Synthesis and epimerization of phenylalanyl 4-aminocyclophosphamides

Article abstract of DOI:10.1016/j.tet.2007.08.009

Peptide and amino acid conjugates of (4R)- and (4S)-4-aminocyclophosphamides (4-NH₂-CPA, 3) were designed as prodrug forms of phosphoramide mustard. Four diastereomers of Boc-Phe-4-NH-CPA (6) were synthesized stereospecifically from homoserine (R or S) and the protection strategy was optimized for the homoserine hydroxyl group during the construction of the 1,3,2-oxazaphosphorinane ring. The Phe-4-NH-CPA isomers of the trans-configuration ((2S,4R)- and (2R,4S)-) were found to be less stable than the corresponding isomers of the cis-configuration ((2R,4R)- and (2S,4S)-) and to undergo epimerization of the C-4 chiral center in the presence of 25% TFA used during Boc deprotection. The synthetic route developed should be applicable to the synthesis of a variety of peptide and amino acid conjugates of 4-aminocyclophosphamide.

Full text of DOI:10.1016/j.tet.2007.08.009

Tetrahedron 63 (2007) 10637–10645
Synthesis and epimerization of phenylalanyl
4-aminocyclophosphamides
Yongying Jiang,a,y Zhoupeng Zhang,a,z Robert S. DiPaolab and Longqin Hua,b,
*
aDepartment of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
160 Frelinghuysen Road, Piscataway, NJ 08854, USA
bThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 20 June 2007; revised 31 July 2007; accepted 1 August 2007
Available online 8 August 2007
Abstract—Peptide and amino acid conjugates of (4R)- and (4S)-4-aminocyclophosphamides (4-NH2-CPA, 3) were designed as prodrug
forms of phosphoramide mustard. Four diastereomers of Boc-Phe-4-NH-CPA (6) were synthesized stereospecifically from homoserine (R
or S) and the protection strategy was optimized for the homoserine hydroxyl group during the construction of the 1,3,2-oxazaphosphorinane
ring. The Phe-4-NH-CPA isomers of the trans-configuration ((2S,4R)- and (2R,4S)-) were found to be less stable than the corresponding iso-
mers of the cis-configuration ((2R,4R)- and (2S,4S)-) and to undergo epimerization of the C-4 chiral center in the presence of 25% TFA used
during Boc deprotection. The synthetic route developed should be applicable to the synthesis of a variety of peptide and amino acid conjugates
of 4-aminocyclophosphamide.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of cyclophosphamide. Tumor-targeted prodrug therapy is
one of the strategies explored to improve the therapeutic in-
dex of cyclophosphamide.7–12 In this strategy, phosphor-
amide mustard is incorporated with a biochemical activation
mechanism specifically associated with tumor cells, such
as hypoxic reduction, enzymatic action, and receptor recog-
nition. Recently, we have reported for the first time that 4-
aminocyclophosphamide (4-NH2-CPA, 3) can be used as
a prodrug form of phosphoramide mustard because of its
structural similarity to 2 and its spontaneous degradation
as a mono-phosphorylated gem-diamine.12 In that communi-
cation we had briefly described the synthesis of phenylalanyl
4-aminocyclophosphamide (H-Phe-4-NH-CPA, 4) and its
Cbz-protected derivative (5). Herein, we wish to report an al-
ternative synthetic route to 4 through its Boc-protected deriv-
ative (6) and an unexpected but interesting epimerization
reaction was observed for the trans-isomers of 4 under acidic
de-Boc conditions. Protecting strategy for the hydroxyl
group of homoserine was also optimized for the construction
of the 1,3,2-oxazaphosphorinane ring. The synthetic routes
developed can be easily adapted for the synthesis of various
4-aminocyclophosphamide conjugates, especially those of
amino acids or peptides.
The oxazaphosphorinane cyclophosphamide (1) is an alkyl-
ating antitumor agent with activity against a broad spectrum
of human cancers including slow-growing solid tumors.1
Over the past four decades, cyclophosphamide has become
one of the most frequently used anticancer agents in clinic.
The clinical significance as well as the unique conforma-
tional and stereochemical aspects of oxazaphosphorinane
derivatives have attracted much interest in the chemistry
community.2–4 To elucidate the mechanism of action of cy-
clophosphamide and to enhance its efficacy as an antitumor
agent, numerous oxazaphosphorinane derivatives were syn-
thesized. The mechanism of action of cyclophosphamide has
been well understood after decades of investigation.5,6
Briefly, cyclophosphamide is oxidized by cytochrome
P450 enzymes in the liver to 4-hydroxycyclophosphamide
(2), which then decomposes into acrolein and the alkylating
species phosphoramide mustard.5,6 Acrolein is responsible
for the hemorrhagic cystitis, a major dose-limiting side effect
Keywords: Cyclophosphamide; Prodrug; Proteolysis; Epimerization.
* Corresponding author at present address: Department of Pharmaceutical
Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State Univer-
sity of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.
Tel.: +1 732 445 5291; fax: +1 732 445 6312; e-mail: longhu@rutgers.edu
URL: http://www.rci.rutgers.edu/wlonghu/
Present address: Department of Pharmaceutical Chemistry, School of
Pharmacy, University of California, San Francisco, CA 94143, USA.
Present address: Department of Drug Metabolism, Merck Research
Laboratories, PO Box 2000, Rahway, NJ 07065, USA.
1O
2P
1O
2P
O
O
4
4
3
3
R
R
N
N(CH2CH2Cl)2
N
H
N
N(CH2CH2Cl)2
H
H
y
1 R = H
2 R = OH
3 R = H
4 R = H-Phe-
5 R = Cbz-Phe-
6 R = Boc-Phe-
z
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.08.009

10638
Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
2. Results and discussion
such as the Curtius rearrangement for its mild reaction con-
ditions, high product yields, and retention of the Ca-chiral
center in homoserine.16,17 The reaction was conveniently
monitored by the disappearance of the starting material on
TLC and the Boc group was stable under the mild acidic
reaction conditions.
The four configurational diastereomers of compounds
4–6 are referred to as (2R,4R)-, (2R,4S)-, (2S,4R)-, and
(2S,4S)- (Fig. 1) according to the C-2 and C-4 absolute con-
figuration. Among them, (2R,4R)- and (2S,4S)- have been
referred to as cis-isomers and (2R,4S)- and (2S,4R)- as
trans-isomers according to the relative orientation of the
C-4 substituent to the oxygen atom of P]O bond in the
oxazaphosphorinane ring (cis¼RR/SS, trans¼SR/RS). Only
the chair conformations are shown in Figure 1 to illustrate
the relative stereochemistry; the actual conformations of
the oxazaphosphorinane ring are more complicated and
are discussed by others.2,4,13 The initial synthesis of com-
pound 6 started from O-benzyl protected Boc-homoserine
(S or R) as shown in Scheme 1. The two diastereomers of
6 with R configuration at C-4 were synthesized stereospecif-
ically from (S)-Boc-Hse(Bn)-OH ((S)-7) that has the same
absolute stereochemistry found in natural amino acids while
the two diastereomers of 6 with the S configuration at
C-4 were synthesized stereospecifically from (R)-Boc-
Hse(Bn)-OH ((R)-7). Amidation of 7 was carried out using
HOBt/EDC activation followed by treatment with saturated
ammonium hydroxide.14,15 After removal of Boc group in 8,
the free amino group was coupled with Boc-Phe-OH to give
the dipeptide amide 9. The bis(trifluoroacetoxy)iodobenz-
ene (BTI)-mediated Hofmann rearrangement was employed
to convert the amide 9 to the corresponding mono-acylated
gem-diamine derivative 10. The BTI-mediated Hofmann
rearrangement method was chosen over other methods
The benzyl group in 10 was removed by catalytic hydroge-
nolysis at 50 psi at pH 3 for 1–2 days. The harsh conditions
were necessary to overcome the problem of catalyst poison-
ing by the free amino group in the product18 and to avoid an
intramolecular O/N benzyl migration side reaction that
would otherwise lead to the formation of a hard-to-remove
benzyl amine side product.19,20 LC–MS analysis indicated
that the Boc group was not affected under these mild acidic
conditions. However, Boc-Phe-NH2 was isolated in 25–30%
yield, indicating significant degradation of the gem-diamine
10 and 11 during the deprotection.21 The amino alcohol 11
was isolated in 67–71% yield by a benzenesulfonic acid-
based cation exchange column using 1% TEA/CH3OH as
the eluant. In an effort to avoid the difficulty of removing
the benzyl group in the presence of an amino group, we re-
moved the benzyl group in 10 prior to the Hofmann rear-
rangement. However, this process led to the formation of
1,3-oxazinan-2-one and was later developed into a useful
method in the construction of 2-oxazolidinone libraries
from b-hydroxylpropionamides.22
To facilitate the synthesis of 4-NH2-CPA conjugates, we ex-
plored other reaction routes that avoided the difficulty of re-
moving the benzyl group in 10 and could be more easily
adapted for scaled-up synthesis. Using (R)-homoserine
(12) as the starting material, we explored various protecting
group strategies of the homoserine hydroxyl group and ob-
tained the key intermediate (R)-11 in much better yields.
As shown in Scheme 2, the HOSu-activated ester of Boc-
Phe-OH reacted with 12 in a mixture of 1 M KHCO3 and
THF (1:1) yielding the dipeptide 13 in 81% yield. The carb-
oxylic acid 13 was subsequently converted to its corre-
sponding amide 14 using the same amidation method as
described above for compounds (S)- and (R)-9. Protection
of the hydroxyl group in 14 was evaluated in parallel using
acetyl, Cbz, TBDMS, and TBDPS groups in compounds
O
O
H
N
H
N
H
N
H
N
N(CH2CH2Cl)2
R-Phe
R-Phe
O
P
P
H
N(CH2CH2Cl)2
H
trans-/(2S, 4R)-
O
cis-/(2R, 4R)-
N(CH2CH2Cl)2
O
P
H
H
P
O
O
O
N(CH2CH2Cl)2
N
H
N
H
R-Phe
N
H
R-Phe NH
cis-/(2S, 4S)-
trans-/(2R, 4S)-
Figure 1. Four diastereomers of phenylalanyl 4-aminocyclophosphamides 4
(R¼H), 5 (R¼Cbz), and 6 (R¼Boc).
O
OBn
NH2
O
OBn
NH2
H
N
H
N
OBn
OBn
iv
(82-84%)
Boc
N
H
i
*
ii, iii
Boc
Boc
(79-84%)
Boc
N
H
OH
*
NH2
*
*
N
H
O
N
H
(87-90%)
O
O
(S)-7
(R)-7
(S)-8
(S)-9
(R)-9
(S)-10
(R)-10
(R)-8
O
OH
NH2
O
O
P
O
O
P
H
N
H
N
O
O
*
*
H2N
vii
vi
*
v
Boc
N
H
*
Boc
N
H
N
N(CH2CH2Cl)2
*
N
H
N
N(CH2CH2Cl)2
H
H
(67-71%)
(13%)
(6%)
(20%)
(8%)
(2R,4R)-6
(2S,4R)-6
(2S,4S)-6
(2R,4S)-6
(2R,4R)-4 (100%)
from (S)-11
from (R)-11
(S)-11
(R)-11
(30%)
(2S,4R)-4
(100%)
(2S,4S)-4
(37%)
(2R,4S)-4
Scheme 1. Stereospecific synthesis of H-Phe-4-NH-CPA (4) from isomers of protected homoserine ((S)-7 and (R)-7). Reagents and conditions: (i) HOBt, EDC,
THF, rt, then added satd NH3 (aq); (ii) 25% TFA, CH2Cl2, rt; (iii) Boc-Phe-OSu, 1 M KHCO3/THF (1:1), rt; (iv) BTI, CH3CN/H2O (1:1), rt; (v) H2 (50 psi), 10%
Pd–C, add 0.2 N HCl to pH 3, MeOH, rt; (vi) Cl2PON(CH2CH2Cl)2, TEA, THF, 0 ꢀC, rt; (vii) 25% TFA, CH2Cl2, rt.

Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
10639
O
OH
OH
O
OH
NH2
O
OPG
NH2
H
N
H
N
H
N
OH
OH
N
H
Boc
N
H
Boc
N
H
Boc
ii
i
iii
O
O
H2N
O
(77%)
(81%)
O
(R)-12
13
14
15 (PG = Ac)
(85%)
(92%)
16 (PG = Cbz)
17 (PG =TBDMS) (90%)
18 (PG = TBDPS) (96%)
O
OPG
NH2
O
OH
NH2
H
N
H
N
N
Boc
v
Boc
N
H
iv
H
19 (PG = Ac)
(90%)
(99%)
(R)-11 (98%) from 20
(77%) from 21
20 (PG = Cbz)
21 (PG = TBDPS) (96%)
Scheme 2. Use of different protecting groups during the synthesis of Boc-Phe-(R)-gHse-NH2 ((R)-11) from (R)-homoserine (12). Reagents and conditions
(PG¼Protecting group): (i) Boc-Phe-OSu, 1 M KHCO3/THF (1/1), rt; (ii) HOBt, EDC, THF, rt, then added satd NH3 (aq); (iii) (a) for compound 15: AcCl,
pyridine, CH2Cl2, rt; (b) for compound 16: Cbz-Cl, pyridine, CH2Cl2; (c) for compound 17: TBDMS-Cl, imidazole, DMF, rt; (d) for compound 18:
TBDPS-Cl, imidazole, DMF, rt; (iv) BTI, CH3CN/H2O¼1:1, rt; (v) (a) from 20: H2 (50 psi), 10% Pd–C, MeOH, rt; (b) from 21: TBAF, THF, rt.
15, 16, 17, and 18, respectively. The acetyl group was intro-
duced using Ac2O/pyridine; the Cbz group was introduced
using Cbz-Cl/pyridine; and the two silyl groups were intro-
duced using the corresponding silyl chloride/imidazole in
DMF. The TBDMS protecting group in 17 was found to be
unstable to the acidic Hofmann rearrangement conditions,
resulting in the formation of the cyclized product, 1,3-
oxazinan-2-one.22 The other three protected dipeptide amides
were successfully converted into their corresponding gem-
diamine derivatives 19–21 in nearly quantitative yields. Sub-
sequently, the Cbz group of 20 was completely removed to
give (R)-11 in quantitative yield by catalytic hydrogenolysis
at 50 psi in 4 h without the need to add HCl. The TBDPS
group of 21 was removed by TBAF at room temperature in
1 h to give (R)-11 in 77% yield with the concurrent formation
of Boc-Phe-NH2 as a side product. Attempt to remove the
acetyl group in 19 by LiOH-catalyzed hydrolysis gave a mix-
ture of (R)-11 and an acetamide resulting from O/N acetyl
migration. The acetamide was resistant to hydrolysis under
the same conditions. Thus, Cbz protection for the homoser-
ine hydroxyl group gave the best yield in the synthesis of (R)-
11 and the carbonate was readily removed in high yield under
mild conditions. TBDPS protection can be used as an alter-
native and was successfully used for the synthesis of Cbz-
Phe-4-NH-CPA (5), when differentiation of the hydroxyl
and the N-terminal amino protecting groups is necessary.12
reaction. Organic carboxylic acid salts of 11 such as those
of TFA were found to adversely affect the cyclization reac-
tion by producing an N-acylated side product of 11, presum-
ably through activation and subsequent amidation of the
carboxylic acid by the bis(dichloroethyl)phosphoramidic di-
chloride reagent used. The configurations of 6 as either cis or
trans were unequivocally assigned according to their NMR
data. The 31P chemical shift for cis-2-oxo-1,3,2-oxazaphos-
phorinanes with an axial P-N(CH2CH2Cl)2 is generally
upfield as compared with that for the trans-isomers with
1
an equatorial P-N(CH2CH2Cl)2.13,24 On H NMR, the cis-
isomers have a more upfield H-4 and a more down field
H-5 as compared to the trans-diastereomers.25 The spectro-
scopic data used to assign the configuration of the four
diastereomers are summarized in Table 1.
To remove the N-terminal Boc group, each of the four diaste-
reomers of 6 was treated with 25% TFA/CH2Cl2 for 30 min
at room temperature and the deprotection reaction was mon-
itored using HPLC and LC–MS. To our surprise, while the
Boc group of each diastereomer was successfully removed
under these conditions, the homogeneity of the deprotected
products varied with the relative configuration of the starting
material. Each of the cis-isomers gave a single product peak
on HPLC, which was confirmed to be the corresponding
amine 4 by LC–MS, but each of the trans-isomers gave
two product peaks on HPLC and LC–MS, both having the
same molecular ions and isotopic patterns corresponding
to 4 (Fig. 2). Similarly, 31P NMR demonstrated that depro-
tection of each cis-isomer gave a single peak, but that of
each trans-isomer gave two peaks with chemical shifts of
about 0.4–0.6 ppm apart (Table 1). The two products from
each trans-isomer were separated by preparative HPLC
and both fit the structure of 4 based on their 1H NMR spectra.
Interestingly, we noticed that each trans-isomer of 6 gave,
upon deprotection, a product mixture that always contained
one component having the same HPLC retention time and
31P NMR chemical shift as the product from the cis-isomer
with the opposite stereochemistry at C-4 under the same
conditions. As shown in Figure 2, deprotection of trans-
isomer (2S,4R)-6 gave two peaks with retention time at
16.2 and 16.8 min; the latter overlapped with the peak corre-
sponding to cis-isomer (2S,4S)-4 (chromatogram a and b).
Cyclization of the g-amino alcohol in (S)- or (R)-11 with
bis(dichloroethyl)phosphoramidic dichloride gave a mixture
of the corresponding cis- and trans-diastereomer of 6, which
were easily separated by silica gel flash column chromato-
graphy, yielding a faster eluting cis-diastereomer in 13–20%
yield and a slower eluting trans-diastereomer in 6–8% yield.
Boc-Phe-NH2 was isolated in 22–32% yield. The low cycli-
zation yield was attributed to the low nucleophilicity of gem-
diamines and the significant degradation of the starting
gem-diamines, a phenomenon that was observed during
the synthesis of other cyclophosphamide analogs.2,7,9,23 Ef-
forts to improve the cyclization yield by using excess re-
agents or extending the reaction time to 72 h were
unsuccessful and yielded more degradation products. The
g-amino alcohols (S)- and (R)-11 were applied in either
the HCl salt form or the free base form in the cyclization

10640
Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
Table 1. Analytical data of diastereomers of Boc-Phe-NH-CPA (6) before and after TFA treatment
Before TFA treatment
After TFA treatment
MSg
MSc
NMR d (ppm)
1H (C-5, 2H)
Producte
31P NMRd
b
b
tR
Compound
Rfa/tR
1H (C-4, 1H)
31Pd
(2R,4R)-6
(2S,4R)-6
0.6/22.5
0.2/22.8
523.1634
523.1629
5.42–5.20
5.43–5.25
2.25–1.87
2.20–1.80
10.7
13.6
(2R,4R)-4
(2S,4R)-4
(2S,4S)-4
(2S,4S)-4
(2R,4S)-4
(2R,4R)-4
15.6 (100%f)
16.2 (52%f)
16.8 (48%f)
16.8 (100%f)
16.0 (41%f)
15.6 (59%f)
423.1
423.1
423.1
423.2
423.3
423.1
11.0
13.7
11.6
11.6
13.5
11.0
(2S,4S)-6
(2R,4S)-6
0.45/22.6
0.2/22.7
523.1653
523.1643
5.34–5.12
5.34–5.30
2.05–1.55
1.8–1.61
11.6
13.7
a
TLC (on silica gel) developing solvents: hexane/CH2Cl2/MeOH¼1:1:0.1.
b
c
d
e
f
HPLC retention time (min) on C18 column (5 mm, 4.6ꢁ250 mm) with a gradient elution of 4–76% methanol in 15 min.
HRMS (FAB), m/z calcd for C21H34Cl2N4O5P [MH]+ 523.1644.
The 31P chemical shift was determined using 5% H3PO4 in D2O in a coaxial insert as an external standard.
Product of 5 after TFA treatment.
Relative composition as determined by peak areas on HPLC chromatogram monitored at 220 nm.
MH+ monoisotopic mass as recorded on ESI LC–MS (isotopic peaks were not listed).
g
(2S,4R)-4
(2S,4S)-4
A220 nm
(mAU)
1000
800
600
400
200
0
(a) TFA treatment of (2S,4R)-6
(b) TFA treatment of (2S,4S)-6
(2R,4R)-4
(2R,4S)-4
(c) TFA treatment of (2R,4R)-6
(d) TFA treatment of (2R,4S)-6
12
13
14
15
16
Retention Time (min)
17
18
19
20
Figure 2. HPLC analyses of products after TFA treatment of each of the four diastereomers of Boc-Phe-NH-CPA (6).
Similarly, deprotection of trans-isomer (2R,4S)-6 gave two
peaks with retention time at 15.6 and 16.0 min; the former
overlapped with the peak corresponding to (2R,4R)-4, the
cis-isomer with opposite stereochemistry at C-4 (comparing
chromatograms d and c, Fig. 2). Similarly, the 31P NMR
chemical shift of cis-isomer (2R,4R)-4 at 11.0 ppm overlap-
ped with one product from trans-isomer (2R,4S)-6 and that
of (2S,4S)-4 at 11.6 ppm with one product from (2S,4R)-6.
This was further confirmed by coinjections of these products
into HPLC and 31P NMR of the mixtures of these products.
the above mixtures were confirmed by its HPLC retention
time and 31P NMR chemical shift. In addition, when each
trans-isomer of 4 was treated with TFA (25–100%) in di-
chloromethane for 30 min, a mixture of trans- and cis-isomer
were obtained as described above while the same treatment
of the two cis-isomers of 4 did not cause any changes in the
composition.
The epimerization of trans-isomers of H-Phe-NH-CPA (4) at
the C-4 chiral center is rationalized by an acid-catalyzed ring
opening of 4-aminocyclophosphamide to a ring-opened
imide form, which would then cyclize to give the corre-
sponding cis-diastereomer, similar to the epimerization of
Based on these evidences from the LC–MS, HPLC, and
NMR, we concluded that deprotection of cis-isomers of 6 us-
ing 25% TFA gave only the corresponding cis-isomers of 4
but the two trans-isomers of 6 gave not only the expected
trans-isomer of 4 but also another cis-isomer of 4 resulting
from epimerization of the C-4 chiral center. Specifically,
the deprotection product of trans-isomer (2S,4R)-6 was
a mixture of trans-(2S,4R)-4 and cis-(2S,4S)-4, and that of
trans-(2R,4S)-6 was a mixture of trans-(2R,4S)-4 and cis-
(2R,4R)-4. This conclusion was further confirmed by the
stereospecific synthesis of the four isomers of 4 from their
Cbz-protected derivatives (5).12 When the Cbz group was re-
moved under neutral hydrogenolysis conditions, each trans-
isomer of 4 was isolated as a single product and its identity in
4-hydroxycyclophosphamide
through
aldophosphor-
amide.26 As shown in Scheme 3, under acidic conditions,
trans-(2S,4R)-4 undergoes a ring opening to form an imide
(2S)-22, which then cyclizes to form either cis-(2S,4S)-4
from si face or back to trans-(2S,4R)-4 from re face. Simi-
larly, trans-(2R,4S)-4 forms cis-(2R,4R)-4 through the cycli-
zation of the imide intermediate (2R)-22 from re face. The
epimerization trend from trans to cis was further confirmed
by the observation that an increasingly high conversion yield
was obtained from trans-isomers of 4 to the corresponding
cis-isomers of 4 when the TFA treatment was prolonged
from 30 min to 9 h. The extended exposure to TFA also

Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
10641
H-Phe
N
N(CH2CH2Cl)2
O
H
P
H+
H
N
"si"
H
N
O
O
O
.
N(CH2CH2Cl)2
H-Phe
P
"re"
N
H
N(CH2CH2Cl)2
H2N
P
H-Phe
N
H
H
H
O
O
(2S,4R)-4
"trans"
(2S,4S)-4
"cis"
(2S)-22
H-Phe
O
N
H+
O
H
H
P
"re"
H
O
O
O
N(CH2CH2Cl)2
.
H-Phe
N
O
N
"si"
P
N
H
H2N
H-Phe NH
P
H
H
N(CH2CH2Cl)2
N(CH2CH2Cl)2
(2R,4S)-4
"trans"
(2R,4R)-4
"cis"
(2R)-22
Scheme 3. TFA-catalyzed epimerization of the trans-isomers of H-Phe-NH-CPA (4) at the C-4 chiral center.
promoted the degradation of 4 as H-Phe-NH2 was observed
as a side product and increased with the reaction time. The
4. Experimental
observation of epimerization only for the trans-isomers indi-
cates that the cis-isomers of 4 are more stable than the trans-
isomers and thus cyclization of 22 favors to the right side.
Previous study has clearly shown that cis- and trans-isomer
of 4-phenyl-cyclophosphamide exist in chair-like conforma-
tion with the phenyl substitute on an equatorial position,
regardless of the phosphorus configuration.24 As the phenyl-
alaninamido group of 4 is more strictly demanding than the
phenyl group, isomers of 4 should exist predominantly in the
similar chair-like conformation with the C-4 substituent on
the equatorial position as shown in Figure 1. Compared to
the trans-isomers, the cis-isomers, therefore, are stabilized
by a more efficient P(2)-N(3) anomeric interaction with
N(CH2CH2Cl)2 and a more efficient intramolecular H bond-
ing between P]O and adjacent NH.13,24 Attempts to avoid
epimerization during Boc deprotection of the trans-isomers
of 6 using milder acidic to neutral conditions included treat-
ment with Me3SiI in chloroform at room temperature,27 bro-
mocatecholborane in dichloromethane,28 AlCl3/PhOCH3 in
dichloromethane,29 and MeSO3H in dioxane.30 However,
none of these conditions were satisfactory, resulting in either
incomplete deprotection or decomposition of the product or
the observed epimerization.
4.1. General
Moisture-sensitive reactions were performed in flame-dried
glassware under a positive pressure of nitrogen or argon.
Air- and moisture-sensitive materials were transferred by
a syringe or cannula under an argon atmosphere. Solvents
were either ACS reagent grade or HPLC grade. Tetrahydro-
furan was dried over sodium/benzophenone. Triethylamine,
dichloromethane, and ethyl acetate were dried over calcium
hydride. Pyridine was dried over potassium hydroxide and
distilled over calcium hydride. N,N-Dimethylformamide
˚
was dried over 4 A molecular sieves at least for one week
prior to use. Unless otherwise stated, all reactions were mag-
netically stirred and monitored by thin layer chromato-
graphy (TLC) using 0.25 mm Whatman precoated silica
gel plates. TLC plates were visualized using either 7% (w/w)
ethanolic phosphomolybdic acid or 1% (w/w) aqueous po-
tassium permanganate containing 1% (w/w) NaHCO3. Flash
column chromatography was performed using silica gel
(Merck 230–400 mesh). Yields refer to chromatographically
and spectroscopically (1H NMR) homogenous material, un-
less otherwise noted. All reagents were purchased at the
commercial quality and used without further purification.
Melting points were determined on a Mel-Temp capillary ap-
paratus and are uncorrected. Infrared spectra were recorded
with a Perkin–Elmer model 1600 series FTIR spectrometer
using polystyrene as an external standard. Infrared absor-
bance is reported in reciprocal centimeters (cmꢂ1) with broad
signals denoted by br. NMR spectra (1H and 13C) were
recorded on a 200 MHz Varian Gemini spectrometer using
residual undeuterated solvents as the internal reference. 31P
NMR spectra were recorded at 121 MHz or 162 MHz using
5% H3PO4 in D2O in a coaxial insert as an external standard.
Chemical shifts are reported in parts per million (d) and
coupling constants (J values) are given in hertz (Hz). The fol-
lowing abbreviations were used to explain the multiplicities:
s¼singlet; d¼doublet; t¼triplet; q¼quartet; m¼multiplet;
br¼broad. High-resolution mass spectral (HRMS) data
were obtained from the University of Kansas Mass Spec-
trometry Laboratory (Lawrence, KS). HPLC analysis was
performed on an HP 1090 system equipped with a Phenom-
enex C18 column (5 mm, 4.6 mmꢁ250 mm) with gradient
elution of 4–76% MeOH containing 0.1% formic acid in
15 min at a flow rate of 1 mL/min and a detection wavelength
3. Conclusion
We successfully synthesized Boc-protected phenylalanine-
conjugated 4-aminocyclophosphamide (6) isomers in a ste-
reospecific manner from homoserine (R or S) and explored
various protection strategies for the hydroxyl group of ho-
moserine for the steps leading to the construction of the
1,3,2-oxazaphosphorinane ring. Among the protecting
groups explored, Cbz was the best for the synthesis of the
key intermediate 10 prior to the formation of the 1,3,2-
oxazaphosphorinane ring and TBDPS can be used as an al-
ternative when Cbz can not be used. The optimized synthetic
route should be applicable to the synthesis of a variety of 4-
aminocyclophosphamide conjugates. The trans-isomers of 6
were found to be unstable under the acidic conditions used to
deprotect Boc and epimerized at the C-4 chiral center to the
corresponding cis-isomers, which were stable under the
same conditions. These findings suggested that acidic treat-
ment of the trans-isomers of 4-aminocyclophosphamide
conjugates should be avoided.

10642
Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
at 220 nm. HPLC purification was performed on a Beckmann
system equipped with a Phenomenex C18 column (10 mm, 9.8
mmꢁ250 mm) with gradient elution of 4–76% MeOH con-
taining 0.1% formic acid in 15 min at a flow rate of 5 mL/
min and a detection wavelength at 220 nm.
30.8, 28.3; IR (KBr) 3296.1, 1667.5, 1520.0, 1366.7,
1167.6 cmꢂ1; HRMS (FAB) m/z calcd for C25H34N3O5
[MH]+ 456.2493, found 456.2467.
4.1.4. Boc-Phe-(S)-Hse(Bn)-NH2 ((S)-9). The compound
was synthesized using the procedure described above in Sec-
tion 4.1.3 from (S)-8 (2.06 g, 6.68 mmol) and Boc-Phe-OH
(1.71 g, 6.68 mmol) as a white solid (2.74 g, 90%); mp
149–150 ꢀC. 1H NMR (200 MHz, CD3OD) d 7.34–7.26
(m, 10H), 4.60–4.44 (m, 3H), 4.32–4.25 (m, 1H), 3.55 (t,
J¼5.0 Hz, 2H), 3.10 (dd, J¼13.9, 5.6 Hz, 1H), 2.85 (dd,
J¼13.4, 8.4 Hz, 1H), 2.20–1.91 (m, 2H), 1.38 (s, 9H); 13C
NMR (50 MHz, CDCl3) d 173.6, 171.4, 156.0, 137.8,
136.2, 129.6, 129.2, 128.6, 128.0, 127.7, 127.3, 80.8, 73.3,
68.3, 56.7, 52.3, 37.9, 30.7, 28.3; IR (CDCl3) 3334.5,
1682.6, 1522.6, 1415.5, 1167.5 cmꢂ1; HRMS (FAB) m/z
calcd for C25H34N3O5 [MH]+ 456.2493, found 456.2467.
4.1.1. (R)-Boc-Hse(Bn)-NH2 ((R)-8). To a solution of (R)-
Boc-Hse(Bn)-OH (2.02 g, 6.54 mmol) in anhydrous THF
(30 mL) were added HOBt (1.33 g, 9.8 mmol) and EDC
(1.25 g, 6.54 mmol). The reaction solution was stirred at
room temperature for 30 min. Saturated ammonium hydr-
oxide solution (5 mL) was added dropwise as the reaction
mixture cleared. The reaction was stirred for 3 h after
the addition was complete. After the removal of solvent in
vacuo, the residue was taken up in ethyl acetate (150 mL),
washed with saturated NaHCO3 and saturated NaCl, and
dried over Na2SO4. Solvent was removed by evaporation
and the residue was purified by flash column chromato-
graphy (CH2Cl2/CH3OH, 20:1) to give a white solid
(1.69 g, 84%); mp 125–127 ꢀC. 1H NMR (200 MHz,
CDCl3) d 7.35–7.28 (m, 5H), 6.56 (br s, 1H), 6.22 (br s,
1H), 5.87 (d, J¼7.0 Hz, 1H), 4.50 (s, 2H), 4.31–4.28 (m,
1H), 3.67–3.57 (m, 2H), 2.07–2.02 (m, 2H), 1.45 (s, 9H);
13C NMR (50 MHz, CDCl3) d 174.8, 155.8, 138.0, 128.5,
128.3, 127.9, 127.8, 127.5, 80.1, 73.4, 67.7, 53.1, 32.2,
28.4; IR (CDCl3) 3334.1 (br), 2976.8, 1683.1, 1506.8,
4.1.5. Boc-Phe-(R)-gHse(Bn)-NH2 ((R)-10). To a stirred
suspension of (R)-9 (1.51 g, 3.32 mmol) in 20 mL of aceto-
nitrile and distilled water (1:1) was added bis(trifluoroace-
toxy)iodobenzene (BTI) (1.57 g, 3.65 mmol). After 4 h of
stirring under N2 at room temperature, a clear solution re-
sulted and no more starting material could be detected by
TLC (CH2Cl2/CH3OH, 20:1). The acetonitrile was removed
under vacuum, and the aqueous phase was lyophilized. The
resulting diamine trifluoroacetate was triturated with Et2O to
1366.6, 1250.8, 1166.9 cmꢂ1
; LC–MS (ESI) 347.0
[M+K]+, 331.0 [M+Na]+, 253.0, 209.0; HRMS (FAB) m/z
give a pale yellow solid (1.51 g, 84%); mp 52–54 ꢀC. H
1
calcd for C16H24N2O4 [MH]+ 309.1809, found 309.1799.
NMR (200 MHz, CD3OD) d 7.36–7.23 (m, 5H), 5.30
(m, 1H), 4.53 (s, 2H), 4.35 (dd, J¼9.2, 6 Hz, 1H), 3.6 (m,
2H), 3.1 (dd, J¼13.6, 9.2 Hz, 1H), 2.8 (d, 2H), 2.2 (m,
2H), 1.35 (s, 9H); 13C NMR (50 MHz, CD3OD) d 173.4,
155.8, 137.4, 136.6, 128.7, 128.5, 127.6, 127.5, 127.0,
126.9, 125.9, 78.9, 72.3, 64.2, 55.7, 55.4, 31.2, 26.7; IR
(CDCl3) 2979.5, 1673.7, 1519.8, 1367.8, 1203.0, 1176.5,
1137.7 cmꢂ1; LC–MS (ESI) 450.1 [M+Na], 428.2 [MH]+,
411.0 [MHꢂ17]+; HRMS (FAB) m/z calcd for
C24H34N3O4 [MH]+ 428.2544, found 428.2525.
4.1.2. (S)-Boc-Hse(Bn)-NH2 ((S)-8). The compound was
synthesized from (S)-Boc-Hse(Bn)-OH (3.0 g, 9.7 mmol)
using the procedure described above in Section 4.1.1 as
a white solid (2.37 g, 79%); mp 128–129 ꢀC. 1H NMR
(200 MHz, CD3OD) d 5.86 (d, J¼7 Hz, 1H), 4.50 (s, 2H),
4.29 (d, J¼5.2 Hz, 1H), 3.62 (m, 2H), 2.05 (m, 2H), 1.45
(s, 9H); 13C NMR (50 MHz, CDCl3) d 175.1, 155.9, 138.0,
128.5, 127.8, 79.9, 77.9, 77.3, 76.7, 73.3, 67.5, 52.9, 32.3,
28.4; IR 3389.6, 3190.0, 2977.5, 2864.7, 1651.0,
1519.0 cmꢂ1; LC–MS (ESI) 331.0 [M+Na]+, 253.0, 209.0.
4.1.6. Boc-Phe-(S)-gHse(Bn)-NH2 ((S)-10). The compound
was synthesized from (S)-9 (3.36 g, 7.37 mmol) using the
procedure described above in Section 4.1.5 as a pale yellow
solid (2.95 g, 82%); mp 101.5–103 ꢀC. 1H NMR (200 MHz,
CD3OD) d 7.34–7.26 (m, 10H), 4.60–4.44 (m, 3H), 4.36–
4.25 (m, 1H), 3.60–3.50 (m, 2H), 3.34–3.31 (m, 1H), 3.10
(dd, J¼13.9, 5.6 Hz, 1H), 2.85 (dd, J¼13.9, 8.4 Hz, 1H),
2.26–2.06 (m, 1H), 2.04–1.84 (m, 1H); 13C NMR
(50 MHz, CD3OD) d 173.3, 155.8, 137.4, 136.6, 128.5,
128.2, 127.6, 127.5, 127.4, 127.0, 126.9, 126.3, 126.1,
125.9, 125.8, 18.9, 72.4, 64.3, 63.3, 55.3, 36.8, 26.7; IR
(CDCl3) 1667.9, 1406.7, 1163.6, 699.2 cmꢂ1; LC–MS
(ESI) 450.1 [M+Na], 428.1 [MH]+, 411.2 [MHꢂ17]+;
HRMS (FAB) m/z calcd for C24H34N3O4 [MH]+ 428.2544,
found 428.2520.
4.1.3. Boc-Phe-(R)-Hse(Bn)-NH2 ((R)-9). Compound (R)-8
(1.40 g, 4.55 mmol) was treated with 50% TFA in CH2Cl2
(5 mL) for 30 min. After evaporation under vacuum the res-
idue was pumped to dryness. The residue was dissolved in
CH2Cl2 (8 mL) and DIEA (1.41 mL) was added. To this
solution was added the preformed activated ester solution
of Boc-Phe-OH, which was generated by Boc-Phe-OH
(1.43 g, 4.55 mmol), HOBT (0.615 g, 4.55 mmol), and
EDC (0.872 g, 4.55 mmol) in CH2Cl2 (10 mL). The mixture
was stirred at room temperature for 4 h and concentrated in
vacuo. The residue was dissolved in EtOAc (150 mL),
washed with 5% citric acid, saturated aqueous NaHCO3,
and saturated aqueous NaCl, dried over Na2SO4, and evapo-
rated under vacuum. Flash column chromatography purifica-
tion (CH2Cl2/CH3OH, 20:1) gave a white solid (1.80 g,
4.1.7. Boc-Phe-(R)-Hse-OH (13). To a solution of Boc-Phe-
OH (5.0 g, 18.8 mol) in 1,2-dimethoxyethane (DME)
(13 mL) at 0 ꢀC were added HOSu (0.868 g, 18.8 mmol)
and DCC (4.28 g, 21 mmol) sequentially. The reaction mix-
ture was stirred at 0 ꢀC overnight and was filtered to remove
DCU. The filtrate was concentrated invacuo and the resulting
white solid was recrystallized from 2-propanol (20 mL) to
give Boc-Phe-OSu as a white solid (5.72 g, 84%). To
1
87%); mp 60–61 ꢀC. H NMR (200 MHz, CDCl3) d 7.33–
7.23 (m, 10H), 7.08 (br d, J¼4.8 Hz, 1H), 5.50 (br s,
1H), 5.06 (d, J¼5.8 Hz, 1H), 4.54–4.36 (m, 4H), 4.10–
3.90 (m, 1H), 3.70–3.45 (m, 2H), 3.10–2.85 (m, 2H),
2.15–1.75 (m, 2H), 1.40 (s, 9H); 13C NMR (50 MHz,
CDCl3) d 173.7, 171.5, 156.0, 137.9, 136.5, 129.2, 128.8,
128.6, 128.0, 127.0, 80.5, 73.5, 67.9, 57.1, 52.1, 37.7,

Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
10643
a solution of H-(R)-Hse-OH (119 mg, 1 mmol) in 1 M
KHCO3 (2.2 mL) and THF (2.2 mL) was added Boc-Phe-
OSu (362.4 mg, 1 mmol) at room temperature. The mixture
was stirred at room temperature overnight. After THF was re-
moved in vacuo, the resulting solution was acidified to pH 3
by adding 10% citric acid and then extracted with ethyl ace-
tate(50mLꢁ4). The organicextractswerecombined, washed
with saturated NaCl, dried over Na2SO4, and evaporated to
give a colorless oil (347.2 mg, 95%). The crude product
was crystallized from a mixture of hexane and ethyl acetate
added imidazole (130 mg, 1.91 mmol) and TBDPS-Cl
(249.3 mL, 0.96 mmol). The solution was stirred at room
temperature for 5 h and was partitioned between ethyl ace-
tate (50 mL) and water (10 mL). The organic phase was
washed with 5% citric acid, saturated NaHCO3, and satu-
rated NaCl, dried over Na2SO4, and evaporated in vacuo.
The residue was purified through flash column chromato-
graphy (CH2Cl2/CH3OH, 40:1) to give a white solid
(506 mg, 96%); mp 71–72.5 ꢀC. 1H NMR (200 MHz,
CDCl3) d 7.68–7.03 (m, 15H), 4.64–4.60 (m, 1H), 4.02–
3.99 (m, 1H), 3.85–3.66 (m, 2H), 3.08–2.90 (m, 2H),
2.06–1.79 (m, 2H), 1.42 (s, 9H), 1.09 (s, 9H); 13C NMR
(50 MHz, CDCl3) d 173.8, 171.6, 162.6, 156.0, 136.4,
135.6, 133.4, 132.9, 129.2, 128.8, 127.9, 127.0, 80.5, 61.7,
57.2, 51.8, 37.8, 36.5, 33.1, 31.5, 28.3, 27.1, 19.3; IR
(CDCl3) 3293.5, 2931.1, 1668.2, 1497.7, 1169.6, 1111.5,
734.4, 701.9 cmꢂ1; LC–MS (ESI) 576.4 [M+H]+ (100%),
559.4, 503.3, 459.3, 312.2; HRMS (FAB) m/z calcd for
C34H46N3O5Si [MH]+ 604.3201, found 604.3163.
1
to give a white solid (296 mg, 81%); mp 66–68 ꢀC. H
NMR (200 MHz, CDCl3) d 7.27–7.21 (m, 5H), 4.54–4.39
(m, 1H), 4.35–4.14 (m, 1H), 3.57–3.49 (m, 2H), 3.13 (dd,
J¼13.5, 6 Hz, 1H), 2.85 (dd, J¼13.5, 9.2 Hz, 1H), 2.08–
1.79 (m, 2H), 1.38 (s, 9H); 13C NMR (50 MHz, CDCl3)
d 174.9, 172.1, 155.6, 136.6, 129.4, 128.8, 128.6, 127.1,
80.7, 65.9, 55.8, 49.0, 38.5, 30.0, 28.3; IR (CDCl3) 3319.1
(br), 2978.2, 1660.5, 1519.7, 1167.9, 1052.0, 733.3 cmꢂ1
;
LC–MS (ESI) 733.4 [2M+H]+, 367.2 [M+H]+, 311.2. Boc-
Phe-OSu: mp 149.5–151.5 ꢀC. 1H NMR (200 MHz, CDCl3)
d 7.32–7.24 (m, 5H), 4.75 (dd, J¼9.9, 4.8 Hz, 1H), 3.78–
3.28 (m, 1H), 3.04 (dd, J¼13.9, 9.8 Hz, 1H), 2.86 (s, 4H),
1.38 (s, 9H); LC–MS (ESI) 363.1 [M+1] (100%).
4.1.11. Boc-Phe-(R)-gHse(Cbz)-NH2$TFA (20). The com-
pound was synthesized from 16 (481 mg, 0.96 mmol) using
the procedure described above in Section 4.1.5. The crude
product was triturated with Et2O to give a white solid
1
4.1.8. Boc-Phe-(R)-Hse-NH2 (14). The compound was
synthesized from 13 (2.15 g, 5.87 mmol) using the proce-
dure described above in Section 4.1.1. The crude product
was purified by flash column chromatography (CH2Cl2/
CH3OH, 20:1 to 5:1) to give a white solid (1.65 g, 77%);
(559.2 mg, 99%); mp 121–122.5 ꢀC. H NMR (200 MHz,
CDCl3) d 7.42–7.34 (m, 5H), 7.23–7.17 (m, 5H), 5.18 (s,
2H), 5.03 (dd, J¼9.6, 5.2 Hz, 1H), 4.27 (t, J¼7.8 Hz, 1H),
4.13–4.05 (m, 1H), 3.86–3.81 (m, 1H), 3.03 (dd, J¼13.5,
7.2 Hz, 1H), 2.89 (dd, J¼13.5, 8.0 Hz, 1H), 2.14–2.00 (m,
2H), 1.41 (s, 9H); 13C NMR (50 MHz, CDCl3, 5% CD3OD)
d 173.4, 155.9, 154.8, 136.3, 135.0, 129.3, 128.7, 128.5,
127.1, 80.7, 70.1, 63.0, 55.9, 50.2, 38.2, 31.4, 28.2; IR
(CDCl3) 2979.2, 1750.7, 1670.8, 1265.3, 1203.2 cmꢂ1; LC–
MS (ESI) 494.3 [M+Na]+, 472.3 [M+H]+; HRMS (FAB) m/z
calcd for C25H34N3O6 [MH]+ 472.2442, found 472.2416.
1
mp 82–83.5 ꢀC. H NMR (200 MHz, CD3OD) d 7.34–7.26
(m, 5H), 4.35–4.27 (m, 1H), 4.25–4.17 (m, 1H), 3.34 (m,
2H), 3.09–2.92 (m, 2H), 2.03–1.66 (m, 2H), 1.41 (s, 9H);
13C NMR (50 MHz, CDCl3) d 174.4, 172.4, 155.9, 136.7,
129.4, 128.7, 127.1, 80.5, 58.9, 56.6, 50.9, 38.3, 34.6,
28.3; IR (CDCl3) 3303.1 (br), 2978.1, 1664.3, 1523.4,
1367.4, 1167.4 cmꢂ1; LC–MS (ESI) 731.5 [2M+H]+,
366.3 [MH]+, 310.2; HRMS (FAB) m/z calcd for
C18H28N3O5 [MH]+ 366.2023, found 366.2008.
4.1.12. Boc-Phe-(R)-gHse(TBDPS)-NH2$TFA (21). The
compound was synthesized from 18 (473.8 mg,
0.79 mmol) using the procedure described above in Section
4.1.5. The crude product was triturated with Et2O to give
a pale yellow solid (519.4 mg, 96%); mp 107–108.5 ꢀC.
1H NMR (200 MHz, CDCl3) d 7.69–7.12 (m, 15H), 6.34
(br s, 1H), 5.08 (br s, 1H), 4.85 (dd, J¼12.6, 5.8 Hz, 1H),
4.22–4.18 (m, 1H), 3.80–3.64 (m, 2H), 3.03–2.98 (m, 2H),
1.70–1.61 (m, 2H), 1.43 (s, 9H), 1.09 (s, 9H); 13C NMR
(50 MHz, CDCl3) d 173.8, 171.6, 162.6, 156.0, 136.4,
135.6, 133.4, 132.9, 130.0, 129.2, 128.8, 127.9, 127.0,
80.5, 61.7, 57.2, 51.8, 37.8, 33.1, 28.3, 27.1, 19.3; IR
(CDCl3) 3316.3, 2932.0, 1674.4, 1531.8, 1429.1, 1203.0,
1114.0 cmꢂ1; LC–MS (ESI) 576.44 [M+H]+, 559.4 [MH]+
(100%), 503.3, 459.3, 312.2; HRMS (FAB) m/z calcd for
C33H46N3O4Si [MH]+ 576.3252, found 576.3215.
4.1.9. Boc-Phe-(R)-Hse(Cbz)-NH2 (16). To a solution of 14
(381.6 mg, 1.04 mmol) in CH2Cl2 (5 mL) and pyridine
(2.5 mL) at 0 ꢀC was added Cbz-Cl (450 mL, 3.13 mmol).
The solution was stirred at 0 ꢀC for 30 min and at room tem-
perature for 6 h. After evaporation of solvent in vacuo, the
residue was dissolved in EtOAc (100 mL), washed with
5% citric acid, saturated NaHCO3, and saturated NaCl, dried
over Na2SO4, and evaporated in vacuo. The residue was pu-
rified by flash column chromatography (CH2Cl2/CH3OH,
50:1) to give a white solid (481 mg, 92%); mp 156.5–
158 ꢀC. 1H NMR (200 MHz, CDCl3) d 7.39–7.09 (m,
12H), 5.36 (d, J¼7.0 Hz, 1H), 5.15 (s, 2H), 4.53–4.43 (m,
1H), 4.22 (dd, J¼14.6, 7.2 Hz, 1H), 3.98 (t, J¼5.6 Hz,
2H), 3.03–2.96 (m, 2H), 2.13–2.03 (m, 1H), 1.95–1.82 (m,
1H), 1.38 (s, 9H); 13C NMR (50 MHz, CDCl3) d 173.7,
172.3, 156.1, 155.0, 136.4, 135.2, 129.2, 128.7, 128.4,
127.0, 80.5, 69.8, 64.6, 56.6, 50.0, 38.1, 30.5, 28.2; IR
(CDCl3) 3296.6, 2977.6, 1747.0, 1666.9, 1498.1, 1455.3,
1266.3, 1167.0, 910.4 cmꢂ1; LC–MS (ESI) 500.3, 444.2,
400.2; HRMS (FAB) m/z calcd for C26H34N3O7 [MH]+
500.2391, found 500.2360.
4.1.13. Boc-Phe-(R)-gHse-NH2 ((R)-11). Method I. To a so-
lution of (R)-10 (1.44 g, 2.66 mmol) in methanol (50 mL)
was added 0.2 N HCl (w5 mL) until pH 3, followed by the
addition of 10% Pd–C (0.15 g). The reaction mixture was
shaken under 50 psi H2 for 48 h and no more starting mate-
rial was detected by TLC (CH2Cl2/CH3OH, 5:1). The cata-
lyst was removed by filtration through Celite 545 and the
filtrate was concentrated in vacuo. The residue (a white
foamy solid) was dissolved in 5 mL of methanol and passed
through a Varian Bond ElutÒ cation exchange cartridge (1 g,
4.1.10. Boc-Phe-(R)-Hse(TBDPS)-NH2 (18). To a solution
of 14 (318.5 mg, 0.87 mmol) in DMF (1 mL) at 0 ꢀC were

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Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
SCX, benzenesulfonic acid sorbent) according to the in-
structions in the manual. The cartridge was washed by meth-
anol and the product was eluted using 1% TEA in methanol
to give a white solid (601 mg, 67%) after solvent evaporation
in vacuo. Method II. To a solution of 20 (435.2 mg,
0.743 mmol) in methanol (30 mL) was added 10% Pd–C.
H2 (60 psi) was established and maintained for 4.5 h. The
catalyst was filtered through Celite 545 and the filtrate was
concentrated in vacuo to give a white solid (307 mg, 98%).
Method III. To a stirred solution of 21 (506 mg,
0.73 mmol) in THF (15 mL) under argon at 0 ꢀC was added
Bu4N+Fꢂ (2.57 mL, 1 M in THF). The solution was stirred at
0 ꢀC for 5 min and at room temperature for 1 h. Solvent was
evaporated in vacuo and the residue was purified by flash
column chromatography (CH2Cl2/CH3OH/TEA, 50:1:0.1)
63.5 (d, J¼5.2 Hz), 57.9, 55.2, 41.1, 37.5, 29.7, 26.8; 31P
NMR (121 MHz, CD3OD) d 11.6 (s); IR (KBr) 2974.4,
2379.5, 1666.7, 1410.3, 1364.1, 1230.8, 1164.1 cmꢂ1; LC–
MS (ESI) 559.2, 557.2, 555.2 [M+Na]+, 523.2 [M+1]+;
HRMS (FAB) m/z calcd for C21H34N4O5PCl2 [MH]+
523.1644, found 523.1653.
Compound (2R,4S)-6: white solid; Rf (hexane/EtOAc/
CH3OH, 1:1:0.1) 0.20. 1H NMR (200 MHz, CD3OD)
d 7.30–7.22 (m, 5H), 5.34–5.30 (m, 1H), 4.25 (m, 3H),
3.68–3.62 (m, 4H), 3.50–3.40 (m, 4H), 3.04–2.90 (m, 2H),
1.80–1.61 (m, 2H), 1.42 (s, 9H); 13C NMR (50 MHz,
CD3OD) d 172.0, 136.4, 128.6, 127.6, 126.0, 78.9, 63.5 (d,
J¼6.1 Hz), 57.7, 55.5, 41.1, 37.7, 29.9, 26.9; 31P NMR
(121 MHz, CD3OD) d 13.6 (s); IR (KBr) 3405.1 (br),
2974.4, 1671.8, 1364.1, 1251.3, 1215.4, 1164.1,
1046.2 cmꢂ1; LC–MS (ESI) 559.2, 557.2, 55.2 [M+Na]+,
527.2, 525.3, 523.2 [M+H]+ (100%); HRMS (FAB) m/z calcd
for C21H34N4O5PCl2 [MH]+ 523.1644, found 523.1643.
1
to give a white solid (95.7 mg, 77%); mp 70.5–72 ꢀC. H
NMR (200 MHz, CD3OD) d 7.38–7.24 (m, 5H), 5.18–5.06
(m, 1H), 4.33–4.26 (m, 1H), 3.60–3.40 (m, 3H), 3.03 (dd,
J¼13.9, 6.6 Hz, 1H), 2.90 (dd, J¼13.9, 8.2 Hz, 1H), 2.01–
1.80 (m, 2H), 1.40 (s, 9H); 13C NMR (50 MHz, CD3OD)
d 172.1, 155.6, 136.6, 128.5, 127.6, 125.9, 78.8, 57.6,
57.0, 55.7, 37.6, 37.0, 26.8; IR (KBr) 3293.2 (br), 2976.9,
1653.4, 1532.8, 1251.0, 1168.3, 1057.0, 753.0,
699.9 cmꢂ1; LC–MS (ESI) 675.5 [2M+1]+, 338.2 [M+H]+,
265.1; HRMS (FAB) m/z calcd for C17H28N3O4 [MH]+
338.2074, found 338.2061.
4.1.16. Boc-Phe-(4R)-4-NH-CPA ((2R,4R)-6 and (2S,4R)-
6). Compound (S)-11 (178 mg, 0.53 mmol) was reacted
with bis(2-chloroethyl)phosphoramidic dichloride (259 mg,
0.58 mmol) in the presence of TEA (163 mL, 1.2 mmol) ac-
cording to the procedure described above in Section 4.1.15.
The products were isolated by flash column chromatography
(hexane/EtOAc/CH3OH, 2:1:0.1) to give Boc-Phe-NH2
(8.7 mg, 25%), (2R,4R)-6 (34 mg, 13%) and (2S,4R)-6
(14 mg, 6%). The yield was calculated after the recovery
of 11% of the starting material.
4.1.14. Boc-Phe-(S)-gHse-NH2 ((S)-11). The compound
was synthesized from (S)-10 (2.23 g, 4.12 mmol) according
to the method I in Section 4.1.13 as a white solid (986 mg,
71%); mp 51–52.5 ꢀC. 1H NMR (200 MHz, CD3OD) d 7.25
(m, 5H), 4.69 (t, J¼6.5 Hz, 1H), 4.22 (dd, 1H, J¼8.0,
6.0 Hz), 3.51 (t, J¼6.6 Hz, 2H), 3.06 (dd, J¼13.4, 6.3 Hz,
1H), 2.84 (d, J¼13.4, 8.0 Hz, 2H), 1.69 (m, 2H), 1.30 (s,
9H); 13C NMR (50 MHz, CD3OD) d 172.2, 155.8, 136.9,
128.7, 127.8, 127.6, 125.9, 78.8, 57.7, 57.0, 55.7, 37.5,
37.2, 26.9; IR (CDCl3) 3297.6 (br), 2977.8, 1657.8, 1531.4,
1367.0, 1250.7, 1168.9, 1055.7, 755.0 cmꢂ1; LC–MS (ESI)
675.5 [2M+1]+, 338.5 [M+H]+ (100%); HRMS (FAB) m/z
calcd for C17H28N3O4 [MH]+ 338.2074, found 338.2062.
Compound (2R,4R)-6: white solid; Rf (hexane/EtOAc/
CH3OH, 1:1:0.1) 0.60. 1H NMR (200 MHz, CD3OD)
d 7.34–7.20 (m, 5H), 5.32 (dt, J¼20.6, 4.8 Hz, 1H), 4.40–
4.18 (m, 3H), 3.76–3.64 (m, 4H), 3.57–3.36 (m, 4H),
3.20–3.10 (m, 1H), 2.92–2.80 (m, 1H), 2.25–2.13 (m, 1H),
1.95–1.87 (m, 1H), 1.40 (s, 9H); 31P NMR (121 MHz,
CD3OD) d 10.7 (s); 13C NMR (50 MHz, CDCl3) d 174.1,
171.5, 155.4, 136.9, 129.5, 129.4, 128.7, 128.6, 127.0,
126.9, 80.2, 63.7, 59.1, 55.6, 48.7 (d, J¼5.0 Hz), 42.3,
38.5, 30.1, 28.4; IR (KBr) 3295.9, 2978.3, 2931.7, 1682.0,
1497.2, 1454.7, 1367.0, 1246.7 (P]O), 1168.4, 910.3,
734.2 cmꢂ1; LC–MS (ESI) 547.0, 545.25 [M+Na]+, 523.1
[M+H]+; HRMS (FAB) m/z calcd for C21H34N4O5PCl2
[MH]+ 523.1644, found 523.1634.
4.1.15. Boc-Phe-(4S)-4-NH-CPA ((2R,4S)-6 and (2S,4S)-
6). To a stirred solution of bis(2-chloroethyl)phosphor-
amidic dichloride (222 mg, 0.86 mmol) in anhydrous THF
(20 mL) under argon at 0 ꢀC was added a solution of (R)-
11 (263 mg, 0.78 mmol) and TEA (241 mL, 1.71 mmol) in
anhydrous THF (20 mL). The reaction solution was stirred
at 0 ꢀC for 30 min and at room temperature for 48 h. The pre-
cipitate in the solution was filtered and the solution was
evaporated in vacuo. The residue was purified by flash col-
umn chromatography (hexane/EtOAc/CH3OH, 2:1:0.1) to
give Boc-Phe-NH2 (65 mg, 38%), (2S,4S)-6 (the faster elut-
ing isomer) (69 mg, 20%) and (2R,4S)-6 (the slower eluting
isomer) (27 mg, 8%). The yield was calculated after the
recovery of 15% of the starting material.
Compound (2S,4R)-6: white solid; Rf (hexane/EtOAc/
CH3OH, 1:1:0.1) 0.20. 1H NMR (200 MHz, CD3OD)
d 7.28 (s, 5H), 5.43–5.25 (m, 1H), 4.37–4.25 (m, 3H),
3.65–3.50 (m, 4H), 3.44–3.33 (m, 4H), 3.08 (dd, J¼13.6,
5.4 Hz, 1H), 2.87 (dd, J¼13.6, 8.6 Hz, 1H), 2.20–1.80 (m,
2H), 1.38 (s, 9H); 13C NMR (50 MHz, CD3OD) d 172.2,
155.8, 136.7, 128.6, 127.6, 126.0, 78.9, 63.6 (d, J¼5.8 Hz),
57.9, 55.5, 41.1, 37.5, 29.9, 26.8; 31P NMR (121 MHz,
CD3OD) d 13.7 (s); IR (CDCl3) 3277.5, 2976.5, 1670.4,
1498.2, 1455.0, 1366.8, 1218.3 (P]O), 1167.4, 988.3,
754.7 cmꢂ1; LC–MS (ESI) 549.1, 547.1, 545.1 [M+Na]+,
527.2, 525.1, 523.2 [MH]+ (100%); HRMS (FAB) m/z calcd
for C21H34N4O5PCl2 [MH]+ 523.1644, found 523.1629.
Compound (2S,4S)-6: white solid; Rf (hexane/EtOAc/
CH3OH, 1:1:0.1) 0.45. 1H NMR (200 MHz, CD3OD)
d 7.34–7.19 (m, 5H), 5.24 (dt, J¼20.6, 4.4 Hz, 1H), 4.35–
4.25 (m, 1H), 4.13–4.04 (m, 2H), 3.68–3.62 (m, 4H),
3.51–3.37 (m, 4H), 3.19–2.78 (m, 2H), 2.05–1.93 (m, 1H),
1.64–1.55 (m, 1H), 1.42 (s, 9H); 13C NMR (50 MHz,
CDCl3) d 172.2, 156.0, 136.7, 128.6, 127.6, 126.0, 78.9,
4.1.17. H-Phe-(2S,4S)-4-NH-CPA$TFA ((2S,4S)-4). Com-
pound (2S,4S)-6 (8.1 mg, 0.016 mmol) was treated with
25% (v/v) TFA in CH2Cl2 for 30 min at room temperature.

Y. Jiang et al. / Tetrahedron 63 (2007) 10637–10645
10645
After evaporation of solvents, the residue was dried under
Acknowledgements
vacuum to give a white semi-solid (8.3 mg, 100%). 1H
NMR (400 MHz, CD3OD) d 7.51–7.39 (m, 5H), 5.29 (dt,
J¼11.8, 2.0 Hz, 1H), 4.47–4.39 (m, 1H), 4.35–4.26 (m,
1H), 4.15 (t, J¼3.8 Hz, 1H), 3.77 (t, J¼3.6 Hz, 4H), 3.61–
3.47 (m, 4H), 3.27 (dd, J¼6.8, 4.0 Hz, 1H), 3.21 (dd,
J¼6.8, 3.8 Hz, 1H), 2.31–2.22 (m, 1H), 1.70–1.66 (m,
1H); 13C NMR (50 MHz, CD3OD) d 167.7, 133.7, 128.6,
128.2, 127.0, 62.7 (d, J¼6.4 Hz), 58.2 (d, J¼3.0 Hz), 53.8,
40.9, 36.6, 28.2 (d, J¼6.5 Hz); 31P NMR (121 MHz,
CD3OD) d 11.8 (s); LC–MS (ESI) 847.2, 427.2, 425.2,
423.2 [MH]+ (100%), 203.2; HRMS (FAB) m/z calcd for
C16H26N4O3PCl2 [MH]+ 423.1120, found 423.1140.
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
CancerCenteroftheCancerInstituteofNew Jersey, andgrant
RSG-03-004-01-CDD from the American Cancer Society.
References and notes
1. Colvin, O. M. Curr. Pharm. Des. 1999, 5, 555–560.
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3. Viljanen, T.; Tahtinen, P.; Pihlaja, K.; Fulop, F. J. Org. Chem.
1998, 63, 618–627.
4.1.18. H-Phe-(2R,4R)-4-NH-CPA$TFA ((2R,4R)-4). The
compound was prepared from (2R,4R)-6 (3.5 mg,
0.0067 mmol) according to the procedure described above
in Section 4.1.17 as a semi-solid (3.6 mg, 100%). 1H NMR
(200 MHz, CD3OD) d 7.38–7.23 (m, 5H), 5.30 (dt,
J¼21.2, 4.8 Hz, 1H), 4.53–4.10 (m, 2H), 3.71–3.64 (m,
5H), 3.50–3.34 (m, 4H), 3.13 (dd, J¼13.9, 5.0 Hz, 1H),
2.88 (dd, J¼13.9, 8.2 Hz, 1H), 2.27–2.12 (m, 1H), 1.91–
1.80 (m, 1H); 13C NMR (50 MHz, CD3OD) d 173.2,
136.3, 128.7, 127.8, 126.1, 63.0 (d, J¼6.5 Hz), 58.0 (d,
J¼2.3 Hz), 40.9, 39.4, 29.0 (d, J¼6.8 Hz); 31P NMR
(121 MHz, CD3OD) d 11.2 (s); LC–MS (ESI) 847.2
[2M+1]+, 425.1, 423.1 [M+1]+ (100%), 203.0; HRMS
(FAB) m/z calcd for C16H26N4O3PCl2 [MH]+ 423.1120,
found 423.1123.
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4.1.19. H-Phe-(2R,4S)-4-NH-CPA$TFA ((2R,4S)-4). Com-
pound (2R,4S)-6 (19.5 mg, 0.0373 mmol) was treated with
25% (v/v) TFA/CH2Cl2 to give a mixture as a semi-solid
(17 mg, 96%). The mixture was separated by preparative
HPLC to give (2R,4S)-4 (6 mg, 37%) and (2R,4R)-4
(3.5 mg, 22%). The remaining amount was recovered as
a mixture of the two compounds. (2R,4S)-4: 1H NMR
(200 MHz, CD3OD) d 7.52–7.39 (m, 5H), 5.54–5.50 (m,
1H), 4.48–4.41 (m, 1H), 4.36–4.27 (m, 1H), 3.78–3.74 (m,
4H), 3.58–3.51 (m, 4H), 2.27–3.2 (m, 2H); 13C NMR
(50 MHz, CD3OD) d 167.3, 133.6, 128.6, 128.2, 127.0,
63.4 (d, J¼5.6 Hz), 58.0, 53.7, 41.0, 36.8, 29.7 (d,
J¼4.6 Hz); 31P NMR (121 MHz, CD3OD) d 11.2 (s); LC–
MS (ESI) 16.0 min, 847.1 [2M+1]+, 455.3, 425.3, 423.3
[MH]+, 203.3; HRMS (FAB) m/z calcd for C16H26N4O3PCl2
[MH]+ 423.1120, found 423.1135.
16. Loudon, G. M.; Radhakrishna, A. S.; Almond, M. R.; Blodgett,
J. K.; Boutin, R. H. J. Org. Chem. 1984, 49, 4272–4276.
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4.1.20. H-Phe-(2S,4R)-4-NH-CPA$TFA ((2S,4R)-4). Com-
pound(2S,4R)-6(12 mg, 0.0229 mmol)wastreatedwith25%
(v/v) TFA/CH2Cl2 to give a mixture as a white solid (11.3 mg,
89%). The mixture was separated by preparative HPLC to
give (2S,4R)-4 (3 mg, 30%) and (2S,4S)-4 (2.5 mg, 25%).
The remaining amount was recovered as a mixture of the
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1
two compounds. (2S,4R)-4: H NMR (200 MHz, CD3OD)
d 7.40–7.23 (m, 5H), 5.45–5.35 (m, 1H), 4.37–4.20 (m,
2H), 3.75–3.57 (m, 5H), 3.51–3.42 (m, 4H), 3.05 (dd,
J¼13.6, 6.2 Hz, 1H), 2.86 (dd, J¼13.6, 7.8 Hz, 1H), 2.09–
1.83 (m, 2H); 13C NMR (50 MHz, CD3OD) d 173.7, 136.5,
128.6, 127.8, 126.1, 63.5 (d, J¼5.7 Hz), 57.8, 55.3, 41.0,
40.0, 29.8 (d, J¼4.6 Hz); 31P NMR (121 MHz, CD3OD)
d 15.5 (s); LC–MS (ESI) 847.2 [2M+1]+, 425.1, 423.1
[M+H]+, 203.1 (100%); HRMS (FAB) m/z calcd for
C16H26N4O3PCl2 [MH]+ 423.1120, found 423.1135.
27. Lott, R. S.; Chauhan, V. S.; Stammer, C. H. J. Chem. Soc.,
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