate the timed-dependent cytotoxicity of compound
3, IC50 assays were performed using both cell lines under dif-
ferent incubation times. As shown in Figure 4A and 4B, the
cytotoxicity of 3 was time-dependent in both cell lines with in-
creased cytotoxicity as incubation time increased. As previ-
ously noted, higher cytotoxicity for 3 was observed in DU145
cells compared to PC-3 under hypoxic conditions. The IC50 of
3 after 6h incubation was determined to be 0.78 µM in DU145
3, 2-NIPAM, demonstrated slightly higher cytotoxicity (IC50
=
1.65 0.09 – 4.70 0.01 µM) compared to the positive control
under hypoxic conditions. However, compound 3 also demon-
strated 3-4 fold higher cytotoxicity under normoxic conditions
relative to compound 2. This suggests that either compound 3
is more easily activated than 2 under normoxic conditions or
that the “inactive” form of 3 is more cytotoxic than
evofosfamide. Interestingly, relative to the positive control, the
3
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cells, and 1.69 µM in PC-3 cells, as shown in Table 2. En-
hanced cytotoxicity with elongated incubation time is likely a
result of the increase in cellular adducts formation.
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Figure 4A & B. Time-dependent cytotoxicity of compound 3 (2-
NIPAM) in DU145 and PC-3 cell lines, values are mean SEM
(n=5).
Table 2. Summary of time-dependent cytotoxicity of com-
pound 3.
IC50(µmol/L; N2)
Compound exposure time
PC-3
DU145
2h
4h
6h
4.70 0.01
3.00 0.22
1.69 0.41
1.65 0.09
1.17 0.15
0.78 0.10
Result (mean SEM) are from 3 or more independent experiments
carried out in quintuplicate.
In order to investigate the conjugation and in vitro trapping
efficacy of 2-NIPAM (3), we radiolabeled 3 with 125I utilizing
a Cu(I)-mediated cycloaddition reaction.31 However, before
proceeding we sought to optimize the cycloaddition reaction
conditions. Our previous unpublished work demonstrated that
2-nitroimidazoles can undergo rapid degradation when exposed
to certain metals (e.g., Cu(II) salts). With that in mind, the sta-
bility of the 2-nitroimidazole reactants and the conjugation ef-
ficacy of the ruthenium-based and Cu(I) catalysts under various
experimental conditions (i.e., temperature and reaction time)
were examined. The coupling of 3 with benzyl azide utilizing
two common ruthenium-based catalysts (Cp*RuCl (PPh3)2 and
Cp*RuCl (COD)) did not result in the desired product as eval-
uated by LC-MS, but instead lead to multiple byproducts at both
room and elevated temperatures. Our initial belief was that the
ruthenium catalysts, under the conditions employed, were lead-
ing to the degradation of the 2-nitroimidazole functionality of
3. To further investigate this, we incubated the ruthenium cata-
lyst in the presence of 2-nitroimidazole-1- acetic acid. LC-MS
demonstrated that exposure of the 2-nitroimidazole analog to
the ruthenium catalysts led to a gradual transformation of the
analog (data not shown), presumably by reduction of the nitro
group to the amine.32,33 Fortunately, the 2-nitroimidazole func-
tionality of 3 remained stable using Cu(I)-mediated cycloaddi-
tion conditions at both room temperature and 60°C with yields
of 81.2 % and 82.7 %, respectively.
Figure 5A. LC-MS chromatogram profile of products of azide-2-
NIPAM cycloaddition reaction. (Eluent: ACN/H2O) B. LC-MS
chromatogram profile of compound 14 aligned with Radio-la-
beled HPLC profile of compound 15. (Eluent: Methanol/H2O)
Following the cycloaddition optimization, we employed a re-
ported procedure by Årstad and colleagues to incorporate 125I
into the resulting triazole ring of the cycloaddition products.38
Briefly, the procedure involves a one-pot reaction that utilizes
copper (II) chloride combined with Et3N to generate the cy-
cloaddition product with the 125I incorporated on the 5-position
of the triazole ring. While some mechanistic studies were per-
formed, the precise mechanism of the 125I-incorporation re-
mains unclear. However, some findings appear to be certain, 1)
the Et3N is able to reduce a portion of the Cu(II) species in so-
lution to Cu(I), thereby generating the needed species to carry
out the cycloaddition reaction and 2) the incorporation of the
125I is carried out by a Cu(I) species. At any rate, we explored
the utilization of this technique with 2-NIPAM (3), as depicted
Scheme 2. Synthesis of 5-[Nat/125I]Iodo-1,2,3-trizoles (13-15)
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in Scheme 2. Utilizing the CuCl2/ Et3N system in a water-ace-
NIPAM), modeled after evofosfamide, that allows easy conju-
tonitrile mixture, we first examined the incorporation of natI
(non-radioactive) into 2-NIPAM utilizing benzyl azide. Upon
completion of the 90min reaction at 60°C, the reaction progress
was evaluated by LC-MS, as shown in Figure 5A. The cycload-
dition product 13, without the incorporation of the iodide, was
observed at 5.0 min, while the single iodinated product 14 was
observed at 7.6 min. As can be seen from the chromatograms,
the incorporation efficiency of the iodide into the triazole was
modest. In order to aid separation and purification of the io-
dinated species (i.e., 14 and 15) the mobile phase was changed
from water:acetonitrile to water:methanol. The chromatogram
of 14 using the water:methanol eluent is depicted in Figure 5B.
Utilizing analogous reaction conditions, the reaction of 3, ben-
zyl azide and 125I was carried out and evaluated by radio-HPLC
using the same gradient. As expected, 15 had an identical reten-
tion times compared to non-radioactive 14. While the overall
radiochemical yield for 15 was poor (10%), the yield was more
than sufficient for further in vitro characterization.
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gation of the hypoxia-selective drug to imaging moieties and
other constructs through an azide-alkyne cycloaddition reac-
tion. Our interest in 2-NIPAM, and other hypoxia-selective
drugs, lies in their potential for selective activation and reten-
tion in hypoxic tissues, such as observed with many cancers.
We envision that this selectivity can be exploited for the pur-
pose of selectively increasing the retention of diagnostic and
therapeutic agents in hypoxic tumor tissues. Our studies demon-
strated that 2-NIPAM had similar cytotoxicity and hypoxia-se-
lectivity compared to evofosfamide, establishing that the incor-
poration of the alkyne functionality did not substantially impact
the activity of the agent. Using a synthesized 125I-labeled 2-
NIPAM conjugate, we observed that the analog demonstrated
greater cellular retention under hypoxic conditions, suggesting
that retention is due to activation and adduct formation with in-
tracellular macromolecules. Given these results, we plan on fur-
ther investigating the potential of 2-NIPAM alone as a hypoxia
diagnostic agent and in conjugation with receptor-targeted
agents for cancer imaging and targeted radiotherapeutic appli-
cations.
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Studies were performed to determine the distribution coeffi-
cient (LogD7.4) of 15. The 125I-labeled conjugate was found to
have a distribution coefficient of 0.55 0.06 which demon-
strated that the compound is hydrophobic enough to readily en-
ter cells through passive diffusion. In order to evaluate the cel-
lular trapping/retention efficacy of compound 15, we investi-
gated the biological performance of this radiolabeled agent us-
ing in vitro efflux studies. The PC-3 and DU145 cells were ini-
tially treated with 15 for 1h at 37℃. Uptake of the 125I-labeled
conjugate in both cell lines ranged from 2% to 4% of the total
radioactivity added under both normoxic and hypoxic condi-
tions, respectively. The efflux results for 15 over an 8 h period
in both PC-3 and DU145 cells under normoxic and hypoxic
conditions are given in Figure 6. For all cell lines and oxygen
conditions, more than 50% of 15 was effluxed from the cells
within the first 2h. However, the 125I-labeled conjugate demon-
strated lower efflux rates under hypoxic conditions in both cell
lines relative to normoxic conditions. At 8h after efflux began,
85.8% and 88.3% of the initial internalized radioactivity of 15
was externalized in normoxic condition in DU145 and PC-3
cells, respectively, compared to 72.2% and 70.5% under hy-
poxic conditions. The increased retention of 15 under hypoxic
conditions, which is likely due to hypoxia activation and adduct
formation with intracellular biomolecules, was found to be sta-
tistically significant (P < 0.05) in both cell lines when compared
to normoxic data.
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures, compounds characterization, tables, and
schemes (PDF).
* Phone: 001-402-559-3453. Fax: 001-402-559-9365. E-mail:
jcgarrison@unmc.edu.
‡These authors contributed equally.
Notes: The authors declare no competing financial interest.
This study was supported by the National Institutes of Health
(5R01CA179059-03). No other potential conflict of interest rele-
vant to this article was reported.
We thank Ed Ezell at the Nuclear Magnetic Resonance (NMR) Fa-
cility at UNMC for assistance in collecting and interpreting the
NMR data for all chemical compounds; Ying Xie and Fei Yu at the
UNMC for assistance using the EVOS FL Cell Imaging System;
Dr. Shana A. Garrison for helpful advice in the preparation of this
manuscript.
TH-302: evofosfamide; 2-NIPAM: 2-nitroimidazole phospho-
ramide nitrogen mustard; equiv: equivalent; Et3N: ethanolamine;
THF: tetrahydrofuran; ACN: acetonitrile; TBAF: tetra-n-bu-
tylammonium fluoride; Et2O: diethyl ester; RCY: radiochemical
yields.
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To summarize, we have designed and successfully synthe-
sized a novel 2-nitroimidazole phosphoramide mustard (2-
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