Labeled oxazaphosphorines for applications in mass spectrometry studies. 2. Synthesis of deuterium-labeled 2-dechloroethylcyclophosphamides and 2- and 3-dechloroethylifosfamides

Article abstract of DOI:10.1002/jlcr.3142

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic α and α

Full text of DOI:10.1002/jlcr.3142

Note
Received 7 August 2013,
Revised 11 October 2013,
Accepted 21 October 2013
Published online 5 December 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3142
Labeled oxazaphosphorines for applications in
mass spectrometry studies. 2. Synthesis of
deuterium-labeled 2-dechloroethylcyclo-
phosphamides and 2- and 3-
dechloroethylifosfamides
James B. Springer,a O. Michael Colvin,a and Susan M. Ludemanb
*
The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a
cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations
at the exocyclic α and α′ carbons, which result in dechloroethylation of CP and IF. The incidence of oxidation at one position
relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and
IF pharmacokinetics. As standards for the mass spectrometry quantification of dechloroethylation, the following were
synthesized: (1) [4,4,5,5-2H4]-2-dechloroethylcyclophosphamide (equivalent to [4,4,5,5-2H4]-3-dechloroethylifosfamide); (2)
[α,α,4,4,5,5-2H6]-2-dechloroethylcyclophosphamide (equivalent to [α,α,4,4,5,5-2H6]-3-dechloroethylifosfamide); and (3)
[α,α,4,4,5,5-2H6]-2-dechloroethylifosfamide. The common precursor to all of the target compounds was [2,2,3,3-2H4]-3-
aminopropanol. A one-pot reaction of this compound with POCl3 and unlabeled or labeled 2-chloroethylamine
hydrochloride gave the d4 and d6 labeled 2-dechloroethylcyclophosphamides. The construction of the 2-dechloro-
ethylifosfamide from the aminopropanol required five discreet steps. Optimization of the synthetic pathways and stability
studies are discussed.
Keywords: dechloroethylcyclophosphamide; dechloroethylifosfamide; deuterium; synthesis
for use as internal standards would facilitate extensions of such
work to the reliable quantification of each dechloroethylation
reaction.
P-450 Oxidation of either the α or α′ carbon in CP yields the
same product: 2-dechloroethylcyclophosphamide (3) (Scheme 1).
Similar oxidation of the nonequivalent α and α′ carbons in IF
give 2-dechloroethylifosfamide (4) and 3-dechloroethylifosfamide
Introduction
The prodrugs cyclophosphamide (1, CP; Scheme 1) and
ifosfamide (2, IF; Scheme 2) each metabolize to an active
alkylating agent through a cytochrome P450-mediated oxidation
at the C-4 position. Competing with this activation pathway are
enzymatic oxidations at the exocyclic α and α′ carbons of CP
and IF.1,2 Each reaction at a side chain produces a hemiaminal
(5), respectively (Scheme 2). Note that the product of 2-dechlo-
(as shown for CP in Scheme 1); rearrangement results in a
roethylation in CP is identical to that for 3-dechloroethylation in IF.
fragmentation of the parent drug to give a dechloroethyl
As standards for the MS quantification of dechloroethylation,
oxazaphosphorine and chloroacetaldehyde. No toxicities have
the following three compounds were synthesized (Schemes 3
and 4): [4,4,5,5-2H4]-2-dechloroethylcyclophosphamide ([4,4,5,
been linked unambiguously to the dechloroethyl metabolites;
5-2H4]-3);
[α,α,4,4,5,5-2H6]-2-dechloroethylcyclophosphamide
however, chloroacetaldehyde is associated with neurotoxicity,
([α,α,4,4,5,5-2H6]-3); and [α,α,4,4,5,5-2H6]-2-dechloroethylifos-
famide ([α,α,4,4,5,5-2H6]-4). Depending on one’s frame of
reference, the 3-d4 and 3-d6 structures can also be named
and this side effect, especially during IF treatment, can be
dose-limiting.2
The incidence of oxidation at one position relative to another
is believed to be at least one factor underlying the high degree
of interpatient variability in both CP and IF pharmacokinetics.
As a result, there is much interest in determining the influence
of different variants, such as race, gender, age, and
polymorphisms, on the extent of C-4 and side chain oxidations
in CP and IF.2–7 There are multiple reports of applications of mass
spectrometry (MS) to such studies, particularly those relating to
the quantification of C-4 oxidation.4,5,7,8 Typically, deuterated
analogs of the C-4 oxidized analytes are used as internal
standards.4,8 The availability of labeled dechloroethyl metabolites
aDuke Comprehensive Cancer Center and Department of Medicine, Duke
University Medical Center, Durham, NC 27710, USA
bDepartment of Basic and Social Sciences, Albany College of Pharmacy and
Health Sciences, Albany, NY 12208, USA
*Correspondence to: Susan M. Ludeman, Albany College of Pharmacy and
Health Sciences, 106 New Scotland Ave., Albany, NY 12208, USA.
E-mail: susan.ludeman@acphs.edu
J. Label Compd. Radiopharm 2014, 57 110–114
Copyright © 2013 John Wiley & Sons, Ltd.

J. B. Springer et al.
3-dechloroethylifosfamides [4,4,5,5-2H4]-5 and [α,α,4,4,5,5-2H6]-
5, respectively.
Experimental
All glassware were dried before use, and reactions were carried out under
N2. Generally, chemicals were purchased from Sigma-Aldrich Corp., St.
Louis, MO, USA, Thermo Fisher Inc., Hampton, NH, USA, or VWR, Bridgeport,
NJ, USA. THF was distilled from potassium/benzophenone ketyl; CH2Cl2,
CH3CN and Et3N were distilled from CaH2. Flash chromatography was
carried out on 230–400 mesh silica gel (Merck). In general, approximately
50 mL of dry silica gel was used per gram crude material, and columns were
6″ in height. Analytical thin layer chromatography plates were hard-coated
with a 250-micron layer of silica gel 60 (Merck). Radial chromatography was
performed on a chromatotron (Harrison and Harrison). NMR spectra were
recorded on a Varian Inova-400 (Agilent Technologies, Santa Clara, CA,
USA) or a GE QE-300 spectrometer (GE NMR Instruments, Freemont, CA,
USA). Chemical shifts are reported relative to TMS (0ppm, 1H), CDCl3
(77 ppm, 13C), or 25% H3PO4 (0ppm, 31P, external reference). NMR solvent
CDCl3 was washed with NaHCO3/D2O prior to use.
Scheme 1. Competing C-4 and side chain oxidations in cyclophosphamide.
[2,2,3,3-2H4]-3-Amino-1-propanol ([2,2,3,3-2H4]-6).
As per a literature report,9 3-hydroxypropionitrile was reacted with NaOD/
D2O (≥98 atom %D, MSD Isotopes, now Cambridge Isotope Laboratories,
Tewksbury, MA, USA) to give [2,2-2H2]-3-deuterioxypropionitrile (DOCH2CD2CN)
in 65% yield [Rf 0.52, EtOAc-hexanes (3:1); 13C NMR (CDCl3) δ 118.4
(CN) and 57.22 (OCH2)]. This nitrile was then reduced with LiAlD4
(98 atom %D, Aldrich) according to the literature9 to give the final
product [96% yield; 13C NMR (CDCl3) δ 61.82 (CH2O)].
[4,4,5,5-2H4]-2-Dechloroethylcyclophosphamide ([4,4,5,5-2H4]-3).
Equivalent to [4,4,5,5-2H4]-3-dechloroethylifosfamide ([4,4,5,5-2H4]-5).
A solution of [2,2,3,3-2H4]-6 (461 mg, 5.8 mmol) in CH2Cl2 (6 mL) was
added dropwise to a cooled (ice bath) solution of freshly distilled POCl3
(0.55 mL, 5.9 mmol) in CH2Cl2 (17 mL). Et3N (1.66 mL, 11.9 mmol) was
then added, and the mixture was stirred for 5 h at ice bath temperature.
2-Chloroethylamine hydrochloride (770 mg, 6.6 mmol) was added as a
solid, in one portion, followed by Et3N (1.66 mL, 11.9 mmol). The reaction
mixture was stirred at room temperature overnight and was then filtered
through a pad (2″) of Celite 545 (Thermo Fisher Scientific, Hampton, NH).
The filtrate was concentrated, and the residue was flash-chromatographed
using CH3OH-CH2Cl2 (5:95). The product was still contaminated, and therefore,
the material was flash-chromatographed again using EtOH-EtOAc (1:9). Pure
product was obtained [527 mg, 45%, Rf 0.19 in EtOH-EtOAc (1:9)]. 1H NMR
(CDCl3) δ 4.40 (dd, 2JHH =11Hz and 3JHP =11Hz, 1H, one C6H), 4.26 (dd, 2JHH
11 Hz and JHP =11Hz, 1H, one C6H), 3.62 (t, J= 6 Hz, 2H, CH2Cl), 3.33–3.25
(m, 2H, CH2CH2Cl), 3.16 (bs, 1H, one NH), and 2.81 (bs, 1H, one NH).
=
3
Scheme 2. Competing C-4 and side chain oxidations in ifosfamide.
Scheme 3. Synthesis of deuterated 2-dechloroethylcyclophosphamides (structurally equivalent to 3-dechloroethylifosfamides).
J. Label Compd. Radiopharm 2014, 57 110–114
Copyright © 2013 John Wiley & Sons, Ltd.
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J. B. Springer et al.
2
3
13CNMR (CDCl3) δ 67.85 (d, JCP =7Hz, C6), 45.88 (d, JCP =5Hz, CH2Cl), and
[α,α,4,4,5,5-2H6]-2-Amino-3-(2′-hydroxyethyl)-2H2-1,3,2-
oxazaphosphorinane-2-oxide ([α,α,4,4,5,5-2H6]-10).
42.98 (CH2CH2Cl). 31P NMR (CDCl3) δ 10.8.
[α,α,4,4,5,5-2H6]-2-Dechloroethylcyclophosphamide ([α,α,4,4,5,5-2H6]- A solution of [α,α,4,4,5,5-2H6]-9 (1.14 g, 4.2 mmol) in 11.8% (v/v) solution
3). Equivalent to [α,α,4,4,5,5-2H6]-3-dechloroethylifosfamide ([α,α,4,4,
of 1,4-cyclohexadiene in absolute ethanol (85 mL) was passed
through a 1 × 6 cm column of freshly prepared palladium black11 at
a rate of 1–2 mL/min. Concentration of the eluent gave a residue that
was flash-chromatographed (CH3OH-CH2Cl2, 1:9) to isolate the product
(0.36 g, 47%, Rf 0.15). Unreacted starting material was also recovered
(0.63 g, 56%, Rf 0.37). 1H NMR (CDCl3) δ 4.60 (bs, 1H, OH), 4.38–4.24
(m, 2H, CH2OP), 3.80–3.60 (m, 2H, NCD2CH2O), and 3.55 (bs, 2H,
NH2). 31P NMR (CDCl3) δ 15.7.
5,5-2H6]-5).
The title compound was synthesized using the same procedure as that given
above for [4,4,5,5-2H4]-3 but instead used [1,1-2H2]-2-chloroethylamine
hydrochloride10 instead of unlabeled 2-chloroethylamine hydrochloride as
a starting material. The reaction mixture was concentrated to ~1/3 of its
original volume, and this turbid mixture was loaded onto a 9″ silica gel
column made with a 50-mL silica gel for flash chromatography. Elution with
EtOH-EtOAc (1:9) gave the product (0.19 g, 33%, Rf 0.19). 1H NMR (CDCl3) δ
[α,α,4,4,5,5-2H6]-2-Dechloroethylifosfamide ([α,α,4,4,5,5-2H6]-4).
2
3
2
4.39 (dd, JHH =11Hz and JHP = 11 Hz, 1H, one C6H) 4.26 (dd, JHH = 11Hz
3
A solution of triphenylphosphine (0.62 g, 2.4 mmol) in CH2Cl2 (5 mL) was
added dropwise to a solution of freshly recrystallized (benzene) N-
chlorosuccinimide (0.33 g, 2.4 mmol) in CH2Cl2 (27 mL). Additional CH2Cl2
(2 mL) was used to rinse the flask and syringe. The turbid mixture was
and JHP =11Hz, 1H, one C6H), 3.61 (s, 2H, CH2Cl), and 3.30 and 3.05 (bm,
1H each, two NH). 13C NMR (CDCl3) δ 67.88 (d, 2JCP =7Hz, C6) and 45.59 (d,
3JCP =5Hz, CH2Cl). 31P NMR (CDCl3) δ 11.2.
[2,2,3,3-2H4]-3-(2′-Benzyloxyacetylamido)-1-propanol ([2,2,3,3-2H4]-7).
stirred vigorously for several minutes, and then
a solution of
[α,α,4,4,5,5-2H6]-10 (0.37 g, 2.0 mmol) in CH2Cl2 (5 mL) was added
quickly with another 2-mL CH2Cl2 being used to rinse the flask and
syringe. The mixture was stirred at room temperature overnight and
then concentrated. The residue was passed through two flash
chromatography columns, each 6″ tall and made with 30-mL silica gel
[first column, EtOH-EtOAc (1:9) eluent; second column, EtOH-benzene
(1:9) eluent]. The still impure product [Rf 0.33 in CH3OH-CH2Cl2 (1:9)]
was then successfully purified using radial chromatography on a 2-mm
silica gel plate with EtOH-EtOAc (1:9) as eluent [0.23 g, 56%, Rf 0.19 in
EtOH-EtOAc (1:9)]. 1H NMR (CDCl3) δ 4.37–4.25 (m, 2H, CH2O), 3.65 (s,
2H, CH2Cl), and 3.05 (bs, 2H, NH2). 13C NMR (CDCl3) δ 67.23 (d, 2JCP = 7 Hz,
C6) and 42.34 (CH2Cl). 31P NMR δ 12.7.
Using a literature procedure for unlabeled material,10 [2,2,3,3-2H4]-6 was
incorporated as a starting material to give the title compound in 60%
yield. The product was obtained as an oil, which solidified upon standing
1
[Rf 0.27 (EtOAc)]. H NMR (CDCl3) δ 7.39–7.31 (m, 5H, aromatic), 6.97 (bs,
1H, OH), 4.56 (s, 2H, CH2Ph), 3.99 (s, 2H, CH2C = O), and 3.60 (s, 2H,
CH2OH); N-H not visible above the baseline. 13C NMR (CDCl3) δ 170.6
(C = O), 136.7, 128.5, 128.2, and 127.9 (aromatic), 73.49 (CH2C = O), 69.22
(CH2Ph), and 59.09 (CH2OH).
1′,1′,2,2,3,3-[2H6]-3-(2′-Benzyloxyethylamino)-1-propanol
([1′,1′,2,2,3,3-2H6]-8).
With minor modification to
a literature preparation of unlabeled
material,10 AlD3 [from LiAlD4 (98 atom %D, 1.0 g, 22.6 mmol)] was reacted
with [2,2,3,3-2H4]-7 (1.5 g, 6.4 mmol) for ~12 h. The flask was then cooled
(ice bath), and the reaction was quenched with the dropwise addition of
1 M sodium potassium tartrate (5.0 mL). The mixture was diluted with
water (100 mL) and CH2Cl2 (50 mL); 40% NaOH (~1.0–1.5 mL) was added
to insure a basic pH. The phases were separated, and the aqueous layer
was extracted with CH2Cl2 (3 × 50 mL). All organic layers were combined,
dried (Na2SO4), filtered, and evaporated to afford the product (86%) as a
pure, colorless oil [Rf 0.50 in NH3-saturated CH3OH : CH2Cl2 (1:9)]. 1H NMR
(CDCl3) δ 7.37–7.28 (m, 5H, aromatic), 4.51 (s, 2H, CH2Ph), 3.79 (s, 2H,
Results and Discussion
Tetratedeuterated 3-aminopropanol ([2,2,3,3-2H4]-6) was the
common precursor to all of the desired dechloroethyl compounds
(Schemes 3 and 4).9 For the 2-dechloroethylcyclophosphamides,
[2,2,3,3-2H4]-6 was reacted with POCl3 followed by either
unlabeled or [1,1-2H2]-2-chloroethylamine hydrochloride10 to
give tetradeuterated or hexadeuterated products (Scheme 3,
[4,4,5,5-2H6]- 3 or [α,α,4,4,5,5-2H6]- 3, respectively). Initially, the
CH2OH), 3.59 (s, 2H, OCH2CD2N), and 3.53 (bs, 2H, NH and OH). 13C work-up included a water wash of the reaction mixture; however,
NMR (CDCl3) δ 138.0, 128.4 and 127.7 (aromatic), 73.20 (CH2Ph), 69.06
(OCH2CD2N), and 64.04 (CH2OH).
2-dechloroethylcyclophosphamide proved to be quite hydrophilic,
and even multiple back-extractions were not successful in
removing all products from the water layer. The water washes
were dropped from subsequent syntheses, and yields improved
by 10–50%. As mentioned previously, the 3-d4 and 3-d6 structures
can also be named 3-dechloroethylifosfamides [4,4,5,5-2H4]-5 and
[α,α,4,4,5,5-2H6]-5, respectively.
As shown in Scheme 4, construction of the remaining
chloroethyl chain in 2-dechloroethylifosfamide [α,α,4,4,5,5-2H6]-4
required multiple steps; the pathway was a modified version of
the one used to make labeled ifosfamides.10 Variations tried during
optimization of the synthetic scheme included the following: (1)
[α,α,4,4,5,5-2H6]-2-Amino-3-(2′-benzyloxyethyl)-2H2-1,3,2-oxazaphos-
phorinane-2-oxide ([α,α,4,4,5,5-2H6]-9).
A solution of [1′,1′,2,2,3,3-2H6]-8 (1.21 g, 5.6 mmol) in CH2Cl2 (5 mL) was
added dropwise to a solution of freshly distilled POCl3 (0.53 mL,
5.7 mmol) in CH2Cl2 (17 mL) at ice bath temperature. Additional CH2Cl2
(3 mL) was used to rinse down the flask and syringe. Et3N (1.57 mL,
11.3 mmol) was then added, and the mixture was stirred in an ice bath
(3 h). The mixture was allowed to warm for ~1–2 min and was then
cooled again. Anhydrous NH3 was bubbled through the mixture at ice
bath temperature for 15 min, and then the flask was capped and
parafilmed, the bath was removed, and the mixture was stirred the oxygen in 6 was protected as an acetal to prevent the
overnight. (Note: The cap blew off overnight as a result of the pressure,
and the solvent evaporated.) The crude reaction mixture was taken up
in minimal CH2Cl2 and loaded onto a 6″ flash chromatography column,
which had been made with 100-mL silica gel and EtOH-EtOAc (1:9)
eluent. The product was obtained as a colorless solid [1.36 g, 88%, Rf
0.37 in CH3OH-CH2Cl2 (1:9)]. 1H NMR (CDCl3) δ 7.38–7.27 (m, 5H,
aromatic), 4.58 (d, J = 12 Hz, 1H, one CH2Ph), 4.51 (d, J = 12 Hz, 1H, one
CH2Ph), 4.35–4.21 (m, 2H, CH2OP), 3.67 (d, J = 10 Hz, 1H, one NCD2CH2O)
3.51 (d, J = 10 Hz, 1H, one NCD2CH2O), and 2.88 (bs, 2H, NH2). 13C NMR
(CDCl3) δ 137.8, 128.4, 127.8, and 127.7 (aromatic), 73.08 (CH2Ph), 68.09
(NCD2CH2O), and 67.44 (d, 2JCP = 7 Hz, C6). 31P NMR (CDCl3) δ 13.2.
formation of any N- and O-bisalkylated product during synthesis
of 7 (no advantage obtained); (2) in place of the benzyloxy group
in 7, 8 and 9, t-butyldimethylsilyl and t-butyldiphenylsilyl ethers
were investigated as moieties, which might be easily converted
to the alcohol group in 10, and synthesis of these compounds
added many steps to the pathway without any net benefits; (3)
catalytic transfer hydrogenations of 9 with 10% Pd/C and formic
acid12 or ammonium formate13 resulted in sluggish reactions at
room temperature and decomposition upon heating; (4) reduction
of 9 with hydrogen and 10% Pd/C at 60psi10 was incomplete
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J. Label Compd. Radiopharm 2014, 57 110–114

J. B. Springer et al.
Scheme 4. Synthesis of deuterated 2-dechloroethylifosfamide.
after 4 h, and allowing the reaction to continue overnight
Under the silylation reaction conditions,8 each dechloroethyl
resulted in decomposition as well as the persistence of metabolite was found to react rapidly and to give multiple
unreacted 9; and (5) direct conversion of 9 to final product 4 products (31P NMR). Mono- or bis-silylated derivatives were
was tried using trimethylsilyl chloride and sodium iodide, and anticipated, but the product mixture was much more complex.
extensive decomposition resulted.
Similar to all CP-related and IF-related compounds, the unchanged (acetonitrile, 70°C, 2 h). Samples for analysis by the
stabilities of dechloroethyl metabolites 3/5 and vary cited GC–MS–MS method would best be split into aliquots, one
In the absence of the silylating agent, the metabolites were
4
significantly depending on conditions. For 3/5 in buffered for silylation and detection of C-4 oxidation products and one
solutions at 20°C, Gilard et al. report half-lives of 2.8 h at pH 3.0 for separate analysis of dechloroethylation products.
and 49 days at pH 5.5; under the same conditions, 4 exhibits
half-lives of 25 min at pH 3.0, 3.8 days at pH 5.5, and 36 days at
pH 6.8.14 According to Kaijser et al., the dechloroethyl Conclusion
metabolites are unchanged after 8 weeks at 4°C in water or ethyl
There is ongoing interest in determining the influence of C-4
versus side chain oxidations on the interpatient variability in
acetate but undergo 10% loss after 4 weeks (4°C) in plasma or
urine (no loss at À20°C).15
Using 31P NMR, we monitored the stabilities of the
response to treatment with the anticancer agents CP and IF.
MS has been applied to both clinical and in vitro studies,
dechloroethyl compounds under conditions that have been
particularly those relating to the quantification of C-4 oxidation.
used for the gas chromatography (GC)–MS–MS and liquid
The syntheses of deuterium-labeled internal standards have been
chromatography–MS–MS quantifications of 4-hydroxy-CP, the
reported for use in C-4 oxidation studies16; deuterated standards
C-4 oxidation product derived from CP (Scheme 1).4,8 In this
for companion investigations of dechloroethylation reactions were
procedure, reaction samples are added to solutions containing
the synthetic goals of this work. Oxidative dechloroethylation of CP
a derivitizing agent, which stabilizes the reactive 4-hydroxy-CP;
gives one possible product (3), whereas the same reaction of IF
the solvent is composed of 2 M ammonium diphosphate (pH
~4.8), methanol, and acetonitrile (20:25:55). Samples may be
stored for months at À80°C prior to analysis. For the GC method,
leads to two possible products (4 and 5; metabolite 5 is structurally
equivalent to 3). Tetra- and/or hexadeuterated analogs of each of
these metabolites were synthesized by multistep but otherwise
the derivitized 4-hydroxy-CP is extracted from the sample and
generally straightforward pathways.
treated with a silylating agent in acetonitrile for 2 h at 70°C. To
determine the suitability of these conditions for isolation and
analysis of the dechloroethyl metabolites, each compound was
dissolved in the ammonium phosphate/methanol/acetonitrile
Acknowledgements
solution and allowed to sit at room temperature. No changes
were observed in 31P NMR spectra taken of 4 periodically
This work was supported in part by Public Health Service
Grant CA16783 (SML/OMC) awarded by the National Cancer
Institute (Department of Health and Human Services). NMR
data were obtained through the Shared Instrumentation Facility
of the Duke University Medical Center and the Department
of Chemistry.
over 10 months. During a 14-month observation time, an
unidentified but prominent 31P signal appeared in spectra of
3/5; however, extraction of the solution yielded near
quantitative recovery of 3/5. Additional studies performed using
liquid chromatography–MS–MS techniques demonstrated that
both labeled metabolite and its unlabeled counterpart had the
same stability: their relative concentrations remained the same in
the derivitizing solution over the length of the experiment (several
days at À80°C).
Conflict of Interest
The authors did not report any conflict of interest.
J. Label Compd. Radiopharm 2014, 57 110–114
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J. B. Springer et al.
[9] S. P. Walsh, E. M. Shulman-Roskes, L. W. Anderson, Y.-H. Chang, S. M.
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