N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide as a potential bioreductively activated prodrug of phosphoramide mustard

Article abstract of DOI:10.1016/j.bmcl.2008.05.099

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were synthesized stereospecifically from Boc-l-Hse(OBn)-OH and the degradation of the corresponding reduced amine 5a was investigated by UV/vis spectroscopy and LC/M

Full text of DOI:10.1016/j.bmcl.2008.05.099

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4059–4063
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide
as a potential bioreductively activated prodrug of phosphoramide mustard
Yongying Jiang a, , Longqin Hu a,b,
*
a Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were
Received 2 May 2008
Accepted 27 May 2008
Available online 13 June 2008
synthesized stereospecifically from Boc-L-Hse(OBn)-OH and the degradation of the corresponding
reduced amine 5a was investigated by UV/vis spectroscopy and LC/MS. The rate of cyclization of 5a
was found to increase with decreasing pH, with half-lives ranging from 3.2 to 54 min at pH 4–7.4, sug-
gesting that the cyclization is catalyzed by the hydronium ions. LC/MS analysis of the degradation prod-
ucts of 5a indicates that 4-aminocyclophosphamide is rapidly released from 4 upon reductive activation
under acidic conditions and further decomposes into the cytotoxic phosphoramide mustard. These
results validated 4-aminocyclophosphamide as a prodrug form of phosphoramide mustard and suggest
that compound 4 can potentially be used as a prodrug of phosphoramide mustard for bioreductive
activation.
Keywords:
Prodrug
Cyclophosphamide
Phosphoramide mustard
Bioreductive activation
Cyclization
Ó 2008 Elsevier Ltd. All rights reserved.
Hypoxia is a unique physiological characteristic of solid tumors
and is, therefore, an important therapeutic target.1 The oxygen
deficiency of solid tumors often leads to resistance to ionizing radi-
ation and to many chemotherapeutic drugs. On the other hand, it
also provides an opportunity for selective anticancer chemother-
apy. Hypoxic tumor cells are known to have a greater capacity
for reductive reactions as compared to well-oxygenated normal
cells. The difference has been used to explore bioreductively acti-
vated prodrugs for higher therapeutic index, some of which are
currently in clinical trials.2,3 Cyclophosphamide (1) is an alkylating
antitumor agent with activity against a broad spectrum of human
cancers including slow-growing tumors.4 However, the clinical
application of cyclophosphamide is limited due to its dose-related
toxic side effects, including the life-threatening hemorrhagic cysti-
tis. Consequently, there has been considerable attention in design-
ing prodrugs that specifically release cytotoxic phosphoramide
mustard at tumor sites, thus minimizing toxic side effects.5–7 We
have recently reported for the first time that 4-aminocyclophos-
phamide (4-NH2-CPA, 3) can be used as a prodrug form of
phosphoramide mustard because of its structural similarity to 4-
hydroxycyclophosphamide (2) and its spontaneous degradation
as a mono-phosphorylated gem-diamine.8 4-Aminocyclophospha-
mide can be easily incorporated into a prodrug through a stable
amide bond, for example, in peptide conjugates for proteolytic acti-
vation. In this communication, we report the synthesis of a pair
of N-(2,2-dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclo-
phosphamide diastereomers (4) and the degradation of the corre-
sponding reduced amine intermediate in buffer. Our results
suggest that compound 4 could potentially be used as a prodrug
of phosphoramide mustard for bioreductive activation in selective
cancer chemotherapy.
O
P
1
2
3
R = H
O
R = OH
R = NH2
N
H
N(CH2CH2Cl)2
R
N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophos-
phamide (4) was designed to release 4-aminocyclophosphamide
(3) through a bioreductive activation mechanism involving reduc-
tion and a spontaneous cyclization–elimination reaction (Scheme
1). Nitroaryl amides are stable compounds; however, their reduced
products, the corresponding amine or hydroxylamine derivatives,
are prone to undergo an intramolecular cyclization–elimination
reaction.9 This feature has been widely explored to develop pro-
drugs for bioreductive activation.10 Because of the conformational
restriction by the two methyl groups, the reduced amine or
hydroxylamine intermediates of the 2,2-dimethyl-2-(2-nitro-
phenyl)acetyl derivatives are known to cyclize much faster than
the corresponding analogs without the two methyl substitu-
* Corresponding author. Address: Department of Pharmaceutical Chemistry,
Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ 08854, USA. Tel.: +1 732 445 5291; fax: +1 732 445 6312.
E-mail address: LongHu@rutgers.edu (L. Hu).
Present address: Hoffman-La Roche Inc., Non-Clinical Safety, Nutley, NJ 07110,
USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.099

4060
Y. Jiang, L. Hu / Bioorg. Med. Chem. Lett. 18 (2008) 4059–4063
R
R
NO2
O
NH
N
O
P
O
P
H3O+
N(CH2CH2Cl)2
O
[H]
N(CH2CH2Cl)2
O
O
+
O
3
N
H
N
H
N
H
N
H
4
5a R = H
6a R = H
6b
5b
R = OH
R = OH
Scheme 1. Proposed mechanism of reductive activation of N-(2,2-dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide (4).
ents.9,11 Our previous work demonstrated that the 2,2-dimethyl-2-
(2-nitrophenyl)acetate of FUDR cyclized within seconds upon
reduction.12 The 2,2-dimethyl-2-(2-aminophenyl)acetamides were
found to be stable under neutral hydrogenation conditions at room
temperature but quickly cyclize upon treatment with acid. The fast
release of 4-aminocyclophosphamide (3) from compound 4 also
provided a system to investigate the degradation pathways of 3.
Similar to the phenylalanyl 4-aminocyclophosphamides,8,14
compound 4 has four configurational stereoisomers which can be
referred to as (2R,4R)-, (2R,4S)-, (2S,4R)-, and (2S,4S)-4. As the con-
figuration of 4-aminocyclophosphamide was expected to have lit-
tle effect on the cyclization reaction of 5a, only one pair of
diastereomers of 4, (2S,4S)- and (2R,4S)-, were synthesized stereo-
the incubation mixtures showed no significant changes over a per-
iod of three days (data not shown). To investigate the mechanism
of reductive activation of compound 4, the corresponding reduced
amine product 5a was synthesized by hydrogenation over 5% Pd–C
in methanol at room temperature for 30 min. The hydrogenation
reaction was easily monitored by TLC. Crude product solutions of
2-nitroaryl amides upon reduction were previously used to inves-
tigate such cyclization reactions.9 However, we found that partial
cyclization of the amine intermediates of 4 could not be avoided
during the hydrogenation process; but, 5a was sufficiently stable
to be purified by flash column chromatography on silica gel. Simi-
lar results were observed for the 2,2-dimethyl-2-(o-nitro-
phenyl)acetamides of amino acids.13 The isolated yields of 5a
were 81–86%. Hydrogenation using platinum catalyst such as
PtO2 was accompanied with significant cyclization reactions, with
both lactam 6a and hydroxylactam 6b isolated as side products.
This is consistent with the observation that platinum-catalyzed
hydrogenation generates more hydroxylamine than the amine
product13 and the hydroxylamine product cyclizes much faster
than the amine product because of its higher nucleophilicity.18
To examine the pH-dependence of the cyclization reaction,
(2S,4S)-5a was incubated at 37 °C in a series of buffers with pH
specifically from a protected L-homoserine and the degradation of
their corresponding amine intermediates was investigated. As
shown in Scheme 2, the synthesis of (2S,4S)- and (2R,4S)-4 started
from Boc-L-Hse(Bn)-OH (7) which was first converted into its
amide 8 by HOBt/EDC activation followed by treatment with satu-
rated ammonium hydroxide. After removal of the Boc group in 8,
the free amino group was allowed to react with 2,2-dimethyl-2-
(2-nitrophenyl)acetyl chloride (DMNA-Cl) to give 10. DMNA-Cl
was prepared by refluxing the corresponding acid in SOCl2. The
bis(trifluoroacetoxy)iodobenzene (BTI)-mediated Hoffmann rear-
rangement was employed to convert the amide 10 to the corre-
sponding monoacylated gem-diamine derivative 11. The benzyl
group in 11 was removed by treatment with BBr3 in CH2Cl2 at
ꢀ50 to 0 °C under argon to give 1,3-aminoalcohol 12. Lactam 6a
was isolated as the major side product from the degradation of
gem-diamines 11 and 12.15 Attempt to remove the benzyl protect-
ing group using trimethylsilyl iodide was not successful.16 Cycliza-
tion of 12 with bis(2-chloroethyl)phosphoramidic dichloride
afforded a mixture of the (2S,4S)- and (2R,4S)-diastereomers of 4,
which were easily separated by flash column chromatography,
Table 1
Cyclization rate constants of (2S,4S)-5a under varying pH conditions at 37 °C
b
c
pHa
kobs (minꢀ1
)
t1/2 (min)
4.0
4.5
5.0
5.4
5.6
6.0
6.5
7.4
8.0
0.22
0.14
0.071
0.04
3.2
4.9
9.8
17.5
27.2
12.6
19.3
53.7
0.025
0.055
0.036
0.013
yielding
a faster-eluting cis-diastereomer ((2S,4S)-4) and a
d
d
slower-eluting trans-diastereomer ((2R,4S)-4).17 Assignment of
the (2S,4S)- and (2R,4S)-configurations was also based on their
1H and 31P NMR shifts and has been extensively discussed
previously.8,14
—
—
a
Buffer components were 100 mM sodium acetate for pH 4.0–5.6, 100 mM
sodium phosphate for pH 6.0–7.4, 100 mM Tris/HCl for pH 8.0.
b
kobs = observed pseudo-first-order rate constants for cyclization reaction. Sub-
strate (10
lM) was incubated in the desired buffer at 37 °C and changes of UV
The stability of the (2S,4S)- and (2R,4S)-diastereomers of 4 un-
der physiological conditions was examined by incubating the iso-
mers at pH 7.4 (phosphate buffer) and 37 °C. HPLC analysis of
absorbance at 249 nm were recorded for the calculation of kobs
.
c
t1/2 (half-life of 5a) = 0.693/kobs
.
d
No cyclization was observed for 3 h.
NO2
OBn
OH
OBn
NH2
OBn
iii
OBn
NH2
ii
H2N
i
O
Boc
Boc
NH2
79%
83%
N
H
N
H
N
(2 steps)
H
O
O
O
O
7
8
9
10
NO2
O
NO2
OH
NH2
iv
91%
OBn
vi
v
vii
81-86%
O
4
5a
66%
N
N
H
NH2
(2S,4S)-4 (16%)
(2R,4S)-4 (6%)
H
11
12
Scheme 2. Synthesis of N-(2,2-dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide (4). Reagents and conditions: (i) HOBt, EDC, THF, rt, then sat’d NH3 (aq); (ii)
TFA/CH2Cl2 (1/3), rt, 1 h; (iii) DMNA-Cl, CH2Cl2, rt, 5 h; (iv) BTI, CH3CN/H2O (1/1), rt, 4 h; (v) BBr3, THF, ꢀ50 to 0 °C, 1.5 h; (vi) Cl2PON(CH2CH2Cl)2, TEA, THF, 0 °C–rt, 48 h; (vii)
H2, 5% Pd–C, MeOH, rt, 30 min.

Y. Jiang, L. Hu / Bioorg. Med. Chem. Lett. 18 (2008) 4059–4063
4061
ranging from 4 to 8 and the rate of cyclization was monitored by
measuring the UV absorbance change at 249 nm corresponding
to the maximum absorption wavelength of the cyclized product
6a.12 Table 1 lists the observed pseudo first order rate constants
of cyclization and the half-lives of compound (2S,4S)-5a under dif-
ferent pH conditions. Kinetics of cyclization of similar systems
have been studied previously by others.9 Consistent with results
reported by others, the cyclization rate constant of (2S,4S)-5a
was found to be proportional to hydronium ion concentration as
indicated by the plot of log(kobs) versus pH shown in Figure 1.
The slight jump in the observed pseudo first order rate constants
in the pH range between 5.6 and 6 was attributed to the change
in the buffer ion species from acetate to phosphate.9 The shortest
half-life was 3.2 min at pH 4 and the longest was 53.7 min at pH
7.4 (Table 1). No significant cyclization was found at or above pH
8.0. These results suggest that the cyclization reaction is catalyzed
by the hydronium ions. Given that the solid tumor cells are often
associated with an acidic microenvironment (pH ꢁ 6),19 half-lives
of (2S,4S)-5a at pH 6.0 and 7.4 indicates that the cyclization of
5a in the solid tumors could be more than four-fold faster than that
under normal physiological pH conditions.
To identify the products of cyclization, compound (2S,4S)-5a
was incubated in 100 mM phosphate buffer at pH 6 and 37 °C
and the reaction progress was monitored by LC/MS at different
time intervals. The incubation conditions were chosen based on
the short half-life (12.6 min) of (2S,4S)-5a under these conditions
and their similarity to the acidic microenvironment of solid tumor
cells (pH ꢁ 6).19 LC/MS analysis indicated that the cyclization reac-
tion was nearly complete within 3 h as indicated by the disappear-
ance of (2S,4S)-5a and two product peaks were observed based on
the UV trace (220 nm) (Fig. 2A). The major product peak (>90%)
was identified as the lactam 6a by the corresponding mass spec-
trum and comparison with the authentic sample. The minor prod-
uct peak (<10%) showed a molecular ion (MH+) of 161, one unit less
than that of the lactam, and was assigned as 3,3-dimethyl-3H-in-
dol-2-ylamine (15) as shown in Scheme 3 (path A). Formation of
15 was attributed to be the decomposition of the intermediate
13 which could be formed through the cyclization–dehydration
(path A) of (2S,4S)-5a. Presence of 13 in the reaction mixture was
confirmed by the selected ion extraction of its molecular ion
(419) and the observation of the corresponding characteristic iso-
topic peaks of two chlorines (Fig. 2B, inset). LC/MS analysis of the
incubation mixtures at different time intervals indicated that com-
pound 13 was formed as a minor component in the incubation
mixture and decreased with the increasing formation of 15. Forma-
tion of 15 from 13 suggested the release of cyclophosphamide-3-
ene (14) as the other product. While 14 was not directly detected
by LC/MS, formation of 14 in the incubation mixture was con-
firmed by the detection of its cyanide and thiolate adducts in the
presence of the corresponding trapping reagents (see below). At-
tempts to amplify the formation of 13 through path A from
-0.4
pH 4-7.4
-0.6
-0.8
-1
y = -0.329x + 0.5447
R2 = 0.8238
Na2HPO4 buffer, pH 6-7.4
y = -0.4527x + 1.4731
R2 = 0.9951
-1.2
-1.4
-1.6
-1.8
-2
NaOAc Buffer, pH 4-5.6
y = -0.5825x + 1.7223
R2 = 0.9819
3.0
4.0
5.0
6.0
7.0
8.0
pH
Figure 1. pH-dependence of the observed pseudo first order rate constants of cy-
clization of (2S,4S)-5a at 37 °C.
mAbs
6a
A
1000
500
UV @ 220nm (1.00)
15
5a
13
0
.
Int.
200e3
Int.
Int.
5a
MS of 2/17
277
MS of 13
419
421
259
300e3
200e3
100e3
B
6.0e6
5.0e6
4.0e6
3.0e6
2.0e6
1.0e6
100e3
2/17
300
391
400
456
450m/z
317
203
221
291
350
200
250
300 m/z
6a
15
13
TIC(1.00)
277.00(5.00)
419.00(5.00)
0
1
2
3
4
5
6
7
Retention Time (min)
Figure 2. LC/MS chromatogram of the incubation mixture of (2S,4S)-5a in 100 mM phosphate buffer at pH 6 and 37 °C for 3 h: (A) UV trace (220 nm) showing peaks
corresponding to amidine 15, intermediate 13, lactam 6a, and starting amine 5a; (B) total ion chromatogram (TIC) showing peaks corresponding to amidine 15, 4-
hydroxylcyclophosphamide (2)/aldophosphamide 17, intermediate 13, lactam 6a, and starting amine (2S,4S)-5a. Selected ion chromatograms of 2/17 (----) and 13 (ꢂ ꢂ ꢂꢂ ꢂ ꢂ) are
also shown with their corresponding MS spectra as inserts.

4062
Y. Jiang, L. Hu / Bioorg. Med. Chem. Lett. 18 (2008) 4059–4063
O
O
P
18
R = CN
R
N
H
N(CH2CH2Cl)2
19 R = SCH2CH2NHBoc
KCN or HSCH2CH2NHBoc
O
O
P
N
O
N
+
NH2
O
P
N
H
13
N
H
N(CH2CH2Cl)2
N
N(CH2CH2Cl)2
A
15
14
h
t
a
P
O
H2O
H2
-
5a
-H2O
P
a
H2O
-NH3
O
P
O
P
t
h
2
3
B
O
O
+
H2N
O
H2N
16
N(CH2CH2Cl)2
H2N
17
N(CH2CH2Cl)2
6a
KCN
NaBH(OAc)3
O
O
P
O
P
NC
HO H2N
O
H2N H2N
20
N(CH2CH2Cl)2
N(CH2CH2Cl)2
21
Scheme 3. Proposed mechanisms of degradation of compound (2S,4S)-5a at pH 6 and 37 °C.
(2S,4S)-5a using dehydration reagents were not successful,20 indi-
cating that the dehydration pathway is not favored in comparison
to the alternative elimination pathway and the dehydrated product
13 is not stable.
phosphoramide mustard, the active metabolite of cyclophospha-
mide.23 Detection of significant formation of 15 indicates that 13
disintegrates into 14 which is known to be in equilibrium with 2.
Formation of 2 in the medium is also supported by its trapping
as cyanide and thiolate adducts. Although detection of 14 and 2
would also support a NH3-elimination pathway of 4-aminocyclo-
phosphamide (3), this route was ruled out for the degradation of
gem-diamines in a previous mechanistic study.15 Instead, detection
of 16 and 17 as trapped intermediates suggests that 4-aminocyclo-
phosphamide disintegrates by the expulsion of the phosphora-
mide, similar to the hydrolysis of its acyl amide counterparts.
The aldophosphamide 17 is known to be in equilibrium with 2
and disintegrate spontaneously into acrolein and phosphoramide
mustard through a base-catalyzed b-elimination.23
In summary, N-(2,2-dimethyl-2-(2-nitrophenyl)acetyl)-4-amino
cyclophosphamide (4) was synthesized as a potential prodrug of
phosphoramide mustard for reductive activation. Compound 4 is
stable under physiological conditions but rapidly releases 4-amino-
cyclophosphamide after reduction of the nitro group to hydroxyl-
amine and/or amine under acidic conditions. The cyclization of the
reduced amine intermediate 5a is catalyzed by acid. LC/MS analysis
of the decomposition products of (2S,4S)-5a suggests that 4-amino-
cyclophosphamide disintegrates into an iminophosphamide which
is further hydrolyzed to aldophosphamide to release the alkylating
agent phosphoramide mustard. These results clearly demonstrate
that 4-aminocyclophosphamide is an effective prodrug form of
phosphoramide mustard and compound 4 could potentially be used
as a prodrug for bioreductive activation to improve the therapeutic
effectiveness of cyclophosphamide in the treatment of cancer.
Observation of >90% lactam in the incubation mixture suggested
that compound (2S,4S)-5a mainly went through the cyclization–
elimination pathway and 4-aminocyclophosphamide (3) was re-
leased (path B). However, attempts to detect compound 3 in the
incubation mixtures at different time intervals by LC/MS were not
successful. Instead, 4-hydroxycyclophosphamide (2) or aldophos-
phamide 17 was unambiguously identified in these mixtures by
the presence of the correct molecular ion of 277 and its major frag-
mentation peak 259 (MH+–H2O), both with their characteristic iso-
topic peaks corresponding to the presence of two chlorines (Fig. 2B
and MS inset). Other potential degradation products of 4-aminocy-
clophosphamide were also examined but not found, including imi-
nophosphamide
16,
cyclophosphamide-3-ene
(14),
and
phosphoramide mustard. To trap these short-lived reactive interme-
diates during the decomposition of 4-aminocyclophosphamide,
cyclization of (2S,4S)-5a was carried out under various conditions
in the presence of different trapping reagents and the corresponding
products were analyzed by LC/MS. The reduced amine product 20
from the imine intermediate 16 was detected when reduction of
(2S,4S)-4 was carried out in the presence of 10% acetic acid by hydro-
genation or incubation of (2S,4S)-5a in a buffer containing sodium
borohydride acetate. Imines are known to be reduced to amines un-
der these conditions.21,22 The amine product was able to be further
trapped by a preformed HOBt ester of Z-Tyr(OBn)-OH. Incubation
of (2S,4S)-5a at 37 °C in phosphate buffer (pH 6.0) containing KCN
or Boc–NH–CH2–CH2–SH also gave the corresponding cyanide or
thiolate adducts of 14 and 17, respectively, as shown in Scheme 3.
The above results clearly suggest that 4-aminocyclophospha-
mide was released from compound (2S,4S)-5a upon cyclization
and further decomposed into the cytotoxic phosphoramide mus-
tard. Based on the intermediates detected, the degradation mech-
anism of (2S,4S)-5a was proposed as shown in Scheme 3.
Compound (2S,4S)-5a decomposes under acidic conditions mainly
through a cyclization–elimination path B to release 4-aminocyclo-
phosphamide, accompanied by a minor cyclization–dehydration
path A to form intermediate 13. Both degradation pathways would
lead to the formation of 4-hydroxycyclophosphamide (2) which is
responsible for the formation of the ultimate alkylating agent
Acknowledgements
We gratefully acknowledge the financial support of Grant SNJ-
CCR 700-009 from the State of New Jersey Commission on Cancer
Research, a pilot grant from the Gallo Prostate Cancer Center of the
Cancer Institute of New Jersey, and Grant RSG-03-004-01-CDD
from the American Cancer Society.
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17. (2S,4S)-4: Rf = 0.35 (CH2Cl2:CH3OH, 20/1); 1H NMR (200 MHz, CD3OD) d 7.87–
7.46 (m, 4H), 5.31 (dt, 1H, J = 20.4 Hz), 4.60–4.50 (m, 1H), 4.31–4.20 (m, 1H),
3.70–3.63 (m, 4H), 3.50–3.36 (m, 4H), 2.20–2.00 (m, 2H), 1.74 (s, 3H), 1.70 (s,
3H); 13C NMR (50 MHz, CD3OD) 175.8, 148.8, 138.4, 132.4, 128.2, 127.3, 124.5,
988.5 cmꢀ1 LC/MS (ESI) 467.1 [MH]+, 469.1; HRMS (FAB) m/z calcd for
;
467.1018, found 467.1042.
18. Sykes, B. M.; Atwell, G. J.; Denny, W. A.; McLennan, D. J.; O’Connor, C. J. J. Chem.
Soc., Perkin Transn 2 1995, 2, 337.
19. Tietze, L. F.; Neumann, M.; Mollers, T.; Fischer, R.; Glusenkamp, K. H.;
Rajewsky, M. F.; Jahde, E. Cancer Res. 1989, 49, 4179.
20. The dehydration conditions used include P2O5, H2SO4 (concd), N,N0-
dicyclohexylcarbodiimide (DCC), and heating in the presence of 4 Å
molecular sieves.
21. Smith, R. G.; Lucas, R. A.; Wasley, J. W. J. Med. Chem. 1980, 23, 952.
22. Wu, P.-L.; Chu, M.; Fowler, F. W. J. Org. Chem. 1988, 53, 963.
23. Borch, R. F.; Millard, J. A. J. Med. Chem. 1987, 30, 427.
2
63.5 (d, J C,P = 25.8 Hz), 59.0, 52.9, 48.4 (d, 2J C,P = 9.2 Hz), 40.9, 29.0 (d,