An expeditious total synthesis of 50-Deoxy-toyocamycin and 50-Deoxysangivamycin

Article abstract of DOI:10.3390/molecules24040737

In present paper, an expeditious total synthesis of naturally occurring 5⁰-deoxytoyocamycin and 5⁰-deoxysangivamycin was accomplished. Because of the introduction of a benzoyl group at N-6 of 4-amino-5-cyano-6-bromo-pyrrolo[2,3-d]pyrimidine, a Vorbrüggen glycosylation with 1,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose afforded a completely regioselective N-9 glycosylation product, which is unambiguously confirmed by X-ray diffraction analysis. All of the involved intermediates were well characterized by various spectra.

Full text of DOI:10.3390/molecules24040737

molecules
Communication
An Expeditious Total Synthesis of
0
0
5
-Deoxy-toyocamycin and 5 -Deoxysangivamycin
Xiangyou Dong, Jie Tang, Chen Hu, Jiang Bai, Haixin Ding * and Qiang Xiao *
Key Laboratory of Organic Chemistry in Jiangxi Province, Institute of Organic Chemistry,
Jiangxi Science & Technology Normal University, Nanchang 330013, China;
dongxiangyou93@yeah.net (X.D.); tangjie93@yeah.net (J.T.); huchen92@yeah.net (C.H.);
mtbaijiang@yeah.net (J.B.)
*
Correspondence: dinghaixin_2010@163.com (H.D.); xiaoqiang@tsinghua.org.cn (Q.X.);
Tel.: +86-791-86422903 (Q.X.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Katherine L. Seley-Radtke and Theodore K. Dayie
Received: 4 February 2019; Accepted: 16 February 2019; Published: 19 February 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
0
Abstract: In present paper, an expeditious total synthesis of naturally occurring 5 -deoxytoyocamycin
0
and 5 -deoxysangivamycin was accomplished. Because of the introduction of a benzoyl group
at N-6 of 4-amino-5-cyano-6-bromo-pyrrolo[2,3-d]pyrimidine, a Vorbrüggen glycosylation with
1,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose afforded a completely regioselective N-9 glycosylation
product, which is unambiguously confirmed by X-ray diffraction analysis. All of the involved
intermediates were well characterized by various spectra.
Keywords: nucleoside; 7-deazapurine; pyrrolo[2,3-d]pyrimidine; total synthesis
1
. Introduction
The synthesis and biological evaluation of nucleoside/tide analogs as agents to treat cancers,
viruses, and parasites, as well as fungal and bacterial infections, spans the past half century [1–3]. The
first steps in this field can be traced back to the emergence of cytarabine to treat for acute myeloid
leukemia and edoxudine for herpes simplex virus, which were both approved by the FDA in 1969.
This strategy has become very successful and there are now more than 30 nucleoside/tide analogues
approved for clinic use, with many more ongoing in preclinical and clinical trials [4–6].
As important analogues of biogenic purine nucleosides, 7-deazapurine nucleosides have shown
diverse biological activities [
more electron rich pyrrole ring more prone to cation–
to increased nucleobase-pairing in DNA or RNA and possibly better binding to proteins [11
7
–
10]. Replacement of the purine’s N-7 atom with a carbon atom results a
or interactions, which subsequently leads
12]. Many
-deazapurine nucleosides were isolated as naturally occurring products, such as toyocamycin (1)
π
π–π
,
7
and sangivamycin (
2
) from Streptomyces cultures (Figure 1). They showed potent cytotoxicity against
) was identified
recently as a potent inhibitor of induced XBP1 mRNA cleavage, which is a key component of the
endoplasmic reticulum stress response [13]. On the other hand, the cytotoxic effect of sangivamycin (
various cancer cell lines with different modes of action. Moreover, toyocamycin (
1
2)
is mainly due to selective inhibition of protein kinase C through inhibition of Erk and Akt signaling
pathway [14,15].
In the course of our continued research interest in the synthesis of 7-deazapurine nucleosides
0
0
and their biological activities, 5 -deoxytoyocamycin (3) and 5 -deoxysangivamycin (4) (Figure 1)
were needed as a control to compare their biological action mechanism with toyocamycin (
1
)
0
and sangivamycin (
2
), respectively. In the published literature, 5 -deoxytoyocamycin (
3
) and
0
5
-deoxysangivamycin (4) were originally synthesized in moderate yields from toyocamycin and
sangivamycin by radical reduction (AIBN and tributyltin hydride in dry THF) of the corresponding
Molecules 2019, 24, 737; doi:10.3390/molecules24040737
www.mdpi.com/journal/molecules

Molecules 2019, 24, 737
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0
0
5
-chloro-5 -deoxynucleosides [16
,
17]. This approach was inefficient, cumbersome and low yielding
0
and the the starting materials very expensive. Later on, 5 -deoxytoyocamycin (
3) was isolated from the
fermentation broth of a Streptomyces sp. from a soil sample collected from Kepong, Malaysia. Although
a manufacturing process for 5’-deoxytoyocamycin using the Streptomyces sp. was patented soon after
its discovery, it was not practical to perform it in our laboratory. Thus, we turned our endeavor to total
0
0
3) and 5 -deoxysangivamycin (4), which could provide enough
syntheses of 5 -deoxytoyocamycin (
material for further biological studies.
Figure 1. Examples of naturally occurring 7-deazapurine nucleosides.
While various glycosylation approaches have been developed for the preparation of
ribonucleosides, the most widely employed method is the silyl-Hilbert-Johnson reaction catalyzed by
equimolar or excess amounts of Lewis acid catalysts (Vorbrüggen glycosylation), which has now been
developed for more than fifty years [18]. However, the Vorbrüggen glycosylation when performed with
7
-deazapurines was found to be much less efficient. Until now, Vorbrüggen glycosylation could only
be used to prepare the corresponding ribonucleosides of 7-deazapurine nucleobases with an electron
withdrawing substitute at C-7 [19]. Specifically, total synthesis of toyocamycin ( ) by Vorbrüggen
glycosylation of ribose and nucleobase was firstly reported by Bobek [20], and further improved
by Townsend [21], but during our many repeats of this approach [22 25], it was found that the N-9
glycosylation product and N-3 glycosylation product were inevitably formed simultaneously
Figure 2). This phenomenon was also reported by several other groups [26 28]. The selectivity and
equilibrium between N-9 glycosylation product and N-3 glycosylation product were subsequently
1
5
6
–
7
8
(
–
7
8
studied in detail [27]. In order to resolve this problem, an improved synthetic approach is reported in
0
the present paper, that was used successfully in the total synthesis of 5 -deoxytoyocamycin (
3) and
0
5
-deoxysangivamycin (4).
Figure 2. Synthesis of 7-deazapurine nucleosides by Vorbrüggen glycosylation.
2
. Results and Discussion
According to the reaction mechanism of the Vorbrüggen glycosylation, we reasoned that N-3
product formation may result from the more electron rich pyrimidine in nucleobase . Thus, the
formation of N-3 products, such as nucleoside 8, could be avoided by reducing the pyrimidine ring’s
electron density. On the other hand, the solubility of nucleobase is very low and its purification
6
6

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is really difficult. We speculated that nucleobase 12 with a benzoyl group at N-6 could fulfill our
requirements. Although, the nucleobase with an acetyl at N-6 was already reported, its use did not
solve the above selectivity and solubility problems [20,29–31].
Preparation of nucleobase from tetracyanoethylene was reported in several references [32
In order to synthesize it on a large scale, the corresponding synthetic procedure was improved in our
following experiments (Scheme 1). Firstly, pyrrole 10 were synthesized from tetracyanoethylene
6
6
9
–35].
9
according to a modification of the protocol developed by Townsend [36]. It should be mentioned that
the intermediate formed after addition of 33% HBr is very hydroscopic and not stable in air. After
transformation to the free pyrrole 10, it could be stored on the bench for a long time. Then, pyrrole 10
was refluxed with ethyl orthoformate in CH CN to give intermediate 11. After removing the volatile
3
byproducts and without further purification, ring annulation to give nucleobase
accomplished in freshly prepared saturated ethanolic ammonia at 105 C in a closed stainless-steel
container for 6 h.
6
in 87% yield was
◦
4
Scheme 1. Synthesis of N -benzoyl-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine.
The subsequent monobenzoylation at N-6 to provide nucleobase 12 proved to be troublesome.
Using pyridine as the base and solvent, at least four products are formed. Other attempts to directly
synthesize the monobenzoylated product 12 also failed. After optimization, complete benzoylation
of nucleobase
6 was conducted firstly to give the corresponding N6,N6,N9-tribenzoyl derivative of
6
using a large excess of benzoyl chloride (5 eq.) and Et N (7.5 eq) in CH CN. Then the tribenzoyl
3
3
intermediate, without isolation, was stirred directly with concentrated ammonia and H O (V/V = 1:1)
2
to afford the desired nucleobase 12 in 76% yield. This modified protocol for the synthesis of nucleobase
12 from 9 was repeated several times at 50 g scale without requiring chromatographic purification.
Furthermore, a single crystal of nucleobase 12 suitable for X-ray crystallographic analysis was obtained
and the X-ray structure shown in Figure 3a was determined [37].
Next, the Vorbrüggen glycosylation between ribose 13 and nucleobase 12 was conducted
(
Scheme 2). After silylation of nucleobase 12 with N,O-Bis(trimethylsilyl)acetamide (BSA) in dry MeCN,
1
,2,3-tri-O-acetyl-5-deoxy-
β
-D-ribofuranose (13) was added, followed by TMSOTf. The resulting
◦
mixture was heated at 80 C for 3.5 h. After purification, 7-deazapurine nucleoside 14 was obtained in
8
7.7% yield. To our delight, the N-3 glycosylation product 15 was not detected. Thus, our introduction
of a benzoyl group at N-6 successfully solved the regioselectivity problem of the glycosylation. At the
same time, a single crystal of nucleoside 14 suitable for X-ray diffraction analysis was also obtained
and its structure is unambiguously confirmed, which showed an anti-glycosylic bond conformation

Molecules 2019, 24, 737
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0
2
and an envelope C2 -endo ( E) sugar conformation (S-type) (Figure 3b) [36]. It is worth mentioning
that X-ray structures of toyocamycin and its analogues have seldom been reported [38].
(
a)
(b)
Figure 3. X-Ray structure of nucleobase 12 (a) and nucleoside 14 (b).
0
0
Scheme 2. Total synthesis of 5 -deoxytoyocamycin and 5 -deoxysangivamycin.
Subsequently, the bromide at C-8 was hydrogenated in a solvent mixture of methanol and
THF using 10% Pd/C as catalyst to give nucleoside 16 in 93% yield. Deprotection of the remaining
four benzoyl groups proved troublesome. When heated with saturated ammonium in methanol,
0
0
3) and 5 -deoxysangivamycin (4) were obtained at the same time along with
5
-deoxytoyocamycin (
other side products.
A variety of other conditions were tried with no improvement. Looking back the X-ray structures
of nucleobase 12 and nucleoside 14, it was obvious that the carbonyl of the N-6 benzoyl was close
and syn to the C-7 nitrile group (Figure 3) [37]. Therefore, the nitrile group of nucleoside 16 could be
activated by the electron-withdrawing benzoyl group, which makes the nitrile group susceptible to

Molecules 2019, 24, 737
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0
0
attack by ammonia. Luckily, 5 -deoxytoyocamycin (
3
) and 5 -deoxysangivamycin (
4
) were both targets,
0
and they could be conveniently separated by chromatography. In the end, 5 -deoxytoyocamycin (
3
)
0
and 5 -deoxysangivamycin (
4
) were obtained in 67% and 24% yield, respectively, after removing all
0
the benzoyl protecting groups with ammonia in dioxane. All the spectra of 5 -deoxytoyocamycin (
3)
were in accordance with those of the naturally occurring product. Because different solvents were
0
used in the original NMR determination of naturally occurring 5 -deoxytoyocamycin (
3) [39], the
corresponding NMR data obtained in various solvents are summarized in this work (Table S1 in the
Supplementary Information).
3
. Conclusions
0
In conclusion, we have accomplished an improved total synthesis of 5 -deoxytoyocamycin
0
(3)
and 5 -deoxysangivamycin
(4)
using
a
Vorbrüggen glycosylation between 1,2,3-tri-
-D-ribofuranose (13) and N -benzoyl-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine
4
O-acetyl-5-deoxy-
β
(12) as the key step. The structure of 7-deazapurine nucleoside 14 was unambiguously confirmed
by X-ray diffraction analysis, which showed an anti glycosylic bond conformation and an envelope
0
2
C2 -endo ( E) sugar conformation. It was demonstrated that the regioselectivity during the Vorbrüggen
glycosylation completely favored the N-9 glycosylation product after introducing a benzoyl group
at the N-6 position of 4-amino-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine. Thus, this newly
developed nucleobase 12 could be used to synthesize other toyocamycin analogues while avoiding the
formation of N-3 glycosylation side products.
4
. Materials and Methods
4
.1. Materials and Instruments
Unless otherwise specified, all the reagents were acquired from commercial sources and used
directly. NMR spectra (400 MHz/100 MHz) were recorded on an Advance DPX spectrometer (Bruker,
Billerica, MA, USA) at room temperature with DMSO-d as solvent. Tetramethylsilane (TMS) was
6
used as an internal reference. Specific rotations were acquired on an Autopol IV polarimeter (Rudolph,
Hackettstown, NJ, USA). High resolution mass spectrometry (HRMS) data were measured with an
AB Sciex TOF 4600 instrument (Billerica, MA, USA). Melting points were measured on an X-4 digital
melting point apparatus (Beijing Taike Corparation, Beijing, China). X-ray diffraction analysis was
performed on a Bruker Smart Apex II system (Bruker, Billerica, MA, USA).
4
.2. Synthesis of 2-amino-5-bromo-3,4-dicyano-1H-pyrrole (10)
Tetracyanoethylene ( , 35.6 g, 277.93 mmol) was dissolved in fresh dried acetone (200 mL) and
9
ethyl acetate (420 mL). Under an Ar atmosphere, 33% HBr in acetic acid (200 mL) was added slowly
◦
while keeping the internal reaction temperature below 2 C. After addition, the reaction mixture was
stirred for another 1.5 h. The formed yellow solid was filtered and washed with cold ethyl acetate
(100 mL). Subsequently, the crude product was suspended in ice cold water (100 mL) and 50% NaOH
in H O (60 mL) was added slowly until a homogenous solution was obtained. After filtration through
2
Celite, the filtrate pH was adjusted carefully to 5 using acetic acid. The thus obtained solid was filtered
and washed thoroughly with water until neutral. After drying under vacuum with P O , a grey solid
2
5
◦
1
of 10 was obtained (38.12 g, 65%). R = 0.6 (DCM/MeOH, V/V = 10:1); mp: 224–226 C; H-NMR δ:
f
13
1
[
2.25 (s, 1H), 6.45 (s, 2H); C-NMR δ: 149.8, 115.1, 114.4, 103.0, 93.5, 71.4; HRMS: calcd. for C H BrN
M − H]− 208.9468; found: 208.9446.
6 2 4
4
.3. Synthesis of 4-amino-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine (6)
2
-Amino-5-bromo-3,4-dicyano-1H-pyrrole (10, 65.5 g, 310.40 mmol) was suspended in fresh dried
CH CN (1.5 L) and ethyl orthoformate (77.44 mL, 465.59 mmol). The suspension was heated to reflux
3
until TLC showed the reaction was finished (about 3 h). The solvent was removed under vacuum to

Molecules 2019, 24, 737
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give a grey solid (82 g). Subsequently, the obtained solid was suspended in freshly prepared saturated
◦
ammonia in ethanol (1150 mL) in a closed stainless-steel container and heated at 105 C for 6 h. Once
cooled to room temperature, the reaction mixture was poured to a 5 L beaker with activated charcoal
(
22 g). After stirring for 30 min, the mixture was filtered through Celite and washed thoroughly with
concentrated ammonia in ethanol (V/V = 1/5, 150 mL 3). The filtration was adjusted to pH = 5
with acetic acid (about 160 mL). The obtained solid was filtered and washed with H O. After dried
×
2
under vacuum with P O ,
6 was obtained as greenish solid (64.28 g, 87%). R = 0.6 (DCM/MeOH,
f
2
5
◦
1
13
V/V = 10:1); mp: 279–281 C; H-NMR
50.1(C-7a), 149.6 (C-2), 124.9 (C-6), 115.9 (CN), 103.7 (C-4a), 84.4(C-5); HRMS: calcd. for C H BrN
5
δ: 8.21 (s, 1H, H-2), 7.20 (s, 2H, NH2); C-NMR δ: 156.0(C-4),
1
7
3
−
[M−H] 235.9577; found: 235.9585.
4
4
.4. Synthesis of N -benzoyl-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine (12)
Deazapurine base 6 (50 g, 210.05 mmol, 1 eq.) was suspended in fresh dried CH CN (450 mL) and
3
Et N (220 mL, 1.58 mol, 7.5 eq.). After the above suspension was stirred for 1 h at room temperature,
3
benzoyl chloride (122 mL, 1.05 mol, 5 eq.) was added and the reaction mixture was stirred overnight.
After TLC showed the 7-deaazapurine base had vanished, the reaction pH was adjusted by acetic acid
to 6 and he reaction mixture was filtered. The filtrate was extracted with DCM (300 mL
×
3). The
combined extracts were washed with H O, saturated NaCl, and dried with anhydrous MgSO . After
2
4
filtration, the solvent was evaporated under vacuum. The obtained crude product was stirred with
concentrated ammonium and H O (1 L, V/V = 1:1) for 5 h to give light yellow protected deazapurine
2
◦
1
1
2
(54.63 g, yield: 76%). R = 0.3 (CH Cl /MeOH, V/V = 10:1); mp 235–237 C; H-NMR
δ: 14.23 (s,
f
2
2
1
H, NH), 11.36 (s, 1H, NH), 8.75 (s, 1H, H-2), 8.06 (d, J = 6.3 Hz, 2H), 7.65 (t, J = 7.2 Hz, 1H), 7.56 (t,
J = 7.5 Hz, 2H); 13C-NMR
δ
: 167.4 (C=O), 153.3 (C-4), 152.7 (C-2), 150.6 (C-7a), 133.5, 132.9, 128.9, 128.8,
−
124.1 (C-6), 114.8 (CN), 111.9 (C-4a), 88.8 (C-5); HRMS: calcd. for C H BrN O [M
−
H] 339.9839;
14
7
5
found: 339.9852.
4
0 0 0
4
.5. Synthesis of N -benzoyl-5-cyano-6-bromo-7-(2 ,3 -bi-O-acetyl-5 -deoxy-β-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (14)
N,O-Bis(trimethylsilyl)acetamide (BSA, 14.27 g, 70.14 mmol) was added to a suspension of
N -benzoyl-5-cyano-6-bromo-7H-pyrrolo[2,3-d]pyrimidine (12, 6.00 g, 17.53 mmol) in dry MeCN
4
(
60 mL) under an argon atmosphere. The reaction mixture was stirred at room temperature for 60 min.
A solution of 1,2,3-tri-O-acetyl-5-deoxy- -D-ribofuranose 13 (5.48 g, 21.04 mmol) in dry MeCN (50 mL)
β
along with TMSOTf (15.59 g, 70.14 mmol) were added to the above reaction mixture under ice-bath
◦
condition. The solution was stirred for 30 min at room temperature before heating to 80 C for 3.5 h.
Then the mixture was cooled and poured into sat. NaHCO solution (150 mL). It was extracted with
3
CH Cl (150 mL
×
3). The combined organic layer was washed with sat. NaHCO (100 mL
×
2), brine
2
2
3
(
100 mL
×
2), and dried over anhydrous MgSO . After filtration, the filtrate was evaporated to dryness
4
under reduced pressure. The residue was purified by silica gel column chromatography (CH Cl
2
2
/
EtOAc 20:1, v:v) to afford 14 as yellow solid (8.34 g, 87.7%). R = 0.4 (CH Cl /EtOAc, V/V = 20:1);
f
2
2
◦
25
1
m.p. 230–232 C;
α
−
22.40 (c = 0.098, CH Cl ); H-NMR
δ
11.58 (s, 1H, NH), 8.89 (s, 1H, H-2), 8.06
2
2
D
0
(
d, J = 7.5 Hz, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 6.23 (d, J = 4.0 Hz, 1H, H-1 ), 6.18–6.14
0
0
0
(
m, 1H, H-2 ), 5.50 (t, J = 6.7 Hz, 1H, H-3 ), 4.33 – 4.25 (m, 1H, H-4 ), 2.13 (s, 3H, OAc), 2.08 (s, 3H,
170.1 (C=O, OAc), 170.0 (C=O, OAc), 167.5 (C=O,
Bz), 153.1 (C-2), 152.0 (C-4), 151.4 (C-7a), 133.1, 129.0, 128.9, 126.6 (C-6), 114.2 (CN), 111.8 (C-4a), 91.4
OAc), 1.39 (d, J = 6.2 Hz, 3H, H-5’); 13C-NMR
δ
0
0
0
0
0
(
C-5), 89.1 (C-1 ), 77.8 (C-4 ), 74.0 (C-3 ), 72.5 (C-2 ), 20.8(OAc), 20.7 (OAc), 18.2 (C-5 ); HRMS: calcd for
+
C H BrN O [M+H] 542.0670; found 542.0679.
23
21
5
6

Molecules 2019, 24, 737
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4
.6. Synthesis of
4
0
0
0
N -benzoyl-5-cyano-7-(2 ,3 -di-O-acetyl-5 -deoxy-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (16)
1
0% Pd/C (0.75 g, 0.70 mmol) and Et N (0.94 g, 9.22 mmol) were added to a solution of nucleoside
3
14 (2.50 g, 4.61 mmol) in the mixture of THF (65 mL) and MeOH (35 mL). After stirring at room
temperature for 2.25 h under H atmosphere (1 atm), the reaction mixture was filtered and concentrated
2
to dryness under reduced pressure. The residue was purified by silica gel column chromatography
(
CH Cl /EtOAc = 20:1, v:v) to afford 16 as light yellow solid (1.99 g, 93%). R = 0.3 (CH Cl /EtOAc,
2
2
f
2
2
◦
25
1
V/V = 20:1); m.p. 92–94 C;
H-2, H-6), 8.07 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.4 Hz, 2H), 6.40 (d, J = 5.4 Hz,
1
3
α
−
45.80 (c = 0.109, CH Cl ); H-NMR δ 11.50 (s, 1H, NH), 8.88 (s, 2H,
2 2
D
0 0 0 0
H, H-1 ), 5.90 (t, J = 5.6 Hz, 1H, H-2 ), 5.32 (t, J = 5.2 Hz, 1H, H-3 ), 4.32–4.26 (m, 1H, H-4 ), 2.13 (s,
0
13
H, OAc), 2.04 (s, 3H, OAc), 1.44 (d, J = 6.3 Hz, 3H, H-5 ); C-NMR δ 170.0 (C=O, OAc), 169.8 (C=O,
OAc), 167.5 (C=O, Bz), 153.2 (C-2), 152.5 (C-4), 152.1(C-7a), 137.1 (C-6), 133.3, 133.0, 128.9, 128.8, 114.9
0 0 0 0
CN), 111.4 (C-4a), 87.5 (C-5), 86.6 (C-1 ), 78.6 (C-4 ), 74.3 (C-3 ,), 73.2 (C-2 ), 20.8 (OAc), 20.6 (OAc),
1
(
0
8.8 (C-5 ); HRMS: calcd for C H N O [M + H] 464.1565; found 464.1579.
23 22 5 6
+
0
0
4
.7. Synthesis of 5 -deoxytoyocamycin (3) and 5 -deoxysangivamycin (4)
Ammonium hydroxide (45 mL) was added to a solution of 5 (1.80 g, 3.88 mmol) in 1,4-dioxane
◦
(
18 mL). The reaction mixture was sealed and heated to 45 C for 21 h. Then the reaction mixture
was evaporated to dryness under reduced pressure and the residue was purified by silica gel column
chromatography (CH Cl /MeOH 20:1, v:v) to afford compounds
3
(716 mg, 67%) and
4
(274 mg,
2
2
2
4%), respectively.
0
4
.8. 5 -Deoxytoyocamycin (3)
◦
◦
25
R = 0.2 (CH Cl /MeOH, V/V = 20:1); m.p. 190–192 C (literature: 187–188 C [16]);
α
87.88
f
2
2
D
1
(
1
4
1
c = 0.099, DMSO); H-NMR
δ
8.40 (s, 1H, H-8), 8.23 (s, 1H, H-2), 6.90 (s, 2H, NH ), 6.04 (d, J = 4.7 Hz,
H, H-1 ), 5.48 (d, J = 5.6 Hz, 1H, 2 -OH), 5.20 (d, J = 5.3 Hz, 1H, 3 -OH), 4.44–4.37 (m, 1H, H-2 ),
.00–3.91 (m, 1H, H-4 ), 3.91–3.84 (m, 1H, H-3 ), 1.30 (d, J = 6.3 Hz, 3H, CH ); C-NMR
54.1 (C-2), 150.8 (C-4), 132.9 (C-8), 115.8 (CN), 101.7 (C-5), 88.4 (C-1 ), 83.8 (C-7), 80.2 (C-4 ), 75.0 (C-3 ),
2
0
0
0
0
0
0
13
δ
0
157.5 (C-6),
3
0
0
0
+
7
4.2 (C-2 ), 19.4 (CH ); HRMS: calcd for C H N O [M + H] 276.1091; found 276.1099.
3
12 14
5
3
0
4
.9. 5 -Deoxysangivamycin (4)
◦
◦
25
D
R = 0.15 (CH Cl /MeOH, V/V = 10:1); m.p. 257–259 C (literature value 260–262 C [16]);
α
f
2
2
1
−
77.05 (c = 0.122, DMSO); H-NMR
δ
8.09 (s, 2H, H-2, H-8), 7.99 (s, 1H, NH), 7.39 (s, 1H, NH), 6.08 (d,
J = 4.2 Hz, 1H, H-1 ), 5.50 (d, J = 5.5 Hz, 1H, 2 -OH), 5.20 (d, J = 5.7 Hz, 1H, 3 -OH), 4.24–4.19 (m, 1H,
H-2 ), 4.00–3.91 (m, 1H, H-4 ), 3.91–3.84 (m, 1H, H-3 ), 1.32 (d, J = 6.3 Hz, 3H, CH ); C-NMR δ 166.8
0
0
0
0
0
0
13
3
0 0
(
7
C=O), 158.5 (C-6), 153.4 (C-2), 151.4 (C-4), 125.4 (C-8), 111.7 (C-7), 101.3 (C-5), 87.7 (C-1 ), 79.5 (C-4 ),
0 0
5.2 (C-3 ), 74.8 (C-2 ), 19.4 (CH ); HRMS: calcd for C H N O [M + H] 294.1197; found 294.1208.
3 12 16 5 4
+
Supplementary Materials: The NMR spectra of compound 3–4, 6, 9, 11–12, 14, 16 are available online at
http://www.mdpi.com/1420-3049/24/4/737/s1.
Author Contributions: Q.X. and H.D. conceived and designed the experiments; X.D., J.T. and C.H. performed the
experiments; C.H. and J.B. analyzed the data; Q.X. and H.D. wrote the paper.
Funding: This research was funded by the National Natural Science Foundation of China (no. 21676131 and
no. 21462019), the Science Foundation of Jiangxi Province (20143ACB20012), and Jiangxi Science & Technology
Normal University (Doctor Startup Fund for Dr. Haixin Ding) for financial support.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 737
8 of 9
References and Note
1
.
Yates, M.K.; Seley-Radtke, K.L. The evolution of antiviral nucleoside analogues: A review for chemists
and non-chemists. Part II: Complex modifications to the nucleoside scaffold. Antivir. Res. 2019, 162, 5–21.
CrossRef] [PubMed]
[
2
.
Seley-Radtke, K.L.; Yates, M.K. The evolution of nucleoside analogue antivirals: A review for chemists and
non-chemists. Part 1: Early structural modifications to the nucleoside scaffold. Antivir. Res. 2018, 154, 66–86.
[CrossRef] [PubMed]
3
4
.
.
Liang, Y.; Wnuk, S.F. Modification of Purine and Pyrimidine Nucleosides by Direct C-H Bond Activation.
Molecules 2015, 20, 4874–4901. [CrossRef] [PubMed]
Jordheim, L.P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucleoside and
nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12, 447–464. [CrossRef]
[
PubMed]
Zhou, X.X.; Littler, E. Nucleoside analogs as anti-HBV agents. Curr. Top. Med. Chem. 2006, 6, 851–865.
CrossRef]
De Clercq, E.; Li, G.D. Approved Antiviral Drugs over the Past 50 Years. Clin. Microbiol. Rev. 2016, 29,
95–747. [CrossRef] [PubMed]
5.
6.
7.
8.
[
6
Perlikova, P.; Hocek, M. Pyrrolo[2,3-d]pyrimidine (7-deazapurine) as a privileged scaffold in design of
antitumor and antiviral nucleosides. Med. Res. Rev. 2017, 37, 1429–1460. [CrossRef]
Townsend, L.B.; Drach, J.C.; Wotring, L.L.; Vittori, S.; Pudlo, J.S.; Swayze, E.E.; Gupta, P.; Maruyama, T.;
Saxena, N.; Coleman, L.A. Design, synthesis, and studies on the structure-activity relationships of certain
pyrrolo[2,3-d]pyrimidine nucleosides and structurally related analogs as potential antineoplastic and
antiviral agents. Farmaco 1991, 46, 113–139. [CrossRef]
9
1
1
.
Dholakia, S.P.; Patel, M.M.; Patel, J.S. Role of pyrrolopyrimidine derivatives as anticancer agent: Mini review.
Indo Am. J. Pharm. Res. 2015, 5, 858–867.
0. De Coen, L.M.; Heugebaert, T.S.A.; Garcia, D.; Stevens, C.V. Synthetic Entries to and Biological Activity of
Pyrrolopyrimidines. Chem. Rev. 2016, 116, 80–139. [CrossRef]
1. Cahova, H.; Panattoni, A.; Kielkowski, P.; Fanfrlik, J.; Hocek, M. 5-Substituted Pyrimidine and 7-Substituted
7
-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence
of Natural dNTPs. ACS Chem. Biol. 2016, 11, 3165–3171. [CrossRef] [PubMed]
1
1
2. Seela, F.; Sirivolu, V.R. DNA containing side chains with terminal triple bonds: Base-pair stability and
functionalization of alkynylated pyrimidines and 7-deazapurines. Chem. Biodivers. 2006, 3, 509–514.
[CrossRef]
3. Ri, M.; Tashiro, E.; Oikawa, D.; Shinjo, S.; Tokuda, M.; Yokouchi, Y.; Narita, T.; Masaki, A.; Ito, A.; Ding, J.; et al.
Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER
stress-induced XBP1 mRNA splicing. Blood Cancer J. 2012, 2. [CrossRef] [PubMed]
1
4. Musumeci, F.; Sanna, M.; Grossi, G.; Brullo, C.; Fallacara, A.L.; Schenone, S. Pyrrolo[2,3-d]Pyrimidines as
Kinase Inhibitors. Curr. Med. Chem. 2017, 24, 2059–2085. [CrossRef]
1
5. Musumeci, F.; Sanna, M.; Greco, C.; Giacchello, I.; Fallacara, A.L.; Amato, R.; Schenone, S.
Pyrrolo[2,3-d]pyrimidines active as Btk inhibitors. Expert Opin. Ther. Pat. 2017, 27, 1305–1318. [CrossRef]
[
PubMed]
6. Wang, Y.; Hogenkamp, H.P.C.; Long, R.A.; Revankar, G.R.; Robins, R.K. A convenient synthesis of
’-deoxyribonucleosides. Carbohydr. Res. 1977, 59, 449–457. [CrossRef]
7. Porcari, A.R.; Ptak, R.G.; Borysko, K.Z.; Breitenbach, J.M.; Vittori, S.; Wotring, L.L.; Drach, J.C.; Townsend, L.B.
Deoxy Sugar Analogues of Triciribine: Correlation of Antiviral and Antiproliferative Activity with
Intracellular Phosphorylation. J. Med. Chem. 2000, 43, 2438–2448. [CrossRef] [PubMed]
1
1
5
1
1
8. Vorbrueggen, H.; Ruh-Pohlenz, C. Synthesis of nucleosides. Org. React. (Hobokennju. S.) 2000, 55. [CrossRef]
9. Seela, F.; Peng, X. Progress in 7-deazapurine-pyrrolo[2,3-d]pyrimidine-ribonucleoside synthesis. Curr. Top.
Med. Chem. 2006, 6, 867–892. [CrossRef] [PubMed]
2
2
0. Sharma, M.; Bloch, A.; Bobek, M. A practical synthesis of the antibiotic toyocamycin. Nucleosides Nucleotides
1
993, 12, 643–648. [CrossRef]
1. Porcari, A.R.; Townsend, L.B. Total synthesis of the naturally occurring antibiotic toyocamycin using new
and improved synthetic procedures. Nucleosides Nucleotides 1999, 18, 153–159. [CrossRef] [PubMed]

Molecules 2019, 24, 737
9 of 9
2
2
2
2
2
2. Ruan, Z.; Gong, Z.; Xiao, Q.; Yang, R. An improved total synthesis of 5’-deoxytoyocamycin. Adv. Mater. Res.
014, 997, 200–202. [CrossRef]
3. Dou, Y.-H.; Ding, H.-X.; Yang, R.-C.; Li, W.; Xiao, Q. A total synthesis of mycalisine A. Chin. Chem. Lett. 2013
4, 379–382. [CrossRef]
4. Hu, C.; Ruan, Z.Z.; Ding, H.X.; Zhou, Y.R.; Xiao, Q. An Expedient Total Synthesis of Triciribine. Molecules
017, 22. [CrossRef] [PubMed]
5. Sun, J.Y.; Dou, Y.H.; Ding, H.X.; Yang, R.C.; Sun, Q.; Xiao, Q. First Total Synthesis of a Naturally Occurring
Iodinated 5 ‘-Deoxyxylofuranosyl Marine Nucleoside. Mar. Drugs 2012, 10, 881–889. [CrossRef] [PubMed]
6. Leonard, P.; Ingale, S.A.; Ding, P.; Ming, X.; Seela, F. Studies on the glycosylation of pyrrolo[2,3-d]pyrimidines
2
,
2
2
with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose: The formation of regioisomers during toyocamycin
and 7-deazainosine syntheses. Nucleosidesnucleotides Nucleic Acids 2009, 28, 678–694. [CrossRef] [PubMed]
7. Kim, Y.-J.; Kwon, S.H.; Bae, I.H.; Kim, B.M. Selectivity between N-1 and N-7 nucleosides: Regioselective
synthesis of BMK-Y101, a potent cdk7 and 9 inhibitor. Tetrahedron Lett. 2013, 54, 5484–5488. [CrossRef]
8. Zhou, L.; Amblard, F.; Zhang, H.; McBrayer, T.R.; Detorio, M.A.; Whitaker, T.; Coats, S.J.; Schinazi, R.F.
Synthesis and evaluation of Janus type nucleosides as potential HCV NS5B polymerase inhibitors. Bioorganic
Med. Chem. Lett. 2013, 23, 3385–3388. [CrossRef]
2
2
2
3
3
9. Tolman, R.L.; Robins, R.K.; Townsend, L.B. Pyrrolopyrimidine nucleosides. III. Total synthesis of
toyocamycin, sangivamycin, tubercidin, and related derivatives. J. Am. Chem. Soc. 1969, 91, 2102–2108.
[CrossRef]
0. Tolman, R.L.; Robins, R.K.; Townsend, L.B. Pyrrolo[2,3-d]pyrimidine nucleoside antibiotics. Total synthesis
and structure of toyocamycin, unamycin B, vengicide, antibiotic E-212, and sangivamycin (BA-90912).
J. Am. Chem. Soc. 1968, 90, 524–526. [CrossRef]
1. Xiao, C.; Sun, C.; Han, W.W.; Pan, F.; Dan, Z.; Li, Y.; Song, Z.G.; Jin, Y.H. Synthesis of 6-(het) ary Xylocydine
analogues and evaluating their inhibitory activities of CDK1 and CDK2 in vitro. Bioorganic Med. Chem. 2011
,
1
9, 7100–7110. [CrossRef] [PubMed]
3
2. Porcari, A.R.;
Townsend, L.B. An improved synthesis of the versatile heterocycle,
4
-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine. Synth. Commun. 1998, 28, 3835–3843. [CrossRef]
3
3. Suh, H.; Choi, K.-W.; Lee, J.; Ryou, C.; Rhee, H.; Lee, C.-H. Effects of a novel carbocyclic analog of
pyrrolo[2,3-d]pyrimidine nucleoside on pleiotropic induction of cell death in prostate cancer cells with
different androgen responsiveness. Bioorganic Med. Chem. Lett. 2016, 26, 1130–1135. [CrossRef] [PubMed]
4. Lee, J.; Seo, H.; Yoon, S.; Choi, K.; Lee, C.-H.; Rhee, H. The synthesis and evaluation of new carbocyclic
pyrrolo[2,3-d]pyrimidine nucleoside analogs. Heterocycles 2014, 89, 1606–1619. [CrossRef]
5. Wilding, B.; Winkler, M.; Petschacher, B.; Kratzer, R.; Glieder, A.; Klempier, N. Nitrile Reductase from
Geobacillus kaustophilus: A Potential Catalyst for a New Nitrile Biotransformation Reaction. Adv. Synth.
Catal. 2012, 354, 2191–2198. [CrossRef]
3
3
3
6. Zemlicka, J.; Owens, J. Nucleic Acid Chemistry, Improved and New Synthetic Procedures, Methods and Techniques,
Part 4; Wiley-Interscience: New York, NY, USA, 1991; pp. 16–18.
3
7. Crystallographic data for nucleobase 12: C14H BrN O, M = 324.16, crystal dimensions 0.08
×
0.12
colorless block, space group, C2/c (No. 15). CCDC 1525337 contains the supplementary crystallographic
data. Crystallographic data for nucleoside 14 C H BrN O , M = 542.35, crystal dimensions
.07 0.20 0.22 mm, monoclinic, space group, P2 (No. 4). CCDC 1895393 contains the supplementary
×
0.20 mm,
8
5
:
2
3
20
5
6
0
×
×
1
crystallographic data. These data can be obtained free of charge from The Cambridge Crystallographic Date
Centre via www.ccdc.cam.ac.uk/date_request/cif.
3
8. Prusiner, P.; Sundaralingam, M. The crystal and molecular structure of toyocamycin monohydrate,
a nucleoside antibiotic. Acta Crystallogr. Sect. B 1978, B34, 517–523. [CrossRef]
3
9. Isaac, B.G.; Ayer, S.W.; Letendre, L.J.; Stonard, R.J. Herbicidal nucleosides from microbial sources. J. Antibiot.
1
991, 44, 729–732. [CrossRef]
Sample Availability: Samples of the compounds 3–16 are available from Prof. Dr. Qiang Xiao.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(