Cofactor-type inhibitors of inosine monophosphate dehydrogenase via modular approach: Targeting the pyrophosphate binding sub-domain

Article abstract of DOI:10.1016/j.bmc.2011.01.042

Cofactor-type inhibitors of inosine monophosphate dehydrogenase (IMPDH) that target the nicotinamide adenine dinucleotide (NAD) binding domain of the enzyme are modular in nature. They interact with the three sub-sites of the cofactor binding domain; the nicotinamide monophosphate (NMN) binding sub-site (N sub-site), the adenosine monophosphate (AMP) binding sub-site (A sub-site), and the pyrophosphate binding sub-site (P sub-site or P-groove). Mycophenolic acid (MPA) shows high affinity to the N sub-site of human IMPDH mimicking NMN binding. We found that the attachment of adenosine to the MPA through variety of linkers afforded numerous mycophenolic adenine dinucleotide (MAD) analogues that inhibit the two isoforms of the human enzyme in low nanomolar to low micromolar range. An analogue 4, in which 2-ethyladenosine is attached to the mycophenolic alcohol moiety through the difluoromethylenebis(phosphonate) linker, was found to be a potent inhibitor of hIMPDH1 (K_i = 5 nM), and one of the most potent, sub-micromolar inhibitor of leukemia K562 cells proliferation (IC₅₀ = 0.45 μM). Compound 4 was as potent as Gleevec (IC₅₀ = 0.56 μM) heralded as a 'magic bullet' against chronic myelogenous leukemia (CML). MAD analogues 7 and 8 containing an extended ethylenebis(phosphonate) linkage showed low nanomolar inhibition of IMPDH and low micromolar inhibition of K562 cells proliferation. Some novel MAD analogues described herein containing linkers of different length and geometry were found to inhibit IMPDH with K_i's lower than 100 nM. Thus, such linkers can be used for connection of other molecular fragments with high affinity to the N- and A-sub-site of IMPDH.

Full text of DOI:10.1016/j.bmc.2011.01.042

Bioorganic & Medicinal Chemistry 19 (2011) 1594–1605
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cofactor-type inhibitors of inosine monophosphate dehydrogenase via
modular approach: Targeting the pyrophosphate binding sub-domain
Krzysztof Felczak a, Liqiang Chen a, Daniel Wilson a, Jessica Williams a, Robert Vince a, Riccardo Petrelli a,
,
Hiremagalur N. Jayaram b, Praveen Kusumanchi b, Mohineesh Kumar b, Krzysztof W. Pankiewicz a,
⇑
a Center for Drug Design, University of Minnesota, 516 Delaware Street S.E., Minneapolis, MN 55455, USA
b Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Richard Roudebush, Veterans Affairs Medical Center,
1481 West Tenth Street, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cofactor-type inhibitors of inosine monophosphate dehydrogenase (IMPDH) that target the nicotinamide
adenine dinucleotide (NAD) binding domain of the enzyme are modular in nature. They interact with the
three sub-sites of the cofactor binding domain; the nicotinamide monophosphate (NMN) binding sub-
site (N sub-site), the adenosine monophosphate (AMP) binding sub-site (A sub-site), and the pyrophos-
phate binding sub-site (P sub-site or P-groove). Mycophenolic acid (MPA) shows high affinity to the N
sub-site of human IMPDH mimicking NMN binding. We found that the attachment of adenosine to the
MPA through variety of linkers afforded numerous mycophenolic adenine dinucleotide (MAD) analogues
that inhibit the two isoforms of the human enzyme in low nanomolar to low micromolar range. An ana-
logue 4, in which 2-ethyladenosine is attached to the mycophenolic alcohol moiety through the difluo-
romethylenebis(phosphonate) linker, was found to be a potent inhibitor of hIMPDH1 (Ki = 5 nM), and
Received 8 December 2010
Revised 14 January 2011
Accepted 21 January 2011
Available online 27 January 2011
Keywords:
IMPDH
Inhibitor design
NAD analogues
Mycophenolic acid
Bis(phosphonates)
one of the most potent, sub-micromolar inhibitor of leukemia K562 cells proliferation (IC50 = 0.45
lM).
Compound 4 was as potent as Gleevec (IC50 = 0.56 M) heralded as a ‘magic bullet’ against chronic mye-
l
logenous leukemia (CML). MAD analogues 7 and 8 containing an extended ethylenebis(phosphonate)
linkage showed low nanomolar inhibition of IMPDH and low micromolar inhibition of K562 cells prolif-
eration. Some novel MAD analogues described herein containing linkers of different length and geometry
were found to inhibit IMPDH with Ki’s lower than 100 nM. Thus, such linkers can be used for connection
of other molecular fragments with high affinity to the N- and A-sub-site of IMPDH.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
514 amino acids and share 84% of amino acid sequences.4,5 The
hIMPDH1 was identified as an anti-angiogenic drug target,6 and
IMPDH, a NAD dependent enzyme, has emerged in recent years
as a major therapeutic target.1,2 It controls de novo synthesis of
purine nucleotides catalyzing oxidation of inosine 50-monophos-
phate (IMP) to xanthosine 50-monophosphate (XMP) which is fur-
ther converted into guanosine 50-monophosphate (GMP) by GMP
synthase. Two isoforms of human IMPDH are known,3 both contain
mycophenolic acid (MPA), a nanomolar inhibitor of hIMPDH, was
found to block tumor-induced angiogenesis in vivo. The hIMPDH2
is selectively up-regulated in neoplastic cells and activated lym-
phocytes and emerges as the dominant form.7 (e.g., 15–42 fold ele-
vated in human myelogenous leukemia compared to normal
leukocytes.)8 It is therefore believed that IMPDH is a key enzyme
in neoplasia and is one of the most sensitive targets for cancer
chemotherapy.
NAD-based inhibitors of IMPDH that target the cofactor binding
site of the enzyme can interact with the three sub-sites of the
cofactor binding domain (Fig. 1); the nicotinamide monophosphate
(NMN) binding sub-site (N sub-site), the adenosine monophos-
phate (AMP) binding sub-site (A sub-site), and the pyrophosphate
binding sub-site (P sub-site or P-groove). Thus, such inhibitors are
modular in nature, and usually consist of two fragments interact-
ing with the N and A binding sub-sites connected via an appropri-
ate linker (accommodated in the P-groove).
Abbreviations: AMP, adenosine 50-monophosphate; IMP, inosine 50-monophos-
phate; IMPDH, inosine monophosphate dehydrogenase; NAD, nicotinamide adenine
dinucleotide; NMN, nicotinamide mononucleotide; TR, tiazofurin; TAD, thiazole-4-
carboxamide adenine dinucleotide; MPA, mycophenolic acid; MAD, mycophenolic
adenine dinucleotide; CML, chronic myelogenous leukemia; AICAR, 5-aminoimi-
dazole-4-carboxamide riboside; PMB, p-methoxybenzyl; DIC, N,N-disopropylcar-
bodiimide; Mycophenolic alcohol, 7-(hydroxy)-6-(2-ethyleneyl)-5-methoxy-4-
methyl-phthalan-1-one with protected or unprotected 7 hydroxyl group.
⇑
Corresponding author. Tel.: +1 612 625 7968; fax: +1 612 624 825.
E-mail address: panki001@umn.edu (K.W. Pankiewicz).
Present address: School of Pharmacy, Medicinal Chemistry Unit, Università di
Camerino, Via S. Agostino 1, 62032 Camerino, Italy.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.042

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1595
Table 1
Inhibition of IMPDH type I and type II, as well as inhibition of K562 cells proliferation
by TAD and MAD analogues
Inhibitors
IMPDH type I
Ki (nM)
IMPDH type II
Ki (nM)
K562 cells proliferation
IC50 (l
M)a
TAD
MPA
110
33
110
7
3.7b
7.7
C2-MAD
1
2
3
330
1
250
14
30 2.7
38
5.7
ND
4.7
13 1.9c
16
1.0
4
5
6
5
87
20
1.1
24 2.1
60
37
0.45
118.4
71.3
7
8
9
95 11.7
99 4.9
5550 350
1100 80
349
255 7.8
23,600
14,200
94 5.4
7900 600
70
84 4.3
73 3.4
3770 240
770 42
167
83 2.0
18,800
6600
10.5
4.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Figure 1. The modular nature of the cofactor binding domain of NAD-dependent
IMPDH (PDB entry 1NFb).
10
11
12
13
14
15
16
17
18
Four potent and selective inhibitors of IMPDH are used in the
clinic. Two of them, ribavirin and mizoribine, are phosphorylated
in the cell to the corresponding 50-monophosphates that interact
with the substrate (IMP) domain of IMPDH. Ribavirin is a broad
spectrum antiviral drug currently used for treatment of hepatitis
C infections and mizoribine found an application in Japan as an
39 3.7
1900 330
44
77
34
a
The values of IC50 for inhibition of K562 cells proliferation are averages from
three experiments.
immunosuppressant. The third, tiazofurin (TR, Fig. 2),
a C-
b
This IC50 value is based on degradation of TAD which releases TR that enters
nucleoside prodrug used for treatment of chronic myelogenous
leukemia (CML) in blast crisis, is metabolically converted into
thiazole-4-carboxamide adenine dinucleotide (TAD), which as a
NAD analogue interacts with all three N, A, and P sub-sites of
NAD-dependent IMPDH. TAD is a selective and potent inhibitor
of IMPDH1 and IMPDH2 (Ki = 110 nM, Table 1).
Finally, mycophenolic acid (MPA, Fig. 3) used in the clinic as an
immunosuppressant, binds to the N sub-site of IMPDH and is one
of the most potent inhibitors of hIMPDH2 (Ki = 7 nM).
In the last decade we and others have shown clearly that the
cofactor binding domain, although conserved in variety NAD-
dependent enzymes, is diversified enough to be a valuable target
for therapeutic intervention with selective cofactor-type inhibi-
tors.9 For example, we used MPA and its fragments (that bind
exclusively at the N sub-site) for construction of mycophenolic
(see abbreviations) adenine dinucleotides (MAD) analogues (such
as C2-MAD, Table 1) by attachment of adenosine to the mycophen-
olic alcohol moiety through methylenebis(phosphonate) linker
(Fig. 3).10 The crystal structure of the C2-MAD/hIMPDH2 complex
showed that indeed the MPA moiety was bound at the N sub-site,
and adenosine was located at the A sub-site mimicking closely
interactions of NAD and TAD with the protein.11 In this article
we focus on IMPDH inhibitors with modified pyrophosphate moi-
eties and their interactions with the P-groove of the enzyme.
cells to be metabolized back to TAD.
c
Standard deviations are reported for new compounds only.
hIMPDH1 (Ki = 1.0 nM) and hIMPDH2 (Ki = 14 nM).12 However, as a
pyrophosphate analogue, compound 1 is metabolically unstable
and cannot penetrate cells membrane. Thus, we report herein that
the replacement of the natural pyrophosphate (P-O-P) moiety of 1
with the isosteric and isopolar difluoromethylenebis(phosphonate)
(P-CF2-P) group afforded TAD analogue 2 (Fig. 4). It still shows (Table
1) low nM inhibition of hIMPDH1 (Ki = 13 nM) and hIMPDH2
(Ki = 30 nM) and, as expected, inhibits proliferation of leukemia
K562 cells (IC50 = 4.7 lM). Earlier, we also prepared the mycophen-
olic alcohol 2-ethyladenosine dinucleotide analogue 3 containing a
methylenebis(phosphonate) group as the replacement for the pyro-
phosphate linkage (Fig. 4).12 Compound 3 showed 20-fold more po-
tent inhibitory activity (Ki = 16 nM) against hIMPDH1 than the
parent C2-MAD (Ki = 330 nM). We have now converted 3 into its
difluoromethylenebis(phosphonate) analogue 4. As expected, we
found an improvement in enzyme inhibitory activity (Ki = 5 nM,
hIMPDH1) and consequently a potent inhibition of K562 cells
growth (IC50 = 0.45 lM). In fact, analogue 4 is the most potent
anti-proliferating agent among IMPDH inhibitors (Table 1); it is
17-fold more potent than MPA showing the inhibitory activity sim-
ilar to that of Gleevec (imatinib) (IC50 = 0.56 lM), the Bcr-abl thyro-
sine kinase inhibitor, used in the clinic for treatment of CML.13
Thus, indeed linking potent N sub-site binders, such as TR or
2. Design of inhibitors and their biological activity
MPA with high affinity
ethyladenosine) through the bis(phosphonate) (P-CF2-P or
P-CH2-P) moiety afforded potent inhibitors of IMPDH.
A sub-site binders (such as 2-
We reported earlier that the analogue of TAD 1 (Table 1) contain-
ing a 2-ethyl group at the adenine ring is a low nM inhibitor of
However, as we found earlier, linkers that are not as close to the
natural architecture of the pyrophosphate group or its methylene-
bis(phosphonate) P-C-P analogue are also of interest. We reported
the synthesis of phosphonophosphate analogues of C2-MAD (5 and
6, Fig. 5), in which an oxygen atom was inserted on either side of
the methylene group of C2-MAD.14 Both analogues 5 and 6 showed
more potent inhibition of hIMPDH1 (Ki = 20 nM and Ki = 87 nM,
respectively) than the parent methylenebis(phosphonate) C2-
MAD (Ki = 330 nM). A brief examination of the P-groove region,
as shown inFigure 1, indicates that the P sub-site is large and inter-
actions of the enzyme with the phosphate groups of the cofactor
CONH2
HO OH
CONH2
S
N
S
N
O
P
O
P
O
N
HO
O
O
O
N
O
O
OH OH
N
N
HO OH
HO OH
NH2
TR
TAD
Figure 2. Tiazofurin (TR), thiazole-4-carboxamide adnine dinucleotide (TAD).

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K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
HO OH
CH3
OH
O
HO
O
P
OH
O
P
OH
OH
O
C
O
O
O
O
CH2
O
N
N
N
H3CO
O
N
CH3
CH3O
CH3
NH2
MPA
C2-MAD
Figure 3. Mycophenolic acid (MPA) and mycophenolic adenine dinucleotide derivative (C2-MAD).
HO OH
HO OH
CONH2
N
O
P
OH
O
P
OH
OH
O
P
OH
O
P
OH
S
O
O
N
O
O
X
O
O
X
O
C2H5
N
C2H5
N
O
N
N
O
N
N
CH3O
N
CH3
HO OH
NH2
NH2
3
1
; X = CH2
; X = O
2; X = CF2
4; X = CF2
Figure 4. The 2-ethyl substituted analogues of TAD and MAD.
HO OH
HO OH
O
P
OH
O
P
OH
OH
O
P
OH
O
P
OH
OH
O
O
O
O
N
O
CH2
O
O
O
O
CH2
O
N
N
N
N
O
O
N
N
CH3O
CH3O
N
CH3
CH3
NH2
NH2
6
5
Figure 5. Phosphonophosphate analogues of MAD.
analogue are weak, maintained mainly by water molecules (repre-
sented by red stars). Thus, we became interested in further exam-
ination of this region.
aromatic mycophenolic moiety of 10 shows some affinity for the
adenine sub-site or interacts favorably with other parts of the cofac-
tor binding domain.
Since compounds 5 and 6 were poorly active in vitro, likely due
to their mixed phospho-phosphonate character, we prepared here-
in ethylenebis(phosphonate) analogue 7 and its 2-ethyl substituted
derivative 8 (Fig. 6) that are strictly phosphonate derivatives that
consequently should be able to penetrate cells membrane well.
Compound 7 inhibited hIMPDH1 (Ki = 95 nM) and hIMPDH2
(Ki = 84 nM) as potently as 5, and as expected showed 10-fold more
potent anti-proliferative activity against K562 cells (IC50 = 10.5 lV).
The ethyl analogue 8 showed similar nanomolar inhibitory activity
against IMPDH and exhibited even more potent anti-proliferative
activity (IC50 = 4.0 lM). The low nanomolar inhibitory activity of
Recently, we reported the synthesis of bis(sulfonamide) isosters
of our bis(phosphonate) MAD analogues.15 We found that the
replacement of the two phosphorus atoms of C2-MAD with geo-
metrically similar (tetrahedral) sulfur atoms afforded sulfonamide
11 with a very similar nanomolar inhibitory activity against
IMPDH to that of the parent bis(phosphonate). Now we examined
the importance of the preservation of the geometry of the pyro-
phosphate linker by preparation of the phosphonoacetamide deriv-
ative of C2-MAD 12 (Fig. 8) in which one of the two phosphorus
atoms was replaced by non-isosteric (planar) carboxyamido group.
We found that compound 12 (Ki = 83 nM) was two-fold more po-
tent inhibitor of hIMPDH2 than the corresponding bis(sulfon-
amide) analogue 11 (Ki = 167 nM).
compounds 7 and 8 against IMPDH indicates high affinity of both
mycophenolic alcohol and adenosine fragments to their correspond-
ing N and A sub-sites. Indeed, we found that mycophenolic alcohol
Unexpectedly, the bis(sulfonamide) analogue of TAD 13 as well
as their phosphonoacetamide derivative 14 (Fig. 9) showed signif-
icantly decreased inhibition of IMPDH as compared to the corre-
sponding MAD analogues (Table 1). Nevertheless, preservation of
the tetrahedral geometry was not important in this case either,
ethylenebis(phosphonate)
9 (without the adenosine moiety)
showed50-fold lesspotent activityagainstIMPDHthanthatof 7 (Ta-
ble 1). The replacement of adenosine of 7 by mycophenolic alcohol
moiety afforded 10-fold less potent inhibitor 10 (Fig. 7). Apparently
HO OH
HO OH
O
P
OH
O
P
OH
OH
O
P
OH
O
P
OH
OH
O
O
O
O
O
CH2 CH2
O
O
CH2 CH2
O
N
C2H5
N
N
N
N
N
O
O
N
N
CH3O
CH3O
CH3
CH3
NH2
7
8
NH2
Figure 6. Ethylenebis(phosphonate) analogues of MAD 7 and 8.

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1597
O
P
OH
O
P
OH
OH
OH
O
P
OH
O
P
OH
OH
O
O
O
HO
CH2 CH2
O
O
CH2 CH2
O
O
O
O
CH3O
OCH3
CH3O
CH3
CH3
CH3
10
9
Figure 7. Mycophenolic alcohol etylenebis(phosphonate) 9 and its symmetrical analogue 10.
HO OH
HO OH
O
C
O
O
S
O
O
S
O
OH
OH
O
O
O
O
N
NH
NH
CH2
NH
CH2
P
OH
O
N
N
N
O
O
N
N
CH3O
N
N
CH3O
CH3
CH3
11
12
NH2
NH2
Figure 8. Bis(sulfonamide) analogue of C2-MAD 11 and phosphonoacetamide derivative 12.
HO OH
CONH2
HO OH
CONH2
O
C
O
P
OH
N
S
O
S
O
O
S
O
N
S
O
O
N
NH
CH2
O
NH
CH2
N
NH
O
N
N
N
O
N
N
N
14
HO OH
13
HO OH
NH2
NH2
Figure 9. Bis(sulfonamide) analogue of TAD 13 and phosphonoacetamide derivative 14.
since phosphonoacetamide analogue of TAD 14 (Ki = 6.6
three-fold more potent against hIMPDH2 than the corresponding
bis(sulfonamide) derivative TAD 13 (Ki = 18.8 M).
l
M) was
indicating that the presence of phosphorous group is not required
for the potent activity against human IMPDH isoforms.
All these results indicate that P-groove of the NAD binding do-
main is quite promiscuous and can accommodate variety of linkers
of different length and geometry.
l
Next, we decided to check whether or not the presence of two
phosphorus atoms (or their structural equivalents such as sulfonyl
group) is crucial in the design of IMPDH inhibitors. Thus we
synthesized mycophenolic alcohol ethylenephosphonate-50-
thioadenosine derivative 15 and the corresponding analogue 16
(Fig. 10) with the sulfur atom of the linker attached to the position
8 of the adenine ring, containing only one phosphorus group. Again
we found a potent inhibitory activity of analogue 15 against
hIMPDH1 (Ki = 94 nM) and hIMPDH2 (Ki = 39 nM). However, mov-
ing of the linker from the 50-position of the sugar moiety to the car-
bon eight of the base resulted in weakly active compound 16
3. Chemistry
The synthesis of difluoromethylenebis(phosphonate) analogues
of TAD 2 and MAD 4 is depicted in Scheme 1. The starting material,
20,30-O-isopropylidene-2-ethylinosine [19, prepared from pro-
tected 5-aminoimidazole-4-carboxamide riboside (AICAR)]17 was
converted into the adenosine analogue 20 by chlorination of 19
at the 6-position with P(O)Cl3 followed by aminolysis. The synthe-
sis of the key bis(phosphonate) intermediate 22 was accomplished
by coupling of 20,30-O-isopropylidene-50-tosyl-2-ethyladenosine
(21) with difluoromethylenebis(phosphonic) acid in a similar
manner as was reported by Poulter and co-workers for coupling
of 50-tosyl adenosine.18 However, we prepared the starting difluo-
romethylenebis(phosphonic) acid differently, that is, by conve-
nient and efficient electrophilic fluorination of tetraisopropyl
methylenebis(phosphonate) with N-fluorobenzenesulfonimide19
followed by hydrolysis.
against the isoform 1 (Ki = 7.9 lM) and the isoform 2 (Ki = 1.9 lM)
(Table 1). The significant decrease of the enzymatic activity of 16 is
likely due to structural differences between isomers 15 and 16
and/or due to conformational change of 16 from anti- to syn-
conformation.
Finally, we recently reported triazole-linked mycophenolic ade-
nine inhibitors of IMPDH.16 Compounds such as 17 and 18 (Fig. 11)
showed low nanomolar inhibition of the two isoforms of the hu-
man enzyme with Ki’s in the range of 34–77 nM (Table 1),
HO OH
HO OH
O
P
OH
OH
O
O
O
OH
S CH2 CH2
O
N
O
P
OH
OH
N
N
N
O
N
N
O
S CH2 CH2
O
N
N
CH3O
O
CH3
NH2
CH3O
NH2
15
16
CH3
Figure 10. Mycophenolic alcohol ethylenephosphonate-50-thioadenosine 15 and the 8-substituted derivative 16.

1598
K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
HO OH
HO OH
N
N
OH
N
N
OH
N
O
O
O
O
N
H
N
N
O
CH2
N
N
N
N
O
O
N
N
N
CH3O
CH3O
17
NH2
18
NH2
CH3
CH3
Figure 11. Triazole-linked MAD analogues 17 and 18.
NH2
O
O
O
N
N
N
NH
O
P
O
P
N
N
C2H5
N
O
RO
N
C2H5
OH
OH
O
O
CF2
HO
a
c
C2H5
N
O
N
OH
N
N
O
O
22
O
O
PMBO
NH2
O
O
HO
d
20; R = H
f
b
19
CH3O
21
; R = Ts
23
CH3
25
RO OR
CONH2
RO OR
S
N
O
P
OH
O
P
OH
O
P
OH
OR'
O
P
OH
O
O
N
O
O
N
O
CF2
C2H5
N
O
O
O
CF2
C2H5
N
O
N
N
N
CH3O
N
RO OR
NH2
CH3
NH2
24; R = isopropylidene
2;
26; R = isopropylidene, R' = PMB
4; R = R' = H
e
e
R = H
Scheme 1. Synthesis of tiazofurin 2-ethyladenosine–, and mycophenolic alcohol 2-ethyladenosine difluoro-methylenebis(phosphonate)s 2 and 4, respectively. Reagents and
conditions: (a) (i) Ac2O, Py, (ii) Et4NCl, N,N-dimethylaniline, POCl3, MeCN, 80 °C 2 h, (iii) 2 M NH3 i-PrOH, 80 °C, overnight; (b) TsCl, Py, ꢀ25 °C; (c) tris-tetrabutylammonium
difluoromethylenebis(phosphonate), MeCN, rt, 24 h; (d) (i) DIC, Py, rt, overnight, (ii) 23, 62 °C, 70 h, (iii) H2O/TEA (9:1), overnight, rt; (e) 50% HCOOH, overnight, rt; (f) (i) DIC,
Py, rt, overnight, (ii) 25, 62 °C, 75 h, (iii) H2O/TEA (9:1), 27 h, 55–65 °C.
N,N-Disopropylcarbodiimide (DIC) promoted coupling of 2030-O-
isopropylidene-2-ethyladenosin-50-yl-difluoromethylenebis
(phosphonate) 22 with 20,30-O-isopropylidenetiazofurin20 (23)
afforded the protected difluoromethylenebis(phosphonate) ana-
logue of TAD 24, which without purification was deisopropylide-
nated by treatment with aq HCOOH to give the desired tiazofurin
2-ethyladenosine difluoromethylenebis(phosphonate) 2. Similarly,
the reaction of 22 with p-methoxybenzyl (PMB) protected myco-
phenolic alcohol derivative 25 (prepared from C2-mycophenolic
alcohol reported earlier by us)10 gave the protected mycophenolic
alcohol 2-ethyladenosine bis(phosphonate) analogue 26. Further
treatment of 26 with formic acid resulted in deprotection of both
p-methoxybenzyl and isopropylidene protecting groups to give
the desired mycophenolic alcohol 2-ethyladenosin-50-yl difluo-
romethylenebis(phosphonate) derivative 4 in moderate yield.
The synthesis of MAD analogues containing a linker extended
by one carbon atom is illustrated in the next two schemes. First,
we prepared ethylenebis(phosphonic dichloride) (28) by DMSO
oxidation of commercially available 1,2-bis(dichlorophosphi-
no)ethane (27) which gave 28 as a white solid in quantitative yield
(Scheme 2). We found this new method much more superior to the
published procedure21 via chlorination of the corresponding ethyl-
enebis(phosphonate)s with PCl5.
mycophenolic alcohol 25 afforded MAD analogue 31, which under
treatment with 50% HCOOH was converted into the desired myco-
phenolic alcohol adenosine ethylenebis(phosphonate) 7.
The synthesis of ethylenebis(phosphonate) MAD analogue 8
was performed in a similar manner (Scheme 3). Briefly, Yoshikawa
phosphorylation of benzyl protected mycophenolic alcohol 3210
with ethenebis(phosphonic dichloride) (28) in the presence of pro-
ton sponge and at the elevated temperature afforded the corre-
sponding mycophenolic alcohol ethylenebis(phosphonic acid) 33
as a major product (73% yield) together with disubstituted ethyl-
enebis(phosphonate) 34 as a minor product (9%). Hydrogenolysis
of 33 and 34 gave the debenzylated compounds 9 and 10, respec-
tively. Finally, DIC coupling of 9 with 20,30-O-isopropylidene-2-
ethyladenosine 20 afforded the protected MAD analogue 35, which
under treatment with 50% HCOOH was converted into the desired
mycophenolic alcohol 2-ethyladenosine ethylenebis(phosphonate)
8.
Next, we prepared phosphonoacetamido MAD analogue 12 and
TADanalogue14thatcontainnon-isosteric carbonylgroupasshown
in Scheme 4. The key intermediate for this synthesis is 20,30-O-iso-
propylideneadenosin-50-deoxy-50-N-acetamidophosphonic acid
(38), which was prepared by coupling of 20,30-O-isopropylidene-
50-aminoadenosine (36)22 with diethyl (phosphono)acetic acid fol-
lowed by a selective deprotection of the phosphonate ethyl groups.
We found that in the presence of N-hydroxybenzotriazole (as the
carboxylic group activator) the yield of the intermediate 37 was
much higher than that reported.23 Coupling of the phosphonic acid
Then, in conditions of Yoshikawa phosphorylation compound
28 reacted with a commercially available 20,30-O-isopropylidene-
adenosine 29 to give the adenosin-50-yl-ethylenebis(phosphonic
acid) 30 as a major product. DIC coupling of 30 with PMB protected

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1599
Cl2P CH2 CH2 PCl2
27
NH2
RO OR
N
N
a
N
O
O
O
P
HO
O
P
OH
O
P
HO
O
P
OH
OR'
CH3
O
O
b
29
N
c
HO
CH2 CH2
O
O
CH2 CH2
O
Cl2P CH2 CH2 PCl2
N
O
N
N
25
O
28
N
CH3O
30
O
O
NH2
31; R = isopropylidene, R' = PMB
d
7
; R = R' = H
Scheme 2. Synthesis of mycophenolic alcohol adenosine ethylenebis(phosphonate) analogue 7. Reagents and conditions: (a) DMSO, benzene, 30 min, 0 °C; (b) (i) 29,
PO(OEt)3, overnight, 50 °C, (ii) 48 h, 72 °C, (iii) TEAB, overnight, rt; (c) 25, DIC, Py, 72 h, 60 °C; (d) 50% HCOOH, overnight, rt.
BnO
O
O
P
HO
O
P
OH
OR
HO
CH3O
O
O
28
a
HO
CH2 CH2
O
O
CH3O
CH3
33
; R = Bn
b
CH3
32
9; R = H
c
20
RO OR
RO
O
P
HO
O
P
OH
OR
O
O
O
P
HO
O
P
OH
OH
O
O
O
O
O
O
CH2 CH2
O
O
CH2 CH2
O
C2H5
N
N
N
OCH3
CH3O
N
CH3O
CH3
CH3
CH3
NH2
34; R = Bn
35; R = isopropylidene
b
d
10
; R = H
8
; R = H
Scheme 3. Synthesis of mycophenolic alcohol 2-ethyladenosine ethylenebis(phosphonate) analogue 8. Reagents and conditions: (a) 28, 1,8-diaminonaphtalene, PO(OMe)3,
72 h, 80 °C; (b) HCOONH4, Pd EnCAT 40 (Aldrich), DMF/MeOH (1:5), overnight, reflux; (c) (i) 20, DIC, Py, 1 h, rt, (ii) 65 °C, 48 h; (d) 50% HCOOH, overnight, rt.
NH2
NH2
RO OR
CONH2
N
N
N
N
N
N
O
C
S
N
O
P
OH
O
C
O
P
RO
O
N
N
N
a
e
23
NH
CH2
O
H2N
RO
CH2
NH
N
O
O
O
N
N
O
O
O
O
RO OR
NH2
d
39; R = isopropylidene
14;
36
37; R = Et
b
R = H
38;
R = H
c
25
RO OR
O
C
O
P
OH
OR'
O
O
N
NH
CH2
O
N
O
N
CH3O
N
CH3
NH2
40;
12;
R = isopropylidene, R' = PMB
R = R' = H
d
Scheme 4. Synthesis mycophenolic alcohol (adenosine-50-N-acetamido)phosphonate 12 and tiazofurin-50-yl-(adenosine-50-N-acetamido)phosphonate 14. Reagents and
conditions: (a) HOBt, diethyl-phosphonoacetic acid, DIC, DMF, 20 h, rt; (b) TMS-Br, 2,6-lutidine, MeCN, overnight, rt; (c) 25, DIC, Py, 48 h, 65 °C; (d) 50% HCOOH, overnight, rt;
(e) 23, DIC, Py, 72 h, 65 °C.
38 in the presence of DIC with 20,30-O-isopropylidenetiazofurin (23)
(adenosine-50-N-acetamido)phosphonate (14). In a similar manner
coupling of the PMB protected mycophenolic alcohol 25 with the
afforded after deisopropylidenation the desired tiazofurin-50-yl-

1600
K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
intermediate 38 gave after deprotection the corresponding myco-
phenolic alcohol (adenosine-50-N-acetamido)phosphonate 12.
Finally, we synthesized MAD analogues 15 and 16 containing
only one phosphorus atom in the linker. Thus, diethyl 20,30-O-iso-
propylideneadenosine-50-thioethylenephosphonate (42) was
prepared by S-alkylation (Scheme 5) of 20,30-O-isopropylidene-50-
mercaptoadenosine (formed in situ from 41) with commercially
available diethyl 2-bromoethylphosphonate.
replacement of the only one phosphorus atom by a geometrically
different (planar) carbonyl group afforded phosphonoacetamide
derivatives MAD 12 and TAD 14. Both MAD analogues 11 and 12
showed enzymatic activity in the nanomolar range. Interestingly,
the non-isosteric phosphonoacetamide analogue 12 (Ki = 0.083
was two-fold more potent than isosteric sulfonamide 11
(Ki = 0.167 M). This indicates that preservation of tetrahedral
lM)
l
geometry of the pyrophosphate bridge is apparently not that crucial.
For unknown reasons, the corresponding sulfonamide TAD analogue
13 and phosphonoamide TAD analogue 14 showed less potent enzy-
The 20,30-O-isopropylideneadenosine-50-thioethylenephosphon-
ic acid (43) was obtained by treatment of 42 with trimethylsilyl
bromide. MAD analogue 15 was prepared by coupling of 43 with
protected mycophenolic alcohol 25 in the presence of DIC to give
44, which was deisopropylidenated with formic acid.
Analogously, 8-thioethylenephosphonate analogue (MAD 16)
was synthesized from 20,30-O-isopropylidene-8-mercaptoadeno-
sine (46),24 as shown in Scheme 6.
Briefly, S-alkylation of 46 with diethyl 2-bromoethylphospho-
nate afforded phosphonate 47 in high yield, which was hydrolyzed
into the free acid 48. Then, the standard tbutyldimethylsilyl
protection gave the 50-silyl derivative 49. Further coupling with
mycophenolic alcohol 25 afforded the protected phosphonate 50.
Treatment of 50 with formic acid resulted in the removal of all
three protecting groups to give the desired final product 16.
matic activity (6.6–23.6 lM). Again the phosphonoamide analogue
14 was more potent than isosteric sulfonamide 13. Finally, com-
pound 15 containing only one phosphonate atom in the linker was
found to be a potent inhibitor of hIMPDH1 (Ki = 94 nM) and
hIMPDH2 (Ki = 39 nM). Recently we reported that triazole-linked
MAD analogues, such as 17 and 18, that do not contain phosphorus
atoms, also showed potent inhibition of IMPDH. Taken together, it
became clear that the pyrophosphate binding sub-domain (P-
groove) of IMPDH is much more promiscuous that it was originally
anticipated. Thus, our studies described herein would encourage fu-
ture attempts of constructing novel inhibitors by using molecular
fragments that fit to N and A sub-domain and linkers that not neces-
sarily resemble well the natural pyrophosphate.
Although, almost all new compounds 1–16 showed a potent
nanomolar inhibition of IMPDH, many, that is, compound 9–16
were not active as anti-proliferative agents in vitro. It was believed,
that negatively charged bis(phosphonate)s could not be able to
cross cells membranes efficiently and we faced such criticism fre-
quently in the past. In contrast, we found herein that bis(phospho-
nates) showed potent anti-proliferative activity in vitro, whereas
neither uncharged sulfonamides 11 and 13 nor negatively charged
phosphonoacetamides 12 and 14, or phosphonate analogue 15
showed such activity. Since sulfonamides, phosphonoacetic ana-
logues, and phosphonate nucleosides are well established pharma-
cophores present in numerous drugs, we are focusing now on
possible explanation of such bizarre behavior of modified NAD
analogues reported in this work.
4. Conclusions
In summary, we synthesized novel cofactor-type inhibitors of
IMPDH in which the pyrophosphate group that connects nicotin-
amide riboside and adenosine moiety in the natural NAD is modified
or replaced by other linkers. For construction of our inhibitors we se-
lected tiazofurin and mycophenolic moiety, the binding fragments
with high affinity to the N sub-site of the cofactor binding domain,
used by us and others for preparation of IMPDH inhibitors such as
TAD and MAD analogues. Adenosine and 2-ethyladenosine were
used to secure binding at the A sub-site of the enzyme. We found
as expected that the replacement of the P-O-P group by its isosteric
and isoelectronic P-CF2-P moiety afforded potent inhibitors of
IMPDH. The most potent compound was MAD analogue 4 containing
2-ethyladenosine. It wasaspotent(IC50 = 0.45
K562 cells proliferation as Gleevec (IC50 = 0.56
as anti-CML drug. MAD analogue 3 containing methylenebis(phos-
l
l
M)againstleukemia
M) used in the clinic
5. Experimental section
5.1. General methods
phonate) linker (P-CH2-P) was only slightly less potent as the en-
zyme inhibitor and the anti-proliferative agent (IC50 = 1.0
l
M). An
All commercial reagents (Sigma-Aldrich, Alfa Aesar) were used
as provided unless otherwise indicated. An anhydrous solvent dis-
pensing system (J.C. Meyer) using two packed columns of neutral
alumina was used for drying THF, Et2O, and CH2Cl2, while two
packed columns of molecular sieves were used to dry DMF. Sol-
vents were dispensed under argon. Analytical HPLC was performed
extension of the length of the linker by one carbon atom as in ethyl-
enebis(phosphonate) (P-CH2-CH2-P) afforded MAD analogues 7 and
8, both2-5-fold less potent against IMPDHisoforms(but still in a low
nanomolar range). They were 10-fold less active as proliferation
agents, as compared with MAD analogue 3. We also found that the
replacement of the methylenebis(phosphonate) group by a geomet-
rically similar (tetrahedral) methylenebis(sulfonyl) moiety resulted
in MAD analogue 11 and TAD analogue 13. On the other hand
on a Varian Microsorb column (C18, 5
flow rate of 0.5 mL/min while preparative HPLC was performed
on a Varian Dynamax column (C18, 8
, 41.4 ꢁ 250 mm) with a
l, 4.6 ꢁ 250 mm) with a
l
NH2
NH2
RO OR
N
N
N
N
N
N
O
P
RO
O
P
OH
OR'
CH3
O
N
N
O
N
AcS
a
c
RO
CH2 CH2 S
S
CH2 CH2
O
O
O
N
O
N
CH3O
N
O
O
O
O
NH2
42;
44;
41
R = Et
R = isopropylidene, R' = PMB
b
d
43; R = H
15; R = R' = H
Scheme 5. Synthesis of 50-thioethylenephosphonate analogue of MAD. Reagents and conditions: (a) diethyl 2-bromoethylphosphonate, MeONa, MeOH, overnight,
ꢀ20 °C ? rt; (b) TMS-Br, 2,6-lutidine, MeCN, overnight, rt; (c) 25, DIC, Py, 24 h, 55 °C; (d) 50% HCOOH, overnight, rt.

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1601
NH2
NH2
O
P
OR
N
N
N
N
N
N
R
O
RO
CH2 CH2
HO
S
O
N
N
HO
O
O
b
d
O
O
45; R = Br
46; R = SH
47; R = Et
48; R = H
a
c
OR OR
NH2
O
P
OH
N
N
N
HO
CH2 CH2
S
O
e
OR''
N
N
O
P
OH
OR'
CH3
TBDMSO
N
N
O
O
25
S
CH2 CH2
O
N
O
49
O
O
NH2
CH3O
50; R = isopropylidene, R' = PMB, R'' = TBDMS
16; R = R' = R'' = H
f
Scheme 6. Synthesis of 8-thioethylenephosphonate analogue of MAD. Reagents and conditions: (a) NaSH24; (b) diethyl 2-bromoethylphosphonate, K2CO3, DMF, overnight, rt;
(c) TMS-Br, 2,6-lutidine, MeCN, overnight, rt; (d) TBDMS-Cl, imidazole, DMF, overnight, rt; (e) 25, DIC, Py, 48 h, 58 °C; (f) 50% HCOOH, overnight, rt.
flow rate of 40 mL/min. An isocratic or linear gradient of 0.1 M tri-
ethylammonium bicarbonate (TEAB) and aq MeCN (70%) were
used. Flash chromatography was performed using Teledyne ISCO
CombiFlash Rf equipped with Teledyne ISCO RediSep Rf flash col-
umn silica cartridges (www.isco.com/combiflash) with the indi-
cated solvent system. Nuclear magnetic resonance spectra were
recorded on a Varian 600 MHz with Me4Si, DDS or signals from
residual solvent as the internal standard for 1H and external
H3PO4 for 31P. Chemical shifts are reported in ppm, and signals
are described as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br s (broad singlet), and dd (double doublet). Values gi-
ven for coupling constants are first order. High resolution mass
spectra were recorded on an Agilent TOF II TOF/MS instrument
equipped with either an ESI or APCI interface. All reactions were
performed under an inert atmosphere of dry Ar in oven dried
(150 °C) glassware. The purity of compounds was P95%, and it
was determined by HPLC using above mentioned analytical col-
umn, and conditions described in the Supplementary data.
added and the mixture was dissolved in dry MeCN (20 mL). Next,
N,N-dimethylaniline (1.1 mL, 8.5 mmol) was added followed by
dropwise addition of POCl3 (4.8 mL, 51 mmol). The resulting mix-
ture was heated at 80 °C for 2 h (preheated bath). The mixture
was cooled, diluted with CH2Cl2, and poured into a stirred mixture
of ice and saturated NaHCO3 (150 mL). After 10 min of stirring,
CH2Cl2 (200 mL) was added, and vigorous stirring was continued
until ice melted. The aqueous fraction was separated and extracted
with CH2Cl2. The combined extracts were dried over MgSO4, fil-
tered, and evaporated to dryness leaving partially crystallizing
oil. The crude 6-chloro derivative was purified by flash chromatog-
raphy on a silica gel with EtOAc/CHCl3 (0–40%) as an eluent to give
the crystalline compound, which was dissolved in 2 M NH3 in
i-PrOH (20 mL) and heated at 80 °C overnight. After evaporation
of solvents the residue was dissolved in a mixture 1,4-dioxane/
concd NH4OH (1:1, 40 mL) and stirred at 50 °C overnight.
Solvents were evaporated and the residue was purified by flash
chromatography on a silica gel with MeOH/CH2Cl2 (0–10%) as the
eluent to give a final compound 20 as a foam (2.1 g 74%). Mp
5.2. 20,30-O-Isopropylidene-2-ethylinosine (19)
159–159.5 °C. 1H NMR (CDCl3)
d 7.76 (s, 1H), 6.82 (d, J =
11.81 Hz, 1H), 5.82 (d, J = 4.97 Hz, 1H), 5.68 (br s, 2H), 5.23 (t,
J = 5.33 Hz, 1H), 5.11 (d, J = 5.70 Hz, 1H), 4.54 (s, 1H), 3.99 (d,
J = 12.73 Hz, 1H), 3.79 (t, J = 12.34 Hz, 1H), 2.80 (q, J = 7.60 Hz,
2H), 1.65 (s, 3H), 1.39 (s, 3H), 1.33 (t, J = 7.60 Hz, 3H). HRMS calcd
for C15H22N5O4 336.1666 (M+H)+, found 336.1681.
It was prepared from 20,30-O-isopropylidene-AICAR according to
the old procedure17 to give 19 as a white crystals, mp 225–6 °C, (lit.
212 °C). 1H NMR (CDCl3) d 12.80 (br s, 1H), 7.81 (s, 1H), 5.84 (d,
J = 4.94 Hz, 1H), 5.29 (d, J = 10.99 Hz, 1H), 5.18 (t, J = 5.44 Hz, 1H),
5.08 (dd, J = 5.89, 1.12 Hz, 1H), 4.52–4.50 (m, 1H), 3.97 (d,
J = 12.29 Hz, 1H), 3.78 (t, J = 11.78 Hz, 1H), 2.88 (q, J = 7.69 Hz,
2H), 1.65 (s, 3H), 1.42 (t, J = 7.69 Hz, 3H), 1.39 (s, 3H).
5.4. 20,30-O-Isopropylidene-50-O-p-tosyl-2-ethyladenosine (21)
Compound 20 (335 mg, 1 mmol) was dissolved in dry pyridine
(6 mL). The mixture was cooled to ꢀ25 °C, TsCl (310 mg,
1.61 mmol) was added in small portions, and after 30 min of stir-
ring the mixture was left in a freezer overnight. It was then poured
into a vigorously stirred mixture of 2 N HCl (25 mL) with ice and
EtOAc, and when ice had melted, the two layers were separated.
The inorganic phase was extracted with EtOAc. Organic fractions
were combined, washed with H2O, dried over MgSO4, filtered,
and evaporated to give the crude product which was quickly
5.3. 20,30-O-Isopropylidene-2-ethyladenosine (20)
Compound 19 (2.86 g, 8.5 mmol) was dissolved in a mixture of
Ac2O (12 mL) and pyridine (8 mL). After stirring for 3 h at rt, the
mixture was diluted with toluene and evaporated to dryness. The
residue was dissolved in EtOH and toluene, evaporated in vacuo,
and then co-evaporated with dry xylene. To the viscous 50-O-acetyl
derivative tetraethylammonium chloride (2.82 g, 17 mmol) was

1602
K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
re-crystallized from EtOAc. The crystals were washed with hexane
and dried under vacuum to give yellow, unstable product which
was used in the next step without further purification (386 mg,
79%). 1H NMR (CDCl3) d 7.72 (s, 1H), 7.57 (d, J = 8.25 Hz, 2H),
7.12 (d, J = 7.96 Hz, 2H), 6.00 (d, J = 1.14 Hz, 1H), 5.36 (dd,
J = 6.25, 1.31 Hz, 1H), 5.12 (dd, J = 6.16, 2.91 Hz, 1H), 4.31 (dd,
J = 10.54, 4.57 Hz, 1H), 4.47 (s, 1H), 2.77 (q, J = 7.69 Hz, 2H), 4.25
(dd, J = 10.52, 7.18 Hz, 1H), 2.38 (s, 3H), 1.59 (s, 3H), 1.37 (s, 3H),
1.29 (t, J = 7.58 Hz, 3H).
product which was purified by crystallization from EtOH (1.8 g).
The mother liquor was concentrated and purified by flash chroma-
tography on a silica gel with MeOH/CHCl3 (0–2%) to give additional
(1 g) of crystalline 25 (total 2.8 g, 73%). 1H NMR (CDCl3) d7.43–7.40
(m, 2H), 6.91–6.88 (m, 2H), 5.25 (s, 2H), 5.17 (s, 2H), 3.81 (s, 3H),
3.79 (s, 3H), 3.70 (q, J = 6.27 Hz, 2H), 2.89 (t, J = 6.47 Hz, 2H), 2.20
(s, 3H), 1.83 (t, J = 5.50 Hz, 1H). HRMS calcd for C20H22NaO6
381.1308 (M+Na)+, found 381.1301.
5.8. Mycophenolic 2-ethyladenosin-50-yl-difluoromethylenebis
(phosphonate) (4)
5.5. 20,30-O-Isopropylidene-2-ethyladenosin-50-yl-difluorometh
ylenebis(phosphonate) (22)
To a solution of 22 (190 mg, 0.27 mmol) in dry pyridine (2 mL),
DIC (250 lL, 1.6 mmol) was added, and the resulting mixture was
A mixture of 21 (385 mg, 0.786 mmol) and tris-tetrabutylammo-
nium difluoromethylenebis(phosphonate) (760 mg; 0.786 mmol) in
dry MeCN (2 mL) was stirred24 h at rt, concentrated, and theresidue
was dissolved in water (2 mL), and this solution was then passed
through a column of Dowex 50WX8-200 (Na+ form). After elution
with water, fractionscontaining product were collected, lyophilized,
and purifiedby preparativeHPLC with 70%MeCN/0.1 M TEAB (5-100
linear gradient) to give 22 as triethylammonium salt (356 mg, 72%).
1H NMR (D2O) d 8.33 (s, 1H), 6.19 (d, J = 3.20 Hz, 1H), 5.32 (dd,
J = 6.03, 3.26 Hz, 1H), 5.13 (dd, J = 6.06, 2.13 Hz, 1H), 4.54–4.50 (m,
1H), 4.16–4.05 (m, 1H), 3.07 (q, J = 7.33 Hz, 12H), 2.71 (q, J = 7.63,
Hz, 2H), 1.55 (s, 3H), 1.33 (s, 3H), 1.19 (t, J = 7.63 Hz, 3H), 1.15 (t,
J = 7.33 Hz, 18H). 31P NMR (D2O) d 5.15 (JP1P2 = 55.8, JP1F = 83.6 Hz),
3.93 (JP1P2 = 55.8, JP2F = 82.2 Hz). HRMS calcd for C16H22F2N5O9P2
528.0866 (MꢀH)ꢀ, found 528.0887.
left overnight at rt. After addition of protected mycophenolic alco-
hol 25 (96 mg, 0.27 mmol) stirring was continued for 75 h at 62 °C.
After cooling to rt, a mixture of water (0.9 mL) and TEA (0.1 mL)
was added, and the reaction was kept at 55–65 °C for 27 h. The
mixture was diluted with toluene and EtOH, evaporated, and co-
evaporated twice. The intermediate 26 was dissolved in 50%
HCOOH (5 mL) and stirred overnight at rt, solvents were evapo-
rated, and the residue was co-evaporated with EtOH and water.
The crude product was purified by preparative HPLC with 70%
MeCN/0.1 M TEAB (20–30 linear gradient). Fractions containing
the desired product were evaporated. The residue was dissolved
in water and then passed through a column of Dowex 50 WX8-
200 (Na+ form). After elution with water, UV active fractions were
collected and lyophilized to give the deprotected 4 (sodium salt) as
a white powder (76 mg, 37%). 1H NMR (D2O) d 8.08 (s, 1H), 5.81 (d,
J = 4.70 Hz, 1H), 4.88–4.79 (m, 2H), 4.40 (t, J = 4.87, Hz, 1H), 4.30 (t,
J = 4.82 Hz, 1H), 4.18–4.07 (m, 3H), 3.93–3.83 (m, 2H), 3.57 (s, 3H),
2.76–2.60 (m, 2H), 2.52 (q, J = 7.69 Hz, 2H), 1.79 (s, 3H), 1.07 (t,
J = 7.69 Hz, 3H). 31P NMR (D2O) d 5.11 (JP1P2 = 75.0, JP1F = 84.0 Hz),
4.95 (JP1P2 = 75.0, JP2F = 86.5 Hz). HRMS calcd for C25H30F2N5O13P2
708.1289 (MꢀH)ꢀ, found 708.1300.
5.6. Tiazofurin-50-yl-2-ethyladenosin-50-yl-difluromethylene
bis(phosphonate) (2)
To the solution of 22 (67 mg, 0.22 mmol) in dry pyridine (2 mL),
DIC (172 lL, 1.11 mmol) was added, and the mixture was stirred
overnight at rt. Next, 20,30-O-isopropylidenetiazofurin (23)20
(156 mg, 0.22 mmol) was added, and stirring was continued for
70 h at 62 °C. After cooling to rt, a mixture of water (0.9 mL) and
TEA (0.1 mL) was added, and the reaction was kept at rt overnight.
After evaporation the intermediate 24 was obtained as a solid. It
was dissolved in 50% HCOOH (5 mL) and stirred overnight at rt, sol-
vents were evaporated, and the residue was co-evaporated with a
mixture of EtOH and water. The crude product 24 was purified by
preparative HPLC with 70% MeCN/0.1 M TEAB (10–15 linear gradi-
ent). Fractions containing product were evaporated. The residue
was dissolved in water and then passed through a column of Dow-
ex 50 WX8-200 (Na+ form). After elution with water, UV active
fractions were collected, and lyophilized to give deprotected 2 (so-
dium salt) as a white powder (32 mg, 20%). 1H NMR (D2O) d 8.28 (s,
1H), 7.83 (s, 1H), 5.96 (d, J = 5.62 Hz, 1H), 4.92 (d, J = 5.65 Hz, 1H),
4.54 (t, J = 5.35 Hz, 1H), 4.38 (dd, J = 4.83, 3.99 Hz, 1H), 4.20–4.17
(m, 2H), 4.17–4.12 (m, 2H), 4.12–4.06 (m, 4H), 2.60 (q,
J = 7.67 Hz, 2H), 1.13 (t, J = 7.67 Hz, 3H). 31P NMR (D2O) d 4.96
(JP1P2 = 51.5 Hz, JP1F = 82.5), 4.62 (JP1P2 = 51.5, JP2F = 84.3 Hz). HRMS
calcd for C22H28F2N7O13P2S 730.0915 (MꢀH)ꢀ, found 730.0926.
5.9. Ethylenebis(phosphonic dichloride) (28)
To a solution of commercially available 1,2-bis(dichlorophosph-
ino) ethane (27, 3 mL, 20 mM) in benzene (160 mL), a solution of
DMSO (3.2 mL) in benzene (160 mL) was added over 30 min under
cooling in ice. 31P NMR showed a complete conversion of 27 (d
191.0) into the desired product 28 (d 41.5). The reaction mixture
was concentrated in vacuo to give 28 as a white solid (5.27 g,
100%). 31P NMR (C6D6) d 41.5 (s).
5.10. 20,30-O-Isopropylideneadenosin-50-yl-ethylenebis
(phosphonate) (30)
To a suspension of tetrachloride 28 (1.06 g, 4 mmol) in triethyl-
phosphate (25 mL), a suspension of 20,30-O-isopropylidene adeno-
sine 29 (614 mg, 2 mmol) in triethylphosphate (25 mL) was
added. The resulting mixture was stirred at 50 °C overnight, and
then at 72 °C for 2 days, cooled, and added to a solution of TEAB
(0.5 M, 20 mL, cooled with ice-water), and stirred at rt overnight.
This mixture was extracted with EtOAc, the aqueous layer was con-
centrated, lyophilized, and purified by preparative HPLC with 70%
MeCN/0.1 M TEAB (10–30 linear gradient) to give 30 as a foam
(350 mg, 26%).1H NMR (D2O) d 8.25 (s, 1H), 8.14 (s, 1H), 6.15 (d,
J = 2.76 Hz, 1H), 5.34 (dd, J = 6.07, 2.78 Hz, 1H), 5.05 (dd, J = 6.06,
1.94 Hz, 1H), 4.57–4.54 (m, 1H), 3.93–3.86 (m, 2H), 3.07 (q,
J = 7.33 Hz, 6H), 1.67–1.56 (m, 2H), 1.54 (s, 3H), 1.45–1.34 (m,
2H), 1.34 (s, 3H), 1.15 (t, J = 7.33 Hz, 9H). 31P NMR (D2O) d 30.26
(d, J = 73.12 Hz), 22.86 (d, J = 70.80 Hz). HRMS calcd for
5.7. 7-(p-Methoxybenzyl)-6-(2-hydroxyethyl)-5-methoxy-4-
methyl-phthalan-1-one (25)
To the solution of 7-hydroxy-6-(2-hydroxyethyl)-5-methoxy-4-
methyl-phthalan-1-one (1.94 g, 8.186 mmol) in 1 N TBAF in THF
(17 mL) p-methoxybenzyl chloride (1.26 mL, 8.89 mmol) was
added and the mixture was stirred at rt overnight, then another
portion of p-methoxybenzyl chloride (0.3 mL) was added, and stir-
ring was continued at rt. When reaction was complete the mixture
was diluted with satd NaHCO3, extracted with EtOAc, extracts were
dried over MgSO4, filtered, and evaporated leaving semi crystalline
C
15H22N5O9P2 478.0898 (MꢀH)ꢀ, found 478.0932.

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1603
5.11. Mycophenolic adenosin-50-yl-ethylenebis(phosphonate)
(7)
5.14. Di-(mycophenolic) ethylenebis(phosphonic acid) (10)
It was prepared from 34 as described for 9. 1H NMR (D2O) d 5.18
(s, 4H), 3.86 (q, J = 6.60 Hz, 4H), 3.77 (s, 6H), 2.92 (t, J = 6.80 Hz,
4H), 2.02 (s, 6H), 1.47 (m, 4H). 31P NMR (D2O) d 26.37 (s). HRMS
calcd for C26H31O14P2 629.1195 (MꢀH)ꢀ, found 629.1224.
The ethylenebis(phosphonate) 30 (185 mg, 0.275 mmol) and 25
(108 mg, 0.303 mmol) were dissolved in dry pyridine (2 mL). To
the resulting mixture DIC (220 L, 1.375 mmol) was added, reac-
l
tion was stirred at 60 °C for 72 h, and concentrated to dryness.
The crude intermediate 31 was dissolved in 50% HCOOH (5 mL)
and stirred overnight at rt, solvents were evaporated, and the res-
idue was purified by preparative HPLC with 70% MeCN/0.1 M TEAB
(15–20 linear gradient). Fractions containing the product were
pooled, evaporated in vacuo, the residue was dissolved in water,
and then passed through a column of Dowex 50 WX8-200 (Na+
form). After elution with water, UV active fractions were collected
and lyophilized to give 7 as a white powder (60 mg, 31%). 1H NMR
(D2O) 8.12 (s, 1H), 7.88 (s, 1H), 5.83 (d, J = 5.44 Hz, 1H), 4.82 (s, 2H),
4.40 (t, J = 5.25, 5.25 Hz, 1H), 4.30–4.26 (m, 1H), 4.19–4.16 (m, 1H),
3.97–3.90 (m, 2H), 3.60–3.53 (m, 2H), 3.49 (s, 3H), 2.61–2.51 (m,
2H), 1.72 (s, 3H), 1.66–1.56 (m, 4H). 31P NMR (D2O) d 28.50 (d,
J = 75.51 Hz), 27.37 (d, J = 75.52). HRMS calcd for C24H30N5O13P2
658.1321 (MꢀH)ꢀ, found 658.1339.
5.15. Mycophenolic 2-ethyladenosin-50-yl-ethylenebis(phos-
phonate) (8)
Bisphosphonate 9 (99.1 mg, 0.194 mmol) and 20 were dissolved
in dry pyridine (2 mL). To the resulting solution, DIC (150 lL,
0.97 mmol) was added. The mixture was stirred at rt for 1 h and
then at 65 °C for 48 h, cooled to rt, evaporated, and co-evaporated
with EtOH to give the crude intermediate 35, which was dissolved
in 50% HCOOH and stirred vigorously overnight at rt, evaporated,
co-evaporated with EtOH, and dissolved in small amount of MeOH.
The product was purified by preparative HPLC with 70% MeCN/
0.01 M TEAB (15–30 linear gradient). Fractions containing the
product were pooled, evaporated to dryness, co-evaporated with
H2O and EtOH, dissolved in small amount of H2O, and passed
through small column of Dowex 50 WX8-200 (Na+). Fractions con-
taining the desired product were combined and lyophilized to give
8 as a white powder (50 mg, 38%). 1H NMR (D2O) d 8.14 (s, 1H),
5.93 (d, J = 5.48 Hz, 1H), 4.92–4.84 (m, 2H), 4.47 (t, J = 5.26 Hz,
1H), 4.38–4.34 (m, 1H), 4.27–4.24 (m, 1H), 4.03–3.96 (m, 2H),
3.73–3.63 (m, 2H), 3.55 (s, 3H), 2.62 (dd, J = 8.48, 7.50 Hz, 2H),
2.56 (q, J = 7.69 Hz, 2H), 1.78 (s, 3H), 1.73–1.62 (m, 4H), 1.10 (t,
J = 7.68 Hz, 3H). 31P NMR (D2O) d 28.42 (d, J = 75.40 Hz), 27.41 (d,
J = 75.61 Hz). HRMS calcd for C26H34N5O13P2 686.1634 (MꢀH)ꢀ,
found 686.1662.
5.12. 7-Benzyl-mycophenolic ethylenebis(phosphonic acid) (33)
and disubstituted analogue (34)
A mixture of ethylenebis(phosphonic dichloride) 28 (530 mg,
2 mmol) and 32 (330 mg, 1 mmol), with addition of the proton
sponge, 1,8-dimethylaminonaphtalene, (210 mg, 1 mmol) in tri-
methylphosphate (5 mL) was stirred at 80 °C for 72 h. The reaction
mixture was cooled, poured on iced water (100 mL), made slightly
basic with TEA, and when ice melted, it was extracted with EtOAc.
The organic fractions were discarded, and water phase was lyoph-
ilized to thick oil. The crude mixture was purified by preparative
HPLC with 70% MeCN/0.1 M TEAB (25–100 linear gradient) to give
benzyl protected monosubstituted bis(phosphonate) 33 as a major
product and benzyl protected disubstituted bis(phosphonate) 34 as
a minor product. Compound 33 was dissolved in MeOH, and then
passed through a column of Dowex 50 WX8-200 (H+ form). After
elution with MeOH, the UV active fractions were concentrated to
give 33 as a white, crystalline powder (360 mg, 73%). 1H NMR
(CD3OD) d 7.49 (d, J = 7.47 Hz, 2H), 7.37 (t, J = 7.42 Hz, 2H), 7.34
(t, J = 7.20 Hz, 1H), 5.30 (s, 2H), 5.28 (s, 2H), 4.10 (q, J = 6.79 Hz,
2H), 3.82 (s, 3H), 3.00 (t, J = 6.91 Hz, 2H), 2.23 (s, 3H), 1.87–1.76
5.16. Diethyl (20,30-O-isopropylideneadenosine-50-N-acetamido)
phosphonate (37)23
To the solution of HOBt (135 mg, 1 mmol), 20,30-O-isopropyli-
dene-50-amino-adenosine22 36 (305 mg, 1 mmol), and diethyl-
phosphonoacetic acid (170
lL, 1 mmol) in DMF (2 mL), DIC
(320 L, 2 mmol) was added dropwise and the mixture was stirred
l
vigorously at rt for 20 h. After filtration, the solvent was evapo-
rated and the residue was co-evaporated with EtOH (3 ꢁ 10 mL).
It was then dissolved in CH2Cl2 (15 mL) and the organic phase
was washed with H2O (20 mL), dried over MgSO4, filtered, and
evaporated to dryness. The resulting oil was purified by flash chro-
matography with MeOH/CH2Cl2 (0–10%) as the eluent to give 37
(410 mg, 85%). 1H NMR (CDCl3) d 8.40 (s, 1H), 7.95 (s, 1H), 8.31
(d, J = 6.47 Hz, 1H), 5.88 (d, J = 4.53 Hz, 1H), 5.76 (s, 2H), 5.38 (dd,
J = 5.92, 4.90 Hz, 1H), 4.87 (dd, J = 6.27, 2.20 Hz, 1H), 4.50–4.43
(m, 1H), 4.20–4.15 (m, 2H), 4.15–4.10 (m, 4H), 4.08 (ddd,
J = 14.16, 8.40, 3.51 Hz, 1H), 3.38–3.32 (m, 1H), 2.97 (qd,
J = 21.07, 14.49 Hz, 2H), 1.61 (s, 3H), 1.35 (s, 3H), 1.30 (q,
J = 7.02 Hz, 6H). 31P NMR (CDCl3) d 23.03 (s). MS calcd for
(m, 2H), 1.75–1.63 (m, 2H). 31P NMR (CD3OD)
d 30.32 (d,
J = 79.21 Hz), 28.14 (d, J = 79.21 Hz). HRMS calcd for C21H25O10P2
499.0928 (MꢀH)ꢀ, found 499.0976. Compound 34 was converted
into free acid form as described for 33 to give a white powder
(75 mg, 9%). 1H NMR (DMSO-d6) d 7.46 (d, J = 7.28 Hz, 4H), 7.35
(t, J = 7.36 Hz, 4H), 7.30 (t, J = 7.22 Hz, 2H), 5.27 (s, 4H), 5.16 (s,
4H), 3.94–3.85 (m, 2H), 3.71 (s, 6H), 2.86 (t, J = 7.12 Hz, 4H), 2.11
(s, 6H), 1.52–1.42 (m, 4H). 31P NMR (DMSO-D6) d 26.76 (s). HRMS
calcd for C40H43O14P2 528.0866 (MꢀH)ꢀ, found 528.0887.
C
16H26N6O7P 446.17 (M+H)+, found 446.18.
5.13. Mycophenolic ethylenebis(phosphonic acid) (9)
5.17. 20,30-O-Isopropylideneadenosine-50-N-acetamidophos
A solution of phosphonate 33 (300 mg, 0.6 mmol) in DMF
(10 mL) was diluted with MeOH (50 mL) and ammonium formate
(1 g) and Pd EnCat 40 from Aldrich (200 mg) was added. The mix-
ture was refluxed overnight, filtered, and filtrate was concentrated
into a thick oil which was purified by preparative HPLC with 70%
MeCN/0.1 M TEAB (10–30 linear gradient) to give 9 as a foam
(290 mg, 79%, triethylammonium salt). 1H NMR (D2O) d 5.10 (s,
2H), 3.88 (q, J = 6.68 Hz, 2H), 3.73 (s, 3H), 2.90 (t, J = 6.77 Hz, 2H),
2.01 (s, 3H), 1.51 (m, 2H), 1.37 (m, 2H); 31P NMR (D2O) d 28.87
(d, J = 73.33 Hz), 25.11 (d, J = 73.24 Hz). HRMS calcd for
phonic acid (38)
To the solution of 37 (355 mg, 0.73 mmol) in dry MeCN (10 mL),
2,6-lutidine (460
lL, 3.65 mmol) was added followed by TMS-Br
(500 L, 3.65 mmol). The mixture was stirred overnight at rt, evap-
l
orated, diluted with EtOH and evaporated. The residue was puri-
fied by preparative HPLC with 0.01 M TEAB/70% MeCN (10–30
linear gradient) to give triethylammonium salt of 38 (420 mg,
91%). 1H NMR (D2O)
d 8.18 (s, 1H), 8.15 (s, 1H), 6.10 (d,
J = 3.12 Hz, 1H), 5.41 (dd, J = 6.43, 3.16 Hz, 1H), 4.95 (dd, J = 6.41,
3.16 Hz, 1H), 4.35 (dt, J = 5.78, 5.59, 3.34 Hz, 1H), 3.43 (dd,
C
14H19O10P2 409.0459 (MꢀH)ꢀ, found 409.0487.

1604
K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
J = 14.40, 6.21 Hz, 1H), 3.33 (dd, J = 14.40, 5.13 Hz, 1H), 3.07 (q,
J = 7.33 Hz, 6H), 2.60–2.51 (m, 2H), 1.52 (s, 3H), 1.31 (s, 3H), 1.15
(t, J = 7.33 Hz, 9H). 31P NMR (D2O) d 14.78 (s). HRMS calcd for
(CDCl3) d 29.21 (s). To the solution of 42 (0.52 g, 1.08 mmol) in dry
MeCN (10 mL) 2,6-lutidine (0.64 mL, 5.4 mmol) was added followed
byTMS-Br(0.73 mL, 5.4 mmol). Themixturewasstirredovernightat
rt, evaporated, diluted with EtOH, and evaporated. The crude prod-
uct was purified by preparative HPLC with 70% MeCN/0.1 M TEAB
(25–50 linear gradient) to give 43 (410 mg, 71% triethylammonium
salt). 1H NMR (D2O) d 8.36 (s, 1H), 8.29 (s, 1H), 6.30 (d, J = 2.79 Hz,
1H), 5.62 (dd, J = 6.48, 2.82 Hz, 1H), 5.18 (dd, J = 6.49, 3.10 Hz, 1H),
4.56 (dt, J = 6.91, 3.10 Hz, 1H), 3.25 (q, J = 7.34 Hz, 6H), 2.92 (d,
J = 6.72 Hz, 1H), 2.77 (dd, J = 15.41, 7.48 Hz, 2H), 1.90–1.82 (m,
2H), 1.71 (s, 3H), 1.49 (s, 3H), 1.33 (t, J = 7.33, Hz, 9H). 31P NMR
(D2O) d 22.61 (s). HRMS calcd for C15H21N5O6PS 430.0956 (MꢀH)ꢀ,
found 430.0983.
C
15H20N6O7P 427.1137 (MꢀH)ꢀ, found 427.1209.
5.18 Mycophenolic (adenosine-50-N-acetamido)phosphonate
(12)
To the phosphonate 38 (85 mg, 0.0135 mmol) the protected
mycophenolic alcohol 25 (49 mg, 0.0135) was added followed by
dry pyridine (2 mL). To the stirred solution DIC (105 lL,
0.675 mmol) was added and a mixture was stirred at 65 °C for
2 days. After cooling to rt the mixture was evaporated and the
crude intermediate 40 was deprotected by treatment with 50%
HCOOH at rt overnight. After evaporation, and co-evaporation with
a mixture of EtOH and water the residue was purified by prepara-
tive HPLC with 0.01 M TEAB/70% MeCN (15–30 linear gradient),
dissolved in small amount of H2O, and passed through a column
of Dowex 50 WX8-200 (Na+). Fractions containing product were
combined and lyophilized to give 12 as a white powder (26 mg,
5.21. Mycophenolic ethylenephosphonate-50-thioadenosine
(15)
To the solution of 25 (86 mg, 0.24 mmol) and phosphonate 43 in
dry pyridine (2 mL) DIC (186 lL, 1.2 mmol) was added. The mix-
ture was heated at 55 °C for 24 h, cooled to rt, evaporated, and
co-evaporated with EtOH to give the intermediate 44 which was
dissolved in 50% HCOOH (8 mL), stirred at rt overnight, evaporated,
co-evaporated with EtOH, and purified by preparative HPLC with
70% MeCN/0.1 M TEAB (20–50 linear gradient). The desired com-
pound 15 was obtained as a triethylammonium salt. It was dis-
solved in water and passed through a small column of Dowex 50
WX8-200 (Na+). Fractions containing product were combined and
lyophilized to give 15 as a white powder (70 mg, 48%). 1H NMR
(D2O) d 8.33 (s, 1H), 8.14 (s, 1H), 6.04 (d, J = 4.77 Hz, 1H), 5.17 (s,
2H), 4.73 (t, J = 5.06, 1H), 4.36 (t, J = 5.29 Hz, 1H), 4.32 (dd,
J = 10.93, 5.03 Hz, 1H), 3.99–3.89 (m, 2H), 3.81 (s, 3H), 3.02 (dd,
J = 14.42, 4.45 Hz, 1H), 2.94 (dd, J = 14.42, 6.36 Hz, 1H), 2.90–2.80
(m, 2H), 2.61–2.51 (m, 2H), 2.05 (s, 3H), 1.79 (ddd, J = 17.02,
11.02, 6.00 Hz, 2H). 31P NMR (D2O) d 25.18 (s). HRMS calcd for
32%). 1H NMR (D2O)
d 8.27 (s, 1H), 8.10 (s, 1H), 5.92 (d,
J = 6.46 Hz, 1H), 5.06 (q, J = 15.35 Hz, 2H), 4.73–4.69 (m, 1H),
4.38–4.34 (m, 1H), 4.26 (dd, J = 5.32, 3.56 Hz, 1H), 4.05–3.99 (m,
2H), 3.85 (dd, J = 14.65, 5.88 Hz, 1H), 3.78 (s, 3H), 3.58–3.51 (m,
1H), 2.91–2.78 (m, 4H), 2.02 (s, 3H). 31P NMR (D2O) d 17.01 (s).
HRMS calcd for C24H28N6O11P 607.1559 (MꢀH)ꢀ, found 607.1578.
5.19. Tiazofurin-50-yl-(adenosine-50-N-acetamido)phosphonate
(14)
To the phosphonate 38 (85 mg, 0.0135 mmol) 20,30-O-isopro-
pylidenetiazofurine 23 (45 mg, 0.0149 mmol) was added followed
by dry pyridine (2 mL). To the stirred solution DIC (105 lL,
0.675 mmol) was added and the mixture was stirred at 65 °C for
72 h. After cooling to rt, the mixture was evaporated, the crude
intermediate 39 was dissolved in 50% HCOOH (5 mL), and the solu-
tion was stirred overnight at rt. Solvents were evaporated, the res-
idue co-evaporated with EtOH and water and purified by
preparative HPLC with 0.01 M TEAB/70% MeCN (3–15 linear gradi-
ent) to give an oil, which was dissolved in small amount of H2O and
passed through a column of Dowex 50 WX8-200 (Na+). Fractions
containing product were combined and lyophilized to give 14 as
a white powder (60 mg, 70%). 1H NMR (D2O) d 8.13 (s, 1H), 8.06
(s, 1H), 7.82 (s, 1H), 5.81 (d, J = 6.14 Hz, 1H), 4.84 (d, J = 5.88 Hz,
1H), 4.58 (t, J = 5.76 Hz, 1H), 4.02–3.90 (m, 2H), 4.09 (t,
J = 3.68 Hz, 2H), 4.18–4.11 (m, 3H), 3.60 (dd, J = 14.65, 5.58 Hz,
1H), 3.35 (ddd, J = 14.54, 3.08, 1.04 Hz, 1H), 2.69 (dd, J = 20.51,
C
24H29N5O10PS 610.1378 (MꢀH)ꢀ, found 610.1419.
5.22. 20,30-O-Isopropylideneadenosine-8-thioethylenephos
phonic acid (48)
To a suspension of K2CO3 (140 mg, 1.02 mmol) in DMF (2 mL)
20,30-O-isopropylidene-8-mercaptoadenosine (46) (prepared from
45 as described24 (288 mg, 0.85 mmol) was added followed by
diethyl 2-bromoethylphopsphonate (187 mg, 1.02 mmol). The
mixture was stirred overnight at rt, evaporated, dissolved in EtOAc
(100 mL), washed with H2O, dried, and evaporated to give interme-
diate 47 as a foam. 1H NMR (CDCl3) d 8.23 (s, 1H), 6.55 (dd,
J = 11.81, 1.27 Hz, 1H), 5.95 (d, J = 5.27 Hz, 1H), 5.53 (s, 2H), 5.21
(t, J = 5.46 Hz, 1H), 5.06 (dd, J = 5.62, 0.60 Hz, 1H), 4.51 (s, 1H),
4.20–4.07 (m, 4H), 3.98–3.93 (m, 1H), 3.77 (dt, J = 12.89, 1.68 Hz,
1H), 3.59–3.41 (m, 2H), 2.34 (ddd, J = 18.31, 8.58, 7.73 Hz, 2H),
1.67 (s, 3H), 1.37 (s, 3H), 1.35 (t, J = 7.08 Hz, 6H). To a solution of
47 (0.428 g, 0.85 mmol) in dry MeCN (10 mL) 2,6-lutidine
(0.5 mL, 4.25 mmol) was added followed by TMS-Br (0.572 mL,
4.25 mmol). The mixture was stirred overnight at rt, evaporated,
diluted with EtOH and evaporated again. The crude product was
purified by preparative HPLC with 70% MeCN/0.1 M TEAB (25-50
linear gradient) to give 48 as a foam (360 mg, 77% triethylammo-
nium salt). 1H NMR (D2O) d 8.10 (s, 1H), 6.22–6.13 (m, 1H), 5.48–
5.40 (m, 1H), 5.18–5.08 (m, 1H), 4.47–4.38 (m, 1H), 3.88 (dd,
J = 12.51, 3.49 Hz, 1H), 3.83 (dd, J = 12.48, 4.75 Hz, 1H), 3.53–3.40
(m, 1H), 3.21 (q, J = 7.33 Hz, 4H), 2.04 (td, J = 17.01, 8.46 Hz, 2H),
1.70 (s, 3H), 1.45 (s, 3H), 1.30 (t, J = 7.33 Hz, 6H)). 31P NMR (D2O)
d 20.57 (s). HRMS calcd for C15H21N5O7PS 446.0905 (MꢀH)ꢀ, found
446.0979.
7.68 Hz, 1H). 31P NMR (D2O)
C
d 17.13 (s). HRMS calcd for
21H26N8O11PS 629.1185 (MꢀH)ꢀ, found 629.1217.
5.20. 20,30-O-Isopropylideneadenosine-50-thioethylenephos
phonic acid (43)
20,30-O-Isopropylidene-50-S-acetyladenosine (41, 0.5 g,
1.37 mmol)25 was dissolved in MeOH (10 mL), the solution was de-
gassed with Ar, cooled to ꢀ20 °C, and diethyl 2-bromoethylphosph-
onate (380 lL, 2.05 mmol) was added followed by MeONa (165 mg).
The mixture was stirred overnight, evaporated, dissolved in EtOAc
(100 mL), washed with H2O, dried over MgSO4, filtered, and evapo-
rated to give the intermediate 42 as a foam (0.53 g, 79%). 1H NMR
(CDCl3) d 8.35 (s, 1H), 7.90 (s, 1H), 6.06 (d, J = 2.10 Hz, 1H), 5.74 (s,
2H), 5.50 (dd, J = 6.40, 2.11 Hz, 1H), 5.06 (dd, J = 6.39, 3.32 Hz, 1H),
4.40–4.34 (m, 1H), 2.89 (dd, J = 13.68, 7.41 Hz, 1H), 2.81 (dd,
J = 13.70, 6.32 Hz, 1H), 2.78–2.72 (m, 2H), 2.02–1.94 (m, 2H), 1.63–
1.59 (m, 3H), 1.39 (s, 3H), 1.29 (dt, J = 7.06, 2.27 Hz, 6H). 31P NMR

K. Felczak et al. / Bioorg. Med. Chem. 19 (2011) 1594–1605
1605
5.23. Mycophenolic ethylenephosphonate-8-thioadenosine (16)
analyzed by GraphPad Prism 4 software (GraphPad Software, San
Diego, CA) as described earlier.30
To the solution of dried 48 in DMF (5 mL) imidazole (103 mg,
1.53 mmol) was added followed by TBDMS-Cl (122 mg, 0.81 mmol).
The mixture was stirred at rt overnight, evaporated, and crude prod-
uct was purified by HPLC with 70% MeCN/0.1 M TEAB (20–50 linear
gradient) to give the intermediate 49 as a foam (not stable—partially
decomposing into substrate) which was used immediately in the
next step. The carefully dried 49 (65 mg, 0.1 mmol) and 25 (36 mg,
0.1 mmol) were dissolved in dry pyridine (1 mL), followed by addi-
Acknowledgments
This research was supported by the Center for Drug Design in
the Academic Health Center of the University of Minnesota and
by the Veterans Affairs Merit Review Awards (HNJ).
Supplementary data
tion of DIC (80 lL, 0.5 mmol). The mixture was stirred at rt for a
few minutes and then heated at 58 °C for 48 h. The crude intermedi-
ate 50 was dissolved in 50% HCOOH (8 mL) and stirred at rt over-
night, evaporated, co-evaporated with EtOH. It was then purified
by preparative HPLC with 70% MeCN/0.1 M TEAB (20–50 linear gra-
dient), dissolved in water, and passed through a small column of
Dowex 50 WX8-200 (Na+). Fractions containing product were com-
bined and lyophilized to give 16 as a white powder (22 mg, 35%). 1H
NMR (D2O) d 8.15 (s, 1H), 6.06 (d, J = 7.12 Hz, 1H), 4.99 (dd, J = 7.03,
5.52 Hz, 1H), 4.97 (s, 2H), 4.50 (dd, J = 5.45, 2.41 Hz, 1H), 4.35 (q,
J = 2.65 Hz, 1H), 4.04 (q, J = 6.40 Hz, 2H), 4.00 (dd, J = 12.92,
2.52 Hz, 1H), 3.91 (dd, J = 12.90, 3.21 Hz, 1H), 3.82 (s, 3H), 3.07–
3.00 (m, 2H), 2.94 (t, J = 6.34 Hz, 2H), 2.03 (s, 3H), 1.95–1.87 (m,
2H). 31P NMR (D2O) d 24.28 (s). HRMS calcd for C24H29N5O11PS
626.1378 (MꢀH)ꢀ, found 626.1327.
Supplementary data (table of HPLC purity of compounds 2–16.
This material is available free of charge via the Internet) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.01.042.
References and notes
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Logarithmically growing K562 cells in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum were plated in 96-well
plates at a density of 800 cells/0.1 mL and incubated at 37 °C in
an atmosphere of air and 5% CO2. Twenty-four hours later, various
concentrations of the compounds were added in 3
mixed and further incubated for 72 h. At the end of the incubation
period, 20
L of cellTiter 96Ò reagent (Promega, Madison, WI) con-
lL volume,
l
taining tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt; MTS] was added and incubated at 37 °C in an atmosphere of
air and 5% CO2 and then the color developed was read at 490 nm
using SpectraMax software in a microplate spectrophotometer
(Molecular Devices Corp., Sunnyvale, CA) and the data were