A bifunctional dimethylsulfoxide substitute enhances the aqueous solubility of small organic molecules

Article abstract of DOI:10.1089/adt.2011.0421

An oxetane-substituted sulfoxide has demonstrated potential as a dimethylsulfoxide substitute for enhancing the dissolution of organic compounds with poor aqueous solubilities. This sulfoxide may find utility in applications of library storage and biological assays. For the model compounds studied, significant solubility enhancements were observed using the sulfoxide as a cosolvent in aqueous media. Brine shrimp, breast cancer (MDA-MB-231), and liver cell line (HepG2) toxicity data for the new additive are also presented, in addition to comparative IC₅₀ values for a series of PKD1 inhibitors.

Full text of DOI:10.1089/adt.2011.0421

A Bifunctional Dimethylsulfoxide Substitute
Enhances the Aqueous Solubility
of Small Organic Molecules
Melissa M. Sprachman and Peter Wipf
ration. Additionally, poor aqueous solubility causes precipitation
6
from aqueous media after dilution of DMSO stock solutions. When
Department of Chemistry, Center for Chemical Methodologies
and Library Development, University of Pittsburgh, Pittsburgh,
Pennsylvania.
compound concentrations in assay media fall below calculated
concentrations, flawed conclusions regarding toxicity, efficacy, or
5
,6
structure–activity relationships are drawn.
Aqueous dissolution of problematic compounds can be enhanced
7
8
by pH adjustment, salt formation, or chemical modification of the
8
ABSTRACT
substrate (formation of pro-drugs). If these methods are not appli-
An oxetane-substituted sulfoxide has demonstrated potential as a di-
methylsulfoxide substitute for enhancing the dissolution of organic
compounds with poor aqueous solubilities. This sulfoxide may find utility
in applications of library storage and biological assays. For the model
compounds studied, significant solubility enhancements were observed
using the sulfoxide as a cosolvent in aqueous media. Brine shrimp,
breast cancer (MDA-MB-231), and liver cell line (HepG2) toxicity data
for the new additive are also presented, in addition to comparative IC50
values for a series of PKD1 inhibitors.
cable, complexing agents or cosolvents can be added to aid in
9
10
dissolution. Some examples include cyclodextrins, dendrimers,
5
low-molecular-weight polyethylene glycols (PEGs, e.g., PEG 400),
2
11
and solvents such as glycerin and N-methyl pyrrolidone (NMP).
Other solubilizing agents have designs based on DMSO; one example
12
is a polymeric sulfoxide derived from poly-L-methionine 2 (Fig. 1).
In this study, we examined the use of sulfoxide 3 (Fig. 1) as a
solubilizer and general compound storage additive. We found that
addition of sulfoxide 3 increased the aqueous solubility of several
model problem compounds, including naproxen, quinine, curcumin,
carbendazim, and griseofulvin. The solubility enhancement sur-
passed that of DMSO at mass fractions > 10%. Herein, we describe our
findings and hypothesis of the mode of the cosolvent effect. A
comparative study of the toxicity and applicability of sulfoxide 3 to
cellular and in vivo bioassays is also discussed.
INTRODUCTION
odern drug development relies on high-throughput
screening assays. Often, these trials use compound
libraries stored in solution for periods of several months
1
M
to as long as 3 years. Dimethylsulfoxide (DMSO) 1 has
been used as the storage solvent of choice, but problems, including
compound degradation and precipitation, are frequently encountered.
MATERIALS AND METHODS
General
In one case study, qualitative compound precipitation was observed in
2
2
6% of test plates. Systematic studies of compound degradation in
Moisture-sensitive reactions were performed under an atmosphere
of nitrogen. 3-Tosyloxymethyl-3-methyl-oxetane was prepared ac-
13
DMSO have indicated that *50% of samples degraded over 12 months
3
,4
when stored in anhydrous DMSO at ambient temperature. Com-
pound storage problems are augmented by low hydrophilicity, since a
large portion of screening libraries is composed of compounds de-
signed for enhanced membrane permeability. The trend toward lipo-
philic, higher-molecular-weight compounds results in libraries of
cording to a literature protocol. Curcumin (Acros, 95%), naproxen
(Acros, 99%), quinine (Acros, 99%), DMSO (Aldrich, 99.9 + %), and
high-performance liquid chromatography (HPLC)-grade water
ꢀ
(Aldrich, Chromasolv ) were purchased from commercial suppliers
and used as received. Carbendazim (Aldrich, 97%) was recrystallized
from absolute ethanol (EtOH), and griseofulvin (Acros, 97%) was
5
materials with lower intrinsic aqueous solubilities. Current estimates
state that 30%–50% of compounds in screening libraries have aqueous
recrystallized from toluene. N-Methyl-2-pyrrolidone (Acros, 99%)
6
˚
solubilities of < 10mM. These lipophilic molecules are more likely to
was distilled from CaH
2
under vacuum and stored over 4 A MS. All
precipitate from DMSO stock solutions, leading to erroneously low
other reagents were used as received unless otherwise stated. Ana-
lytical thin-layer chromatography was performed on precoated silica
assay concentrations when using the DMSO stock for sample prepa-
ABBREVIATIONS: ATR, attenuated total reflection; DMSO, dimethylsulfoxide; ES, electrospray; EtOH, ethanol; GI50, 50% growth inhibition; HPLC, high-performance liquid
chromatography; HRMS, high resolution mass spectrometry; IR, infrared; MeOH, methanol; NBP, 4-(p-nitrobenzyl)pyridine; NMP, N-methylpyrrolidinone; NMR, nuclear
magnetic resonance; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PKD1, protein kinase D isoform 1; rt, room temperature; TsCl, p-toluenesulfonyl chloride; UV/VIS,
ultra-violet/visible spectra.
DOI: 10.1089/adt.2011.0421
ª MARY ANN LIEBERT, INC.
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SPRACHMAN AND WIPF
-
1 1
8
29 cm ; H NMR (300 MHz, CDCl ) d 4.47 (d, J = 5.7 Hz, 4 H), 4.38
3
13
(
d, J = 6.0 Hz, 4 H), 2.93 (s, 4 H), 1.38 (s, 4 H); C NMR (75 MHz, CDCl3)
d 81.9, 43.7, 40.3, 23.0; HRMS electrospray ionization (ES) m/z calc
for C10H O NaS (M + Na) 225.0925, found 225.0908.
18 2
0
3
,3 -Sulfinylbis(methylene)bis(3-methyloxetane) (3)
A 1-L round-bottom flask was charged with a solution of 6 (14.9 g,
3.6 mmol) in methanol (MeOH; 240 mL) and cooled to 0ꢁC. A so-
lution of NaIO (16.5 g, 77.3 mmol) in water (180 mL) was added via
7
4
Fig. 1. Structures of sulfoxides used for compound storage or
aqueous solubility enhancement.
addition funnel for *15 min. The ice bath was removed and the
slurry was warmed to rt. MeOH (2 · 50 mL, added 20 min apart) was
added, and the mixture was stirred for 12 h at rt. The mixture was
filtered through a fritted funnel, and the white precipitate was
washed with MeOH. The combined filtrate and washings were
concentrated in vacuo, and the concentrate was coevaporated with
toluene (200 mL). CH Cl (400 mL) was added to the residue, followed
gel 60 F-254 plates (particle size 0.040–0.050mm, 230–400 mesh) and
1
visualized by staining with KMnO
nuclear magnetic resonance (NMR) spectra (CDCl
spectra (CDCl ) were referenced to residual chloroform (7.27 ppm, H,
4
or p-anisaldehyde solutions. H
1
3
3
) and C NMR
1
3
13
2
2
7
7.00 ppm, C). Chemical shifts (d) are reported in ppm using the
4
by MgSO . The mixture was filtered, and the filtrate was concentrated
following convention: chemical shift, multiplicity (s= singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, b = broad), coupling
constants, and integration. Infrared (IR) spectra were collected as
attenuated-total-reflection infrared (ATR-IR) spectra. Mass spectra
were obtained on a Micromass Autospec double focusing instrument.
ultraviolet/visible (UV/VIS) spectra were recorded on a Perkin Elmer
Lambda EZ210 spectrophotometer. pH Determinations were made
in vacuo to afford crude 3 (15.82 g) as a yellow solid. To the flask
containing the crude solid was added toluene (200 mL), and the slurry
was heated to 60ꢁC to affect complete dissolution. Decolorizing
carbon was added, and the mixture was filtered by gravity into a 1-L
Erlenmeyer flask. To the colorless solution was slowly added distilled
hexanes (* 100 mL total) until cloudiness/precipitation occurred. The
mixture was allowed to stand at rt overnight. Upon filtration and
drying under high vacuum, 3 (10.49 g) was collected as a white solid.
Material recovered from the mother liquor was recrystallized to af-
ford an additional 2.68 g of 3 as white solid for a total yield of 82%.
TM
using a 3-mm Ross glass combination micro-pH electrode (model
8220BNWP) after calibration in standard buffer solutions (pH 4.0, 7.0,
and 10.0) at room temperature (rt).
Reported analytical data refer to that of the first crop: Mp 92.8–
Bis((3-methyloxetan-3-yl)methyl)sulfane (6)
4.1ꢁC; IR (ATR) 2,939, 2,863, 1,451, 1,381, 1,227, 1,026, 971 cm-
1
;
9
A three-necked 3-L round-bottom flask equipped with an over-
head stirrer, internal thermometer, and a third arm bearing an argon
balloon was charged with 3-tosyloxymethyl-3-methyl-oxetane 4
1
H NMR (400 MHz, CDCl
3
) d 4.80 (d, J = 6.0 Hz, 2 H), 4.61 (d,
J = 5.6 Hz, 2 H), 4.50 (d, J = 5.4 Hz, 2 H), 4.45 (d, J = 6.0 Hz, 2 H), 3.38
13
(
d, J = 12.9 Hz, 2 H), 2.75 (d, J = 12.9 Hz, 2 H), 1.61 (s, 6 H); C NMR
100 MHz, CDCl ) d 82.4, 82.0, 61.9, 38.4, 23.4; HRMS (ES) m/z calc
NaS (M + Na) 241.0874, found 241.0885.
(
2
45.4 g, 177 mmol) and backfilled with N (3 · ). To the flask was
(
3
added acetonitrile (900 mL) via cannula. The reaction apparatus was
placed in a large heating mantle. The argon balloon was replaced
with a 250-mL addition funnel containing a solution of Na S$9H O
18 3
for C10H O
To unambiguously characterize 3 as the sulfoxide, the corre-
2
2
sponding sulfone was synthesized from sulfide 6.
(
94.5 g 386 mmol) in degassed H O (100 mL). The solution was added
2
drop-wise over 25 min. Once the addition was complete, the reaction
0
3
,3 -Sulfonylbis(methylene)bis(3-methyloxetane)
mixture was heated to 70ꢁC over 45 min and maintained at 70ꢁC for
A suspension of oxone (650mg, 1.06 mmol) in water (2.0 mL) was
1
h. The mixture was cooled to 20ꢁC (internal temp), the resulting
cooled to 10ꢁC and treated (dropwise) with a solution of 6 (108 mg,
.533mmol) in MeOH (2.0mL). The solution was warmed to rt and
stirred for 1 h. MeOH was removed in vacuo, and the aqueous layer was
diluted with water (5 mL) and extracted with CH Cl (4 ·10 mL). The
combined organic layers were washed with brine (5mL), dried (MgSO4),
and concentrated in vacuo to afford the sulfone (120mg, 96%) as a
white solid: Mp 93.4–95.1ꢁC; IR (ATR) 2,949, 2,867, 1,456, 1,301, 1,277,
67 cm ; H NMR (400 MHz, CDCl
J =6.4 Hz, 4 H), 3.43 (s, 4 H), 1.69 (s, 6 H); C NMR (100 MHz, CDCl
2.2, 62.6, 37.9, 23.3; HRMS atmospheric pressure chemical ionization
APCI) m/z calc for C10 S (M+ H) 235.1004, found 235.1032.
white precipitate was filtered by gravity, and to the filtrate was added
EtOAc (1 L). The resulting precipitate was removed by aspirator fil-
tration, and the filtrate was divided into two 1-L batches. To each
batch was added water (500 mL), the layers were separated, and the
aqueous portion was extracted with EtOAc (2 · 200 mL). The com-
bined organic layers were washed with brine (100 mL), and the EtOAc
0
2
2
layers from the batches were combined and dried (Na
2
SO
4
) overnight,
-1 1
9
3
) d 4.68 (d, J =6.4 Hz, 4 H), 4.46 (d,
filtered, and concentrated. Kugelrohr distillation was performed on
the concentrate. One fraction (T <100ꢁC, 15 Torr) was discarded, and
subsequent product collection (140ꢁC < T <160ꢁC) yielded a yellow
distillate. The distillate was taken up in EtOAc (200 mL), washed with
water (100 mL) and brine (100 mL), dried (Na SO ), and concentrated.
13
3
) d
8
(
19 4
H O
Determination of LogP Value of 3
2
4
Kugelrohr distillation (140ꢁC, 15 Torr) afforded 6 (14.2 g, 79%) as a
yellow-green oil: IR (ATR) 2956, 2924, 2861, 1450, 1236, 973,
The logP (octanol-water partition coefficient) was determined
using the shake-flask method. Three determinations were made. A
2
70 ASSAY and Drug Development Technologies JUNE 2012

A BIFUNCTIONAL DMSO SUBSTITUTE
representative procedure is as follows: a 250-mL separatory funnel
was charged with a solution of 3 (50.0 mg) in water (50.0 mL) and
n-octanol (50.0 mL). The funnel was capped and inverted 100 times.
The funnel and contents were left to stand at rt (23.5ꢁC) for 40 h.
Aliquots of both phases were analyzed by UV/VIS (214 nm for the
aqueous layer and 218 nm for the octanol layer), and the concen-
tration in each layer was determined using previously generated
calibration curves. In the case of the aqueous layer, a 10-fold dilution
was necessary before measurement. All measurements were made in
triplicate. The logP was determined as log ([3]octanol/[3]aqueous), and
the average logP value from the three trials was - 0.87.
with an appropriate volume of either absolute EtOH (quinine, na-
proxen, griseofulvin) or MeOH (carbendazim). Concentrations were
calculated using previously generated calibration curves. Appro-
priate blanks were prepared by diluting aliquots (0.800 mL) of the
DMSO/water solutions (or HPLC-grade water in the case of the
control) in analogous fashion to the sample being measured.
General Procedure for Solubility Determination
in Mixtures of Sulfoxide 3 in pH 7.0 Buffer
Preparation of sulfoxide/buffer solutions. In the case of a 10% w/w
solution of sulfoxide 3 in 0.01 M pH 7.0 phosphate buffer, 3 (700 mg)
2 4 2 4
was dissolved in 0.01 M Na HPO /NaH PO buffer (6.30 mL) and
General Procedure for Determination of Solubility
in Solutions of 3 and HPLC-Grade Water
equilibrated on a platform shaker at 200 rpm for 30 min at rt. In the case
of a 25% w/w solution of sulfoxide 3 in 0.01 M pH 7.0 phosphate buffer,
Preparation of sulfoxide/water solutions. Solutions of 3 and HPLC-
grade water were prepared in 1-dram vials by dissolving the appro-
priate amount of sulfoxide 3 in HPLC-grade water (2.00 mL). In the
2 4 2 4
3 (1.50 g) was dissolved in 0.01 M Na HPO /NaH PO buffer (4.50 mL)
and equilibrated on a platform shaker at 200 rpm for 30 min at rt.
case of the 25% solution of 3 in H O (w/w), 3 (500 mg) was dissolved
Solubility measurements. Eppendorf vials (1.5 mL size) were charged
with model compounds in excess. Vials were charged with either pH
7.0 phosphate buffer (1.00 mL) or the appropriate 3/buffer solution.
The vials were briefly vortexed and equilibrated in an end-over-end
rotator at 30.0ꢁC for 20 h. The vials were centrifuged directly after
removal from the rotator (4,000 rpm, 1,300 g, 15 min, rt), and aliquots
of the supernatant (0.800 mL) were filtered through 0.45-mm syringe
filters. The pH of each solution was measured using a Thermo-
Scientific electrode (3 mm tip) after calibration. Each solution was
diluted with an appropriate volume of either absolute EtOH (quinine,
naproxen) or MeOH (carbendazim). Concentrations were determined
using standard additions of a stock solution of the compound in
either absolute EtOH (quinine, naproxen) or MeOH (carbendazim) to
aliquots of the diluted filtrates.
2
in water (1.50 mL). Each vial was placed on a platform shaker and
shaken at 200 rpm for 30 min.
Solubility measurements. Eppendorf vials (1.5 mL size) were charged
with model compounds in excess. Vials were charged with HPLC-
grade water (0.500 mL) or the appropriate 3/water solution
(
0.500 mL). The vials were briefly vortexed and equilibrated in an
end-over-end rotator at 30.0ꢁC for 20 h. The vials were centrifuged
4,000 rpm, 1,300 g, 15 min, rt) directly after removal from the rotator,
and aliquots of the supernatant (0.400 mL) were filtered through
.45-mm syringe filters. The pH of each solution was measured using a
(
0
ThermoScientific electrode (3mm tip). Each solution was diluted with
an appropriate volume of either absolute EtOH (quinine, naproxen,
griseofulvin) or MeOH (carbendazim). Concentrations were calculated
by using previously generated calibration curves. Appropriate blanks
were prepared by diluting aliquots (0.400mL) of the 3/water solutions
General Procedure for Kinetic Solubility Measurements
A polypropylene tube was charged with phosphate-buffered saline
(PBS; 490 mL). To the buffer was added a 10-mM stock solution of
compound (10 mL). The tube was vortexed and equilibrated on an
end-over-end rotator for 15 min at rt. Aliquots (400 mL) were filtered
through 0.45-mm syringe filters and diluted to 5.0 mL with absolute
MeOH. Concentrations were determined by UV/VIS analysis.
(
or HPLC-grade water in the case of the control) in an analogous
fashion to the sample being measured.
General Procedure for Determination of Solubility
in Solutions of DMSO and HPLC-Grade Water
Preparation of DMSO/water solutions. Solutions of DMSO and
HPLC-grade water were prepared in 1-dram vials by dissolving the
appropriate amount of DMSO in HPLC-grade water (2.00 mL). Each
vial was placed on a platform shaker and shaken at 200rpm for 30min.
1
4
General Procedure for Brine Shrimp Toxicity Assays
Sample preparation. Stock solutions of 3 were prepared by dissol-
ving 3 (50.0 mg) in HPLC-grade water (5.0 mL) (solution A) and 3
(
2.50 g) in HPLC-grade water (10.0 mL) (solution B). Stock solutions
Solubility measurements. Eppendorf vials (1.5 mL size) were charged
with model compounds in excess. Vials were charged with either
HPLC-grade water (1.00 mL) or the appropriate DMSO/water solution
of DMSO were prepared by diluting DMSO (45 mL) with HPLC-grade
water (5.0 mL) (solution C) and DMSO (2.27 mL) with HPLC-grade
water (10.0 mL) (solution D).
(
1.00 mL). The vials were briefly vortexed and equilibrated in an end-
In each case, five replicates were performed. Each replicate was
performed in a 2-dram vial marked at the 4 and 5 mL volume points.
To each vial was added artificial seawater (3 mL) followed by the
appropriate volume of stock solution. For set 1 (1.0 mg/mL), solution
A (0.500 mL) or solution C (0.500 mL) was added. For set 2 (5.0 mg/
mL), solution B (0.100 mL) or solution D (0.100 mL) was added to each
over-end rotator at 30.0ꢁC for 20 h. The vials were centrifuged di-
rectly after removal from the rotator (4,000 rpm, 1,300 g, 15 min, rt),
and aliquots of the supernatant (0.800 mL) were filtered through
0
.45-mm syringe filters. The pH of each solution was measured using
a ThermoScientific electrode (3 mm tip). Each solution was diluted
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SPRACHMAN AND WIPF
vial. For set 3 (20.0 mg/mL), solution B (0.400 mL) or solution D
(
(
0.400 mL) was added to each vial. For set 4 (50.0 mg/mL), solution B
1.00 mL) or solution D (1.00 mL) was added to each vial. Controls
containing HPLC-grade water (0.100 mL, 0.400 mL, and 1.00 mL)
were prepared in the same manner, and five replicates of each control
were prepared.
Brine shrimp hatching. Brine shrimp eggs (San Francisco Bay Brand)
were hatched in a commercial salt mixture (Instant Ocean). Constant
aeration was provided using a pump and airstone, and illumination
was maintained using a desk lamp. The shrimp were collected in a
separate tank after 48 h and used within 3–4 h of collection.
Fig 2. Synthesis of water-soluble sulfoxide 3.
Assay. Brine shrimp (10) were added to each vial using a plastic
transfer pipet. After the shrimp were transferred, artificial seawater
was added until the volume reached the 5-mL mark. One drop of a
yeast suspension prepared by suspending 11 mg yeast in seawater
We took advantage of the stability and hydrophilicity of 3 by
exploring its use for the solubility enhancement of poorly aqueous-
soluble compounds. Oxetanes are attractive functional groups for
increasing aqueous solubility due to their rigid geometries and ex-
posed polar surface area at the ring ether. Among unsubstituted
cyclic ethers, oxetane has the greatest basicity, which may be at-
tributed to a smaller carbon-to-oxygen ratio and a larger dipole
moment than its larger counterparts (e.g., tetrahydrofuran and
(
20 mL) was added to each vial. The shrimp were counted at t = 24 h.
Another drop of freshly prepared yeast solution (6 mg in 10 mL
seawater) was added, the vials were maintained under illumination,
and shrimp were counted at t = 48 h.
1
6
tetrahydropyran). Recently, M u¨ ller, Carreira, and coworkers dem-
onstrated that replacing a gem-dimethyl group with an oxetane
moiety can increase a scaffold’s aqueous solubility by up to three
General Procedure for Calculation of Water Absorption
Oven-dried flasks capped with septa were cooled under an N
mosphere and charged with a volume of the appropriate solutions
DMSO, 3.0 mL; NMP, 600 mL; 25% 3/NMP, 600 mL). The water con-
tent was determined by Karl Fischer titration using *100-mL aliquots
t = 0 measurement). Each septum was pierced with a 1.5-inch 18-
2
at-
17
orders of magnitude while also enhancing metabolic stability. We
reasoned that sulfoxide 3 may have sufficient aqueous solubility to
be completely miscible with water at useful cosolvent concentrations
while also disrupting the hydrogen-bonding network of water, thus
aiding in solubilization of lipophilic drug candidate compounds.
To evaluate the utility of sulfoxide 3 as a solubilizing agent, we
chose a test set of poorly aqueous-soluble compounds, beginning
with curcumin 7 (Fig. 3). Curcumin has shown promise in treating
(
(
gauge needle and left to stand at rt for 7 days. The water content was
measured at the end of this period (t = 7 days measurement) by Karl
Fischer titration. All measurements were made in duplicate.
RESULTS AND DISCUSSION
Water-soluble sulfoxide 3 was prepared in three steps from alco-
hol 4, which is commercially available or readily prepared from
1
5
inexpensive 2-(hydroxymethyl)-2-methylpropane-1,3-diol. Briefly,
treatment of 4 with p-toluenesulfonyl chloride in pyridine afforded
13
tosylate 5. Dimeric sulfide formation using Na
2
S followed by oxi-
dation with NaIO provided sulfoxide 3 (Fig. 2). The synthesis is
4
amenable to large-scale preparation and requires no chromatography.
Initially, we aimed to use sulfoxide 3 as a substitute for DMSO in
chemical transformations such as modified Swern and Kornblum
oxidations. We reasoned the resulting sulfide 6, being less volatile
and odorous than dimethyl sulfide, would be a more tractable by-
product on industrial scale. Additionally, bisoxetanyl sulfoxide 3 is
water soluble, and we found that the sulfide byproduct could be
removed from reaction mixtures by an oxidative work-up and
aqueous washing procedure. Surprisingly, however, sulfoxide 3 was
not a viable oxidizing agent in Kornblum oxidations. For example, a-
bromoacetophenone was stable to an excess of 3 when monitored in
CD CN over the course of several weeks; under the same conditions,
3
a-bromoacetophenone was reactive with DMSO within 2 days of
treatment.
Fig. 3. Compounds screened in the aqueous solubility study.
2
72 ASSAY and Drug Development Technologies JUNE 2012

A BIFUNCTIONAL DMSO SUBSTITUTE
Fig. 4. Solubility of quinine 8 in aqueous solutions with sulfoxide 3
( ) and DMSO ( + ) as cosolvents. Each trial was run in duplicate
and each point represents the average of the duplicate trials. In the
Fig. 6. Solubility of carbendazim 10 in aqueous solutions with
sulfoxide 3 ( ) and DMSO ( + ) as cosolvents. Each trial was run in
duplicate and each point represents the average of the duplicate
trials. In the case of sulfoxide 3, the pH ranged from 6.7 (with no
additive) to 7.0 (with a 0.15 weight fraction additive). In the case of
DMSO, the pH ranged from 6.8 (with no additive) to 7.4 (with a
0.25 weight fraction additive).
case of sulfoxide 3, the pH ranged from 8.7 (with no additive) to
.4 (with a 0.25 weight fraction additive). In the case of DMSO, the
9
pH ranged from 8.9 (with no additive) to 9.5 (with a 0.25 weight
fraction additive). DMSO, dimethylsulfoxide.
colon cancer and various other disorders, but its use is limited in part
by its low aqueous solubility (0.6 mg/mL at ambient temperature, as
high as 7.4 mg/mL upon heating) and consequently limited bio-
availability. We measured the solubility of curcumin by UV/VIS
spectroscopy after equilibration for 20 h at ambient temperature in an
end-over-end rotator. We were pleased to see an increase in aqueous
solubility at ambient temperature to 60 – 20 mg/mL using a 25 wt%
solution of 3 in water. Although the aqueous solubility remained low,
the *100-fold enhancement factor was encouraging.
sulfoxide 3 in water. Gratifyingly, up to 10-fold increases in aqueous
solubility were observed for 8–11, and almost twofold improvements
in aqueous solubility were observed in comparison to equivalent
DMSO/water solutions (Figs. 4–7).
18
Based on the solubility curves (Figs. 4–7), the sulfoxide 3 and
DMSO function as cosolvents rather than complexing agents. Ac-
7
cording to the model derived by Yalkowsky, an exponential increase
in observed solubilities occurs with increasing the volume fraction
w
solvent according to equation 1, where Smix and S are the solubility
We further examined the solubility enhancement of 8–12 by
equilibrating the compounds in solutions of increasing amounts of
Fig. 7. Solubility of griseofulvin 11 in aqueous solutions with sulf-
oxide 3 ( ) and DMSO ( + ) as cosolvents. Each trial was run in
duplicate and each point represents the average of the duplicate
trials. In the case sulfoxide 3, the pH ranged from 6.3 (with no
additive) to 6.8 (with a 0.20 weight fraction additive). In the case of
DMSO, the pH ranged from 6.3 (with no additive) to 6.8 (with a
0.20 weight fraction additive).
Fig. 5. Solubility of naproxen 9 in aqueous solutions with sulfoxide
3 ( ) and DMSO ( + ) as cosolvents. Each trial was run in duplicate
and each point represents the average of the duplicate trials. In the
case of sulfoxide 3, the pH ranged from 4.6 (with no additive) to
4.2 (with a 0.25 weight fraction additive). In the case of DMSO, the
pH ranged from 4.8 to 4.4.
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Table 1. Solubility of Estrone 12 in Media Buffered at pH 9.0
Table 3. Kinetic Solubility Measurements
in Phosphate-Buffered Saline
a
Entry
Medium
Solubility (lg/mL)
30 – 10
Medium of
Kinetic
1
3:1 pH 9.0 buffer/3
a
Entry
Compound
stock solution solubility (lM)
2
3
3:1 pH 9.0 buffer/NMP
63 – 5
b
1
10
DMSO
140 – 13
160 – 5
150 – 9
170 – 16
180 – 13
190 – 7
3:1:1 pH 9.0 buffer/NMP/3
3:1:1 pH 9.0 buffer/NMP/DMSO
160 – 20
230 – 30
2
3
4
5
10
10
11
11
11
NMP
4
25% 3/NMP
DMSO
a
Solubility was determined after equilibration for 20 h at 30ꢁC. Data were
obtained by UV/VIS analysis of the saturated solutions and were confirmed by
analysis of independently prepared estrone standards. Each entry represents a
mean solubility – standard deviation (n = 3).
NMP
DMSO, dimethylsulfoxide; NMP, N-methylpyrrolidinone.
6
25% 3/NMP
a
Data are reported as mean – standard deviation (n = 3).
Data are reported as mean – standard deviation (n = 5).
b
of the solute in the cosolvent mixture and water, respectively, r is the
solubilizing power of the cosolvent, and f is the volume fraction of
c
the cosolvent. The slope of a semi-log plot (r) is related to the co-
solvent’s ability to disrupt the intermolecular hydrogen bond net-
work of water and form a less polar solvent mixture. Thus, the
solubilizing power of sulfoxide 3 and DMSO can be accounted for
when comparing the difference in the experimental log P of sulfoxide
We can tentatively rule out complexation as a solubilization
mechanism assuming that additive–solute complexes would form in
a 1:1 ratio. If that were the case, a linear correlation between additive
fraction and solubility would be observed.
1
9
3
( - 0.87) and the reported log P of DMSO ( - 1.3).
20,21
The aqueous solubility of estrone 12 is low (0.8 mg/mL),
and
Log Smix = log Sw + rfc
(1)
we were unable to quantify the aqueous solubility or observe an
increase in solubility in the case of a 25% (w/w) mixture of 3 and
water; however, increasing the pH of the media using a pH 9.0 buffer
(
Borax) as well as addition of NMP to generate a ternary mixture was
Table 2. Solubility of Selected Model Substrates in Solutions
of Sulfoxide 3 in 0.01 M pH 7.0 Phosphate Buffer
useful. In this case, the ternary mixture of 3:1:1 pH 9.0 buff-
er:NMP:DMSO was more effective at solubilization than the 3:1:1 pH
9.0 buffer:NMP:3 mixture (Table 1, entries 3 and 4).
Weight
Measured
solubility
percent 3 in
pH 7.0 buffer
Solution
We also explored the solubility of test compounds with ionizable
functionalities in 0.01 M pH 7.0 phosphate buffer (Table 2). In the
case of naproxen, the sulfoxide had little effect on the ionized sub-
strate even at 25% w/w concentrations. The effect on the solubility of
quinine was diminished at 10% w/w, but an advantage in using 3 was
observed in 25% w/w solutions. The solubility of carbendazim in the
buffered medium was the same as in solutions made from unbuffered
HPLC-grade water.
a
b
Entry Compound
pH
(lg/mL)
c
1
2
3
4
5
6
7
8
9
8
8
0
10
25
0
6.0
5.8
5.2
7.6
8.0
8.2
6.8
6.9
7.1
1,100 (780)
c
c
1,900 (1,300)
8
6,200 (3,900)
d
9
950
We also determined the kinetic solubility of two test compounds,
carbendazim 10 and griseofulvin 11, in PBS solution after adding
stock solutions prepared in three media: DMSO, NMP, and 25% 3/
NMP (Table 3). The test compounds were prepared at concentrations
of 10 mM and added to the buffer at room temperature such that
cosolvent concentration was fixed at 2%. The measured solubility
was consistent for both test compounds across the three stock solu-
tions. While the kinetic solubility of griseofulvin was found to be
9
10
25
0
1,100
1,400
10
9
10
10
10
10
25
34
77
a
The pH was measured electrochemically after excess compound had been
filtered from the solution.
22
higher than reported (and nearing the threshold solubility of
b
200 mM), there was no statistical difference in the kinetic solubilities
of the test compounds among the three different media.
Measurements were performed in duplicate unless otherwise noted.
The two numbers represent data from separate trials where entries 1–3 were
c
run in parallel. There was some variability in the results from the two trials.
d
Average of five trials.
Recently, we disclosed a series of compounds with nanomolar to
micromolar inhibitory activity against the serine/threonine protein
2
3–25
kinase D isoform 1 (PKD1) (Fig. 8).
Several lead structures,
2
74 ASSAY and Drug Development Technologies JUNE 2012

A BIFUNCTIONAL DMSO SUBSTITUTE
models have indicated that despite having comparable
26
6
strain energy to oxirane (26.8kcal/mol), oxetane is 10
times less susceptible to nucleophilic addition than ox-
2
7
etane. Animal studies have implicated the potential
carcinogenicity of oxetane and 3,3-dimethyloxetane; it
was shown that both compounds induce tumor forma-
2
8,29
tion at the site of injection in rats.
However, a recent
report studying the alkylating ability of oxetane, 3,3-
dimethyloxetane, and 3-methyl-3-oxetanemethanol (1)
demonstrated that these oxetanes are neither mutagenic
3
0
nor genotoxic.
Furthermore, alkylation of 4-(p-
nitrobenzyl)pyridine was only observed at acidic pH,
implicating that oxetanes do not act as alkylating agents
3
0
at physiological pH.
Fig. 8. Compounds assayed for PKD1 inhibitory activity. The structures have been
reported previously.
To assess the systemic toxicity of oxetane-substituted
23–25
PKD1, protein kinase D isoform 1.
sulfoxide 3, we performed a brine shrimp assay (Table
1
4
4
). Brine shrimp floating in water containing con-
especially those containing a pyrimidine moiety, suffered from poor
solubility not only in aqueous media but also in DMSO. To test the
feasibility of using sulfoxide 3 as a cosolvent for in vitro assays, we
centrations of 3 up to 20 mg/mL showed < 10% mortality after 48 h.
Shrimp incubated in water containing 50 mg/mL of 3 had 85%
mortality after 24 h and 100% mortality within 48 h. These data in-
dicate an LC50 of *32 mg/mL (i.e., at 147 mM). In comparison, brine
shrimp treated with DMSO at the same concentrations showed no
mortality after 24 h and only 15% mortality after 48 h at 50 mg/mL of
DMSO. These results indicate that sulfoxide 3 may be tolerated in
biological assays in concentrations by mass of up to 2%.
2
3–25
examined its use in the radiometric PKD1 inhibition
assay.
Inhibitory activity at two concentrations (1 mM and 10 mM) was
measured using compound stock solutions in three formulations:
DMSO, NMP, and 25% 3/NMP (Figs. 9 and 10). Comparable bio-
logical efficacy was observed for stock solutions in 3/NMP versus
DMSO. Most significantly, sulfoxide 3 did not interfere with the
standard PKD1 inhibition assay.
The cellular toxicity of sulfoxide 3 was further determined for a
breast cancer cell line (MDA-MB-231) and a liver cell line (HepG2). In
both cases, the 50% growth inhibition (GI50) for 3 was *200 mM,
whereas the GI50 for DMSO was determined as *800 mM. Interest-
ingly, both cell lines exhibited the same threshold effect as observed
in the case of the brine shrimp, showing only very limited toxicity at
concentrations up to *100 mM.
Toxicity
2
6
The strain energy of the oxetane ring (25.2 kcal/mol) raises
concerns about the electrophilicity and related toxicity and muta-
genicity of molecules containing an oxetane moiety. Computational
Fig. 9. Plot of PKD1 activity with compound concentrations of 1 mM.
Stock solutions were prepared in three different media [DMSO ( ),
NMP (,), and 25% 3/NMP (-)] at concentrations of 10 mM, and
dilutions were performed using the same media. The % PKD1 ac-
tivity is reported as the mean, and error bars represent SEM (n = 3).
Fig. 10. Plot of PKD1 activity with compound concentrations of
10 mM. Stock solutions were prepared in three different media
[DMSO ( ), NMP (,), and 25% 3/NMP (-)] at concentrations of
10 mM, and dilutions were performed using the same media. The %
PKD1 activity is reported as the mean, and error bars represent
SEM (n = 3). The % PKD1 activity was determined as previously
24
The % PKD1 activity was determined as previously described.
24
NMP, N-methylpyrrolidinone; SEM, standard error of the mean.
described.
ª MARY ANN LIEBERT, INC.
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JUNE 2012 ASSAY and Drug Development Technologies 275

SPRACHMAN AND WIPF
time, we noted that 3 partly precipitated from the solution at this
temperature, but no change in model compound concentration was
observed after thawing of the storage vessels.
Table 4. Results from a Brine Shrimp Assay to Assess
the Toxicity of Sulfoxide 3
a
a
Concentration % Mortality % Mortality
A study performed at Abbott indicated that water absorption
might induce more significant compound degradation than oxygen
Entry Compound
(mg/mL)
after 24 h
after 48 h
3
2
1
2
3
4
5
6
7
8
9
0
3
0
<10
10
exposure. To ascertain the degree of water absorption, we moni-
tored a 25% w/w 3/NMP solution for 1 week at ambient temperature
3
1
5
0
0
0
0
(
Table 5). Although significant water absorption was observed
3
(*7,000 ppm over the course of 7 days), the 3/NMP solution ab-
sorbed less water than NMP alone. This result indicates that the hy-
groscopicity of sulfoxide 3 is low relative to NMP.
3
20
50
0
0–10
85
<10
0
0–10
100
10
3
CONCLUSION
DMSO
DMSO
DMSO
DMSO
DMSO
We have demonstrated the utility of oxetane-substituted sulfoxide
3 as a cosolvent for enhancing the aqueous solubility of model drug
compounds. Although the relative acute toxicity of 3 was higher than
that of DMSO in brine shrimp and cell-based assays, it was suffi-
ciently low to permit its use in cellular and in vivo assay development
in up to 2% final concentrations. Furthermore, sulfoxide 3 proved
experimentally to be far less oxidizing than DMSO, and this property
could provide greater stability to long-term compound storage so-
lutions. The amount of water absorption will likely depend on the
choice of cosolvent, but the nature of 3 (being a solid) may allow the
assay developer to choose cosolvents that either do not absorb as
much water as DMSO (or NMP) or do not undergo the dramatic
changes in physical properties observed in the case of wet DMSO
solutions. As shown in our PKD1 assays, 3 does not alter the bio-
chemical readout in standard in vitro assays.
1
0–10
0–6
0
5
0
20
50
0
1
0
15
a
Percent mortality was determined for an average of five trials. Percent
mortality was determined by estimating the number of shrimp showing no
motility after several minutes of observation.
Properties of 3/NMP Solutions
Because sulfoxide 3 is a solid, it would have to be mixed with an
appropriate water-soluble cosolvent to act as a compound storage
additive. We chose NMP for exploring this potential application due to
its thermal stability and low toxicity. Furthermore, it had been dem-
onstrated that NMP had a greater solubilizing power than ethanol and
To the best of our knowledge, this is the first report of the incor-
poration of an oxetane moiety into a cosolvent structure for solu-
bility enhancement. The oxetane motif allows for the design of more
lipophilic cosolvents that still maintain good aqueous miscibility due
to the dipole moment at the oxetane oxygen. This study on the utility
of sulfoxide 3 as an aqueous-soluble cosolvent prompts further de-
velopment of additives bearing oxetanes or related heterocycles.
11
propylene glycol, and NMP was previously used for solubility en-
3
1
hancements both in bioassays and commercial pharmaceutical ap-
ꢀ
plications such as the Eligard formulation for delivery of leuprolide
to prostate cancer patients. We stored the compound test set (Fig. 2) in
25% w/w solutions of 3 and NMP for 6 weeks at - 20ꢁC. During this
ACKNOWLEDGMENTS
This work was supported by a grant from the National Institutes of
Health: NIGMS Centers for Chemical Methodologies and Library
Development (P50GM067082). The authors thank Profs. Andreas
Vogt and Q. Jane Wang and Dr. Manuj Tandon (Department of
Pharmacology and Chemical Biology, University of Pittsburgh) for
PKD1, MDA-MB-231, and HepG2 assays.
Table 5. Comparative Water Absorption Measurements
of Possible Compound Storage Media
Water
Water
content at
content at
a
a
Entry
Medium
t = 0 (ppm)
t = 7 d (ppm)
b
b
1
2
3
DMSO
160, 170
1,080, 1,130
DISCLOSURE STATEMENT
No competing financial interests exist.
c
c
NMP
150, 130
12,090, 17,000
c
c
25% 3/NMP (w/w)
160, 200
6,500, 8,600
REFERENCES
a
Water content was analyzed by Karl Fischer titration. The two values listed
represent individual vessels, the first value in each column corresponding to the
same vessel at each time-point.
1
2
3
.
.
.
Janzen WP, Popa-Burke IG: Advances in improving the quality and flexibility of
compound management. J Biomol Screen 2009;14:444–451.
Waybright TJ, Britt JR, McCloud TG: Overcoming problems of compound storage
in DMSO: solvent and process alternatives. J Biomol Screen 2009;14:708–715.
Talaga P: Compound decomposition: a new drug discovery tool? Drug Discov
Today 2004;9:51–53.
b
Average of three measurements.
Average of two measurements.
c
2
76 ASSAY and Drug Development Technologies JUNE 2012

A BIFUNCTIONAL DMSO SUBSTITUTE
4
.
.
Kozikowski BA, Burt TM, Tirey DA, et al.: The effect of room-temperature storage
on the stability of compounds in DMSO. J Biomol Screen 2003;8:205–209.
Stegemann S, Leveiller F, Franchi D, de Jong H, Lind e´ n H: When poor solubility
becomes an issue: from early stage to proof of concept. Eur J Pharm Sci
21. Shareef A, Angove MJ, Wells JD, Johnson BB: Aqueous solubilities of estrone,
17b-estradiol, 17a-ethynylestradiol, and bisphenol A. J Chem Eng Data
2006;51:879–881.
5
22. BioFocus. Available at: http://biofocus.com/offerings/adme-pk-laboratory/
solubility (last accessed: September 21, 2011).
2
007;31:249–261.
6
7
8
9
.
.
.
.
Di L, Kerns EH: Application of pharmaceutical profiling assays for optimization
of drug-like properties. Curr Opin Drug Discov Devel 2005;8:495–504.
Yalkowsky SH: Solubility and Solubilization in Aqueous Media. Oxford
University Press, New York, 1999.
23. Bravo-Altamirano K, George KM, Frantz MC, et al.: Synthesis and structure-
activity relationships of benzothienothiazepinone inhibitors of protein kinase D.
ACS Med Chem Lett 2011;2:154–159.
24. George KM, Frantz MC, Bravo-Altamirano K, et al.: Design, synthesis, and
biological evaluation of PKD inhibitors. Pharmaceutics 2011;3:186–228.
25. Sharlow ER, Giridhar KV, LaValle CR, et al.: Potent and selective disruption of
protein kinase D functionality by a benzoxoloazepinolone. J Biol Chem
2008;283:33516–33526.
Strickley RG: Solubilizing excipients in oral and injectable formulations. Pharm
Res 2004;21:201–230.
Oda M, Saitoh H, Kobayashi M, Aungst BJ: b-Cyclodextrin as a suitable
solubilizing agent for in situ absorption study of poorly water-soluble drugs. Int
J Pharm 2004;280:95–102.
26. Eigenmann HK, Golden DM, Benson SW: Revised group additivity parameters
for the enthalpies of formation of oxygen-containing organic compounds.
J Phys Chem 1973;77:1687–1691.
27. Banks HD: A computational study to elucidate the extraordinary reactivity of
three-membered heterocycles in nucleophilic substitution reactions. J Org
Chem 2003;68:2639–2644.
1
0. Ouyang L, Ma L, Jiang B, Li Y, He D, Guo L: Synthesis of novel dendrimers having
aspartate grafts and their ability to enhance the aqueous solubility of model
drugs. Eur J Med Chem 2010;45:2705–2711.
1
1. Sanghvi R, Narazaki R, Machatha SG, Yalkowsky SH: Solubility improvement of
drugs using N-methyl pyrrolidone. AAPS PharmSciTech 2008;9:366–376.
2. Pitha J, Szente A, Greenberg J: Poly-L-methionine sulfoxide: a biologically inert
analogue of dimethyl sulfoxide with solubilizing potency. J Pharm Sci
1
28. Druckrey H, Kruse H, Preussmann R, Ivankovic S: Cancerogene Alkylierende
Substanzen. Z Krebsforsch 1970;74:241–273.
1
983;72:665–668.
3. Rose NG, Blaskovich MA, Evindar G, et al.: Preparation of 1-[N-benzyloxycarbonyl-
1S)-1-amino-2-oxoethyl]-4-methyl-2,6,7-trioxabicyclo[2.2.2]octane. Org Synth
002;79:216–221.
29. Dickens F, Jones HE: Further studies on the carcinogenic and growth-
inhibitory activity of lactones and related substances. Br J Cancer 1963;17:
100–108.
30. G o´ mez-Bombarelli R, Palma BB, Martins C, et al.: Alkylating potential of
oxetanes. Chem Res Toxicol 2010;23:1275–1281.
31. Uch AS, Hesse U, Dressman JB: Use of 1-methyl-pyrrolidone as a solubilizing
agent for determining the uptake of poorly soluble drugs. Pharm Res
1999;16:968–971.
1
(
2
1
4. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL:
Brine shrimp: a convenient general bioassay for active plant constituents.
Planta Med 1982;45:31–34.
5. Zhdanko AG, Nenajdenko VG: Nonracemizable isocyanoacetates for
multicomponent reactions. J Org Chem 2009;74:884–887.
6. Bellon L, Taft RW, Abboud JM: Structural effects on the reactivity of ethers in
donor-acceptor reactions. J Org Chem 1980;45:1166–1168.
7. Wuitschik G, Carreira EM, Wagner B, et al.: Oxetanes in drug discovery:
structural and synthetic insights. J Med Chem 2010;53:3227–3246.
8. Kurien BT, Singh A, Matsumoto H, Scofield RH: Improving the solubility and
pharmacological efficacy of curcumin by heat treatment. Assay Drug Dev
Technol 2007;5:567–576.
1
1
1
1
32. Cheng X, Hochlowski J, Tang H, et al.: Studies on repository compound stability
in DMSO under various conditions. J Biomol Screen 2003;8:292–304.
Address correspondence to:
Peter Wipf, PhD
Department of Chemistry
Center for Chemical Methodologies and Library Development
University of Pittsburgh
1
9. Yu X, Li Y, Wu D: Enzymatic synthesis of gallic acid esters using
microencapsulated tannase: effect of organic solvents and enzyme
specificity. J Mol Catal-B Enzym 2004;30:69–73.
Pittsburgh, PA 15260
2
0. Yalkowsky SH, He Y: Handbook of Aqueous Solubility Data. CRC Press, New
York, 2003.
E-mail: pwipf@pitt.edu
ª MARY ANN LIEBERT, INC.
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JUNE 2012 ASSAY and Drug Development Technologies 277