Facile functionalization at the C4 position of pyrimidine nucleosides via amide group activation with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and biological evaluations of the products

Article abstract of DOI:10.1039/C6OB02334G

Reactions of O-t-butyldimethylsilyl-protected thymidine, 2′-deoxyuridine, and 3′-azidothymidine (AZT) with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) leads to activation of the C4 amide carbonyl by formation of putative O⁴-(benzotriazol-1-yl) derivatives. Subsequent substitution with alkyl and aryl amines, thiols, and alcohols leads to facile functionalization at this position. Reactions with amines and thiols were conducted either as a two-step, one-pot transformation, or as a one-step conversion. Reactions with alcohols were conducted as two-step, one-pot transformations. In the course of these investigations, the formation of 1-(4-pyrimidinyl)-1H-benzotriazole-3-oxide derivatives from the pyrimidine nucleosides was identified. However, these too underwent conversion to the desired products. Products obtained from AZT were converted to the 3′-amino derivatives by catalytic reduction. All products were assayed for their abilities to inhibit cancer cell proliferation and for antiviral activities. Many were seen to be active against HIV-1 and HIV-2, and one was active against herpes simplex virus-1 (HSV-1).

Full text of DOI:10.1039/C6OB02334G

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Facile functionalization at the C4 position of
pyrimidine nucleosides via amide group activation
with (benzotriazol-1-yloxy)tris(dimethylamino)
phosphonium hexafluorophosphate (BOP) and
biological evaluations of the products†
Cite this: DOI: 10.1039/c6ob02334g
Hari K. Akula,a,b Hariprasad Kokatla,a Graciela Andrei,c Robert Snoeck,c
Dominique Schols,c Jan Balzarini,c Lijia Yanga and Mahesh K. Lakshman*a,b
Reactions of O-t-butyldimethylsilyl-protected thymidine, 2’-deoxyuridine, and 3’-azidothymidine (AZT)
with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) leads to acti-
vation of the C4 amide carbonyl by formation of putative O4-(benzotriazol-1-yl) derivatives. Subsequent
substitution with alkyl and aryl amines, thiols, and alcohols leads to facile functionalization at this position.
Reactions with amines and thiols were conducted either as a two-step, one-pot transformation, or as a
one-step conversion. Reactions with alcohols were conducted as two-step, one-pot transformations. In
the course of these investigations, the formation of 1-(4-pyrimidinyl)-1H-benzotriazole-3-oxide deriva-
tives from the pyrimidine nucleosides was identified. However, these too underwent conversion to the
desired products. Products obtained from AZT were converted to the 3’-amino derivatives by catalytic
reduction. All products were assayed for their abilities to inhibit cancer cell proliferation and for antiviral
activities. Many were seen to be active against HIV-1 and HIV-2, and one was active against herpes
simplex virus-1 (HSV-1).
Received 26th October 2016,
Accepted 16th December 2016
DOI: 10.1039/c6ob02334g
www.rsc.org/obc
Introduction
Nucleosides are central to the control and treatment of viral dis-
eases, and with the emergence of new viral diseases and devel-
opment of drug resistance, easy access to novel structural motifs
is critical. Within this context, the classical method for modify-
ing the C4 position of pyrimidine nucleosides is via introduc-
tion of a leaving group at this position, followed by a displace-
ment with nucleophiles. Typically, electrophilic nucleoside
derivatives used for this purpose contain a C4 thione, methyl-
thio, chloro, 1,2,4-triazol-1-yl, 1,2,3,4-tetrazol-1-yl, arylsulfonyl,
or N-methylpyrrolidinyl functionality (Fig. 1), all generated from
the amido group in the nucleobase.
Fig. 1 Partial structures of various electrophilic pyrimidine nucleosides
developed for C4 functionalization.
aDepartment of Chemistry and Biochemistry, The City College of New York,
160 Convent Avenue, New York, New York, 10031, USA.
E-mail: mlakshman@ccny.cuny.edu
Thiones, which can be synthesized by reaction of the amide
linkage with P2S5
bThe Ph.D. Program in Chemistry, The Graduate Center of the City University of
New York, New York, New York 10016, USA
1,2
or Lawesson’s reagent,3 have been used
for direct displacement reactions.1–3 However, such reactions
usually require harsh conditions. Alternatively, the thiones can
be S-methylated and subsequently reacted with nucleophiles.4–6
Thiones can also be reacted with dimethyldioxirane and nucleo-
cLaboratory of Virology and Chemotherapy, Rega Institute for Medical Research,
Herestraat 49, Postbus 1043 3000 Leuven, Belgium
†Electronic supplementary information (ESI) available: Experimental procedures
and copies of NMR spectra of the products. See DOI: 10.1039/c6ob02334g
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phile, usually an amine or alcohol, leading to modification at amide linkages in pyrimidine nucleosides (Fig. 1), as a facile
the C4.7,8 The C4 chloro derivatives can be prepared by one of approach to modification at the C4 position.
two methods, either chlorination with SOCl2/DMF,9–12 or via
the Appel reaction with PPh3/CCl4.13–15 However, such chloro
compounds seem labile16 and have been used immediately
upon preparation. For instance, the C4 chloro derivatives
Results and discussion
were converted to the C4 ethoxy compounds, which were then
used in displacement reactions with nucleophiles.10,12 C4
triazoles17–21 and tetrazoles22–26 are common, stable reactive
pyrimidine derivatives that have seen wide applications for
nucleoside and nucleic acid modifications. A range of aryl sulfo-
nates (Ar = p-toluenesulfonyl, 2-mesitylenesulfonyl, 2,4,6-tri-
isopropylbenzenesulfonyl),27–32 have also been explored as reac-
tive reagents, not only for direct substitution reactions, but also
for cross-coupling chemistry.16,33 Among these, the 2,4,6-tri-
isopropylbenzenesulfonyl derivatives have been shown to be
reasonably stable but the 2-mesitylenesulfonyl analogues are
not quite as stable.27 By contrast, a p-toluenesulfonyl pyrimidine
nucleoside was used directly after preparation.32 Pyrimidine C4
tosylates have also been converted to N-methylpiperidinyl and
N-methylpyrrolidinyl salts, as reactive nucleoside analogues.34
We35–41 and others42 have evaluated the use of (benzo-
triazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) for the activation of amide linkages of purine
nucleosides, and this approach has now seen general applica-
bility for nucleoside modification.43–47 In our studies, we were
the first to report O6-(benzotriazol-1-yl)purine nucleosides as
stable, isolable, but reactive nucleoside derivatives.35–37,39–41 In
this work, we have evaluated the utility of BOP for activation of
Our studies commenced with the evaluation of conditions for
the activation of unprotected thymidine (1) using 2 equiv. each
of BOP and a base. Although the reaction with DBU (entry 1 in
Table 1) as base proceeded to completion in THF at room
temperature, chromatographic purification of the O4-(benzo-
triazol-1-yl) product 3 was difficult due to its instability
towards silica gel chromatography. Next, a one-pot reaction
was considered (entry 2) with 4 equiv. of morpholine as a
representative nucleophile. This reaction also proceeded but
purification of product 5 was difficult due to its polarity and
co-elution of impurities.
This made us consider protected derivative 2. Here, the
reaction with BOP proceeded rapidly but isolation of inter-
mediate 4 was not readily possible due to degradation upon
chromatography (entry 3). Interestingly, the silyl groups did
not offer any stabilization against decomposition, which con-
trasts with the stabilizing influence of the silyl groups on
8-fluoro-2′-deoxyadenosine.48 Next, formation of the O4-(benzo-
triazol-1-yl) intermediate 4 was first allowed to proceed to com-
pletion (0.5 h), followed by addition of morpholine (entry 4).
After an additional 2 h reaction time, a good 76% yield of
product 6 was obtained. 1,2-Dimethoxyethane (DME) proved
inferior (entry 6) and formation of intermediate 4 was in-
Table 1 Activation of the amide linkage in thymidine with BOP and basea
Entry
Substrate
Conditions
Conversionb
Product: yieldc
1
1
1
2
2
2
2
2
2
2
2
THF, BOP, DBU, 3 h
THF, BOP, DBU, 3 h, then morpholine, 12 h
THF, BOP, DBU, 0.5 h
THF, BOP, DBU, 0.5 h, then morpholine, 2 h
THF, BOP, Cs2CO3, 24 h
DME, BOP, DBU, 2 h
MeCN, BOP, Cs2CO3, 0.5 h, then morpholine, 2 h
MeCN, BOP, DBU, 0.5 h, then morpholine, 2 h
THF, BOP, DBU, morpholine, 20 min
MeCN, BOP, Cs2CO3, morpholine, 24 h
100%
100%
100%
100%
ca. 50%
ca. 50%
100%
100%
100%
3: decompd
5: ca. 58%f
4: decompd
6: 76%
2e
3
4g
5h
6
4: inci
4: inci
6: 94%
6: 73%
7h
8g
9j
10h, j
6: 90%
ca. 10%
6: inci
a Reactions were conducted at room temperature with 0.424 M solutions of 1 or 2 in the anhydrous reaction solvent (except when noted other-
wise), BOP (2 equiv.), and base (2 equiv.). b As assessed by TLC. c Where reported, yield is of isolated and purified product. d Product was not iso-
lated due to decomposition over silica. e Morpholine (4 equiv.) was added to the reaction mixture after complete conversion of 1 to 3. f Product
isolation was difficult as it co-elutes with impurities. g Morpholine (4 equiv.) was added to the reaction mixture, after complete conversion of 2 to
4. h Reaction was conducted with a 0.132 M solution of 2. i Product was not isolated due to incomplete reaction. j After stirring a mixture of 2,
BOP (2 equiv.), and base (2 equiv.) in the appropriate solvent for 5 min, morpholine (4 equiv.) was added to the reaction mixture.
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complete. Then, in a modification of entry 4, MeCN and Cs2CO3 alcohol, and benzyl alcohol, 1-alkoxy-1H-benzotriazoles51 were
were used (entry 7), resulting in an excellent 94% yield of observed to be byproducts (8–20%).
product 6. By replacing Cs2CO3 with DBU, a 73% yield of 6 was
Reagents such as BOP can contain phosphorus triamides
obtained, comparable to that obtained in THF (compare and this is pertinent to some observations that were made.
entries 4 and 8). Interestingly, an excellent 90% yield of 6 was First, an azido group is tolerated under the reaction con-
obtained in a very short 20 min reaction time when the entire ditions, despite possible PIII contaminants, and good product
process was conducted as one-step transformation, with all yields were obtained with substrate 8 (see Tables 2 and 3).
reactive components present (entry 9). However, this reaction During the synthesis of product 11j, the O4-(benzotriazol-1-yl)
appears sensitive to base and solvent because the MeCN and intermediate 4 was generated in situ by reaction with 2 equiv.
Cs2CO3 combination resulted in very little conversion even each of BOP and DBU, in THF. Then 2 equiv. of MeSNa was
after 24 h (entry 10).
added to the reaction mixture, but the second step did not
On the basis of the optimizations conducted in Table 1, reach completion, possibly due to the insolubility of MeSNa in
further generality assessments were conducted. TBDMS- THF. In order to circumvent the solubility issue, polar solvents
protected thymidine, 2′-deoxyuridine, and 3′-azido-2′-deoxy- were utilized for the second step. This was done by evaporating
thymidine (AZT), 2, 7, and 8, respectively, were selected. Two THF after the formation of 4 and use of polar solvents such as
approaches were undertaken with amines and thiols. A two- MeCN, DMF, DMSO, and EtOH. Reactions in MeCN and DMF
step, one-pot method that involved preformation of the did not reach completion and the yields of 11j were very low.
O4-(benzotriazol-1-yl) intermediate, at room temperature (0.5 h The reaction in DMSO gave a moderate yield (49%) of 11j
reaction). This was followed by addition of the nucleophile along with byproducts, identified as 17 and 18 (Fig. 2).
and the reaction was conducted until completion. In a second, N-Oxide 17 has the same molecular mass as intermediate 4
one-step method, all reactants were added and the reaction but is slower eluting on TLC. Compound 18 has one less
was allowed to attain completion. The following conditions oxygen atom in comparison to 4. Increasing the amount of
were used for the various nucleophiles. Other details are pres- MeSNa from 2 to 4 equiv. did not improve the yield of 11j.
ented under Table 2. (a) For all reactions of alkyl amines,
Formation of byproducts 17 and 18 was also observed when
amide-activation was conducted with 2 equiv. each of BOP and reactions were conducted in MeCN and DMF but no attempt
DBU, and 4 equiv. of the amine, in THF. (b) With p-toluidine, was made to isolate them, as they were relatively minor. When
for the two-step, one pot reaction, formation of the O4-(benzo- EtOH was used as solvent, product 19 was observed. This
triazol-1-yl)pyrimidine derivative was accomplished with could result either by displacement of BtO− from 4 or of MeS−
2 equiv. each of BOP and DBU in THF, at room temperature. from 11j, with EtOH. Compound 11j is known to be suscep-
The mixture was evaporated and then the second step was con- tible to substitution reactions by suitable nucleophiles.4–6
ducted in EtOH (2 mL) with 2 equiv. each of the aryl amine
Compound 17 could originate from a unimolecular isomeri-
and iPr2NEt. For the one-step method, 2 equiv. of BOP, zation of the O4-(benzotriazol-1-yl) intermediate 4 or en route
4 equiv. of DBU, and 2 equiv. of the aryl amine were used, in to 4 (see discussion below). In reactivity studies of phosphory-
MeCN. (c) With EtSH and PhCH2SH, for the two-step one-pot lating reagents in the phosphotriester approach to oligonucleo-
reaction, after amide activation the solvent was evaporated, as tides,
a reagent derived from 1-hydroxy-1H-benzotriazole
with p-toluidine. The second step was conducted with 2 equiv. showed significant reaction at the amide residue of thymi-
each of Cs2CO3 and the thiol in DME (2 mL). The one step dine.52 In that work, O4-(benzotriazol-1-yl)-3′,5′-di-O-acetyl-
reaction was conducted with 2 equiv. each of BOP and thiol, thymidine was isolated and it was shown to undergo
and 4 equiv. of DBU, in DME (2 mL). (d) For the two-step, one- rearrangement to the 3′,5′-di-O-acetyl equivalent of compound
pot reaction with MeSNa, as with p-toluidine, amide activation 17, upon heating with 1-methylimidazole in dry pyridine at
was performed and the solvent was evaporated. The second 50 °C.52
step was conducted with MeSNa (2 equiv.) in anhydrous DMSO
Compound 18 is likely a reduction product, and we have
(1 mL). The one-pot reaction was not attempted in this case. shown that 1-hydroxy-1H-benzotriazoles as well as O6-(benzo-
The collection of results from these experiments is shown in triazol-1-yl)purine nucleosides can be reduced with B2(OH)4
Table 2.
and (pinB)2, respectively.53,54 As mentioned earlier, because
Next, we queried the applicability of this method for the benzotriazole-based peptide-coupling agents such as BOP
synthesis of C4 ether derivatives of pyrimidine nucleosides. As could contain PIII contaminants, these could potentially act as
we have previously shown, these reactions cannot be con- reducing agents for a species such as 17 or even intermediate
ducted as one-step processes, but as two-step, one-pot reac- 4. We, therefore, conducted an assessment of BOP obtained
tions.49,50 We had also shown that after formation of the inter- from two different sources to evaluate the ratios of these
mediate benzotriazol-1-yl derivative, evaporation of the reac- various products (supplier 1: Chem Impex and supplier 2:
tion solvent, addition of the alcohol, and the base allows for Sigma-Aldrich).
conversion to the desired ethers. Thus, such an approach was
BOP from supplier 2 out performed that from supplier 1 in
evaluated in the present cases (Table 3). Although Cs2CO3 was a 0.5 h reaction time, in that desired intermediate 4 was
used previously, DBU was also effective and was used here, formed in ca. 60% from the former. Nearly 20% of the N-oxide
and good yields were obtained. In reactions with MeOH, allyl 17 and 0–1% of reduction product 18 was formed from both
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Table 2 Results from the two-step, one-pot, and one-step transformations of three pyrimidine nucleosidesa
Two step, one potb
One stepc
Entry
Substrate
Nucleophile
Time, temp.
Product: yieldd
Time, temp.
Product: yieldd
1
2
3
4
5
6
2
2
7
2
2
8
MeNH2
rt, 0.5 h
rt, 1.5 h
rt, 2 h
rt, 2 h
rt, 2 h
rt, 1 h
11a: 84%e
11b: 72%
12a: 78%
11c: 76%e
11d: 78%
13a: 57%
rt, 45 min
rt, 1 h
rt, 30 min
rt, 1 h
rt, 1 h
rt, 1 h
11a: 90%(97%)e, f
11b: 98%
PhCH2NH2
PhCH2NH2
Me2NH
Et2NH
Et2NH
12a: 92%
11c: 93%e
11d: 98%
13a: 80%
7
2
7
8
2
7
8
7
8
2
rt, 2 h
11e: 75%
12b: 71%
13b: 53%
11f: 75%
12c: 75%
13c: 63%
12d: 76%
13d: 57%
11g: 62%g
rt, 20 min
rt, 30 min
rt, 1 h
11e: 95%
12b: 89%
13b: 80%
11f: 92%
12c: 91%
13c: 90%
12d: 95%
13d: 85%
11g: 67%h
8
rt, 2 h
9
rt, 2 h
10
11
12
13
14
15
rt, 2 h
rt, 1 h
rt, 2 h
rt, 30 min
rt, 40 min
rt, 30 min
rt, 1 h
rt, 1.5 h
rt, 2 h
rt, 2 h
50 °C, 16 h
Reflux, 12 h
16
7
50 °C, 16 h
12e: 64%g
Reflux, 12 h
12e: 69%h
17
18
19
2
2
2
EtSH
PhCH2SH
MeSNa
rt, 3.5 h
rt, 4 h
50 °C, 2 h
11h: 68%g
11i: 70%g
11j: 49%i
rt, 3 h
rt, 3 h
Not done
11h: 76%h
11i: 82%h
NA j
a Reactions were conducted at 0.424 M nucleoside concentration in the reaction solvent, using 0.212 mmol of 2, 0.219 mmol of 7, or 0.262 mmol
of 8. b The substrates were converted to the respective O4-(benzotriazol-1-yl) derivatives using BOP (2 equiv.) and DBU (2 equiv.) in anhydrous
THF, and then nucleophile (4 equiv.) was added to the reaction mixture. c The nucleoside, BOP (2 equiv.), DBU (2 equiv.), and nucleophile
(4 equiv.) were reacted in the appropriate solvent. d Yields are of isolated and purified products. e The following amine solutions were used: 2 M
MeNH2 in THF and 40 wt% Me2NH in H2O. f The yield was based on recovered 2, the reaction was incomplete and ca. 7% of 2 was reisolated.
g After formation of the benzotriazolyl derivative, the solvent was evaporated. Then nucleophile (2 equiv.), and base (2 equiv.) were used in the
appropriate solvent (2 mL). h DBU (4 equiv.), nucleophile (2 equiv.), and appropriate solvent (2 mL) were used. i After formation of the benzo-
triazolyl derivative, the solvent was evaporated. Then MeSNa (2 equiv.) was used in anhydrous DMSO (1 mL). j A one-step approach was not
attempted in this case.
within the short reaction time. At 24 h, a substantial amount then evaluated whether DBU had any role in this process, and
of 17 had resulted from both, with an increased amount of the with the better-performing BOP, the amount of DBU was
reduction product (more so with BOP from supplier 1). We increased to 4 equiv. After 24 h, none of intermediate 4 was
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Table 3 C4 ether derivatives of pyrimidine nucleosides with BOP and alcoholsa
Entry
Substrate
Alcohol
Time
Product: yieldb
1
2
3
2
7
2
MeOH
MeOH
0.5 h
1 h
1 h
14a: 91%c
15: 85%c
14b: 88%c
4
5
8
8
CH2vCHCH2OH
PhCH2OH
1 h
1 h
16a: 56%d
16b: 85%d
a Unless noted otherwise, reactions were conducted at 0.424 M nucleoside concentration in the reaction solvent, using 0.212 mmol of 2,
0.219 mmol of 7, or 0.262 mmol of 8. The substrates were converted to the respective O4-(benzotriazol-1-yl) derivatives using BOP (2 equiv.) and
DBU (2 equiv.) in anhydrous THF. After evaporation, alcohol and additional DBU were added. b Yields are of isolated and purified products.
c DBU (2 equiv.) and the alcohol (20 equiv.) were used for the second step. d The reaction was conducted at 0.115 M nucleoside concentration in
the reaction solvent, and after the formation of O4-(benzotriazol-1-yl) derivative, 2 equiv. of DBU and 2 equiv. of alcohol were added to the reac-
tion mixture.
observed, the amount of 17 was lower, and the amount of the
reduction product 18 increased. It has been empirically shown
that an N-oxide type intermediate from uridine can also react
with nucleophiles.52 Thus, one must surmise that DBU can
react with 4 and/or 17 (there are numerous examples in the lit-
erature where DBU undergoes ring opening to a caprolactam by
action as nucleophile). From this, the displaced BtO− could
undergo reduction, possibly with HMPT from BOP, forming the
Fig. 2 Formation of side products from precursor 2 en route to 11j.
PG = TBDMS.
benzotriazolide. The benzotriazolide can then function as nucleo-
phile, resulting in compound 18 from any reactive intermediate
Fig. 3 Partial proton NMR spectra of compounds 17 (in black) and 18 (in blue) in CDCl3. Benzotriazolyl resonances are indicated by dots and
TBDMS resonances are not shown.
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to confirm that byproducts 17 and 18 are indeed as anti-
cipated. We have previously shown that 1-hydroxy-1H-benzo-
triazoles can be reduced to 1H-benzotriazoles by
reaction with B2(OH)4/Et3N in MeCN.53 Because product 17 is
anticipated to be an N-oxide, exposure to just B2(OH)4 in
MeCN should result in its reduction. This was indeed the
case and a high-yield conversion 17 to 18 was observed
(Fig. 3).
From a mechanistic standpoint, there are three possible
routes to the O4-(benzotriazol-1-yl)pyrimidine derivatives,
represented by pathways a, b, and c shown in Scheme 1.
Among these, pathway a proceeds through phosphonium ion I.
Pathways b and c produce the O4-(benzotriazol-1-yl)pyrimidine
derivative II directly, either via direct displacement of HMPA
from the benzotriazolyl N1 atom or a SN2′-like process at the
N3 atom of that moiety.
In our previous reports on purine nucleoside modification,
clear formation of a phosphonium ion was evident via 31P{1H}
NMR studies, and no N-oxide-type species could be identi-
fied.35,40 In contrast, in the present cases, exposure of the
pyrimidine nucleoside to BOP and DBU led to concomitant
formation of the O4-(benzotriazol-1-yl) intermediate and the
Scheme 1 Some mechanistic considerations.
formed along the reaction path, including a species formed from
the reaction of DBU with 4 and/or 17. This would be consistent
with a mechanism we have recently proposed for the reduction of
O6-(benzotriazol-1-yl)purine nucleosides by (pinB)2.54
Mechanistic considerations
With the reactions and analysis of plausible modes for for-
mation of byproducts completed, we performed one reaction
Table 4 Evaluating the various products formed with BOP from two sourcesa
Yieldc
BOPb
DBU
2 equiv.
2 equiv.
2 equiv.
2 equiv.
4 equiv.
Time
4
17
18
Recovered 2
Supplier 1: 99.7% pure
0.5 h
24 h
0.5 h
24 h
24 h
33%
14%
59%
22%
—
18%
32%
23%
46%
8%
1%
10%
—
3%
16%
14%
—
—
—
—
Supplier 2: 97% pure
a Reactions were conducted with 2 equiv. of BOP in THF. b Purities are as stated by the suppliers. c Yields are of isolated and purified products.
Fig. 4 31P{1H} NMR spectra in CD3CN to observe for changes during the course of the reaction at room temperature.
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N-oxide, but the former was the predominant product as of the data in Table 4 (increase in the N-oxide at the expense of
observed by TLC. Although we cannot comment on whether the O4-(benzotriazol-1-yl) species over a protracted reaction
the N-oxide is independently formed in the reaction, it can time). Also, we cannot comment on the role of DBU, if any, in
result from the O4-(benzotriazol-1-yl) intermediate on the basis this isomerization process. Nevertheless, both species II and
Fig. 5 31P{1H} NMR spectra in CD3CN to observe for changes when BOP is in contact with DBU and pyrrolidine at room temperature.
Fig. 6 Structures of products prepared, desilylation conditions, reaction times, and yields. Conditions A: n-Bu4NF, THF, rt; B: KF, MeOH, 80 °C; C:
TASF, MeCN, 0 °C to rt.
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III ultimately result in product formation. Phosphonium ion I, 3′-Amino-2′,3′-dideoxy-5-methylcytidine, cytidine, and 5-fluoro-
if formed, can also lead directly to product. cytidine have previously been prepared via the C4 triazolyl
In order to gain additional insight in plausible pathways intermediates
and for comparison to the reactions of purine nucleosides,35,40 derivatives.21
obtained
from
corresponding
azido
several 31P{1H} experiments were undertaken. In one experi-
Compounds shown in Fig. 6 and Scheme 2 were assayed for
ment BOP (δ = 43.8 ppm, PF6− δ = −144.5 ppm) was exposed to their abilities to inhibit proliferation of murine leukemia
precursor 2 (Fig. 4), with no observable change. However, (L1210), human cervix carcinoma (HeLa), and human
upon addition of DBU, formation of HMPA was immediately T-lymphocytic (CEM) cell lines. Modest activity (90 5 to 219
observed (δ
diminished.
=
23.5 ppm), and this increased as BOP 48 µM) was observed for thymidine-derived compounds 5, 20b,
d, e, f–i, and AZT-derived 25b (see Table 5). Notably, removal
In another experiment, BOP was exposed to DBU and pyrro- of the 5-methyl group abrogated activity in compounds 21a,
lidine. Here a trace of HMPA was observed upon contact of 21b, 21d, and 21e.
all reagents, and after 1 h (Fig. 5). Eventually though, BOP
All compounds were also tested for their antiviral activities
diminishes but HMPA formation remains low. This appears to against: (a) cytomegalovirus (CMV), varicella-zoster virus (VZV),
indicate that DBU and pyrrolidine will cause consumption of herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2),
BOP. But in the presence of the nucleoside, because reaction vaccinia virus, and vesicular stomatitis virus (VSV) in human
of the amide linkage is fast (0.5 h), this secondary reaction embryonic lung (HEL) cells, (b) VSV, coxsackie virus B4, and
may be inconsequential. Individually, DBU and pyrrolidine respiratory syncytial virus in human cervix carcinoma (HeLa)
also cause consumption of BOP with formation of HMPA, but
those reactions (data not shown) are slower than that with
the combination. Importantly though, at least as assessed by
Table 5 Inhibitory effects of compounds against proliferation of
murine leukemia (L1210), human T-lymphocyte (CEM), and human
31P{1H} NMR, the mechanism by which BOP reacts with amide
groups in the pyrimidine nucleosides studied here, appears to cervix carcinoma (HeLa) cellsa
differ from the corresponding reactions of purine nucleosides
where discernible phosphorus resonances of nucleoside phos-
IC50b (µM)
phonium salts can be observed (δ ∼ 35 ppm) as the reactions
Compound
L1210
CEM
HeLa
progress.35,40
5
150 56
116 32
99 33
141 40
130 32
106 15
>250
120
≥250
>250
>250
103
231 27
219 48
198 75
120
>250
201
188 49
207 14
181 23
20b
20d
20e
20f
20g
20h
20i
25b
8
1
Biological results
4
In order to obtain materials for biological assays, compounds
were subjected to desilylation (Fig. 6). Three different con-
ditions were used for this purpose: n-Bu4NF in THF at room
temperature, KF in MeOH at 80 °C, and tris(dimethylamino)
sulfonium difluorotrimethylsilicate (TASF) in MeCN from 0 °C
to room temperature. Specific conditions used and product
yields are summarized in Fig. 6. Notably desilylation of 11h
with KF/MeOH yielded 23a by substitution of the SEt group.
Because C4-modified azido nucleosides were available, we
decided to reduce the N-substituted 3′-azido-2′,3′-dideoxy-
5-methylcytidine derivatives to the corresponding amino com-
pounds for assessment of biological activities (Scheme 2).
7
90
103
5
9
≥250
≥250
166 94
123 15
a Only compounds showing some activity for any of the three cell lines
are shown. The other synthesized compounds were not inhibitory at
the higher concentration evaluated, i.e. 250 µM. b IC50
= 50%
Inhibitory concentration or compound concentration required to
inhibit tumor cell proliferation by 50%.
Table 6 Activities of compounds against HIV-1 and HIV-2a
EC50b (µM)
Compound
HIV-1
HIV-2
59 46
13 8.7
≥250
23 2.1
1.6 0.57
0.64 0.064
0.74 0.16
20d
22a
22b
22c
22d
25a
25b
151 85
11 7.9
176 105
8.6 2.1
0.83 0.38
0.44 0.21
0.66 0.087
a Only compounds showing some anti-HIV activity are shown. The
other synthesized compounds were not inhibitory at the higher con-
centration evaluated, i.e. 250 µM. b EC50 = 50% Inhibitory concen-
tration or compound concentration required to inhibit tumor cell pro-
liferation by 50%.
Scheme 2 Reduction of the 3’-azido-2’,3’-dideoxy nucleosides to the
corresponding amines.
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cells, (c) parainfluenza-3 virus, reovirus, sindbis virus, cox- corresponding amines. The ensuing 29 compounds were assessed
sackie B-4 virus, and punta toro virus in green monkey kidney for their abilities to inhibit cancer cell proliferation using L1210,
(VERO) cells, (d) feline corona and feline herpes viruses in HeLa, and CEM cell lines, where some showed modest activity.
feline kidney (CRFK) cells, and (e) influenza A H1N1, influenza Antiviral assessments indicated several to possess anti-HIV-1 and
B H3N2, and influenza B viruses in canine kidney (MDCK) HIV-2 activities, and one compound showed anti-HSV-1 activity. In
cells. The activity data are summarized in Table 6.
summary, the method described herein offers a generally facile,
Several compounds showed activities against HIV-1 and broadly applicable approach for the C4 functionalization of pyri-
HIV-2 (Table 6), particularly interesting are azido derivatives midines and pyrimidine nucleosides, which can lead to the dis-
22a (compare with 20d), 22c, 22d, 25a, and 25b. In comparison covery of new pharmacologically active agents as well as for other
to diethylamino derivative 20d, presence of a 3′-azide greatly applications.
enhances activity in 22a. Piperidinyl derivative 22c was com-
parable to 22a and both showed higher activity over the pyrroli-
dinyl analogue 22b. Inclusion of an oxygen atom in the six-
membered ring in 22d increased activity by an order of magni-
Acknowledgements
tude. Ether derivatives 25a and 25b also displayed significant This work was supported by National Science Foundation
activity, with the former being higher. In these cases, release Grant CHE-1265687 to MKL. Infrastructural support at CCNY
of AZT by loss of the ether moiety may be possible.
was provided by National Institutes of Health Grant
Among the entire set of compounds tested, none of them G12MD007603 from the National Institute on Minority Health
showed activity against the other virus tested except for a and Health Disparities. Dr Padmanava Pradhan (CCNY) is
single C4 ether derivative; compound 23a displayed anti-HSV-1 thanked for assistance with some NMR experiments.
activity. This compound inhibited HSV-1 Kos strain with an
EC50 of 5.4 2.0 µM while the minimum cytotoxic concen-
tration required to cause a microscopically detectable altera-
tion of normal cell morphology was >100 µM. Removal of the
References
5-methyl unit of 23a, however, caused loss of anti-HSV-1
activity as observed with compound 24. Notably, compound
23a lacked activity against HSV-2 and an acyclovir-resistant
(thymidine-kinase deficient) HSV-1 virus.
1 J. J. Fox, D. Van Praag, I. Wempen, I. L. Doerr, L. Cheong,
J. E. Knoll, M. L. Eidinoff, A. Bendich and G. B. Brown,
J. Am. Chem. Soc., 1959, 81, 178–187.
2 I. Wempen, R. Duschinsky, L. Kaplan and J. J. Fox, J. Am.
Chem. Soc., 1961, 83, 4755–4766.
3 J.-F. Griffon, C. Mathé, A. Faraj, A.-M. Aubertin, E. De
Clercq, J. Balzarini, J.-P. Sommadossi and G. Gosselin,
Eur. J. Med. Chem., 2001, 36, 447–460.
Conclusions
Herein, we have demonstrated a procedure for the facile intro-
duction of substituents at the C4 position of pyrimidine
nucleosides. These include amines, thiols, and alcohols. The
approach involves reaction of the amide, but not the urea
moiety, with BOP and base, leading to an intermediate O4-
(benzotriazol-1-yl)pyrimidine nucleoside derivative, within a
short reaction time. With amines and thiols, two-step, one-pot,
and one-step processes have been investigated. The latter was
marginally to significantly superior to the former in every case
studied. With alcohols, only a two-step, one-pot approach is
feasible. The method is tolerant of a 3′-azido group in the
4 T. Ueda and J. J. Fox, J. Med. Chem., 1963, 6, 697–701.
5 I. Wempen, N. Miller, E. A. Falco and J. J. Fox, J. Med.
Chem., 1968, 11, 144–148.
6 M. E. Perlman, K. A. Watanabe, R. F. Schinazi and J. J. Fox,
J. Med. Chem., 1985, 28, 741–748.
7 R. Saladino, E. Mincione, C. Crestini and M. Mezzeti,
Tetrahedron, 1996, 52, 6759–6780.
8 R. Saladino, C. Crestini, R. Bernini, G. Frachey and
E. Mincione, J. Chem. Soc., Perkin Trans. 1, 1994, 3053–3054.
9 J. Žemlička and F. Šorm, Collect. Czech. Chem. Commun.,
1965, 30, 2052–2067.
nucleoside. It appears that prolonged reactions times with 10 M. J. Robins and S. R. Naik, Biochemistry, 1971, 10, 3591–
BOP and DBU lead to conversion of the O4-(benzotriazol-1-yl)
3597.
pyrimidine nucleoside to an isomeric benzotriazolyl N-oxide 11 T.-S. Lin and W. R. Mancini, J. Med. Chem., 1983, 26, 544–
form, although the latter is also expected to be reactive 548.
towards displacement by nucleophiles. The N-oxide could 12 A. Matsuda, H. Itoh, K. Takenuki, T. Sasaki and T. Ueda,
undergo reduction with PIII species in BOP, resulting in 4-(ben-
Chem. Pharm. Bull., 1988, 36, 945–953.
zotriazol-1-yl)pyrimidine nucleosides. That the byproducts 13 R. Appel, Angew. Chem., Int. Ed. Engl., 1975, 14, 775–843.
from the reactions of the amide groups in pyrimidine nucleo- 14 L. De Napoli, A. Messere, D. Montesarchio, G. Piccialli and
sides with BOP are plausibly the benzotriazolyl N-oxide and
the 4-(benzotriazol-1-yl) nucleoside derivatives was shown by 15 C. Lou, A. Dallmann, P. Marafini, R. Gao and T. Brown,
reduction of the former to the latter by B2(OH)4. Mechanism of Chem. Sci., 2014, 5, 3836–3844.
the reaction was queried by NMR experiments. All products 16 U. Hennecke, D. Kuch and T. Carell, Synthesis, 2007, 929–
C. Santacroce, Nucleosides Nucleotides, 1991, 10, 1719–1728.
were desilylated and the azido compounds were reduced to the
935.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
17 C. B. Reese and A. Ubasawa, Nucleic Acids Symp. Ser., 1980, 37 S. Bae and M. K. Lakshman, J. Org. Chem., 2008, 73, 3707–
7, 5–21. 3713.
18 K. J. Divakar and C. B. Reese, J. Chem. Soc., Perkin Trans. 1, 38 S. Bae and M. K. Lakshman, Org. Lett., 2008, 10, 2203–
1982, 1171–1176. 2206.
19 C. B. Reese and P. A. Skone, J. Chem. Soc., Perkin Trans. 1, 39 S. Bae, S. Chaturvedi and M. K. Lakshman, Curr. Protoc.
1984, 1263–1271.
Nucleic Acid Chem., 2009, 36, 1.22.1–1.2.23.
40 M. K. Lakshman and J. Frank, Org. Biomol. Chem., 2009, 7,
2933–2940.
41 S. K. Sakilam, P. K. Vuram, S. S. Relangi, V. Gurram,
H. Zhou, R. J. Kreitman, M. Martinez Montemayor,
L. Yang, M. Kaliyaperumal, S. Shama, N. Pottabathini and
M. K. Lakshman, Molecules, 2015, 20, 18437–18463.
42 Z.-K. Wan, S. Wacharasindhu, E. Binnun and T. Mansour,
Org. Lett., 2006, 8, 2425–2428.
43 M. K. Lakshman, M. K. Singh, D. Parrish, R. Balachandran
and B. W. Day, J. Org. Chem., 2010, 75, 2461–2473.
44 T. D. Ashton and P. J. Scammells, Aust. J. Chem., 2008, 61,
49–58.
45 S. M. Devine, L. T. May and P. J. Scammells, Med. Chem.
Commun., 2014, 5, 192–196.
20 W. L. Sung, J. Org. Chem., 1982, 47, 3623–3628.
21 T.-S. Lin, Y.-S. Gao and W. R. Mancini, J. Med. Chem., 1983,
26, 1691–1696.
22 W. L. Sung and S. A. Narang, Can. J. Chem., 1982, 60, 111–
120.
23 K. Kamaike, M. Takahashi, K. Utsugi, K. Tomizuka and
Y. Ishido, Tetrahedron Lett., 1995, 36, 91–94.
24 X. Ariza and J. Vilarrasa, J. Org. Chem., 2000, 65, 2827–
2829.
25 A. Kobori, K. Miyata, M. Ushioda, K. Seio and M. Sekine,
J. Org. Chem., 2002, 67, 476–485.
26 S. K. Mahto and C. Chow, Bioorg. Med. Chem., 2008, 16,
8795–8800.
27 N. Bischofberger, Tetrahedron Lett., 1987, 28, 2821–2824.
28 N. Bischofberger and M. D. Matteucci, J. Am. Chem. Soc., 46 S. Krüger and C. Meier, Eur. J. Org. Chem., 2013, 1158–1169.
1989, 111, 3041–3046.
47 M. Printz and C. Richert, Chem. – Eur. J., 2009, 15, 3390–
3402.
48 A. Ghosh, P. Lagisetty and B. Zajc, J. Org. Chem., 2007, 72,
8222–8226.
49 H. P. Kokatla and M. K. Lakshman, Org. Lett., 2010, 12,
4478–4881.
50 H. P. Kokatla and M. K. Lakshman, Curr. Protoc. Nucleic
Acid Chem., 2012, 49, 1.26.1–1.26.16.
29 Y. Ding, Q. Habib, S. Z. Shaw, D. Y. Li, J. W. Abt, Z. Hong
and H. An, J. Comb. Chem., 2003, 5, 851–859.
30 S. Verma and P. S. Miller, Bioconjugate Chem., 1996, 7, 600–
605.
31 C. Cismaş and T. Gimisis, Tetrahedron Lett., 2008, 49, 1336–
1339.
32 S. C. Holmes and M. J. Gait, Eur. J. Org. Chem., 2005, 5171–
5183.
33 S. B. Kang, E. De Clercq and M. K. Lakshman, J. Org.
Chem., 2007, 72, 5724–5730.
51 M. K. Lakshman, M. K. Singh, M. Kumar, R. R. Chamala,
V. R. Yedulla, D. Wagner, E. Leung, L. Yang, A. Matin and
S. Ahmad, Beilstein J. Org. Chem., 2014, 10, 1919–1932.
34 K. Tsuchiya and H. Komatsu, Nucleic Acids Res. Suppl., 52 C. B. Reese and K. H. Richards, Tetrahedron Lett., 1985, 26,
2002, 135–136. 2245–2248.
35 S. Bae and M. K. Lakshman, J. Am. Chem. Soc., 2007, 129, 53 V. Gurram, H. K. Akula, R. Garlapati, N. Pottabathini and
782–789. M. K. Lakshman, Adv. Synth. Catal., 2015, 357, 451–462.
36 S. Bae and M. K. Lakshman, J. Org. Chem., 2008, 73, 1311– 54 V. Basava, L. Yang, P. Pradhan and M. K. Lakshman, Org.
1319.
Biomol. Chem., 2016, 14, 7069–7083.
Org. Biomol. Chem.
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