Synthesis of C -Pseudonucleosides Bearing Thiazolidin-4-one as a Novel Potential Immunostimulating Agent

Article abstract of DOI:10.1021/ml200155k

Several novel C-pseudonucleosides bearing thiazolidin-4-one were synthesized by one-pot three-component condensation using unprotected sugar aldehyde at room temperature, and their effects on T cells, B cells, the cytokine secretion of IL-2, IL-4, and IFN-γ, T cell-associated molecules (CD3, CD4, CD8), and B cell-associated molecules (CD19) were first evaluated. The experimental data demonstrated that such thiazolidin-4-one C-pseudonucleosides hold potential as immunostimulating agents.

Full text of DOI:10.1021/ml200155k

LETTER
pubs.acs.org/acsmedchemlett
Synthesis of C-Pseudonucleosides Bearing Thiazolidin-4-one as a
Novel Potential Immunostimulating Agent
Hua Chen,† Qingmei Yin,† Chunxiao Li,‡ Enkai Wang,† Fang Gao,† Xiaobo Zhang,† Zhi Yin,† Sinan Wei,†
Xiaoliu Li,*,†,§ Ming Meng,*,‡ Pingzhu Zhang,† Na Li,‡ and Jinchao Zhang†
†Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science and ‡Medical School,
Hebei University, Baoding 071002, China
§State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
S Supporting Information
b
ABSTRACT: Several novel C-pseudonucleosides bearing thiazolidin-4-one were synthe-
sized by one-pot three-component condensation using unprotected sugar aldehyde at room
temperature, and their effects on T cells, B cells, the cytokine secretion of IL-2, IL-4, and
IFN-γ, T cell-associated molecules (CD3, CD4, CD8), and B cell-associated molecules
(CD19) were first evaluated. The experimental data demonstrated that such thiazolidin-4-
one C-pseudonucleosides hold potential as immunostimulating agents.
KEYWORDS: Immunostimulating agents, C-pseudonucleosides, thiazolidin-4-one
mmunostimulators, as adjuvant therapeutic agents for the
treatment of many tumors and infectious diseases such as
I
the AIDS epidemic, have aroused great attention due to their
highly potential value to enhance the ability of the human
immune system.1 Many different compounds, such as biological
macromolecules, including glycoproteins,2 polynucleotides,3 and
polysaccharides,4 and small molecules, including heterocycles,5
nucleosides,6 glycosylceramides (glycolipids),7 and iminosugars,8
have been demonstrated to possess immunostimulatory proper-
ties. Some of them have been applied in the clinic as immunoad-
juvants or immunomodulating drugs, such as interferon,2 β-glucan,4
levamisole,9 and pidotimod10 (Figure 1A, B), and so on. However,
the drawbacks, including side-effects, low titer, and high cost, limited
their widely applications in clinical trails.11 It is still of great interest
and challenge to explore more effective and safe immunostimulants
for treating immunologic disorders.
Figure 1. Some chemical small molecule immunostimulating agents.
strong immunostimulating activities. More recently, we have
found that the thiazolidin-4-one linked pseudodisaccharides
(Figure 1E) showed very good immunostimulatng activity,18
which prompted us to develop the novel C-pseudonucleosides
having a thiazolidin-4-one moiety for investigating their immune
activity. Herein, as the continuation of our studies on function-
alized nucleoside analogues,19 we would like to report a simple
and convenient synthesis of novel C-pseudonucleosides bearing
thiazolidin-4-one (4 and 5) by one-pot, three-component con-
densation from the unprotected sugar aldehyde 1 (Scheme 1),
and their biological effects on T cells, B cells, and the cytokine
secretion of IL-2, IL-4, and IFN-γ from mouse splenocytes.
Furthermore, the effects on the membrane expression of T cell-
associated molecules (CD3, CD4, CD8) and B cell-associated
molecules (CD19) were also investigated.
The requisite sugar aldehyde 1 has been readily prepared in
one step from D-glucosamine hydrochloride in the high yield of
85%.20 The C-pseudonucleosides 4 and 5 were synthesized by
one-pot, three-component condensation from the unprotected
sugar aldehyde 1, aliphatic primary amines or anilines 2aꢀe, and
mercaptoacetic acid 3 at room temperature, as shown in Scheme 1.
The one-pot synthesis, following our reported precedure,19 but with-
out using the condensation reagent N,N0-dicyclohexylcarbodiimide
Nucleoside-based small molecule immunostimulants that
affect the proliferation and potentiation of the cytokine-produ-
cing humoral cells have offered considerable promise in drug
development.6 Among them, the guanosine derivatives (e.g.,
Loxoribine, Figure 1D) have been extensively studied and proved
to hold potential as immunostimulating agents.12 Nonclassic
base-modified nucleosides represent a large group of pseudonu-
cleosides in which the natural pyrimidine or purine base was
replaced by heterocycles such as thiazole, imidazole, triazole,
triazine, etc.13 However, such nucleoside analogues are scarcely
explored for their immunomodulating activity, although they
have been well documented as promising anti-infective and
antitumor agents.14,15
The thiazolidin-4-one ring is a core substructure in various
synthetic pharmaceuticals which are associated with diverse
biological activities,16 such as anticancer, antiviral, and anti-
inflammatory, and some thiazolidine derivatives; for instance,
levamisole, pidotimod, and CGP52608 (Figure 1C)17 exhibited
Received: June 27, 2011
Accepted: September 7, 2011
Published: September 07, 2011
r
2011 American Chemical Society
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ACS Medicinal Chemistry Letters
LETTER
Scheme 1. Reagents and Conditions: (a) 1 (1 mmol),
2 (1 equiv), 3 (2 equiv), dry EtOH (3 mL), rt, 1 h
Figure 3. Effects of compounds 4a, 5a, 4e, and 5e on IL-4 (A), INF-γ
(B), and IL-2 (C) in mouse splenocyte cultures. The concentrations of
compounds were 10, 50, 5, and 5 μM, respectively. Data are means (
SEM of at least three independent experiments. *p < 0.05, **p < 0.01,
***p < 0.001.
Figure 2. Effects of compounds 4a, 5a, 4e, and 5e on Con A-induced
splenocyte proliferation. Data are means ( SEM of at least three
#
independent experiments. p < 0.001 with respect to the untreated
cells; *p < 0.05, **p < 0.01, ***p < 0.001 with respect to the Con
A-treated cells.
also found in compounds 4e and 5e, which showed the most
pronounced effects on T cell proliferation, at 5 and at 50 μM,
respectively. However, the other compounds, 4b, 5b, 4c, 5c, 4d,
and 5d, did not express significant effects on T cells proliferation
at the tested concentrations, and all the proliferation rates were
below 50% (not shown). The results suggested that the immu-
nostimulating activity decreased with the elongation of the alkyl
chain on the N-3 position at the thiazolindin-4-one ring. In
addition, the similar activity of the diastereomeric compounds 4a
and 5a (or 4e and 5e) implied that the configurations in C-2 did
not have significant effects on their immunostimulatory activity.
In the absence of Con A, compounds 4a, 5a, 4e, and 5e could
not induce cell proliferation, implying that the compounds
synergistically induced the immune response with Con A. Next
we assayed the effects of these four compounds on the secretion
of various cytokines in the culture of total splenocytes. Using a
mice ELISA kit, the levels of the cytokines, such as interleukin-4
(IL-4), IL-2, and interferon-γ (IFN-γ), were measured from the
supernatant of cell cultures in the presence of Con A (20 μM) in
combination with the compounds synthesized above. Com-
pounds 4a, 5a, and 4e at their optimized effective concentrations
of 10, 50, and 5 μM were tested respectively. Since 5e at 5 μM
(166%) and 50 μM (171%) demonstrated similar effects on Con
A-induced cell proliferation, 5 μM of 5e were tested.
Compared with Con A alone, a combination of Con A and 4a,
5a, 4e, or 5e increased the levels of IL-4 secretion by 43, 42, 39,
and 41% (p < 0.001), respectively (Figure 3 A), which indicated
that the compounds had similar proliferation effects on IL-4.
With regard to IFN-γ, compounds 4a and 5a obviously increased
the secretion levels of this cytokine by 21 and 30% (p < 0.001),
respectively, while compounds 4e and 5e had no effects on
IFN-γ secretion (Figure 3B). Compounds 4a, 4e, and 5e
also markedly induced the secretion levels of IL-2 by 78, 99,
and 138% (p < 0.001), respectively, and compound 5a increased
slightly the IL-2 level by 37% (Figure 3C). Clearly, compounds
4e and 5e had more pronounced effects on IL-2 secretion than
compounds 4a and 5a.
(DCC) and the promoter 4-dimethylaminopryidine (DMAP),
directly afforded the diastereomeric products 4 (less polar) and 5
(more polar), respectively, in the overall yields of 36ꢀ43%, after
simple working up and purification by reverse silica (C18) gel
column chromatography. This modified reaction provided a more
direct, simple, and efficient access to the target C-pseudonucleosides
containing thiazolidin-4-one using the unprotected sugar aldehyde,
although the yields in the condensation step were moderate com-
pared to those reported,21,22 in which the protected sugars were
used and a deprotection must be done to give the target molecules.
The structures of the C-pseudonucleosides 4 and 5 were
assigned on the basis of the analyses of their spectral data of
NMR and HRMS(ESI) (see the Supporting Information). The
X-ray crystallographic analysis of compound 5a indicated that the
configuration of the new generated chiral carbon (C-2) is in the
(R) form. Accordingly, its diastereomer 4a should have the (S)
form. The circular dichroism (CD) spectra of compounds 4
showed positive Cotton effects nearly at 230 nm and negative
Cotton effects at 205 nm, while 5 showed opposite Cotton effects
at the corresponding regions, providing a further support to the
configurations of C-2 in the diastereomers 4 (S) and 5 (R).
The effects of the C-pseodonucleosides 4 and 5 on the
concavalin A (Con A)-induced proliferation of mouse splenocyte
were assessed by the MTT method. The cells were harvested
after 72 h incubation at 37 °C, 5% CO2. As shown in Figure 2,
compounds 4a, 5a, 4e, and 5e promoted Con A-induced T cell
proliferation significantly at certain concentrations. Compared to
the Con A-treated sample, the treatment with the combination of
Con A and 4a (5, 10, 25, 50, and 100 μM) increased the
proliferation by 28, 290, 138, 35, and 245%, respectively, and
the concentration dependence was not obvious. Compound 5a
(5, 10, 25, 50, and 100 μM) also increased the Con A-induced cell
proliferation irregularly by 111, 98, 91, 327, and 230%, respec-
tively. Similarly concentration-independent phenomena were
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LETTER
the body’s natural response to microbial infection. IFN-γ, the
hallmark cytokine of the TH1 cells, shows antiviral, immunor-
egulatory, and antitumor properties and is used to treat infectious
diseases, although it may precipitate autoimmunity. The imbal-
ance between TH1 and TH2 cytokines is closely related with the
outcome of many diseases. TH1 responses predominate in organ-
specific autoimmune disorders, in acute allograft rejection, and in
some chronic inflammatory disorders. In contrast, TH2 responses
predominate in Omann’s syndrome, transplantation tolerance,
chronic graft-versus-host disease, systemic sclerosis, and allergic
diseases.
Compounds 4a and 5a (R = CH3) had a significant capability
to augment all of the secretions of IL-4, IFN-γ, and IL-2.
Therefore, they might have immunopotentiating efficiency via
both TH1- and TH2-mediated cellular as well as humoral immune
activation. On the other hand, compounds 4e and 5e (R =
phenyl) increased IL-4 and IL-2 secretions and inhibited IFN-γ,
which indicated that they might have a bias via TH2-mediated
cellular immunity. Furthermore, compounds 4a and 5a specifi-
cally promoted CD3+ and CD4+T cell proliferation, while com-
pounds 4e and 5e could enhance the proliferation of CD3+,
CD4+T, and B cells. The results would suggeste that (1) such
C-pseudonucleosides bearing thiazolidine-4-ones 4a, 5a, 4e, and
5e were good immunostimulators in controlling infection and
triggering the response to antibodies and (2) the difference of the
substituent at N-3 on the thiazolindin-4-one ring would lead to
different mechanisms for the compounds to proliferate different
cells, possibly due to the different structural regions.
Figure 4. Proliferative effects on CD3+ (A), CD4+ (B), CD8+ (C), and
CD19+ (D) cells of compounds 4a, 5a, 4e, and 5e with the concentra-
tion of 10, 50, 5, and 5 μM, respectively. Data are means ( SEM of at
least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
In conclusion, we have reported a simple and convenient
synthesis of C-pseudonucleosides bearing thiazolidin-4-one as
base from the unprotected sugar aldehyde, aliphatic and alkyl
primary amines, and mercaptoacetic acid at room temperature.
Biological tests revealed that compounds 4a, 5a, 4e, and 5e had
significant effects on T cell proliferation at certain concentra-
tions. The preliminary structureꢀactivity relationship (SAR)
analysis indicated that the configuration difference in C-2
between 4 (S) and 5 (R) had little effect on their immunosti-
mulating activity, and as the alkyl chain on the N-3 position at the
thiazolindin-4-one ring lengthened, the immunostimulating ac-
tivity reduced. Given that the compounds act synergistically with
the mitogen Con A, they may be suitable adjuvants with immun-
boosting drugs and vaccines. The interesting findings may open a
new avenue in the development of a new class of pseudonucleo-
side drugs possessing immunoregulatory activity.
Figure 5. Effects of compounds 4a, 5a, 4e, and 5e on 10 μg/mL LPS-
induced splenocyte proliferation. The concentrations of compounds
were 10, 50, 5, and 5 μM, respectively. Data are means ( SEM of at least
three independent experiments. #p < 0.01 with respect to the untreated
cells; *p < 0.05, **p < 0.01, ***p < 0.001 with respect to the LPS-
treated cells.
The effects of the compounds on the T cells expressing CD3,
CD4, and CD8 and on the B cells expressing CD19 were assayed
by the flow cytometer (FCM).23 Compared to the Con A-treated
cells, the four compounds 4a, 5a, 4e, and 5e had proliferative
effects on CD3+T cells and CD4+T cells (Figure 4A, B).
However, they had no effects on CD8+T cells (Figure 4C).
On the other hand, compounds 4e and 5e demonstrated stronger
proliferative effects on the CD19+B cell population than com-
pounds 4a and 5a did (Figure 4D), which indicated that
compounds 4e and 5e preferred to promote B cell proliferation
more than compounds 4a and 5a (Figure 5, compounds 4e and
5e at 5 μM increased the LPS-induced cell proliferation by 117
and 238%, respectively). These observations suggested that the
C-nucleosides 4a and 5a had potentials in proliferative effects on
the CD4+T cells, and compounds 4e and 5e specifically stimu-
lated the proliferation of both CD4+T cells and B cells.
CD4+T cells which recognize the antigen peptide and MHC
II complex include T helper type 1 and 2 (TH1 and TH2) cells.
TH1 cells secrete IFN-γ and IL-2, while TH2 cells secrete IL-4.
IL-4, the hallmark cytokine of the TH2 cells, stimulates B cells to
secrete antibodies and improves the proliferation and differentia-
tion of T-cells as a key regulator in humoral and adaptive
immunity.24 IL-2 secreted by the TH1 cells is instrumental in
’ ASSOCIATED CONTENT
S
Supporting Information. Full experimental procedures
b
and characterization data for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone and Fax: (+86)-312-5971116. E-mail: lixl@hbu.edu.cn
or mengming127@163.com.
Author Contributions
The manuscript was written through contributions of all authors.
Funding Sources
The financial supports from the National Basic Research 973
Program of China (2010CB534913), the National Natural Science
847
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ACS Medicinal Chemistry Letters
LETTER
Foundation of China (NSFC) (20972039), the Natural Science
Foundation of Hebei (B2011201169), the Program of Science and
Technology (S&T) of Hebei (09276418D-13), the Natural Science
Foundations of the Education Department of Hebei (2009309,
ZH2011110), and the Open Research Fund of the State Key
Laboratory of Natural and Biomimetic Drugs, Peking Uni-
versity (20080205), are gratefully acknowledged.
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