New conjugates of mycophenolic acid and their antiproliferative activity

Article abstract of DOI:10.1080/10286020.2016.1184653

The new conjugates of mycophenolic acid (MPA) were obtained in the reaction of N⁶-(ω-aminoalkyl)adenosines with MPA in the presence of EDCI as a coupling reagent. New compounds 4a–h were evaluated on leukemia cell line (Jurkat) and PBMC from healthy donors. Length of the linker influenced observed activity. The compound 4b possessing 1,3-diamine spacer exhibited the most promising results and can be considered to further investigations.

Full text of DOI:10.1080/10286020.2016.1184653

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
New conjugates of mycophenolic acid and their
antiproliferative activity
Michał Prejs, Grzegorz Cholewinski, Agnieszka Siebert, Piotr Trzonkowski &
Krystyna Dzierzbicka
To cite this article: Michał Prejs, Grzegorz Cholewinski, Agnieszka Siebert, Piotr Trzonkowski &
Krystyna Dzierzbicka (2016): New conjugates of mycophenolic acid and their antiproliferative
activity, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2016.1184653
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1184653
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Date: 19 June 2016, At: 10:12

Journal of asian natural Products research, 2016
ꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1184653
ꢀ
New conjugates of mycophenolic acid and their
antiproliferative activity
a
a
a
b
Michał Prejs , Grzegorz Cholewinski , Agnieszka Siebert , Piotr Trzonkowski and
a
Krystyna Dzierzbicka
ꢈ
b
dꢉpꢈꢅꢁmꢉꢆꢁ ꢃꢇ oꢅgꢈꢆꢄꢌ cꢀꢉmꢄꢊꢁꢅy, Gꢂꢈꢆꢊk uꢆꢄvꢉꢅꢊꢄꢁy ꢃꢇ tꢉꢌꢀꢆꢃꢋꢃgy, 80-233 Gꢂꢈꢆꢊk, Pꢃꢋꢈꢆꢂ; dꢉpꢈꢅꢁmꢉꢆꢁ ꢃꢇ
cꢋꢄꢆꢄꢌꢈꢋ immꢍꢆꢃꢋꢃgy ꢈꢆꢂ tꢅꢈꢆꢊpꢋꢈꢆꢁꢃꢋꢃgy, Mꢉꢂꢄꢌꢈꢋ uꢆꢄvꢉꢅꢊꢄꢁy ꢃꢇ Gꢂꢈꢆꢊk, 80-211 Gꢂꢈꢆꢊk, Pꢃꢋꢈꢆꢂ
ABSTRACT
ARTICLE HISTORY
The new conjugates of mycophenolic acid (MPA) were obtained in the
rꢉꢌꢉꢄvꢉꢂ 17 dꢉꢌꢉmbꢉꢅ 2015
aꢌꢌꢉpꢁꢉꢂ 26 apꢅꢄꢋ 2016
6
reaction of N -(ω-aminoalkyl)adenosines with MPA in the presence of
EDCI as a coupling reagent. New compounds 4a–h were evaluated on
leukemia cell line (Jurkat) and PBMC from healthy donors. Length of
the linker influenced observed activity. The compound 4b possessing
KEYWORDS
Myꢌꢃpꢀꢉꢆꢃꢋꢄꢌ ꢈꢌꢄꢂ; MPa;
ꢈꢂꢉꢆꢃꢊꢄꢆꢉ; ꢊyꢆꢁꢀꢉꢊꢄꢊ;
ꢈꢆꢁꢄpꢅꢃꢋꢄꢇꢉꢅꢈꢁꢄvꢉ ꢈꢌꢁꢄvꢄꢁy
1
,3-diamine spacer exhibited the most promising results and can be
considered to further investigations.
1
. Introduction
Mycophenolic acid (MPA) 1 (Scheme 1) is widely reported as an uncompetitive inhibitor
of inosine-5′-monophosphate dehydrogenase (IMPDH) and its derivatives, mycophenolate
mofetil (MMF, CellCept) and mycophenolate sodium (MPS, Myfortic), are used in preven-
tion of organ rejection transplant and autoimmune disorders treatment [1–4]. However,
numerous side effects such as myelosuppression, vomiting, diarrhea, nausea limit usage
of these prodrugs, and various structural modifications were reported [5–14]. One of the
alterations is a conjugate forming, where MPA is connected to other bioactive molecules
[
15–17]. Pankiewicz and coworkers rationalized series of inhibitors of IMPDH as a nico-
tinamide adenine dinucleotide-dependent enzyme. Mycophenolic adenine dinucleotides
include derivatives revealing higher potency toward both isoforms of IMPDH than reference
tiazofurin adenine dinucleotide and in some cases comparable to MPA, which is one of the
most potent hIMPDH1 and 2 inhibitor [18–22].
In this paper, we report synthesis of the new conjugates 4a–h (Scheme 1) possessing
adenosine attached with a simple diamine linker and MPA, followed by their preliminary
antiproliferative activity investigations.
CONTACT Kꢅyꢊꢁyꢆꢈ dzꢄꢉꢅzbꢄꢌkꢈ
kꢅyꢂzꢄꢉꢅ@pg.gꢂꢈ.pꢋ
tꢀꢉ ꢊꢍppꢋꢉmꢉꢆꢁꢈꢅy mꢈꢁꢉꢅꢄꢈꢋ ꢇꢃꢅ ꢁꢀꢄꢊ pꢈpꢉꢅ ꢄꢊ ꢈvꢈꢄꢋꢈbꢋꢉ ꢃꢆꢋꢄꢆꢉ ꢈꢁ ꢀꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1184653.
©
2016 iꢆꢇꢃꢅmꢈ uK lꢄmꢄꢁꢉꢂ, ꢁꢅꢈꢂꢄꢆg ꢈꢊ tꢈyꢋꢃꢅ & fꢅꢈꢆꢌꢄꢊ Gꢅꢃꢍp

2
M. PꢀEJS ET AL.
O
Cl
HN
N
NH2
n
HO
N
N
N
N
N
N
O
HO
HO
HO
O
N
H N
NH2
O
2
n
1
O
O
NEt , EtOH,
3
o
60
C, 18 h
DMAP, EDCI, DMF
OH OH
OH OH
o
0
C - rt, 48 h
2
3a-h
O
HN
N
N
H
n
O
N
N
HO
O
HO
N
O
O
OH OH
4
a-h
n = 2, 3, 4, 5, 6, 8, 10, 12
Scheme 1. syꢆꢁꢀꢉꢊꢄꢊ ꢃꢇ ꢌꢃꢆjꢍgꢈꢁꢉꢊ ꢃꢇ ꢈꢂꢉꢆꢃꢊꢄꢆꢉ ꢂꢉꢅꢄvꢈꢁꢄvꢉꢊ wꢄꢁꢀ myꢌꢃpꢀꢉꢆꢃꢋꢄꢌ ꢈꢌꢄꢂ 4a–h.
2
. Results and discussion
2
.1. Chemistry

e synthetic pathway included aromatic nucleophilic substitution of the relevant diamine
6
to 6-chloropurine ryboside 2 [23] and coupling of N -(ω-aminoalkyl) adenosines 3a–h
with MPA 1 (Scheme 1). e second stage was optimized with several condensing reagents,
and the best results were achieved using 1-ethyl-3-(3′-dimethylaminipropyl) carbodiimide
(
EDCI) with 4-(dimethyloamino)pyridine (DMAP). Although the yields of these reactions
were relative low, they did not need any protection of hydroxyl groups. e final products
1
4
a–h were purified with preparative TLC, and their composition was confirmed by H NMR
and MALDI-TOF mass analysis.
General procedure for the preparation of derivatives of adenosine 3a–h and its conjugates
with MPA 4a–h was described in Supplementary Information.
2
.2. Biological assays
Biological potency was established as antiproliferation activity specified against lymphoid
cell line Jurkat and activated peripheral blood mononuclear cells (PBMC) from healthy
donors as in vitro model of immunosuppression. Jurkat is a model lymphocyte cell line
which can be bought similarly to any other consumables, see the link to the most popular
source [24]. We have used lymphocyte model cell line as the analyzed effects of the tested
compounds are mainly seen in the lymphocytes. Main idea of using these compounds as
immunosuppressants is to stop proliferation of the lymphocytes. All transplantology focuses
on the inhibition of lymphocytes when it comes to the treatment of rejection. Using human
blood as the most important tissue for testing covers PBMC cells, not only in transplan-
tation but in any somatic diseases diagnostics or treatment. ese results were presented
in Table 1 and expressed as micromolar EC concentrations. EC values were calculated
5
0
50

JꢁꢂꢀNAL ꢁꢃ ASIAN NATꢂꢀAL PꢀꢁDꢂCTS ꢀESEAꢀCꢄ
3
Table 1. ec (μM) vꢈꢋꢍꢉꢊ ꢃꢇ ꢈꢂꢉꢆꢃꢊꢄꢆꢉ ꢂꢉꢅꢄvꢈꢁꢄvꢉꢊ 3a–h ꢈꢆꢂ ꢌꢃꢆjꢍgꢈꢁꢉꢊ 4a–h ꢇꢃꢅ ꢌꢉꢋꢋ ꢋꢄꢆꢉ Jꢍꢅkꢈꢁ ꢈꢆꢂ
5
0
ꢈꢌꢁꢄvꢈꢁꢉꢂ PBMc, ꢃbꢁꢈꢄꢆꢉꢂ ꢄꢆ ꢈꢆꢁꢄpꢅꢃꢋꢄꢇꢉꢅꢈꢁꢄꢃꢆ ꢁꢉꢊꢁ.
Jurkat
PBMC
Entry
EC50
±
p
F
EC50
±
p
F
3
3
3
3
3
3
3
3
a
b
c
d
e
f
24.49
90.03
1.48
28.09
279.5
69.71
0.71
15.54
1952.9
10.28
1.28
10.77
11.00
111.6
16.36
0.41
7.93
53.96
0.68
6.38
81.9
5.42
0.71
5.149
72.59
1.355
0.138
0.468
0.46
38.58
2.15
0.138
0.11
0.005
0.024
0.102
0.049
0.03
0.022
0.025
0.043
20,144
884.5
47.23
16.08
50.8
100.4
75.43
268.7
0.967
427
0.59
1.42
0.82
0.249
141.5
0.34
0.42
0.54
7.71
5.96
3.33
31.54
7.15
10.98
3.81
4.673
15.1
0.015
0.038
0.014
0.056
0.043
0.012
0.015
0.08
6.59
11.78
6.67
4.12
10.19
7.01
6.5
25.86
44.49
8.43
129.5
8.49
17.71
11.87
9.93
61.16
13.63
46.49
114.1
0.001
g
h
3.28
aꢂꢉꢆꢃꢊꢄꢆꢉ
4
4
4
4
4
4
4
4
a
b
c
d
e
f
0.033
0.086
0.0497
0.019
0.031
0.054
0.0128
0.025
39.65
1.751
1.524
130.2
47.24
12.44
300.3
77.1
0.000002
0
0
0
0
0
90.18
590.8
178
1900
596.6
704.6
62.86
1962
5.07
g
h
0.759
1.793
0.0002
0.000007
0
0.26
12.17
MPa 1
3.34
nꢃꢁꢉꢊ: p—ꢊꢁꢈꢁꢄꢊꢁꢄꢌꢈꢋ ꢊꢄgꢆꢄfiꢌꢈꢆꢌꢉ, F—fꢄꢊꢀꢉꢅ ꢁꢉꢊꢁ.
3
from incorporation of H-TdR. EC was the concentration of a drug that gave halfmaximal
5
0
response during scintillation measurement (radiation).
e data presented in Table 1 show that the all compounds 3a–h, 4a–h are more active
than adenosine both against Jurkat cell line and PBMC as well. Several derivatives 3c, 3g,
b, 4g, 4h exhibited considerably higher activity against Jurkat cell line than MPA 1; how-

4
ever, MPA 1 gave the best activity within experiments against PBMC. e most interesting
conjugate 4b revealed better activity than its starting adenosine derivative 3b against both
Jurkat cell line and PBMC and than MPA 1 against Jurkat cell line.

e cytotoxic properties of compounds 3a–h, 4a–h were evaluated with colorimetric
MTT (λ = 570 nm), and the data were presented in Table 2. Results are expressed as micro-
molar IC concentrations and reported as the compound dose required to reduce the via-
5
0
bility of the tested cells by 50% in regard to control sample. e highest value in this series
of measurements was assigned to adenosine both against Jurkat cell line and PBMC. Similar
values of IC against Jurkat cell line gave MPA 1, and derivatives 3a, 3b, 4b. Adenosine
5
0
derivative 3b and conjugate 4 g indicated the highest IC values against PBMC.
5
0
In order to identify analogs of MPA which showed the most favorable parameters, we
appointed selectivity index (SI). SI gives the idea which is the real action of the compound,
means, what is the effect (EC ) in the context of relatively non-toxic concentration (IC ).
5
0
50
Proposed comparison or ratio of native PBMC to the cell line has no biological value for
immunosuppressive drug candidate. ese results were collected in Table 3. According to
these data, the shortest alkyl chain is favorable in the case of adenosine derivatives 3a–h,
since the best results gave 3a; SI against Jurkat was 92.8, and 185 against PBMC, where
adenosine exhibited SI = 1.5 against Jurkat, and 23.2 against PBMC. However, SI index
received for MPA 1 occurred to be much higher against PBMC (SI = 15,920). Noteworthy,
conjugate forming with MPA 1 did not change observed activity additively, as compounds
4
a–h provided SI values different from MPA 1, especially against PBMC. Length of diamine
linker influenced the activity of 4a–h, but the correlation was not clear. e best SI index

4
M. PꢀEJS ET AL.
Table 2. ic50 (μM) vꢈꢋꢍꢉꢊ ꢃꢇ ꢈꢂꢉꢆꢃꢊꢄꢆꢉ ꢂꢉꢅꢄvꢈꢁꢄvꢉꢊ 3a–h ꢈꢆꢂ ꢌꢃꢆjꢍgꢈꢁꢉꢊ 4a–h ꢇꢃꢅ ꢌꢉꢋꢋ ꢋꢄꢆꢉ Jꢍꢅkꢈꢁ ꢈꢆꢂ
ꢈꢌꢁꢄvꢈꢁꢉꢂ PBMc, ꢃbꢁꢈꢄꢆꢉꢂ ꢄꢆ Mtt ꢁꢉꢊꢁ.
Jurkat
PBMC
Entry
IC50
±
p
F
IC50
±
p
F
3
3
3
3
3
3
3
3
a
b
c
d
e
f
2273.2
1796.6
15.07
9.93
26.74
13.94
35.26
29.07
2912.4
477
1109.5
589.2
68.27
73.12
73.77
92.3
305.5
92.5
7.093
4.37
0.047
0.371
0.014
0.144
0.113
0.044
0.351
0.066
221.8
3.127
2603
23.56
38.39
252.8
3.563
115.2
179.2
718.4
21.28
10.78
18.01
12.93
118.1
25.52
3010.4
125.5
80.9
5.15
5.19
69.53
232.2
721.9
319.8
15.92
127.6
36.35
6.8
1.561
5.185
7.276
11.12
6.88
911.5
29.54
11.63
1.171
1.298
1.495
19.95
38.49
1.99
0.04
0.023
0.002
0.103
0.113
0.003
0.1
305.4
910.9
165,514
46.31
38.7
44,052
49.22
2.402
3.802
15.15
8.656
548.6
106.4
47.08
43.72
3.16
6.579
37.03
36.84
3.56
g
h
0.01
4895.7
aꢂꢉꢆꢃꢊꢄꢆꢉ
4
4
4
4
4
4
4
4
a
b
c
d
e
f
0.054
0.068
0.049
0.001
0.007
0.018
0.005
0.12
168.9
108.8
206.3
270,746
9352
1538.6
17,048
33.96
0.385
0.36
2.868
3.35
34.73
179.4
56.93
186
0.119
0.053
0.093
0.052
0.141
0.114
g
h
24.59
37.87
13.15
1553.3
MPa 1
204.2
8.05
nꢃꢁꢉꢊ: p—ꢊꢁꢈꢁꢄꢊꢁꢄꢌꢈꢋ ꢊꢄgꢆꢄfiꢌꢈꢆꢌꢉ, F—fꢄꢊꢀꢉꢅ ꢁꢉꢊꢁ.
Table 3. sꢉꢋꢉꢌꢁꢄvꢄꢁy ꢄꢆꢂꢉx ꢃꢇ ꢈꢂꢉꢆꢃꢊꢄꢆꢉ ꢂꢉꢅꢄvꢈꢁꢄvꢉꢊ 3a–h ꢈꢆꢂ ꢌꢃꢆjꢍgꢈꢁꢉꢊ 4a–h ꢇꢃꢅ ꢌꢉꢋꢋ ꢋꢄꢆꢉ Jꢍꢅkꢈꢁ ꢈꢆꢂ
ꢈꢌꢁꢄvꢈꢁꢉꢂ PBMc.
Jurkat
PBMC
Entry
IC /EC
IC /EC
50
5
0
50
50
3
3
3
3
3
3
3
3
a
b
c
d
e
f
92.8
20
10.2
0.35
0.096
0.2
49.7
1.9
1.5
46.4
867
54.7
6.2
0.66
4.5
225
50.6
128
185
1.7
36
7.6
22
0.5
2.6
3
23.2
14.8
4.6
0.43
0.52
1.1
17
15.5
2.8
g
h
aꢂꢉꢆꢃꢊꢄꢆꢉ
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
MPa 3
15,920
value of 867 was received in case of compound 4b (n = 3), whereas MPA 1 provided SI = 128.
Derivative 4g (n = 10) also gave SI better than MPA against Jurkat cell line (225).
3
. Conclusions
6
To sum up, obtained N -(ω-aminoalkyl)adenosines 3a–h and conjugates with MPAs 4a–h
revealed antiproliferative activity against Jurkat cell line comparable to MPA 1, a very potent
immunosuppressant. e most promising result was received in case of analog 4b (n = 3),

JꢁꢂꢀNAL ꢁꢃ ASIAN NATꢂꢀAL PꢀꢁDꢂCTS ꢀESEAꢀCꢄ
5
where SI value against Jurkat cell line reached 867. is compound possessing short
1
,3-diamine linker between the two bioactive molecules can be proposed to further
investigations.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was supported by the Polish National Science Center (NCN) [grant number 2013/11/B/
NZ7/04838].
References
[
[
1] L. Hedstrom, Chem. Rev. 109, 2903 (2009).
2] F. Schmidt, K. Eckardt, M. Shakibaei, P. Glander, and R. Stahlmann, Arch. Toxicol. 87, 361
(
2013).
[
3] P.A. Brookes, J. Cordes, A.J.P. White, and A.G.M. Barret, Eur. J. Org. Chem. 2013, 7313 (2013).
4] S. Mitsuhashi, J. Takenaka, K. Iwamori, N. Nakajima, and M. Ubukata, Bioorg. Med. Chem.
[
1
8, 8106 (2010).
5] E. Habib, F. Leo
1, 1915 (2008).
[
[
́n , J.D. Bauer, R.A. Hill, P. Carvalho, H.G. Cutler, and S.J. Cutler, J. Nat. Prod.
7
6] P.H. Nelson, S.F. Carr, B.H. Devens, E.M. Eugui, F. Franco, C. Gonzalez, R.C. Hawley, R.G.
Loughhead, D.J. Milan, E. Papp, J.W. Patterson, S. Rouhafza, E.B. Sjogren, D.B. Smith, R.A.
Stephenson, F.X. Talamas, A.N. Waltos, R.J. Weikert, and J.C. Wu, J. Med. Chem. 39, 4181
(
1996).
[
[
7] Z. Chen, Z. Zheng, H. Huang, Y. Song, X. Zhang, J. Ma, B. Wang, C. Zhang, and J. Ju, Bioorg.
Med. Chem. Lett. 22, 3332 (2012).
8] W.J. Watkins, J.M. Chen, A. Cho, L. Chong, N. Collins, M. Fardis, W. Huang, M. Hung,
T. Kirschberg, W.A. Lee, X. Liu, W. omas, X. Xu, A. Zeynalzadegan, and J. Zhang, Bioorg.
Med. Chem. Lett. 16, 3479 (2006).
[
9] M. Fardis, M. Mertzman, W. omas, T. Kirschberg, N. Collins, R. Polniaszek, and W.J.
Watkins, J. Org. Chem. 71, 4835 (2006).
[
[
[
[
[
10] N. Yang, J. Wang, Z.W. Wang, Q.H. Wang, H.G. Yang, X.J. Wang, and M.S. Cheng, Chem. Biol.
Drug Des. 79, 1063 (2012).
11] K. Sunohara, S. Mitsuhashi, K. Shigetomi, and M. Ubukata, Bioorg. Med. Chem. Lett. 23, 5140
(
2013).
12] G. Cholewinski, M. Malachowska-Ugarte, and K. Dzierzbicka, Curr. Med. Chem. 17, 1926
(
2010).
13] G. Cholewiński, D. Iwaszkiewicz-Grześ, M. Prejs, A. Głowacka, and K. Dzierzbicka, J. Enzyme
Inhib. Med. Chem. 30, 550 (2015).
14] D. Iwaszkiewicz-Grzes, G. Cholewinski, A. Kot-Wasik, P. Trzonkowski, and K. Dzierzbicka,
Eur. J. Med. Chem. 69, 863 (2013).
[
[
15] X. Wang, Y. Lin, Y. Zeng, X. Sun, T. Gong, and Z. Zhang, Int. J. Pharm. 456, 223 (2013).
16] M. Malachowska-Ugarte, G. Cholewinski, K. Dzierzbicka, and P. Trzonkowski, Eur. J. Med.
Chem. 54, 197 (2012).
[
[
[
17] H. Wu, J. Pagadala, C.R. Yates, D.D. Miller, and R.I. Mahato, Bioconjugate Chem. 24, 2036
(
2013).
18] K. Felczak, L. Chen, D. Wilson, J. Williams, R. Vince, R. Petrelli, H.N. Jayaram, P. Kusumanchi,
M. Kumar, and K.W. Pankiewicz, Bioorg. Med. Chem. 19, 1594 (2011).
19] L. Chen, D. Wilson, H.N. Jayaram, and K.W. Pankiewicz, J. Med. Chem. 50, 6685 (2007).

6
M. PꢀEJS ET AL.
[
[
20] K. Felczak, R. Vince, and K.W. Pankiewicz, Bioorg. Med. Chem. Lett. 24, 332 (2014).
21] D. Rejman, M. Olesiak, L. Chen, S.E. Patterson, D. Wilson, H.N. Jayaram, L. Hedstrom, and
K.W. Pankiewicz, J. Med. Chem. 49, 5018 (2006).
[
[
[
22] L. Chen, G. Gao, K. Felczak, L. Bonnac, S.E. Patterson, D. Wilson, E.M. Bennett, H.N. Jayaram,
L. Hedstrom, and K.W. Pankiewicz, J. Med. Chem. 50, 5743 (2007).
23] R. Ottria, S. Casati, E. Baldoli, J.A.M. Maier, and P. Ciuffreda, Bioorg. Med. Chem. 18, 8396
(
2010).
24] http://www.lgcstandards-atcc.org/products/all/TIB-152.aspx?geo_country=pl.