Novel bile acid derived H-phosphonate conjugates: Synthesis and spectroscopic characterization

Article abstract of DOI:10.1002/hc.20447

The H-phosphonate bioconjugates of bile acids, conjugated with various alcohols and nucleosides, were obtained in one pot by a tandem transesterification with diphenyl phosphite (DPP). The synthesis of cholic acid derived phosphoramide from the correspond

Full text of DOI:10.1002/hc.20447

Heteroatom Chemistry
Volume 19, Number 4, 2008
Novel Bile Acid Derived H-Phosphonate
Conjugates: Synthesis and Spectroscopic
Characterization
Yan Li, Weijun Chu, and Yong Ju
Key Laboratory of Bio-organic Phosphorus Chemistry & Chemical Biology, Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China
Received 9 October 2007; revised 25 December 2007, 27 February 2008
make these compounds attracting building blocks
ABSTRACT: The H-phosphonate bioconjugates of
for the construction of environmentally responsive
bile acids, conjugated with various alcohols and nu-
amphiphiles [5] and functional supramolecular sys-
cleosides, were obtained in one pot by a tandem trans-
tems [6].
esterification with diphenyl phosphite (DPP). The syn-
It is especially true for the case of cholic acid
thesis of cholic acid derived phosphoramide from
1, which is one of the primary bile acids found
the corresponding H-phosphonate was also demon-
in human and a rare naturally occurring molecule
strated. The structures of these novel conjugates were
exhibiting facial amphiphilicity [5]. It possesses
confirmed on the basis of IR,31P NMR, 1H NMR,
one carboxylic acid and three hydroxyl groups,
and mass spectra. The synthesized bile acid conju-
which render various chemical modifications possi-
gates were mixtures of diastereoisomers due to the
ble. However, one of the major problems associated
ꢀ
C
chirality of the phosphorus.
2008 Wiley Periodicals,
with the synthesis of cholic acid conjugates is that
these “natural” hydroxyl groups are relatively inert,
especially the 7α- and 12α-OH [6]. A number of ar-
ticles have reported the conversion of the hydrox-
yls to other functional groups. For instance, Davis
et al. have used 11 high yielding steps to replace
three hydroxyls by amine groups [7]. The “triamino-
analogue” of methyl cholate is explored as a pre-
cursor to design anion receptors and a scaffold for
combinatorial chemistry [8]. Savage and his cowork-
ers have reported cholic acid derived anionic facial
amphiphiles with three carboxyl groups, which can
aggregate at very low concentration [9]. To develop
new cross-linking reagents in polymeric gels, Zhu’s
group has prepared cholic acid derivatives with mul-
tiple methacrylate groups by the use of methacrylic
acid, methacryloyl chloride, and methacryloyl anhy-
dride as acylating agents [10].
Inc. Heteroatom Chem 19:402–407, 2008; Published on-
line in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hc.20447
INTRODUCTION
Bile acid is a class of biogenetic compounds and
plays a prominent role in biological systems [1].
The medicinal properties of bile acids and their con-
jugates have been discussed in the literature with
promising results [2–4]. Besides their biological im-
portance, the rigid concave of the hydrophobic back-
bone and the unique disposition of hydroxyl groups
Correspondence to: Yong Ju; e-mail: juyong@tsinghua.edu.cn.
Contract grant sponsor: National Science Foundation of China.
Contract grant number: 20772071.
Contract grant sponsor: Specialized Research Fund for the
Doctoral Program of Higher Education.
H-Phosphonate has been proven to be a ver-
satile building block in organic synthesis. It can
be further transferred to phosphate, phosphoroth-
ioate, and phosphoroselenoate by oxidation [11]
Contract grant number: 20050003104.
c
ꢀ
2008 Wiley Periodicals, Inc.
402

Novel Bile Acid Derived H-Phosphonate Conjugates: Synthesis and Spectroscopic Characterization 403
RESULTS AND DISCUSSION
The general synthetic strategy for bile acid H-
phosphonates conjugates is shown in Schemes 2 and
3. Our approach starts from methyl cholate because
it can be prepared easily in large quantities and has
much better solubility in pyridine than cholic acid.
Many other bile acid derived esters and amides are
also appropriate starting materials. The reaction was
monitored by ESI-MS and 31P NMR. The first step
of transesterification is fast, and all hydroxyl groups
were converted to mono-phenyl H-phosphonates 4
in 5 h under anhydrous condition, evidenced by the
peak of ion [M + Na]+at 866 m/z on the ESI-MS spec-
trometry. However, this intermediate is very unsta-
ble and could not to be isolated. The second step
transesterification between the mono-phenyl phos-
phonates and the corresponding alcohol is relatively
slow and is sensitive to steric hindrance, thereby ef-
fectively preventing the formation of di-steroid H-
phosphonate.
The deoxycholic acid derived H-phosphonate
conjugates were also synthesized in a similar proce-
dure. It is worth mentioning that some bioactive nu-
cleosides, such as 3ꢁ-azido-3ꢁ-deoxythymidine (AZT)
and 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxythymidine (D4T),
possess one primary alcohol group. These nucleo-
sides can also be attached to the deoxycholic acid
methyl ester using similar tandem transesterifica-
tion with DPP. Bile acids are interesting carriers that
target a specific organ and improve the lipophilicity
for drug molecules [2]. These compounds contain-
ing both steroid and nucleosides have the potential
to act as prodrugs or drugs by themselves. However,
SCHEME 1 Chemical structure of cholic acid and diphenyl
phosphite.
as well as phosphoramidate by the Atherton–Todd
reaction in high yield [12]. In addition, phosphate
derivatives are important intermediates in biologi-
cal metabolism [13] and are widely used in phar-
maceutical industry [14]. Diphenyl phosphite (DPP,
2; see Scheme 1), which can undergo fast trans-
esterification with various alcohols in pyridine, is
used as an effective phosphorylation reagent in
the synthetic process [15]. To obtain mono-phenyl
H-phosphonates, a large excess of DPP (7 equiv.)
was required to prevent the formation of double-
exchange H-phosphonates [16]. However, our previ-
ous work showed that if one of the hydroxyl com-
ponents is sterically hindered moiety, the desired
mono-phenyl H-phosphonate can be obtained in
high yield [17]. The hydroxyls on the bile acids are
oriented along one face and are typical steric hin-
dered groups. Taking account of these facts, bile
acids are ideal starting structures to prepare multi-
functional scaffolds containing phosphorus. In this
paper, we report the synthesis and characterization
of H-phosphonates derived bile acid conjugates.
SCHEME 2 Synthesis and chemical structure of H-phosphonate derivatives of cholic acid. (i) DPP, py, 0◦C, 5 h; (ii) propargyl
alcohol, py, 8 h; (iii) i-PrOH, py, 8 h.
Heteroatom Chemistry DOI 10.1002/hc

404 Li et al.
SCHEME 3 Chemical structure of H-phosphonate derivatives of deoxycholic acid.
three relatively large nucleoside moieties cannot be
attached to the steroid nucleus simultaneously due
to the steric hindrance. When the same reaction con-
dition was applied to cholic acid methyl ester, only
a complex mixture was obtained and could not be
purified by chromatography on a silica gel.
Furthermore, the related bile acid derivatives
could be easily obtained from the corresponding
H-phosphonate. The synthesis of phosphoramidate
with three molecules of L-phenylalanine methyl es-
ter was demonstrated as an example, as shown in
Scheme 4. Despite the large steric hindrance, the re-
action proceeded well using the improved Atherton–
Todd method [12]. The end of the reaction can
be easily detected by the observation of the disap-
pearance of the 1 J coupling constant (710 Hz) in
the 31P NMR spectrum (coupled mode). The result-
ing phosphoramidate is tolerant to moisture and
is much more stable than the corresponding H-
phosphonates.
The structures of these newly synthesized H-
phosphonate and phosphoramidate bile acid con-
jugates have been well confirmed by IR, 31P NMR,
1H NMR, and HRMS. Because the phosphorus in
every H-phosphonate group is chiral and can read-
ily change configuration between R and S, one bile
acid H-phosphonates conjugate was in fact the mix-
ture of several diastereoisomers [17,18]. Consider-
ing the fact that the corresponding carbons on the
steroid nucleus (3-C, 7-C, and 12-C) are also chi-
ral atoms, the splitting caused by the presence of
diastereoisomers can be observed in both 31P and
1H NMR spectra. Figure 1 presents the character-
istic resonance peaks in the cholic acid derivatives.
From the NMR spectra, it was found that the 3-H,
7-H, and 12-H moved toward the lower field after
SCHEME 4 Synthesis and chemical structure of cholic acid derived phosphoramide conjugate.
Heteroatom Chemistry DOI 10.1002/hc

Novel Bile Acid Derived H-Phosphonate Conjugates: Synthesis and Spectroscopic Characterization 405
FIGURE 1 Comparison of the 600 MHz 1H NMR spectra of compounds 3, 5, and 12 in CDCl3. The insert shows the detailed
resonance peaks of 5 in the downfield. The signal of 7-H in compounds 5 and 12 was overlapped with propargyl protons
according to 1H-1H COSY spectra.
phosphorylation. However, a slight reverse shift was
observed when H-phosphonate was transferred into
the phosphoramide group. In addition, the sharp
peaks of 12-H and 7-H in the steroid nucleus were re-
placed by broader peaks in the H-phosphonate con-
jugate 5 and in the phosphoramidate conjugate 12,
indicating the presence of diastereoisomers.
tons connected with phosphorus. Every small group
was further splitted into two peaks, also suggesting
the existence of diastereoisomers.
In conclusion, bile acids, which possess up to
three steric hindered hydroxyl groups, have pro-
vided a unique structure for the synthesis of H-
phosphonate derivatives. The above protocols for the
preparation of H-phosphonate and phosphorami-
date conjugates involve mild and efficient chemi-
cal transformations, which make multiple modifi-
cations at the steroid nucleus possible. According to
NMR studies, the protons directly connected with
phosphorus show distinguishable signals and the
resulting products were a mixture of several di-
astereoisomers. To the best of our knowledge, this
is the first report about direct phosphorylation on
The protons directly connected with phosphorus
1
are easily identified by the JP−H coupling constant
1
(about 700 Hz) in the downfield of H NMR spec-
trum. In 600 MHz NMR spectrum, resonance peaks
of the three protons show two main groups due to
the large 1 JP−H coupling. It is interesting to note that
each of the two groups can be divided into three
small groups with same integral area, indicating that
the distinguishable chemical shift of the three pro-
Heteroatom Chemistry DOI 10.1002/hc

406 Li et al.
the hydroxyl groups of bile acids. On the basis of the
preliminary results, the extension of this method-
ology may find applications in pharmacology and
supramolecular chemistry.
J = 710 Hz, 3ꢁ-PH), 6.91, 6.93∗ (d, 1H, J = 710 Hz, 7ꢁ-
PH), 6.96, 6.99∗ (d, 1H, J = 710 Hz, 12ꢁ-PH); HRMS
(ESI) found: 946.2994, [C34H51O11P3 + NH4]+ calcd:
946.2982. (*The chemical shift of the diastereoiso-
mer)
EXPERIMENTAL
1H NMR spectra were recorded at 300 MHz or
600 MHz for protons on JOEL JNM-ECA300 or
JNM-ECA600 spectrometer. Chemical shifts (δ) are
given in ppm relative to TMS (δ = 0.0) and cou-
pling constants (J) are given in Hz. 31P NMR spec-
tra were recorded on a Bruker 200 MHz operating
at 81 MHz with 85% phosphoric acid (δ = 0.0) as
external standard. The ESI-HRMS was measured
on Bruker APEX spectrometer in positive mode. IR
spectra were recorded on AVATAR 360 ESP FTS
spectrophotometer with KBr pellets. Melting points
were determined with XRC-1 micro melting point
apparatus and were uncorrected.
Cholic acid, deoxycholic acid, and diphenyl
phosphite (DPP) were purchased from Sigma Com-
pany (Beijing-order Branch, People’s Republic of
China) and used without further purification. Pyri-
dine was purchased from Beijing Chemicals Co.
(Beijing, People’s Republic of China) and redistilled
prior to use. Cholic acid methyl ester (CAME, 3) and
deoxycholic acid methyl ester (DCAME, 7) were pre-
pared according to a previous reported procedure
[19]. A typical procedure for the synthesis of bile
acid derived H-phosphonates and phosphoramidate
is represented by conjugates 5 and 12, respectively.
Methyl 3α,7α,12α-tri(isopropyl-H-phosphonate)-
5β-cholan-24-ate (6)
Yield: 52%; colorless oil; IR (cm−1): 2940, 2872, 2424,
2338, 1735, 1376, 1256, 974; 31P NMR (81 MHz,
CDCl3): δ = 4.93, 5.79, 6.33; 1H NMR (600 MHz,
CDCl3): δ = 0.66 (s, 3H, 18-CH3), 0.84 (s, 3H, 19-
CH3), 0.90–0.91 (d, 3H, J = 6 Hz, 21-CH3), 0.97–1.94
(m, 38H, aliphatic H), 2.14–2.32 (m, 4H, 2,4-CH2),
3.59 (s, 3H, OCH3), 4.04–4.09 (m, 1H, 3ꢁ-POCH),
4.21 (br, s, 1H, 3α-CH), 4.53–4.55 (m, 1H, 7α-CH),
4.63–4.70 (m, 2H, 7ꢁ,12ꢁ-POCH), 4.74–4.76 (m, 1H,
12α-CH), 6.75 (m, 3H, J = 690 Hz, 3 × PH); HRMS
(ESI) found: 958.3910, [C34H63O11P3 + NH4]+ calcd:
958.3921.
Methyl 3α,12α-di(propargyl-H-phosphonate)-5β-
deoxycholan-24-ate (8)
Yield: 57%; light-yellow oil; IR (cm−1): 3459, 3293,
3224, 2943, 2868, 2435, 2125, 1735, 1251, 976; 31P
NMR (81 MHz, CDCl3): δ = 6.94, 7.94; 1H NMR (300
MHz, CDCl3): δ(ppm) = 0.67 (s, 3H, 18-CH3), 0.86
(s, 3H, 19-CH3), 0.92 (d, 3H, J = 6 Hz, 21-CH3),
0.98–1.97 (m, 26H, aliphatic H), 2.57–2.61 (m, 2H,
2 × C H), 3.64 (s, 3H, OCH3), 4.43 (br, s, 1H,
Methyl 3α,7α,12α-tri(propargy-H-phosphonate)-
5β-cholan-24-ate (5)
3α-CH), 4.64–4.69 (m, 4H, 2 × CH C CH), 4.79–
2
4.85 (m, 1H, 12α-CH), 6.86 (s, 1H, J = 710 Hz, 3ꢁ-
PH), 7.00, 7.01∗ (d, 1H, J = 710 Hz, 12ꢁ-PH); ESI-
MS (+): m/z 633 [M + Na]+, 649 [M + K]+; HRMS
(ESI) found: 628.3163, [C31H48O8P2 + NH4]+ calcd:
628.3163.
Under an argon atmosphere, CAME (254 mg, 0.6
mmol) dissolved in pyridine (5 mL) was added drop-
wise to DPP (534 mg, 2.0 mmol) solution in 4 mL dry
pyridine at −10◦C. The resulting mixture was stirred
at room temperature for 5 h, and propargyl alcohol
(0.129 mL, 2.2 mmol) was syringed. After stirring for
additional 7 h, the solvent was evaporated under vac-
uum and the obtained residue was purified on a col-
umn (silica gel; CH2Cl2: AcOEt = 7:2) to provide the
desired product (244 mg, 0.34 mmol, light-yellow oil)
in 56% yield. IR (cm−1): 3449, 3293, 3228, 2947, 2946,
2873, 2434, 2125, 1735, 1251, 977; 31P NMR (81 MHz,
CDCl3): δ = 6.92, 7.25, 7.99, 8.08; 1H NMR (600 MHz,
CDCl3): δ = 0.67(s, 3H, 18-CH3), 0.85 (s, 3H, 19-CH3),
0.91–0.92 (d, 3H, J = 6 Hz, 21-CH3), 0.98–1.97 (m,
20H, aliphatic H), 2.14–2.32 (m, 4H, 2,4-CH2), 2.56–
2.64 (m, 3H, 3 × C H), 3.60 (s, 3H, OCH3), 4.26
Methyl 3α,12α-di(isopropyl-H-phosphonate)-5β-
deoxycholan-24-ate (9)
Yield: 60%; colorless oil; IR (cm−1): 2978, 2868,
2429, 1736, 1375, 1240, 965; 31P NMR (81 MHz,
CDCl3): δ = 5.19, 6.20, 6.62; 1H NMR (300 MHz,
CDCl3): δ = 0.66 (s, 3H, 18-CH3), 0.84 (s, 3H, 19-CH3),
0.91 (d, 3H, J = 6 Hz, 21-CH3), 0.96–2.10 (m, 38H,
aliphatic H), 3.61 (s, 3H, OCH3), 4.07–4.10 (m, 1H, 3ꢁ-
POCH (CH3)2), 4.36 (br, s, 1H, 3α-CH), (m, 1H, 12ꢁ-
2
POCH (CH3)2), 4.75–4.78 (br. s, 1H, 12α-CH), 6.77
2
(m, 1H, 3α-CH), 4.59–4.68 (m, 7H, 3 × CH C CH,
(m, 2H, J = 693 Hz, 2 × PH); HRMS (ESI) found:
2
7α-CH), 4.80–4.84 (m, 1H, 12α-CH), 6.86 (d, 1H,
636.3789, [C31H56O8P2 + NH4]+ calcd: 636.3787.
Heteroatom Chemistry DOI 10.1002/hc

Novel Bile Acid Derived H-Phosphonate Conjugates: Synthesis and Spectroscopic Characterization 407
Methyl 3α,12α-di(3ꢁ-azido-3ꢁ-deoxythymidine-
H-phosphonate)-5β-deoxycholan-24-ate (10)
(413 mg, 0.33 mmol, hygroscopic white solid) in
82% yield; Mp = 58–62◦C; IR (cm−1): 2961, 2925,
1734, 1260, 1018, 799; 31P NMR (121 MHz, CDCl3):
Yield: 48%; colorless solid; IR (cm−1): 2949, 2870,
2108, 1693, 1253, 974; 31P NMR (81 MHz, CDCl3):
δ = 7.38, 7.63, 8.13, 9.57; 1H NMR (300 MHz, CDCl3):
δ = 0.66 (s, 3H, 18-CH3), 0.83 (s, 3H, 19-CH3), 0.91 (d,
3H, J = 6 Hz, 21-CH3),1.10–1.90 (m, 28H, aliphatic
H), 1.91 (s, 6H, AZT-CH3), 2.05–2.45 (m, 6H, 23-CH2,
AZT-CH2), 3.60 (s, 3H, OCH3), 3.94–4.00 (m, 2H,
2×AZT-CHN3), 4.20–4.40 (m, 7H, 3α-CH, 2×AZT-
OCH, POCH2), 4.77–4.85 (br, m, 1H, 12α-CH),
5.92–6.00 (m, 2H, 2 × NCH), 6.86 (s, 1H, J = 687
Hz, 3ꢁ-PH), 6.89, 6.93∗ (d, 1H, J = 687 Hz, 12ꢁ-PH),
7.38–7.80 (2d, 2H, 2× CH C (CH3)CO), 9.30–9.45
(br, d, 2×CONH); HRMS (ESI) found: 1050.4567,
[C45H66N10O14P2 + NH4]+ calcd: 1050.4573.
1
δ = 7.19, 7.25, 7.30, 7.62, 8.19, 8.54; H NMR (600
MHz, CDCl3): δ = 0.62 (s, 3H, 18-CH3), 0.80 (s,
3H, 19-CH3), 0.92–0.93 (d, 3H, J = 6 Hz, 21-CH3),
1.18–2.48 (m, 24H, aliphatic H), 2.39–2.45 (m, 3H,
3 × C H), 2.78–3.03 (m, 6H, 3×PhCH2), 3.12–3.48
(m, 3H, 3 × NH), 3.56–3.68 (q, 12H, 4 × OCH3), 4.00
(m, 1H, 3α-CH), 4.08–4.44 (m, 3H, 3 × CHNH),
4.50–4.58 (m, 6H, 3 × CH C CH), 4.48, 4.55∗ (d,
2
1H, 7α-CH), 4.62, 4.68∗ (d, 1H, 12α-CH), 7.07–7.23
(m, 25H, Ar-H); HRMS (ESI) found: 1277.5357,
[C64H84N3O17P3 + NH4]+ calcd: 1277.5352.
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Methyl 3α,12α-di(2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxy-
thymidine-H-phosphonate)-5β-deoxycholan-
24-ate (11)
Yield 49%; colorless solid; IR (cm−1): 2963, 1663,
1261, 1093, 1020, 800; 31P NMR (81 MHz, CDCl3):
δ = 7.31, 7.76, 7.78, 8.86; 1H NMR (300 MHz, CDCl3):
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CH2), 3.55 (s, 3H, OCH3), 4.13–4.35 (m, 5H, 3α-
CH, 2 × POCH2), 4.79–4.85 (br, m, 1H, 12α-CH),
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2 × CH ), 6.13–6.19 (2d, 2H, 2 × CHCHN), 6.82
(s, 1H, J = 690 Hz, 3ꢁ-PH), 6.84, 6.87∗ (d, 1H, J = 687
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Cholic Acid Phosphoramidate Conjugate (12)
The compound 5 (291.2 mg, 0.4 mmol) in CH3CN
(2 mL) was added dropwise to the solution of l-
phenylalanine methyl ester hydrochloride (295 mg,
1.2 mmol) in Et3N (0.5 mL)–CCl4 (0.5 mL)–H2O
(0.1 mL)–CH3CN (4 mL) at 0◦C. Then the result-
ing mixture was stirred at room temperature for
30 min and the solvent was concentrated under vac-
uum below 40◦C. The obtained residue was puri-
fied on a column (silica gel; CH2Cl2:CH3OH = 20:1)
to yield the corresponding phosphoramidate 12
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Heteroatom Chemistry DOI 10.1002/hc