Artemisinin-derived dimer phosphate esters as potent anti-cytomegalovirus (anti-CMV) and anti-cancer agents: A structure-activity study

Article abstract of DOI:10.1016/j.bmc.2013.04.027

We recently reported the anti-cancer and anti-cytomegalovirus (CMV) activity of artemisinin-derived trioxane diphenylphosphate dimer 838. To probe the relationship between chemical structure and anti-CMV and anti-cancer activities, we now report synthesis and evaluation of a series of eight new dimer phosphate ester analogs of 838. This series of novel molecules was screened against human foreskin fibroblasts (HFFs) infected with CMV and against the human Jurkat T cell acute lymphoblastic leukemia cell line. This SAR study confirms the very high anti-CMV and anti-cancer potencies of dimer diphenyl phosphate ester 838 without its being toxic to normal cells.

Full text of DOI:10.1016/j.bmc.2013.04.027

Bioorganic & Medicinal Chemistry 21 (2013) 3702–3707
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Artemisinin-derived dimer phosphate esters as potent
anti-cytomegalovirus (anti-CMV) and anti-cancer agents:
A structure–activity study
Bryan T. Mott a, Ran He b, Xiaochun Chen c,d, Jennifer M. Fox c,d, Curt I. Civin c,d, Ravit Arav-Boger b,
Gary H. Posner a,e,
⇑
a Department of Chemistry, School of Arts and Sciences, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, United States
b Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD 21205, United States
c Center for Stem Cell Biology & Regenerative Medicine, Departments of Pediatrics, University of Maryland, Baltimore, MD 21201, United States
d Center for Stem Cell Biology & Regenerative Medicine, Departments of Physiology, University of Maryland, Baltimore, MD 21201, United States
e Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins School of Medicine, Baltimore, MD 21205, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported the anti-cancer and anti-cytomegalovirus (CMV) activity of artemisinin-derived tri-
oxane diphenylphosphate dimer 838. To probe the relationship between chemical structure and anti-
CMV and anti-cancer activities, we now report synthesis and evaluation of a series of eight new dimer
phosphate ester analogs of 838. This series of novel molecules was screened against human foreskin
fibroblasts (HFFs) infected with CMV and against the human Jurkat T cell acute lymphoblastic leukemia
cell line. This SAR study confirms the very high anti-CMV and anti-cancer potencies of dimer diphenyl
phosphate ester 838 without its being toxic to normal cells.
Received 21 March 2013
Revised 9 April 2013
Accepted 15 April 2013
Available online 22 April 2013
Keywords:
Artemisinin-derived dimer
CMV
Ó 2013 Elsevier Ltd. All rights reserved.
Leukemia
Phosphate ester
1. Introduction
species of Plasmodium parasites, derivatives of artemisinin have
shown also significant activity against a variety of other diseases,
Despite increased spending over the past two decades, the
number of FDA approved drugs per dollar spent continues to de-
cline.1 As such, focus has increasingly shifted to finding new uses
for therapeutics already evaluated in humans in order to lower
the cost of drug development.1,2 In addition to this decline in suc-
cess, malaria parasites and some cancer cell lines have developed
resistance to several currently used drugs.3–5 Chemotherapy of
cancer and other diseases, such as cytomegalovirus (CMV), is often
associated with undesirable side effects. Therefore, the need for
continued drug discovery and development is of paramount
importance. The natural product artemisinin (1, Fig. 1) is a well-
known antimalarial drug that is the active ingredient of an ancient
Chinese remedy discovered by the lab of Dr. Youyou Tu.6 The nat-
ural product has been functionalized to improve the pharmacoki-
netics of the molecule, and first generation derivatives such as
dihydroartemisinin (DHA), artemether and sodium artesunate (2,
3, and 4, respectively, Fig. 1) are currently part of artemisinin com-
bination therapy (ACT), the first line of malaria chemotherapy.7 In
addition to their potent and rapid antimalarial efficacy against all
including helminth infections,8 CMV,9,10 and many different cancer
cell lines.10–16
A number of successful synthetic strategies have been employed
to further improve the natural product or harness the activity of the
essential peroxide linkage, ranging from semisynthetic17,18 to fully
synthetic analogs.19 It is important to note that many of the most po-
tent artemisinin synthetic analogs that have been reported by our
group and by others, against malaria and other diseases, are pre-
pared by coupling two artemisinin units into one dimeric mole-
cule.9,11,12,20,21 For example, we showed that artemisinin dimer
primary alcohol 5 (Fig. 2) had significantly greater anti-CMV activity
than artemether (3) and artesunate (4)9 and that the deoxy version
of dimer primary alcohol 5 (lacking the peroxide pharmacophore)
was not active against CMV.10 Additionally, bis-benzylic alcohol di-
mer 6 (Fig. 2) was >100-fold more active than DHA (2) against cervi-
cal cancer HeLa cells without showing toxicity to normal cells.11
Trioxane dimer diphenyl phosphate ester 838 (the name is derived
from the compound’s molecular weight, Fig. 2) has especially high
potency and is more potent than the standard drug doxorubicin at
inhibiting growth of prostate cancer LNCaP cells.12 We also recently
demonstrated that dimer 838 has a selectivity index (anti-CMV/
cytotoxicity) of approximately 1500, about 15 times higher than
⇑
Corresponding author. Tel.: +1 410 516 4670; fax: +1 410 516 8420.
E-mail address: ghp@jhu.edu (G.H. Posner).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.027

B. T. Mott et al. / Bioorg. Med. Chem. 21 (2013) 3702–3707
3703
H
O
H
H
H
O
O
O
O
O
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
Na
O
artemisinin - 1
dihydroartemisinin
artemether - 3
sodium artesunate - 4
(DHA) - 2
Figure 1. Artemisinin and its first generation derivatives.
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
P
OPh
OPh
OH
O
HO
OH
5
6
838
Figure 2. Highly potent artemisinin-derived trioxane dimers.
that of the standard anti-CMV drug ganciclovir (GCV).10 The high po-
tency of this molecule spurred us to explore structure-activity rela-
tionships surrounding the diphenylphosphate moiety to probe what
effect different substitutions might have on both the anti-CMV and
anti-cancer activities. We show here the synthesis of this series of
phosphate esters and their activities against CMV, a human leuke-
mia cell line, and human normal cells.
the dicycohexylphosphoryl chloride 20 (Scheme 2b). Each synthe-
sized phosphoryl chloride was then coupled to dimer primary alco-
hol 5, using the procedure shown for the preparation of 838
(Scheme 1), to provide the desired dimeric phosphate products.
3. Results and discussion
The series of trioxane phosphate dimers were screened in vitro
against both CMV-infected cells and leukemia cells. For CMV, HFFs
were infected and treated with the compounds in a dose-depen-
dent manner, and at 72 h post infection (hpi) luciferase activity
was measured as an indication for late CMV gene expression.9
The anti-leukemia effect of these dimers against Jurkat cells (T-
cell acute lymphoblastic leukemia cell line from American Type
Cell Collection, VA) was assessed after 48 h of drug treatment using
the AlamarBlue assay (Invitrogen, CA).24–26 To measure toxicity to
non-malignant cells, peripheral blood mononuclear cells (PBMCs)
were isolated from four consenting healthy volunteers and cul-
2. Chemistry
The syntheses of dimer primary alcohol 522 and dimer diphenyl-
phosphate ester 83812 have been previously reported. The lactone
moiety of artemisinin (1) is reduced by diisobutylaluminum hy-
dride (DIBALH) and is subsequently trapped in situ with acetic
anhydride (Ac2O) to provide exclusively a-dihydroartemisinin ace-
tate (DHA-OAc, 7, Scheme 1). Sakurai bis-allylation using tin(IV)
chloride (SnCl4) and bis-trimethylsilyl isobutylene linker 8 at
ꢀ78 °C provides the isobutylene dimer 9 in excellent yield. The al-
kene is converted into primary alcohol 5 via hydroboration/oxida-
tion, and coupling with diphenylphosphoryl chloride (10) affords
the lead phosphate ester dimer 838. This lead compound 838 was
partially hydrolyzed using sodium hydroxide (NaOH) to give the
monophenylphosphate ester derivative 11 (Scheme 1). We wished
to vary substitution patterns on the aromatic rings, requiring func-
tionalized diphenylphosphoryl chlorides. Many of these were not
commercially available and were therefore synthesized following
literature procedure.23 Substituted phenols were reacted with
phosphoryl chloride (POCl3, or thiophosphoryl chloride, PSCl3) in
the presence of aluminum trichloride (AlCl3) to afford the desired
phosphoryl chlorides 12–17 (Scheme 2a). Functionality was chosen
to have both electron donating (12) and electron withdrawing (13)
effects on the phosphate, and to incorporate a sterically crowded
system (14). We also wished to explore the activity of thiophos-
phates (15 and 16) and rigidified systems (16 and 17). Finally, to test
the effect of aromaticity on biological activity, a fully saturated
phosphoryl chloride was prepared. Cyclohexanol (18) was treated
with sodium hydride (NaH) and phosphorus trichloride (PCl3),
yielding tricyclohexyl phosphite (19) which was subsequently re-
acted with triphosgene and dimethylimidazolidinone to provide
tured with phytohemagglutinin (PHA, Sigma, MO; 10 lg/ml) in
multiwell plates. After 24 h, drug or vehicle was added for 48 addi-
tional hours before Alamar Blue assays were performed.27,28
The results from these assays are summarized in Table 1. As
previously determined, both artemether (3) and artesunate (4)
showed weak activity against CMV.9 In sharp contrast, however,
all of the trioxane dimer phosphates reported here were signifi-
cantly more potent than artemether (3) and artesunate (4) against
CMV replication, having IC50 activities in the low-nanomolar range.
Lead compound 838 displayed the most potent activity, and dic-
yclohexylphosphate 851 also was very potent.
The anti-leukemic activities also were in the low-nanomolar
range for most of this series of phosphates, and important informa-
tion was gained from the SAR. First, partial hydrolysis of diphenyl-
phosphate 838 into the monophenylphosphate ester 762 resulted
in a 70-fold loss in potency (Table 1). This is likely due to the
diminished ability of the more hydrophilic compound 762 to pen-
etrate the leukemia cells. A second point of note is the replacement
of the phosphoryl oxygen with sulfur. These compounds (853 and
855) showed ꢁ9- and ꢁ18-fold loss in potency, respectively, com-
pared to their oxygenated counterparts. The aryl phosphate with

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B. T. Mott et al. / Bioorg. Med. Chem. 21 (2013) 3702–3707
H
H
O
O
O
O
TMS TMS
H
H
O
a
O
b
+
O
O
O
H
OAc
1
7
8
H
O
O
O
O
H
H
ART ART
ART ART
O
O
c
O
O
OH
9
5
ART ART
ART ART
d
O
e
O
P
O
P
O
O
O
O
OH
838
11
Scheme 1. Synthesis of artemisinin dimer phosphates. Reagents and conditions: (a) (i) DIBALH, CH2Cl2, ꢀ78 °C; (ii) DMAP, py, Ac2O, ꢀ78 °C to rt, 95%; (b) SnCl4, CH2Cl2,
ꢀ78 °C, 85%; (c) (i) BH3/SMe2, THF, 0 °C; (ii) NaBO3/4H2O, 0 °C to rt, 85%; (d) diphenylphosphoryl chloride 10, DMAP, py, CH2Cl2, rt, 95%; (e) NaOH, DMF, 50 °C, 56%.
one alkyl substituent (compound 923) was fivefold less potent
than dimer 838, while the aryl phosphate with two alkyl substitu-
ents (compound 895) had comparable potency to dimer 838. Rigid-
ified phosphate ester 836 and saturated dicyclohexyl phosphate
dimer 851were about half as efficacious as dimer 838. Finally,
artesunate was nearly 70-fold less efficacious than the lead com-
pound 838, which was the most active dimer against leukemia,
while artemether was essentially inactive.
5. Experimental
All reactions were performed under argon in oven-dried or
flame-dried glassware. Microwave reactions were performed in a
Biotage Initiator microwave. Dichloromethane was dispensed from
an LC Technology Solutions SPBT-1 bench top solvent purification
system. All commercially available reagents were purchased from
Sigma Aldrich and used as received. All experiments were moni-
The therapeutic index was determined for each compound by
measuring the activity against phytohemagglutinin (PHA)-stimu-
lated peripheral blood mononuclear cells (PBMCs) and determining
the ratio of activities (PBMC IC50/Jurkat cell IC50). While lead com-
pound 838 was the most active compound in this series against
leukemia cells, it had a reduced therapeutic window (TI = 14.8)
as compared to the standard drug doxorubicin (TI = 28.2, Table 1).
Two other analogs (compounds 855 and 853) had comparable
therapeutic indices (TI = 22.9 and 20.7, respectively), while isopro-
pylphenyl dimer 923 had a noteworthy therapeutic index of 78.3.
Importantly, 4-chloro-substituted dimer 907 was both active
against leukemia cells (IC50 = 152 nM) and was essentially inactive
against PHA-stimulated PBMCs (IC50 >40,000), providing a thera-
peutic index of >263, approximately 10-fold greater than that of
doxorubicin.
tored by analytical thin layer chromatography (TLC) performed on
Silicycle silica gel 60 ÅA glass supported plates with 0.25 mm thick-
ness. Flash chromatography was performed with EMD silica gel
0
(40–63 lM). Yields are not optimized. Infrared (IR) spectra were re-
corded on a Perkin Elmer 1600 FT-IR spectrometer. Nuclear mag-
netic resonance (NMR) spectra were recorded on a Bruker Avance
400 MHz FT-NMR spectrometer (400 MHz for 1H, 100 MHz for
13C). The following abbreviations are used in the Section 5 for the
description of 1H NMR spectra: singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), broad singlet (br s), doublet of doublets
(dd), doublet of triplets (dt), and doublet of quartets (dq). Low res-
olution mass spectra (electrospray ionization) were acquired on an
Agilent Technologies 6130 quadrupole spectrometer coupled to an
Agilent Technologies 1200 series HPLC. High resolution mass spec-
trum-electron ionization sprary (HRMS-ESI) were obtained on an
Agilent Technologies 1200 series Dual Absorbance Detector HPLC
system equipped with a Phenomenex Luna 75 ꢂ 3 mm, C18, 3
lm
4. Conclusion
column at 45 °C (UV detection at 220 nm, BW 8 nm, and 254 nm
BW 8 nm, flow rate: 0.8 mL/min (increasing), Injection volume:
It is imperative to continue the pursuit of new therapies, and it
may be beneficial to find multiple uses for the same therapeutic.
We have previously demonstrated that trioxane dimer diphenyl-
phosphate ester 838 is highly potent against both cancer and
CMV-infected cells.9,10 Additionally, dimer 838 has a significant
therapeutic window in each case.9,10 Herein we report an SAR study
surrounding this lead compound. The structure–activity relation-
ships based on this study confirm and emphasize the very high anti-
leukemic and anti-CMV activities of the lead compound 838 and its
low toxicity to human normal cells. This study also identified bis(4-
chlorophenyl)phosphate dimer 907 as a highly active compound
against leukemia that is also non-toxic to normal human cells.
1.0
lL, sample solvent: 100% Methanol, sample conc.: ꢁ0.01 mg/
mL, mobile phase A: Water with 0.1% acetic acid, mobile phase B:
Acetonitrile with 0.1% acetic acid) coupled to a Agilent 6210 time-
of-flight mass spectrometer (ion source: Duel ESI, min range:
115 m/z, max range: 1400 m/z, scan rate: 0.9 s, gas temp: 340 °C,
gas flow: 10 L/min, nebulizer:50 Psi, ion polarity: positive, VCap:
3500 V, fragmentor: 175 V, skimmer1: 65 V, OctopoleRFPeak:
250 V, ref mass: enabled (Agilent P/N G1969-85001). Data were
analyzed using Agilent Masshunter Workstation Data Acquisition
(v B.02.00, Patch 1,2,3) and Agilent Masshunter Qualitative Analysis
(v B.02.00, Build 2.0.197.7, Patch 3). List of abbreviations are as

B. T. Mott et al. / Bioorg. Med. Chem. 21 (2013) 3702–3707
3705
X
P
O
dropwise over several minutes. The heat was turned off, allowing
the reaction to slowly cool to rt, and the reaction mixture was stir-
red at rt overnight. The reaction was deemed complete by TLC, and
the solvent was removed under reduced pressure and the residue
was dissolved in hexanes (50 mL). The solution was filtered
through celite and the solvent was again removed under reduced
pressure. The resulting crude oil was taken on to the next step
without further purification.
O
a
Cl
OH
a
R
R
R
P(X)Cl3
Substituted Phenols Equivs.
Compound Substitution
OH
2
X = O
12
R = 4-iPr
5.3. Synthesis of dicyclohexyl phosphorochloridate (20)
Tricyclohexyl phosphite 19 (2.3 g, 7.0 mmol, 1.0 equiv) and 1,3-
dimethylimidazolidin-2-one (24 mg, 0.21 mmol, 0.13 equiv) and
dichloromethane (30 mL) were added to a round bottom flask,
and the stirring solution was cooled to 0 °C in an ice bath. A sepa-
rate solution of triphosgene (706 mg, 2.38 mmol, 0.34 equiv) in
dichloromethane (5 mL) was prepared and added dropwise to the
original stirring solution over 10 min. The reaction mixture was
stirred at 0 °C for 30 min, the ice bath was removed, and the mix-
ture was heated to 35 °C for 3 h. The reaction was deemed com-
plete by TLC, and the solvent was removed under reduced
pressure. The residue was distilled in a Buchi Kugelrohr, affording
a colorless oil: (1.68 g, 5.98 mmol, 85%).
Cl
OH
2
X = O
13
R = 4-Cl
2
2
X = O
X = S
14
15
R = 2,6-diMe
OH
OH
R = H
S
O
P
OH OH
OH OH
Cl
O
X = S
X = O
1
1
16
17
5.4. General procedure for the synthesis of dimer phosphate
esters
O
Primary alcohol 18 (1.0 equiv) was dissolved in dichlorometh-
ane (2 mL) under argon at room temperature. To the stirring solu-
tion were added pyridine (5.0 equiv), dimethylaminopyridine
(DMAP, 1.0 equiv) and the appropriate phosphoryl chloride
(5.0 equiv) in that order. The reaction mixture was allowed to stir
at rt for 3 h, at which point the reaction was deemed complete by
analytical TLC. The reaction mixture was diluted with dicholorom-
ethane and washed consecutively with 10% citric acid and brine.
The organic layer was dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified directly on silica gel. Gradient
elution (10–40% ethyl acetate in hexanes) afforded the desired
products as a colorless, amorphous solid.
O
P
Cl
O
b
OH
O
O
b
c
P
O
P
O
Cl
3
18
19
20
Scheme 2. Synthesis of phosphoryl chlorides. Reagents and conditions: (a) AlCl3,
PXCl3, 150 °C, microwave, 1 h 62–93% crude; (b) (i) NaH, THF, rt to 50 °C; (ii) PCl3;
(c) dimethylimidazolinone, triphosgene, CH2Cl2, 85% over two steps.
5.5. Synthesis of dimer diphenylthiophosphate ester 855
The title compound was prepared as described in the general pro-
cedure, using diphenylthiophosphoryl chloride (15): yield (7.5 mg,
0.009 mmol, 27%), 1H NMR (400 MHz, CDCl3) d 7.36–7.17 (m, 12H),
5.33 (s, 1H), 5.30 (s, 1H), 4.55–4.37 (m, 3H), 4.27–4.24 (m, 1H),
2.73–2.65 (m, 1H), 2.59–2.52 (m, 1H), 2.37–2.28 (m, 3H), 2.04–1.98
(m, 3H), 1.94–1.82 (m, 3H), 1.79–1.63 (m, 6H), 1.52–1.35 (m, 12H,
including singlets at 1.40 and 1.38), 1.31–1.23 (m, 4H), 1.00–0.82
(m, 12H); 13C NMR (100 MHz, CDCl3) d 150.7, 129.7, 125.1, 120.2,
120.1, 103.1, 102.4, 89.9, 89.0, 81.2, 81.0, 74.2, 72.1, 70.8, 52.4,
52.1, 44.5, 44.2, 38.8, 38.0, 36.6, 36.5, 35.0, 34.5, 30.0, 30.0, 29.9,
29.4, 26.1, 25.9, 24.9, 24.9, 24.7, 20.2, 20.1, 13.2, 12.6; ESI-HRMS for
follows: dichloromethane—DCM; dimethylformamide—DMF; satd
aq NaCl—brine; ethyl acetate—EtOAc.
5.1. General procedure for the synthesis of diaryl chlorophosate
esters (12–17)
To a 0.5–2 mL microwave vial were added 4.46 mmol of substi-
tuted phenol (1.0 equiv), 2.23 mmol of phosphorus oxychloride
(POCl3, 0.5 equiv), and 0.446 mmol of aluminum chloride (AlCl3,
0.1 equiv). The reaction mixture was heated in the microwave at
150 °C for 1 h. Upon completion, the mixture was diluted with
CH2Cl2, filtered through a pad of celite, and purified directly on sil-
ica. Gradient elution (5–15% ethyl acetate in hexanes) afforded the
desired products as colorless amorphous solids or oils.
C
46H63NaO11PS (M+Na)+ calcd = 877.3721, found = 877.3741; ½a 2D3
ꢃ
62 (c 0.39, CHCl3); FT-IR (cmꢀ1) 2919, 2861, 1589, 1485, 1455,
1376, 1290, 1225, 1194, 1110, 1009, 943, 760.
5.6. Synthesis of dimer biphenylphosphate ester 836
5.2. Synthesis of tricyclohexyl phosphite (19)
The title compound was prepared as described in the general
procedure, using biphenylphosphoryl chloride (17): yield (7.0 mg,
0.008 mmol, 34%), 1H NMR (400 MHz, CDCl3) d 7.55–7.26 (m, 8H),
5.29 (s, 1H), 5.28 (s, 1H), 4.62–4.46 (m, 2H), 4.39–4.35 (m, 1H),
4.24–4.19 (m, 1H), 2.73–2.65 (m, 1H), 2.60–2.52 (m, 1H), 2.37–
2.26 (m, 3H), 2.08–1.97 (m, 2H), 1.92–1.80 (m, 3H), 1.79–1.70 (m,
2H), 1.68–1.55 (m, 5H), 1.50–1.35 (m, 12 H, including singlets at
Cyclohexanol (18, 2.26 g, 22.57 mmol, 3.1 equiv) and THF
(40 mL) were added to a round bottom flask. Sodium hydride
(NaH, 542 mg, 22.57 mmol, 3.1 equiv) was added and the reaction
mixture was heated to 50 °C for 3 h. At that time, phosphorus
trichloride (PCl3, 0.635 mL, 7.28 mmol, 0.32 equiv) was added

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B. T. Mott et al. / Bioorg. Med. Chem. 21 (2013) 3702–3707
Table 1
In vitro results from anti-CMV and anti-leukemia studiesa
b
c
d
Key structural unit
Trioxane dimer
Anti-CMV EC50 (nM)
Anti-leukemia IC50 (nM)
PHA-simulated PBMC IC50 (nM)
Tle
X = O
X = S
838
855
39
78
3
4
100
1750 132
9
1477 639
>40,000g
14.8
>22.9
X
P
OPh
OPh
X = O
X = S
836
853
72
70
4
9
220 16
1933 278
2281 710
>40,000g
10.4
>20.7
X
P
O
O
R = 4-Cl
R = 4-iPr
R = 2,6-diMe
907
923
66
300
71
4
4
152
511 63
296 37
7
>40,000g
>40,000g
>263
>78.3
>135
O
P
2
895
851
762
>40,000g
O
R
2
2
O
P
44
5
287 10
2031 2192
>40,000h
7.1
O
O
P
OH
>300
7436 536
-5.4
OPh
Monomer
Monomer
Artemether
Sodium artesunate
Doxorubicn
Ganciclovir
Dimer alcohol 5
5300 2700f
18,500 5200f
—
>30,000
6887 770
9.6 0.6
—
>100,000g
27,431 20,993
271 163
—
—
—
4.0
28.2
—
2700 5200i
160 8i
—
—
a
b
c
d
e
f
Average of triplicate experiments.
Activity determined in HFF cells.
Activity determined in Jurkat T-ALL cells.
Average of 4 experiments, each in triplicate.
Therapeutic index, ratio of IC50 in PHA-stimulated PBMCs to the IC50 in JURKAT cells.
Data were determined in Ref. 9.
50% Growth inhibiton was not achieved only at the highest tested dose.
50% Growth inhibiton was achieved only at the highest tested dose.
Data were determined in Ref. 9.
g
h
i
1.40 and 1.36), 1.31–1.27 (m, 6H), 1.00–0.92 (m, 6H), 0.89–0.80 (m,
6H); 13C NMR (100 MHz, CDCl3) d 134.4, 129.9, 129.6, 129.5, 105.3,
102.8, 89.3, 88.5, 81.2, 81.1, 74.1, 72.0, 52.5, 52.2, 43.6, 43.4, 37.4,
37.3, 34.5, 31.0, 26.1, 26.0, 24.8, 20.6, 20.2, 20.1, 17.1, 13.5; ESI-
7.28 (d, J = 8.65 Hz, 4H), 7.15–7.21 (m, 4H), 5.28 (s, 1H), 5.25 (s,
1H), 4.45–4.54 (m, 2H), 4.41 (dd, J = 9.28, 6.76 Hz, 1H), 4.26 (dd,
J = 9.38, 6.54 Hz, 1H), 2.57–2.71 (m, 1H), 2.39–2.51 (m, 2H), 2.29
(t, J = 13.80 Hz, 4H), 1.96–2.04 (m, 2H), 1.82–1.94 (m, 2H), 1.70–
1.80 (m, 4H), 1.55–1.68 (m, 4H), 1.39–1.50 (m, 4H), 1.36 (d,
J = 10.74 Hz, 6H), 1.16–1.31 (m, 8H), 0.93 (dd, J = 9.54, 4.93 Hz,
6H), 0.80 (t, J = 8.27 Hz, 4H); 13C NMR (100 MHz, CDCl3) d 149.2,
149.1, 130.7, 129.8, 121.6, 121.5, 121.5, 103.1, 102.8, 89.7, 89.0,
81.2, 77.4, 77.1, 76.8, 73.5, 72.3, 72.2, 70.2, 52.3, 52.1, 44.4, 44.0,
37.5, 37.4, 36.7, 36.6, 35.4, 35.4, 34.5, 34.4, 30.6, 30.5, 30.3, 29.7,
26.1, 26.1, 24.9, 24.9, 24.8, 24.7, 20.2, 20.1, 13.0, 12.5; ESI-HRMS
for C46H61NaO12P (M+Na)+ calcd = 929.3170, found = 929.3186;
HRMS
for
C
46H61NaO12
ꢃ
P
(M+Na)+
calcd = 859.3793,
found = 859.3814; ½a D23
73 (c 0.42, CHCl3); FT-IR (cmꢀ1) 2930,
2927, 1582, 1489, 1376, 1175, 1130, 1009, 965, 941, 878, 852, 752.
5.7. Synthesis of dimer biphenylthiophosphate ester (853)
The title compound was prepared as described in the general
procedure, using biphenylthiophosphoryl chloride (16): yield
(5.5 mg, 0.006 mmol, 26%), 1H NMR (400 MHz, CDCl3) d 7.54–
7.31 (m, 8H), 5.30 (s, 1H), 5.28 (s, 1H), 4.57–4.54 (m, 2H), 4.41–
4.38 (m, 1H), 4.24–4.20 (m, 1H), 2.71–2.65 (m, 1H), 2.59–2.51
(m, 1H), 2.37–2.28 (m, 2H), 2.07–1.98 (m, 4H), 1.96–1.81 (m,
4H), 1.79–1.60 (m, 4H), 1.60–1.42 (m, 12H, including singlets at
1.40 and 1.39), 1.35–1.24 (m, 6H), 1.00–0.92 (m, 6H), 0.88–0.80
(m, 6H); 13C NMR (100 MHz, CDCl3) d 134.4, 131.6, 131.9, 129.6,
103.3, 102.8, 89.3, 81.2, 81.1, 73.1, 71.0, 55.5, 52.2, 44.6, 44.2,
38.4, 37.4, 34.5, 30.4, 26.1, 26.0, 24.8, 20.6, 20.2, 17.1; ESI-HRMS
½
a 2D5 +60.09 (c 0.27, CHCl3); FT-IR (cmꢀ1) 2923, 2918, 1580, 1486,
ꢃ
1375, 1301, 1197, 1092, 1009, 949, 878, 834, 754.
5.9. Synthesis of dimer bis-(4-isopropylphenyl)phosphate ester
923
The title compound was prepared as described in the general
procedure, using bis(4-isopropylphenyl)phosphoryl chloride (12):
yield (8.6 mg, 0.009 mmol, 56%), 1H NMR (400 MHz, CDCl3) d
7.16–7.09 (m, 8H), 5.33 (s, 1H), 5.28 (s, 1H), 4.56–4.99 (m, 1H),
4.84–4.37 (m, 2H), 4.27–4.20 (m, 1H), 2.91–2.83 (m, 2H), 2.71–
2.64 (m, 1H), 2.54–2.48 (m, 1H), 2.33–2.26 (m, 3H), 2.03–1.95
(m, 2H), 1.92–1.81 (m, 4H), 1.78–1. 70 (m, 4H), 1.68–1.55 (m,
4H), 1.54–1.43 (m, 4H), 1.42–1.35 (m, 9H), 1.29–1.20 (m, 15H),
0.99–0.89 (m, 6H), 0.85–0.78 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 149.2, 149.1, 130.7, 129.8, 121.6, 121.5, 121.5, 103.1, 102.8,
89.7, 89.0, 81.2, 77.4, 77.1, 76.8, 73.5, 72.3, 72.2, 70.2, 52.3, 52.1,
52.1, 44.4, 37.5, 37.4, 36.7, 36.6, 35.4, 34.5, 34.4, 30.9, 30.3, 29.7,
26.3, 26.1, 24.9, 24.9, 24.8, 24.7, 20.2, 20.1, 13.0, 12.5; ESI-HRMS
for C46H62O11PS (M+H)+ calcd = 853.3745, found = 853.3747; ½a 2D3
ꢃ
124 (c 0.40, CHCl3); FT-IR (cmꢀ1) 2930, 2924, 2915, 1582, 1481,
1376, 1279, 1189, 1130, 1009, 965, 941, 878, 852, 752.
5.8. Synthesis of dimer bis-(4-chlorophenyl)phosphate ester
(907)
The title compound was prepared as described in the general
procedure, using bis(4-chlorophenyl)phosphoryl chloride (13):
yield (21.3 mg, 0.023 mmol, 71%); 1H NMR (400 MHz, CDCl3) d

B. T. Mott et al. / Bioorg. Med. Chem. 21 (2013) 3702–3707
3707
for C52H76O12P (M +H)+ calcd = 923.5069, found = 923.5036; ½a 2D3
ꢃ
J = 13.93, 5.34 Hz, 6H), 0.88–0.75 (m, 6H); 13C NMR (100 MHz,
CDCl3) d 129.5, 124.7, 120.2, 103.5, 89.4, 88.8 81.1, 81.1, 74.7,
71.9, 69.7, 52.4, 52.2, 44.6, 44.2, 37.4, 37.1, 36.5, 34.4, 30.2, 29.5,
57 (c 0.14, CHCl3); FT-IR (cmꢀ1) 2923, 2918, 1580, 1486, 1375,
1301, 1197, 1092, 1009, 949, 878, 834, 754.
25.8, 24.8, 20.2, 20.1, 14.2, 13.3, 12.7; ESI-HRMS for C40H60O12
P
5.10. Synthesis of dimer bis-(2,6-dimethylphenyl)phosphate
ester 895
(M+H)+ calcd = 763.3840, found = 763.3835; ½a 2D5
ꢃ
+70.38 (c 1.315,
CHCl3); FT-IR (cmꢀ1) 2952, 2876, 1739, 1593, 1491, 1453, 1377,
1208, 1159, 1103, 1012, 938, 876, 824, 757, 690.
The title compound was prepared as described in the general
procedure, using bis(2,6-dimethylphenyl)phosphoryl chloride
(14): yield (18.0 mg, 0.02 mmol, 61%); 1H NMR (400 MHz, CDCl3)
d 7.07–6.91 (m, 6H), 5.29 (s, 1H), 5.25 (s, 1H), 4.53–4.44 (m, 1H),
4.42–4.30 (m, 2H), 4.19–4.11 (m, 1H), 2.72–2.62 (m, 1H), 2.56–
2.46 (m, 1H), 2.34 (s, 6H), 2.32 (s, 6H), 2.29–2.18 (m, 2H), 2.02–
1.93 (m, 2H), 1.92–1.79 (m, 5H), 1.72 (d, J = 15.54 Hz, 4H), 1.66–
1.49 (m, 6H), 1.36 (d, J = 8.46 Hz, 8H), 1.34–1.16 (m, 8H), 0.94 (d,
J = 6.00 Hz, 3H), 0.88 (d, J = 4.67 Hz, 3H), 0.79 (t, J = 6.41 Hz, 4H);
13C NMR (100 MHz, CDCl3) d 157.7, 148.5, 148.4, 130.4, 129.0,
125.1, 103.2, 102.8, 89.4, 88.6, 81.2, 81.1, 74.0, 72.1, 72.0, 70.9,
52.5, 52.2, 44.6, 44.2, 37.5, 37.3, 36.7, 36.6, 35.4, 35.4 34.6, 34.5,
30.5, 30.4, 30.2, 29.2, 26.1, 26.1, 24.9, 24.7, 20.2, 20.1, 17.2, 13.3,
Acknowledgments
Financial support is acknowledged from the following agencies:
NIH Grant R37 AI34885 (G.H.P.), NIH grant R01 AI093701 (R.A.B.),
National Foundation for Cancer Research (C.I.C.), NIH grant PO1
CA70970 (C.I.C.), the Maryland Stem Cell Research Foundation/
TEDCO 2010-MSCRFII-0065 (C.I.C.), the Curing Kids Cancer Founda-
tion (X.C.) and the William Lawrence & Blanche Hughes Founda-
tion (X.C.).
References and notes
12.7; ESI-HRMS for
C
50H71NaO12
P
(M+Na)+ calcd = 917.4575,
found = 917.4589; ½a D25
ꢃ
+60.33 (c 0.69, CHCl3); FT-IR (cmꢀ1) 2937,
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5.11. Synthesis of dimer dicyclohexylphosphate ester 851
The title compound was prepared as described in the general
procedure, using dicyclohexylphosphoryl chloride (20): yield
(7.1 mg, 0.008 mmol, 25%); 1H NMR (400 MHz, CDCl3) d 5.30 (s,
2H), 4.63–4.53 (m, 1H), 4.50–4.34 (m, 2H), 4.30 (d, J = 4.86 Hz,
2H), 4.24 (dd, J = 9.76, 6.16 Hz, 1H), 2.74–2.64 (m, 1H), 2.63–2.54
(m, 1H), 2.38–2.25 (m, 2H), 2.25–2.18 (m, 1H), 2.04–1.97 (m,
3H), 1.95–1.85 (m, 7H), 1.82–1.69 (m, 8H), 1.68–1.59 (m, 6H),
1.58–1.47 (m, 6H), 1.47–1.38 (m, 10H), 1.37–1.29 (m, 6H), 1.28–
1.19 (m, 6H), 0.95 (d, J = 6.06 Hz, 6H), 0.85 (t, J = 7.14 Hz, 4H); 13C
NMR (100 MHz, CDCl3) d 154.8, 103.2, 102.9, 89.37, 81.1, 81.1,
73.8, 71.4, 52.5, 52.2, 44.6, 44.2, 37.4, 36.6, 34.5, 34.4, 31.6, 30.6,
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HRMS
for
C
46H75NaO12
ꢃ
P
(M+Na)+
calcd = 873.4888,
found = 873.4890; ½a D23
49.95 (c 0.355, CHCl3); FT-IR (cmꢀ1) 2936,
2925, 1737, 1451, 1376, 1254, 1105, 1038, 1008, 963, 880.
5.12. Synthesis of monophenylphosphate ester 762
To a 1 dram vial were added 83812 (62.0 mg, 0.074 mmol) and
DMF (1 mL), followed by 1 N NaOH (89 lL, 0.089 mmol, 1.2 equiv).
The reaction mixture was heated to 50 °C for 3 h. The reaction was
deemed complete via analysis on LCMS, and the reaction mixture
was by direct injection onto reverse phase chromatography sys-
tem. The fractions containing the desired product were lyophilized
overnight, providing the title compound as a colorless, amorphous
solid: yield (31.5 mg, 0.041 mmol, 56%); 1H NMR (400 MHz, CDCl3)
d 11.22 (br s, 1H), 7.47–7.02 (m, 5H), 5.46–5.24 (m, 2H), 4.41–4.22
(m, 2H), 4.21–4.06 (m, 2H), 2.83–2.65 (m, 1H), 2.65–2.50 (m, 1H),
2.29 (t, J = 11.87 Hz, 2H), 2.21–2.09 (m, 1H), 2.08–1.95 (m, 2H),
1.93–1.81 (m, 2H), 1.75 (d, J = 12.13 Hz, 2H), 1.69–1.50 (m, 6H),
1.37 (d, J = 8.65 Hz, 12H), 1.25 (t, J = 7.07 Hz, 5H), 0.92 (dd,
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