Nucleolipids of the Nucleoside Antibiotics Formycins A and B: Synthesis and Biomedical Characterization Particularly Using Glioblastoma Cells

Article abstract of DOI:10.1002/cbdv.201900012

Two lipophilic derivatives of formycin A (1) and formycin B (5) carrying an O-2′,3′-(ethyl levulinate) ketal group have been prepared. These were base-alkylated at N(1) (for 1) and N(1) and N(6) (for 5) with both isopentenyl and all-trans-farnesyl residues. Upon the prenylation, side reactions were observed, resulting in the formation of nucleolipids with a novel tricyclic nucleobase (→4a, 4b). In the case of formycin B, O-2′,3′-(ethyl levulinate) (6) farnesylation gave the double prenylated nucleolipid 7. All new compounds were characterized by ¹H-, ¹³C-, UV/VIS and fluorescence spectroscopy, by ESI-MS spectrometry and/or by elemental analysis. Log P determinations between water and octanol as well as water and cyclohexane of a selection of compounds allowed qualitative conclusions concerning their potential blood-brain barrier passage efficiency. All compounds were investigated in vitro with respect to their cytotoxic activity toward rat malignant neuroectodermal BT4Ca as well as against a series of human glioblastoma cell lines (GOS 3, U-87 MG and GBM 2014/42). In order to differentiate between anticancer and side effects of the novel nucleolipids, we also studied their activity on PMA-differentiated human THP-1 macrophages. Here, we show that particularly the formycin A derivative 3b possesses promising antitumor properties in several cancer cell lines with profound cytotoxic effects partly on human glioblastoma cells, with a higher efficacy than the chemotherapeutic drug 5-fluorouridine.

Full text of DOI:10.1002/cbdv.201900012

Title: Nucleolipids of the Nucleoside Antibiotics Formycin A and B:
Synthesis and Biomedical Characterization particularly using
Glioblastoma Cells
Authors: Helmut Rosemeyer, Christine Knies, Katharina
Hammerbacher, Eugenia Bender, Gabriel A. Bonaterra,
Ricarda Hannen, Jörg W. Bartsch, Christopher Nimsky, and
Ralf Kinscherf
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900012
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900012

10.1002/cbdv.201900012
Chemistry & Biodiversity
1
Nucleolipids of the Nucleoside Antibiotics Formycin A and B: Synthesis
and Biomedical Characterization particularly using Glioblastoma Cells#)
Helmut Rosemeyer 1), Christine Knies 1), Katharina Hammerbacher 2), Eugenia Bender 1),
Gabriel A. Bonaterra2), Ricarda Hannen 3), Jörg W. Bartsch 3), Christopher Nimsky 3), and
Ralf Kinscherf 2)
1) Organic Chemistry I - Bioorganic Chemistry, Institute of Chemistry of New Materials,
University of Osnabrück, Barbarastr. 7, D-49069 Osnabrück
2) Institute for Anatomy and Cell Biology, Department of Medical Cell Biology, Philipps-
University Marburg, Robert-Koch-Str. 8, D-35032 Marburg
3) Department of Neurosurgery, Philipps-University Marburg, Baldingerstr. D-35032
Marburg, Germany
E-Mail: Helmut.Rosemeyer@uos.de
#)Dedicated to the memory of the lateKlaus Mühlegger, Penzberg and Polling, Bavaria,
Germany
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
2
Abstract.Two lipophilic derivatives of formycin A (1) and formycin B (5) carrying an
O-2’,3’-ethyllevulinate ketal group have been prepared. These were base-alkylated at N(1)
(for 1) and N(1) and N(6) (for 5) with both, isopentenyl or all-trans farnesyl residues. Upon
the prenylation side reactions were observed, resulting in the formation of nucleolipids with
a novel tricyclic nucleobase (→ 4a,b). In case of formycin B O-2’,3’-ethyllevulinate (6)
farnesylation gave the double prenylated nucleolipid 7. All new compounds were
characterized by 1H-, 13C-, UV-Vis, and fluorescence spectroscopy, by ESI mass
spectrometry and/or by elemental analysis.LogP determinations between water and n-
octanol as well as water and cyclohexane of a selection of compounds allowed qualitative
conclusions concerning their potential blood-brain barrier passage efficiency. All
compounds were investigated in vitrowith respect to their cytotoxic activity toward rat
malignant neuroectodermal BT4Ca as well as against a series of human glioblastoma cell
lines (GOS 3, U-87 MG and GBM 2014/42). In order to differentiate between anticancer and
side effects of the novel nucleolipids, we also studied their activity on PMA-differentiated
human THP-1 macrophages. Here we show, that particularly the formycin A derivative 3b
possesses promising antitumor propertiesin several cancer cell lines with profound
cytotoxic effects partly on human glioblastoma cells, with a higher efficacy than the
chemotherapeutic drug 5-fluorouridine.
Key words: Formycin A and B, Nucleolipids, Drug Profiling, Glioblastoma
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
3
1. Introduction.The C-nucleoside antibiotic formycin A (from Nocardia interforma),
an adenosine analogue, is a fluorescent nucleoside with an extremely stable glycosylic
bond.For a comprehensive overview about formycin A and B, its chemistry, biochemistry
and fluorescence spectroscopy, see [1]. Formycin A exhibits activity against various tumor
cell lines such as Ehrlich carcinoma, HeLa cells,and Yoshida rat sarcoma [2].Besides the
anti-tumoral impact of formycin A,an insulinotropic action was reported [3]. It is rapidly
deaminated to its inosine analogue, formycin B, by the action of adenosine deaminase [1,
2].J. Giziewiczet al.[4] synthesized a series of base- and ribose-methylated formycin A
derivatives and tested their antiviral and antitumor activity. In the course of this work it
turned out that N(1)-methylformycin A proved to be extraordinarily resistant towards the
enzymatic deamination by adenosine deaminase. F. Seela et al. [5] prepared the N(1)-
methylated 2’-deoxy-ß-D-ribonucleosides of formycin A and B and their 2’-
cyanoethylphosphoramidites as well as an N(1)-carboxymethyl-functionalized formycin A
compound and digoxigenin derivatives thereof for the detection of nucleic acids [6].
In this paper we report on the preparation of nucleolipids of formycin A(1,
NS_8.0.0.0) and B (5, NS_9.0.0.0)and similar tricyclic formycin A derivatives carrying
additional lipophilic moieties at N(1) and exhibiting strong fluorescence.Formycin B (5,
NS_9.0.0.0) was prepared from formycin A by deamination with adenosine deaminase.
2. Syntheses of Formycin A and B Compounds. Reaction of formycin A (1) with
ethyllevulinate in the presence of triethylorthoformate and 4M HCl in 1,4-dioxane in dry and
amine-free DMF gave – after 24 h – the O-2’,3’-ketal derivative 2 as main product which
was purified by column chromatography. Subsequent reaction of 2 with trans,trans-farnesyl
bromide in the presence of K2CO3 in DMF gave compound 3b as main product besides a
small amount of a highly blue-fluorescent, faster migrating by-product, in the following
identified as compound 4b(Formula Scheme 1) - formed upon prenylation- carrying a novel
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
4
heterocyclic system. Both compounds were analyzed by 1H-(see Supplementary Material)
and 13C-NMR spectroscopy; assignment of the resonances was made with the help of
DEPT-135, [1H,1H]- and [1H,13C]-correlation spectroscopy. Moreover, ESI mass
spectrometry as well as UV- and fluorescence spectroscopy (see Supplementary Material)
proved the structure and integrity of the compounds.
NH2
N
N
R1
NH2
R1
N
H
N
N
N
N
N
N
N
N
N
N
(ii)
HO
HO
HO
+
O
O
O
O
O
O
O
O
O
(i)
O
O
O
O
O
O
NH2
NL_8.1.0.0
NL_8.1.1X.0
NL_8.1.1X.0P
H
N
N
N
2
3 a,b
4 a,b
N
HO
O
(iii)
O
O
N
O
N
R1
OH OH
Formycin A
NS_8.0.0.0
H
N
H
R1
N
N
N
NH
NH
N
N
N
N
1
(ii)
(i)
HO
HO
HO
O
O
O
OH OH
O
O
O
O
O
O
Formycin B
NS_9.0.0.0
O
O
NL_9.1.0.0
NL_9.1.1363.0
5
6
7
Formula scheme 1. Syntheses of Formycin A- and Formycin B-Nucleolipids. (i) DMF,
Ethyllevulinate, HC(OEt)3 H+/1.4-Dioxane; (ii) at FA: DMF, K2CO3, X = 1 = R1 =
Isopentenylbromide, X = 3 = R1 = Farnesylbromide; (ii) at FB: DMF, K2CO3, R1 = Farnesyl-
; (iii) H2O, ADA. Red colored new heterocyclic system: 1,7-dihydropyrazolo[3,4-
e]pyrimido[1,2-c]pyrimidine (red highlighted).
Formycin B (5) was prepared from formycin A (1) by deamination with adenosine
deaminase. Of particular interest was the detection of compound 4b, the formation of which
is conceivable by reaction of compound 3bwith an excess of farnesyl bromide by an
unknown mechanism. In Formula Scheme S2 (Supplementary Material) two variants of
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
5
thisside reaction are tentatively postulated.Compound 4b contains a novel N-heterocyclic
ring system, namely a 9-methylated 1,7-dihydropyrazolo[3,4-e]pyrimido[1,2-c]pyrimidine
carrying a glyconic residue with an O-2’,3’-ethyllevulinate moiety at O-2’,3’ and a
trans,trans-farnesyl residue at N(1). A reaction of compound 2 with isopentenyl bromide
gave two analogous reaction products (compounds 3a and 4a).
Both farnesylated compounds (3b, 4b) exhibit similar Rf values; the slightly faster
migrating spot, however, shows a blue fluorescence upon irradiation at 366 nm, while the
slower migrating nucleolipid does not. In both cases (isopentenylation, 3a, 4a; farnesylation,
3b, 4b) the products were separated chromatographically and characterized (see
experimental part). A presentation of the corresponding TLC plates, of the UV/Vis spectra,
the ESI mass spectra as well as of the 1H-NMR spectra can be found in the Supplementary
Material Section (S1-S6). Figure 1 shows an energy-minimized computer model of
compound 4bformed upon prenylation; Figure 2presents an optical comparison of the
fluorescence of compounds 1, 2, 3b, 4band 4a (0.1 mM in methanol) upon irradiation at
254 nm.
FFigure 1. Computer Modelling of Compound
Figure 2. Comparison of fluorescence at a
concentration of 0.1 mM in MeOH.
Irradiation 254 nm, cut-off filter: 280 nm. A:
formycin A (1), B: NL_8.1.0.0 (2), C:
NL_8.1.13.0 (3b), D: NL_8.1.13.0P(4b), E:
NL_8.1.11.0P(4a).
bNL_8.1.13.0P (4b, Formula Scheme 1).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
6
Figure 3. Presentation of the topological polar surface area of compound 4b.
Figure 3 displays the topological surface area of compound 4bwhich amounts to
126.96 Å2. According to D. E. Clark [7] and M. D. Wessel [8] this value lies in a range which
is acceptable for an intestinal drug absorption.
3. Biophysical Studies.
In order to evaluate how much lipophilization of formycin A (1) changes its
lipophilicity, the 10logPOW values of all nucleolipids were determined in silico using the
http://eadmet.com/de/physprop.php website with ePhysChem that contains ALOGPS
v.3.0. [9,10]. In addition, the values of NS_8.0.0.0 (1) and NL_8.1.13.0P (4a)were
determined experimentally (see experimental. part). Moreover, the corresponding 10logPCh
data of the latter nucleolipids were measured in water-cyclohexane (Table 1). According to
the work of P. Seiler [11] and later of R. C. Young [12], the difference of the two
10logPvaluesallows a rough assessment of the ability of various low-molecular-weight
drugs to cross the blood-brain barrier, applying eq. 1:
10log(CBrain/CBlood) = -0.604 (± 0.169) ∆10logP + 1.23 (± 0.56) (eq. 1)
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
7
Table 1. Biophysical data of NS_8.0.0.0(1) and its nucleolipids NL_8.1.13.0 (3b)
andNL_8.1.13.0P(4b).
Compound
logS
logPOW
logPOW
logPChW
logPOW-logPChW CBrain/CBlood
(calc.)
(measured) (measured)
NS_8.0.0.0, 1 -1.28 -1.25 ± 0.74
NL_8.1.13.0, 3b -4.93 2.78 ± 0.74
NL_8.1.13.0P, 4b -4.34 3.05 ± 0.74
S: water solubility
-2.11
2.20
2.83
-2.29
2.06
2.69
0.185
0.140
0.140
13.13
14.00
14.00
Inspection of the data shown in Table 1 exhibits a slighttheoretical improvement of
the blood-brain passage of Formycin A upon O-2’,3’-ketalization with an ethyllevulinate
residue and a further hydrophobization at the heterocyclic base. However, from Table 1 it
can be seen that there is no difference in the CBrain/CBlood values between compounds 3b
and 4b.
4. Biological Studies.
To differentiate anticancer-fromsideeffects [20], we treated either brain tumor cell
lines or human macrophages as non-tumor cell line with compounds as indicatedin
vitro.As brain tumor cells, rat malignant neuroectodermal BT4Ca[13], human
astrocytoma/oligodendroglioma GOS-3, human glioblastoma U-87MG (ATCC HTB-14)
and patient-derived human primary glioblastoma cells GBM 2014/42 were studied. As non-
tumor cell line, human THP-1 monocytic cells were used that had been differentiated with
phorbol 12-myristate 13-acetate (PMA) to represent a macrophage phenotype. The effects
of 5-fluorouridine (5-FUrd; NS_4.0.0.0 [14]; ~positive control), formycin A (NS_8.0.0.0, 1)
and its previously described derivativesNL_8.1.0.0 (2), NL_8.1.13.0 (3b),
NL_8.1.13.0P(4b), NL_8.1.11.0P(4a)plus formycin B (NS_9.0.0.0, 5)
NL_9.1.0.06), and
,
NL_9.1.1363.0 (7)on cell viability were determined after a 48h incubation with thesenovel
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
8
compounds(Figures. 4-13).The experimental setting of our viability/cytotoxicity assays was
chosen according to previous results using other novel anti-tumor derivatives, where we
found 100 % cytotoxicity already at 50 µM after 48 h treatment (e. g. Farhat et al. 2015;
Knies et al., 2015a, 2015b, 2016). Additionally, under these experimental conditions,
active derivatives revealed an IC50 between 1.56 µM and 50µM in different tumor cell
lines (e. g. Farhat et al. 2015; Knies et al., 2015a, 2015b, 2016).
After treatment (48h) of rat malignant neuroectodermal BT4Ca cells with NS_4.0.0.0
(5-FUrd), we observed a significant(p<0.001) inhibition of cell viability at all concentrations
testedby -66.3 % (1.56 µM), -69.3 % (3.12 µM), -75.4 % (6.25µM), -77.9 % (12.5µM), -81.9
% (25 µM) and -83.3 %(50 µM) compared to DMSO that was always used as control
(~100% viability, Figures. 5& 6). Formycin A (1, NS_8.0.0.0) itself showed an almost
concentration-dependent effect and diminished cell viability significantly by -23.9 % (12.5
µM, p<0.05), -46.7 % (25 µM, p<0.01) and -63.1 % (50 µM, p<0.001)compared to DMSO
(Figure. 4).
Treatment of BT4Ca cells with NL_8.1.0.0 (2) at 25 µM resulted in a significant
increase of cellviability by +16.1 % (p<0.05)compared to DMSO (Fig. 5).In contrast, at all
concentrations tested,NL_8.1.13.0 (3b) significantly inhibited cell viability by -37.9 % (1.56
µM; p<0.01), -65.1 % (3.12 µM; p<0.01), -80.3 % (6.25 µM; p<0.01), -87.5 %(12.5 µM;
p<0.001), - 92.4 % (25 µM; p<0.001) and -95.1 % (50 µM; p<0.01) compared to DMSO
(Fig. 4).
In contrast, the derivative NL_8.1.13.0 P(4b) significantly enhanced cell viability by
+18.2 % (1.56 µM, p<0.001), +20.0 % (3.12 µM, p<0.01), +32.9 % (6.25 µM, p<0.001) and
+ 13.7 % (12.5 µM, p<0.001) compared to DMSO. Only at a concentration of 50
µM,NL_8.1.13.0P(4b) revealed a significant (p<0.001) inhibition of cell viability by -75.8 %
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
9
compared to DMSO (Fig. 5). In addition, derivative NL_8.1.11.0 P(4a) showed at 50 µM a
significant (p<0.05) reduction of the viability by -10.6 % compared to DMSO (Fig. 4).
NS_4.0.0.0 (5-FUrd)
NS_8.0.0.0
160
NL_8.1.0.0
NL_8.1.(1)3.0
NL_8.1.(1)3.0 P
NL_8.1.(1)1.0 P
140
120
100
80
***
**
*
***
***
*
*
**
60
**
40
**
***
***
***
***
***
**
***
***
***
***
***
20
**
0
1.56
3.12
6.25
12.5
25
50
µM
Figure 4.Viability (in %) of rat BT4Ca astrocytoma/oligodendroglioma cells after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin A (NS_8.0.0.01) or its derivatives (NL_8.1.0.0, 2, NL_8.1.13.0,
3a, NL_8.1.13.0P, 4a, and NL_8.1.11.0P, 4b) is shown. Values are given (in % viability of control
[incubation with medium alone = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001,
significance vs. DMSO control (equal concentration of DMSO to 50 µM substance solution), n=5
independent experiments assayed in quadruplicates.
Formycin B (NS_9.0.0.0, 5) at a concentration of 12.5 µMsignificantly increased
viability of BT4Ca cells (p<0.05) by +10.4 %compared to DMSO (Fig. 5).At the same
concentration (12.5 µM), its derivative NL_9.1.1363.0 (7) also led to a significant (p<0.05)
increase of cell viability by +17.9 %compared to the DMSO control (Fig.5), while derivative
NL_9.1.0.0 (6) insignificantly diminished viability by -32.4 % (50 µM) (Fig.5).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
10
NS_4.0.0.0 (5-FUrd)
NS_9.0.0.0
160
140
120
100
80
NL_9.1.0.0
NL_9.1.(1)3(6)3.0
*
*
60
40
***
***
20
0
***
***
***
***
1.56
3.12
6.25
12.5
25
50
µM
Figure 5.Viability (in %) of rat BT4Caastrocytoma/oligodendroglioma cells after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin B (NS_9.0.0.0, 7) or its derivatives (NL_9.1.0.0, 6, and
NL_9.1.1363.0, 7) is shown. Values are given (in % viability of control [incubation with medium
alone = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs. DMSO
control (equal concentration of DMSO to 50 µM substance solution), n=5 independent experiments
assayed in quadruplicates.
Next, we tested the human GOS-3 glioblastoma cells in response to the
compounds. Treatment (48h) of GOS-3 cellswith NS_4.0.0.0 (5-FUrd)resulted in a
significant reductionof cell viability (Figs.6, 7). 5-FUrd significantly decreased the viabilityat
all concentrations testedby -16.7 % (1.56 µM; p<0.01), -15.9 % (3.12 µM; p<0.05), -20.0 %
(6.25 µM; p<0.001), -20.1 % (12.5 µM; p<0.001), -26.4 % (25 µM; p<0.001) and -29.7
%(50 µM; p<0.001) compared to DMSO (~100% viability) (Figs. 6, 7).
Formycin A (NS_8.0.0.0, 1) reduced cell viability significantly at 3.12-50 µMby -13.2
% (3.12 µM; p<0.05), -15.0 % (6.25 µM; p<0.01), -16.5 % (12.5 µM; p<0.01), -26.3 % (25
µM; p<0.001) and -38.0 % (50 µM; p<0.001)compared to DMSO (Fig. 6).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
11
In contrast, derivative NL_8.1.0.0 (2) at 25 µM caused a significant (p<0.01)
increase of viability by +14.0 % (p<0.01)compared to DMSO (Fig. 6).NL_8.1.13.0 (3a)
significantly diminished viability of GOS-3 cellsin a concentration-dependent manner by -
26.0 % (6.25 µM, p<0.01), -61.5 % (12.5 µM, p<0.01), -97.0 % (25 µM, p<0.001) and -100
% (50 µM, p<0.001) compared to DMSO (Fig. 6). Moreover, NL_8.1.13.0 (3a) significantly
inhibited the viability by -41.4 % (12.5 µM, p<0.001), -70.6 % (25 µM, p<0.001) and -71.6
% (50 µM, p<0.01) compared to 5-FUrd(Fig. 6).
However, in contrast to NL_8.1.13.0 (3a), derivative NL_8.1.13.0P(4a) (1.56-50 µM)
significantly raised cell viability at all concentrations by +26.8 % (1.56 µM; p<0.001), +29.7
% (3.12 µM; p<0.001), +32.7 % (6.25µM; p<0.001), +22.4 % (12.5 µM; p<0.01), +31.2 %
(25µM; p<0.001) and + 16.4 % (50 µM; p<0.001) compared to DMSO (Fig. 6).
Substance NL_8.1.11.0P(4b)revealedno significant effects on cellviability of GOS-3
cells (Fig. 6).
NS_4.0.0.0 (5-FUrd)
NS_8.0.0.0
160
NL_8.1.0.0
NL_8.1.(1)3.0
NL_8.1.(1)3.0 P
NL_8.1.(1)1.0 P
140
120
100
80
***
***
***
**
**
***
**
***
*
**
**
**
*
***
***
***
***
***
***
60
40
**
###
20
***
###
0
##
***
1.56
3.12
6.25
12.5
25
50
-20
µM
Figure 6.Viability (in %) of human GOS-3 glioblastoma cells after incubation (48h) with NS_4.0.0.0
(5-FUrd), formycin A (NS_8.0.0.0, 1) or its derivatives (NL_8.1.0.0,2, NL_8.1.13.0,3a, NL_8.1.13.0 P,
4a, and NL_8.1.11.0P, 4b) is shown. Values are given (in % viability of control [incubation with
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
12
medium alone; = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs.
#
DMSO control (equal concentration of DMSO to 50 µM substance solution), p<0.05, ##p<0.01,
###p<0.001, significance vs. 5-FUrd at equal concentration; n=5 independent experiments assayed
in quadruplicates.
Formycin B (NS_9.0.0.0, 5) increased the cellviability of GOS-3 cells at 1.56, 3.12,
6.25 and 12.5 µMby +26.1 % (p<0.001), +16.8 % (p<0.05), +15.3 % (p<0.05) and +19.4 %
(p<0.05), respectively,compared to DMSO (Fig. 7).Derivative NL_9.1.1363.0(7) only at a
concentration of 1.56 µM led to a significant (p<0.01) increase of the cell viability by
+18.0%compared to DMSO (Fig.7).Also NL_9.1.0.0 (6) at a concentration of 1.56 µM
increased the viability of GOS-3 cellssignificantly (p<0.01) by +16.9 %, but at the
concentration of 50 µM it significantly (p<0.05) reduced cell viability by -6.4 % (Fig.7).
NS_4.0.0.0 (5-FUrd)
NS_9.0.0.0
160
NL_9.1.0.0
NL_9.1.(1)3(6)3.0
140
***
**
*
*
120
100
80
60
40
20
0
*
*
*
*
**
*
***
***
***
***
1.56
3.12
6.25
12.5
25
50
µM
Figure 7.Viability (in %) of human GOS-3 glioblastoma cells after incubation (48h) with NS_4.0.0.0
(5-FUrd), Formycin B (NS_9.0.0.0, 5) or its derivatives (NL_9.1.0.0, 6, and NL_9.1.1363.0, 7) is
shown. Values are given (in % viability of control [incubation with medium alone; = 100 % viability])
as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs. DMSO control (equal concentration
of DMSO to 50 µM substance solution), n=5 independent experiments assayed in quadruplicates.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
13
After incubation (48h) of human U-87MG glioblastoma cells with NS_4.0.0.0 (5-
FUrd), we observed a significant inhibition of cellviability (Figs. 8, 9). At all concentrations
tested, 5-FUrd significantly (p<0.001) reduced viabilityby -68.8 % (1.56 µM), -71.6 % (3.12
µM), -67.8 % (6.25 µM), -79.7 % (12.5 µM), -71.1 % (25 µM) and -69.6 %(50 µM)
compared to DMSO (~100% viability) (Figs. 8, 9).
Formycin A (NS_8.0.0.0, 1)significantly reduced cell viability by -17.2 % (1.56 µM,
p<0.01), -47.9 % (3.12 µM, p<0.001), -70.6 % (6.25 µM, p<0.001), -84.2 % (12.5 µM,
p<0.01), -93.3 % (25 µM, p<0.001) and -97.1 % (50 µM, p<0.001)compared to DMSO (Fig.
8).Interestingly, NS_8.0.0.0(1) significantly (p<0.001) reduced the viability of U-87 MG
cells by -11.6 % (12.5 µM), -20.2 % (25 µM) and -26.2 % (50 µM) in comparison to 5-FUrd
(Fig. 8).
Moreover, at all concentrations tested, derivative NL_8.1.13.0(3a) significantly
inhibitedcell viability by -23.0 % (1.56 µM; p<0.001), -39.1 % (3.12 µM; p<0.001), -51.1 %
(6.25 µM; p<0.001), -55.3 % (12.5 µM; p<0.001), - 86.2 % (25 µM; p<0.01) and -90.2 %
(50 µM; p<0.01)compared to DMSO(Fig. 8). Interestingly, NL_8.1.13.0(3a)
significantlydiminished the cell viability by -15.1 % (25 µM, p<0.01) and -20.7 % (50 µM,
p<0.05) compared to 5-FUrd (Fig. 8).
NL_8.1.11.0P(4b) showed only at 3.12 µMa significant (p<0.01) inhibition of cell
viability by -6.0 % compared to DMSO (Fig. 8).In contrast, compounds NL_8.1.0.0 (2), and
NL_8.1.13.0P(4a)revealedno significant effects on viability of U-87 MG cells compared to
DMSO (Fig. 8).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
14
NS_4.0.0.0 (5-FUrd)
NS_8.0.0.0
160
140
120
100
NL_8.1.0.0
NL_8.1.(1)3.0
NL_8.1.(1)3.0 P
NL_8.1.(1)1.0 P
**
***
80
60
40
20
0
***
***
***
***
***
***
***
***
##
**
***
***
***
**
###
#
***
**
###
***
###
1.56
3.12
6.25
12.5
25
50
µM
Figure 8.Viability (in %) of human U-87 MG glioblastoma cells after incubation (48h) with NS_4.0.0.0
(5-FUrd), Formycin A (NS_8.0.0.0, 1) or its derivatives (NL_8.1.0.0,2, NL_8.1.13.0, 3a,
NL_8.1.13.0P(4a) and NL_8.1.11.0P, 4b) is shown. Values are given (in % viability of control
[incubation with medium alone; = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001,
significance vs. DMSO control (equal concentration of DMSO to 50 µM substance solution),
#p<0.05, ##p<0.01, ###p<0.001, significance vs. 5-FUrd at equal concentration; n=5 independent
experiments assayed in quadruplicates.
At concentrations of 25 and 50 µM,Formycin B (NL_9.0.0.0, 5) significantly
(p<0.001) reduced viability of U-87 MG cells by -24.5 % and -37.5 % compared to DMSO
(Fig. 9).At concentrations of 12.5, 25 and 50 µM NL_9.1.1363.0 (7),a significant (p<0.001)
inhibition of viability of U-87 MG cellsby -11.2 %, -69.5 % and -99.2 % in comparison to
DMSO (Fig. 9) was observed. Additionally,NL_9.1.1363.0(7) significantly (p<0.001)
decreased viabilityof human U-87 MG glioblastoma cellsby -28.3 % at 50 µMcompared to
5-FUrd (Fig. 9) whereas NL_9.1.0.0 (6) showed no significant effects on the viability of
human U-87 MG glioblastoma cells compared to DMSO (Fig. 9).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
15
5-FUrd (NS_4.0.0.0)
NS_9.0.0.0
160
140
120
100
80
NL_9.1.0.0
NL_9.1.(1)3(6)3.0
***
***
***
***
***
60
40
***
***
***
***
***
***
20
0
###
1.56
3.12
6.25
12.5
25
50
µM
Figure 9.Viability (in %) of human U-87 MG glioblastoma cells after incubation (48h) with NS_4.0.0.0
(5-FUrd), Formycin B (NS_9.0.0.0, 5) or its derivatives (NL_9.1.0.0, 6and NL_9.1.1363.0, 7) is
shown. Values are given (in % viability of control [incubation with medium alone; = 100 % viability])
as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs. DMSO control (equal concentration
of DMSO to 50 µM substance solution), n=5 independent experiments assayed in quadruplicates.
Next, we used a primary Glioblastoma patient-derived cell line GBM 2014/42 for cell
viability assays. After treatment (48h) of GBM 2014/42cellswith NS_4.0.0.0 (5-FUrd), we
observed that5-FUrd significantly (p<0.001) reduced the viabilityat alltested concentrations
by -71.3 % (1.56 µM), -74.1 % (3.12 µM), -71.1 % (6.25 µM), -72.7 % (12.5 µM), 73.2 %
(25 µM) and -70.9 % (50 µM) in comparison to DMSO (~100% viability) (Figs. 10, 11).
Furthermore, Formycin A (NS_8.0.0.0, 1) significantly (p<0.001) decreased viability
of GBM 2014/42 cellsat all concentrationstestedby -58.2 % (1.56 µM), -80.0 % (3.12 µM), -
92.3 % (6.25 µM), -95.3 % (12.5 µM), -100 % (25 µM) and -99.6 % (50 µM)compared to
DMSO (Fig. 10).Additionally, NS_8.0.0.0 (1) significantlyreducedthe viability by -5.9 %
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
16
(3.12 µM, p<0.05), -21.1 % (6.25 µM, p<0.001), -22.7 % (12.5 µM, p<0.001), -29.9% (25
µM, p<0.001) and -28.6 % (50 µM, p<0.001) in comparison to 5-FUrd (Fig. 10).
At all concentrations tested,NL_8.1.13.0(3a) significantly diminished the viability of
human primary glioblastoma cells GBM 2014/42 by -20.8 % (1.56 µM, p<0.001), -55.4 %
(3.12 µM, p<0.01), - 65.4 % (6.25 µM, p<0.01), -80.3 % (12.5 µM, p<0.001), -100 % (25
µM, p<0.001) and -100 % (50 µM, p<0.001) in comparison to the DMSO control (Fig. 10).
Moreover, NL_8.1.13.0 significantly inhibited the viability by -7.6 % (12.5 µM, p<0.01), -
40.9 % (25 µM, p<0.001) and -39.5 % (50 µM, p<0.001) compared to 5-FUrd (Fig. 10).
Only at a concentration of 50µM,NL_8.1.13.0P(4a)caused a significant reduction in
viability of GBM 2014/42 cellsby -18.4 % (p<0.01) compared to DMSO (Fig. 10).
Compounds NL_8.1.0.0 (2), and NL_8.1.11.0P(4b)revealedno significant effects on the
viability of GBM 2014/42cells compared to DMSO (Fig. 10).
NS_4.0.0.0 (5-FUrd)
NS_8.0.0.0
160
NL_8.1.0.0
140
120
100
80
NL_8.1.(1)3.0
NL_8.1.(1)3.0 P
NL_8.1.(1)1.0 P
***
**
60
***
***
**
40
**
***
***
***
***
***
20
***
##
***
#
###
***
###
###
***
###
***
***
0
***
###
***
-20
-40
###
1.56
3.12
6.25
12.5
25
50
µM
Figure 10.Viability (in %) of human primary glioblastoma cells (GBM 2014/42) after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin A (NS_8.0.0.0, 1) or its derivatives (NL_8.1.0.0, 2,
NL_8.1.13.0,3a, NL_8.1.13.0P, 4a, and NL_8.1.11.0P, 4b) is shown. Values are given (in % viability
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
17
of control [incubation with medium alone; = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01,
***p<0.001, significance vs. DMSO control (equal concentration of DMSO to 50 µM substance
#
solution), p<0.05, ##p<0.01, ###p<0.001, significance vs. 5-FUrd at equal concentration; n=5
independent experiments assayed in quadruplicates.
At concentrations of 12.5, 25 and 50 µM,Formycin B (NL_9.0.0.0, 5) significantly
(p<0.001) inhibited the viability of GBM 2014/42 cells by -15.4 %, -48.4 % and -59.2 %
compared to DMSO (Fig. 11).Only at a concentration of 50 µM,the derivative
NL_9.1.1363.0 (7)led to a significant (p<0.01) reduction of cell viability in GBM 2014/42
cellsby -87.6% compared to DMSO (Fig. 11). Additionally,NL_9.1.1363.0 (7)significantly
(p<0.001) diminished the viability of by -16.3 % (50 µM) in comparison to 5-FUrd(Fig. 11).
NL_9.1.0.0 (6) showedno significant effects on cellviability compared to DMSO (Fig. 11).
5-FUrd (NS_4.0.0.0)
NS_9.0.0.0
160
NL_9.1.0.0
NL_9.1.(1)3(6)3.0
140
120
100
***
***
80
60
40
20
0
***
***
***
***
***
***
***
###
**
1.56
3.12
6.25
12.5
25
50
µM
Figure 11.Viability (in %) of human primary glioblastoma cells (GBM 2014/42) after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin B (NS_9.0.0.0, 5) or its derivatives (NL_9.1.0.0, 6, and
NL_9.1.1363.0, 7) is shown. Values are given (in % viability of control [incubation with medium alone;
= 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs. DMSO control
(equal concentration of DMSO to 50 µM substance solution), n=5 independent experiments assayed
in quadruplicates.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
18
Moreover, the higher concentrations (12.5, 25 and 50 µM) of the most effective
formycin A derivative NL_8.1.13.0 were auxiliary tested on BT4Ca, GOS-3 and U-87 MG
by using a second viability/cytotoxicity assay (sulforhodamin B assay), indeed confirming
the cytotoxic effects of Formycin A derivative NL_8.1.13.0 (data not shown).
In addition to the significant cytotoxic effects of Formycin A derivative NL_8.1.13.0;
we found that this substance is also cytotoxic (at various concentrations; after incubation
for 48h) for other tumor cell lines, i.e. human colorectal carcinoma (HT29), human
hepatocellular carcinoma (HEPG2), human pancreas carcinoma cells (Panc-1) and murine
renal carcinoma (RenCa) cells (data not shown).
As previously published concerning other anti-tumor derivatives (Knies et al.,
2015a, 2015b, 2016; Hammerbacher et al. 2018), the novel derivatives were tested on
PMA-differentiated human THP-1 macrophages to estimate potential (cytotoxic) side
effects on immune cells.Incubation (48h) of PMA-differentiated human THP-1
macrophageswith NS_4.0.0.0 (5-FUrd) revealed a marginal inhibition of cell
viability(Figs.12, 13). At all concentrations tested, 5-FUrd significantly reduced the
viabilityof PMA-differentiated human THP-1 macrophages by -0.8 % (p<0.05), -2.7 %
(p<0.01), -5.2 % (p<0.01), -5.0 % (p<0.01), -6.1 % (p<0.001) and -6.8 %(p<0.01) in
comparison to DMSO (~100% viability) (Figs. 13, 14).Unlike, Formycin A (NS_8.0.0.0, 1)
significantly diminished cell viability of THP-1 macrophagesat alltested concentrations by -
11.5 % (1.56 µM, p<0.001), -19.4 % (3.12 µM, p<0.01), -28.1 % (6.25 µM, p<0.01), -43.0
% (12.5 µM, p<0.01), -50.5 % (25 µM, p<0.001) and -64.4 % (p<0.001)compared to
DMSO (Fig. 12).
Derivative NL_8.1.13.0(3a) decreased the viability of PMA-differentiated human
THP-1 macrophages by -26.0 % (6.25 µM, p<0.01), -61.5 % (12.5 µM, p<0.01), -97.0 %
(25 µM, p<0.001) and -100 % (50 µM, p<0.001) compared to the DMSO control (Fig.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
19
7).Moreover, NL_8.1.13.0(3a) significantlyinhibited the viability of PMA-differentiated
human THP-1 macrophagesby -41.4 % (12.5 µM, p<0.001), -70.6 % (25 µM, p<0.001) and
-71.6 % (50 µM, p<0.01) in comparison to 5-FUrd (Fig. 12)
At concentrations of 6.25, 12.5, 25 and 50 µM, NL_8.1.13.0(3a) led to a significant
reduction of viability in human THP-1 macrophagesby -5.5 % (p<0.01), -57.9 % (p<0.01), -
89.2 % (p<0.001) and -99.6 % (p<0.001) compared to the DMSO control (Fig. 12).
Additionally, NL_8.1.13.0(3a) significantly reduced the viability of PMA-differentiated
human THP-1 macrophagesby -52.9 (12.5 µM, p<0.01), -83.1 % (25 µM, p<0.001) and -
92.8 % (50 µM, p<0.01) compared to 5-FUrd (Fig. 12).
In comparison with derivative NL_8.1.13.0 (3a), at all tested concentrations,
NL_8.1.13.0P (4a) showed a proliferative effect, i.e.significantly increased the viability of
human THP-1 macrophages by +13.2 % (1.56 µM; p<0.05), +14.0 % (3.12 µM; p<0.01),
+23.5 % (6.25 µM; p<0.05), +26.9 % (12.5µM; p<0.05), +43.3 % (25µM; p<0.01) and +
47.4 % (50µM; p<0.001) compared to DMSO (Fig. 12).The compoundsNL_8.1.0.0 (2), and
NL_8.1.11.0P(4b) revealedno significant effects on the viability of PMA-differentiated
human THP-1 macrophages in comparison to DMSO (Fig. 12).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
20
NS_4.0.0.0 (5-FUrd)
NS_8.0.0.0
160
140
NL_8.1.0.0
**
***
**
NL_8.1.(1)3.0
NL_8.1.(1)3.0 P
NL_8.1.(1)1.0 P
*
*
120
*
**
**
*
100
**
**
***
###
**
***
80
**
##
**
##
60
40
20
0
##
**
###
***
**
##
###
***
###
***
##
***
1.56
3.12
6.25
12.5
25
50
µM
Figure 12. Viability (in %) of PMA-differentiated human THP-1 macrophages after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin A (NS_8.0.0.0, 1) or its derivatives (NL_8.1.0.0,2,
NL_8.1.13.0,3a, NL_8.1.13.0P, 4a, and NL_8.1.11.0P, 4b) is shown. Values are given (in % viability
of control [incubation with medium alone; = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01,
***p<0.001, significance vs. DMSO control (equal concentration of DMSO to 50 µM substance
#
solution), p<0.05, ##p<0.01, ###p<0.001, significance vs. 5-FUrd at equal concentration; n=5
independent experiments assayed in quadruplicates.
Only at a concentration of 50 µM,Formycin B (NL_9.0.0.0, 5) significantly (p<0.05)
diminished the viability human THP-1 macrophages by -19.8 % compared to the DMSO
control (Fig. 13). NL_9.1.0.0 (6) and NL_9.1.1363.0(7)revealed no significant effects on the
viability of PMA-differentiated human THP-1 macrophages compared to DMSO(Fig. 13).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
21
NS_4.0.0.0 (5-FUrd)
NS_9.0.0.0
160
140
120
NL_9.1.0.0
NL_9.1.(1)3(6)3.0
100
80
60
40
20
0
*
**
**
*
**
**
***
1.56
3.12
6.25
12.5
25
50
µM
Figure 13. Viability (in %) of PMA-differentiated human THP-1 macrophages after incubation (48h)
with NS_4.0.0.0 (5-FUrd), Formycin B (NS_9.0.0.0, 5) or its derivatives (NL_9.1.0.0, 6, and
NL_9.1.1363.0, 7) is shown. Values are given (in % viability of control [incubation with medium
alone; = 100 % viability]) as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, significance vs. DMSO
control (equal concentration of DMSO to 50 µM substance solution); n=5 independent experiments
assayed in quadruplicates.
Because Formycin Aderivative NL_8.1.13.0 inhibited the viability of glioblastoma cells, we
have exemplarily analyzed a possible molecular mechanism to explain the cytotoxic effects,
and we found that Formycin Aderivative NL_8.1.13.0 significantly increased the active
caspase-3 activity in BT4Ca cells, which is interpreted as apoptosis induction (data not
shown).
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
22
5. Experimental Part.
5.1. Nomenclature. For the general numbering of nucleosides and nucleolipids, a
novel denomination system was developed, which allows an easy comparison of compound
data among the various publications of our groups and which is disclosed in ref. [14].
Moreover, it was deposited sustainably in the repository of the library of the University of
Osnabrück under the following unique registration number (URN) and URL:
https://repositorium.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2015110413639.
Nucleolipids are abbreviated as“NL”, nucleosides are abbreviated as“NS”. The first number
refers to the nucleosides. The second number refers to the moiety at the 2’,3’-position at
the glyconic ring; cyclic moieties are abbreviated by “cycl” before the number. The third
number refers to the lipophilic moiety at the base [N(3) for pyrimidines, N(1) for purines].
The forth number refers to a lipophilic moiety at the 5’-O position. Identical residues carry
the same number; “0” stands for a molecule without a residue at this position. For a
translation of the NS/NL nomenclature to the plain compound numbers used throughout the
text, schemes and figures see Table 2.
Table 2. Translation of plain compound numbers (Schemes 1 and 2) into the NS/NL
nomenclature.
1
2
NS_8.0.0.0
NL_8.1.0.0
3a
3b
4a
4b
5
NL_8.1.11.0
NL_8.1.13.0
NL_8.1.11.0P
NL_8.1.13.0P
NS_9.0.0.0
6
NL_9.1.0.0
7
NL_9.1.13.63.0
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
23
5.2. Chemistry.
5.2.1.General Remarks. All chemicals were purchased from Sigma-Aldrich (DE-
Deisenhofen) or TCI Europe (BE-Zwijnrecht). Formycin A was a generous gift of Mr. Klaus
Mühlegger, Roche Diagnostics DE-Penzberg). Solvents were laboratory grade and were
distilled before use. Column chromatography was performed on silica gel 60 (CAS 112926-
00-8). Thin layer chromatography (TLC) was performed using aluminum sheets and silica
gel 60 F254; 0.2 mm layer (Merck, Germany). NMR spectra (1H, 13C, DEPT-135, gated-
decoupled 13C) were obtained using an AMX-500 instrument (Bruker, DE-Rheinstetten,
1
Germany): H: 500.14 MHz, 13C: 125.76 MHz; chemical shifts (δ) are reported in ppm
referenced to an internal standard of residual proteosolvent [D6]DMSO (2.50. 39.50 ppm,
relative to tetramethylsilane (TMS) as internal standard). Multiplicity is quoted as br (broad),
s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), Ψt (pseudotriplet),
dd (doublet of doublet), ddd (doublet of doublet of doublets). J values are reported in Hz.
2D [1H,1H] and [1H,13C] correlation spectra (heteronuclear single quantum coherence,
HSQC) and Cosy Long Range spectra (pulse program: cosygpmfph) were measured with
the same instrument. Sample preparation was performed as follows: an appropriate amount
of compound (usually 20 – 25 mg) was dissolved in [D6]DMSO (0.5 ml) and placed in the
NMR quartz tube (diameter, 5 mm). Before measurement the solutions were degassed by
1
ultrasonication for several minutes. Number of scans: H: 64, 13C: 12.000, DEPT-135:
5.000. ESI MS was performed by a Bruker Daltronics Esquire HCT instrument (Bruker
daltronics, DE-Leipzig); ionization was performed with a 2% formic acid (HCOOH) solution
in MeCN. UV/Vis spectra were obtained using a Cary 1E spectrophotometer (Varian, DE-
Darmstadt). Compound samples of about 1mg were dissolved either in MeOH, or an
appropriate buffer solution (pH 3, 7 or 9, 100 ml, each). Aliquots of the fully dissolved
compounds (warming, ultrasonication) were subjected to UV/Vis spectrometry in MT4
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
24
quartz cuvettes (Hellma, DE-Darmstadt). Elemental analyses (C, H, N) were performed on
a VarioMICRO instrument (Fa. Elementar, DE-Hanau). 10logPOW values were determined in
silico using the http://eadmet.com/de/physprop.phpwebsite with ePhysChem that contains
ALOGPS v.3.0. [15,16]. Experimental determination of 10logPChW values of compounds
were performed as follows: samples of compounds (2 mg, each) were dissolved in a
heterogenic mixture of cyclohexane (25 ml) and water (25 ml) by ultrasonication (10 min)
under slightly warming. After separation of the layers from each phase aliquots of 1 ml were
withdrawn, and their UV spectra were run in 1cm quartz cuvettes. From the ratio of maximal
extinctions of both layers at λmax the corresponding 10logPChW values were calculated.
5.3. Syntheses.
Formycin A (1,NS_8.0.0.0).1H-NMR (500.13 MHz, DMSO-d6):12.68 (s, br. NH); 8.15
(s, H-C(5)); 7.36 (s, br., NH2); 5.85 (s, H-C(1‘)); 4.94-4.93 (m, HO-C(5’), HO-C(2’)); 4.87 (d,
1H, 3J(HO-C(3’),H-C(3’)) = 3.5, HO-C(3’)); 4.52-4.49 (m, 1H, H-C(2’)); 4.11-4.09 (m, 1H, H-
3
3
C(3’)); 3.93 (ψq, 1H, J(H-C(4’),H2-C(5’)) = 3.0, J(H-C(4’), H-C(3’)) = 3.0, H-C(4’)); 3.67-
3.64 (m, 2H, H2-C(5’)).13C-NMR (125.76 MHz, DMSO-d6):151.26 (C(5)); 150.73 (C(7));
144.04 (C(3)); 138.81 (C(3a)); 122.15 (C(7a)); 85.93 (C(4‘)); 78.40 (C(1‘)); 75.00 (C(2‘));
72.36 (C(3‘)); 62.54 (C(5‘)).
3-((2S,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (5, NS_9.0.0.0; Formycin B) by enzymatic
deamination: 0.8 g (3.75 mmol) of Formycin A (1, NS_8.0.0.0), suspended in ultrapure
H2O(27 ml), were charged dropwise with a solution of adenosine desaminase (0.75 ml, 90
units, calf intestine). The reaction mixture was stirred for 5 d at ambient temperature (TLC-
control). The milky-white suspension formed was evaporated to dryness yielding a
colorless powder of formycin B (0.75 g, 3.75 mmol, 100 %). TLC (SiO260,
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
25
CH2Cl2/MeOH,75:25; v/v)): Rf, 0.48. UV (MeOH):λmax 278.3 nm (ε = 7.200 mol-1 cm-1). logP
(ALOGPS 3.01): -1.40 ± 0.74.1H-NMR (500.13 MHz, DMSO-d6): 7.87 (H-C(5)); 4.92 (d,
3
3
3
1H, J(H-C(1’),H-C(2’)) = 7.0, H-C(1’)); 4.43 (dd, 1H, J(H-C(2’),H-C(1’)) = 6.5, J(H-
C(2’),H-C(3’)) = 5.5, H-C(2’)); 4.06 (dd, 1H, 3J(H-C(3’),H-C(2’)) = 5.0, 3J(H-C(3’),H-C(4’)) =
3
3
4.0, H-C(3’)); 3.88 (ψq, 1H, J(H-C(4‘),H-C(3‘)) = 3.5, J(H-C(4‘),H2-C(5‘)) = 3.5, H-C(4‘));
13
3.63-3.46 (m, partially superimposed by HOD, H2-C(5‘)). C-NMR (125.76 MHz, DMSO-
d6): 153.86 (C(7)); 143.06 (C(5)); 142.87 (C(3)); 136.43 (C(3a)); 129.52 (C(7a)); 85.55
(C(4‘)); 77.35 (C(1‘)); 74.76 (C(2‘)); 71.99 (C(3‘)); 62.46 (C(5‘)). ESI-MS: 269.04 [M+H]+,
537.18 [2M+H]+; calculated: 268.23.
Ethyl3-[4-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-6-hydroxymethyl-2-methyl-
tetrahydro-furo[3,4-d][1,3] dioxol-2-yl]-propionate(2, NL_8.1.0.0). Anhydrous formycin A(1,
NS_8.0.0.0; 0.75 g; 2.805 mmol) was dissolved in dry dimethylformamide (12 ml).
Subsequently, ethyl levulinate (0.75 ml; 5.332 mmol), triethyl orthoformate (0.69 ml; 4.211
mmol) and 4 M HCl in 1.4-dioxane (2.55 ml) were added. After stirring for 24 h at room
temperature the reaction mixture was partitioned betweenCH2Cl2 (120 ml) and a saturated
aqueous NHCO3 solution (35 ml). The aqueous layer was back-extracted thrice (30, 20
and 15 ml CH2Cl2). The combined organic layers were evaporated on a rotary evaporator
and then co-evaporated repeatedly from CH2Cl2 to remove residual DMF. The residue was
dried overnight in high vacuo at 40 °C. Purification of the title compound was performed by
silica gel chromatography(SiO260, column: 6.5 x 11 cm; CH2Cl2/MeOH 85:15; v/v). After
pooling of appropriate fractions and evaporation of the solvent the product was isolated as
a colorless foam (0.705 g, 1.792 mmol, 64 %; diastereoisomeric mixture, [1R]/[1S]= 8.6:1).
TLC (SiO260,CH2Cl2/MeOH 85:15; v/v):Rf, 0.71. UV (MeOH):λmax 293 nm (ε = 11.150 mol-1
1
cm-1), λmax 304.5 nm (ε= 7.400 mol-1 cm-1). logPOW(ALOGPS 3.01): 0.18 ± 0.74. H-NMR
(500.13 MHz, DMSO-d6),12.85 (s, br., 1H, NH); 8.19 (s, H-C(5)); 7.36 (s, br., 2H, NH2);
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
26
5.40 (s, br., 1H, HO-C(5’)); 5.28 (s, br., H-C(1‘)); 5.16-5.16 (m, 1H, H-C(2’), (1R)+(1S));
3
3
4.88 (dd, 1H, J(H-C(3‘),H-C(4‘)) = 3.0, J(H-C(3’),H-C(2’)) = 7.0, H-C(3‘)(1R)); 4.85 (dd,
3
3
3
1H, J(H-C(3‘),H-C(4‘)) = 3.0, J(H-C(3’),H-C(2’)) = 7.0, H-C(3‘)(1S)); 4.14 (ψq, 1H, J(H-
3
3
C(4’),H-C(3’)) = 4.0, J(H-C(4’), H2-C(5’)) = 4.0, H-C(4’)(1S)); 4.10 (ψq, 1H, J(H-C(4’),H-
C(3’))
=
4.0, 3J(H-C(4’), H2-C(5’))
=
4.0, H-C(4’)(1R)); 4.07 (q, 2H,
3
3J(CH2(ester),CH3(ester)) = 7.0, CH2(ester)(1R)); 4.01 (q, 2H, J(CH2(ester),CH3(ester)) =
7.0, CH2(ester)(1S)); 3.54-3.48 (m, 2H, H2-C(5‘) (1R)+(1S)); 2.79 (t, 2H,
3
3J(CH2(C=O),CH2(acetal)) = 7.3, CH2(C=O)(1R)); 2.30 (t, 2H, J(CH2(C=O),CH2(acetal)) =
7.3, CH2(C=O)(1S)); 2.69 (t, 2H, 3J(CH2(acetal),CH2(C=O)) = 7.3, CH2(acetal)(1R)); 1.81(t,
2H, 3J(CH2(acetal),CH2(C=O)) = 7.3, CH2(acetal)(1S)); 1.43 (s, 3H, CH3(acetal)(1S)); 1.23
3
(s, 3H, CH3(acetal)(1R)); 1.10 (t, 3H, J(CH3(ester),CH2(ester)) = 7.0, CH3(ester)(1R));
3
1.13 (t, 3H, J(CH3(ester),CH2(ester)) = 7.0, CH3(ester)(1S)).13C-NMR (125.76 MHz,
DMSO-d6),172.64 (C=O(ester)(1R)); 172.53 (C=O(ester)(1S)); 151.89 (C(5)); 150.75
(C(7)); 142.65 (C(3)); 139.15 (C(3a)); 122.17 (C(7a)); 114.12 (C(acetal)(1S)); 113.99
(C(acetal)(1R)); 85.36 (C(4’)(1S)); 85.02 (C(4’)(1R)); 84.06 (C(1’)(1S)); 83.30 (C(1’)(1R));
83.07 (C(3’)(1S)); 82.39 (C(3’)(1R)); 80.30 (C(2’)(1S)); 79.69 (C(2’)(1R)); 61.00 (C(5’)(1S));
61.87 (C(5’)(1R)); 59.87 (CH2(ester)(1R)); 59.82 (CH2(ester)(1S)); 33.74 (CH2(C=O)(1R));
33.57 (CH2(C=O)(1S)); 29.11 (CH2(acetal)(1S)); 28.35 (CH2(acetal)(1R)); 25.32
(CH3(acetal)(1S));
23.70
(CH3(acetal)(1R));
14.03
(CH3(ester)(1R));
13.96
(CH3(ester)(1S)).ESI-MS: 394.16 [M+H]+, 787.10 [2M+H]+; calculated:393.39. Anal. Calc.
for C17H23N5O6 (393.39): C, 51.90; H, 5.89; N, 17.80. Found: C, 51.87; H, 5.79; N,18.01.
Ethyl 3-((2S,3aS,4S,6R,6aR)-4-(7-amino-1-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-
trien-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-6-(hydroxymethyl)-2-methyltetra hydrofuro[3,4-
d][1,3]dioxol-2-yl)propanoate (3b, NL_8.1.13.0) and Ethyl 3-((2S,3aR,4R,6S,6aS)-4-
(hydroxymethyl)-2-methyl -6-(9-methyl-1-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
27
yl)-1,7-dihydropyrazolo[3,4-e]
pyrimido[1,2-c]
pyrimidin-3-yl)tetrahydrofuro[3,4-d]
[1,3]dioxol-2-yl) propanoate(4b, NL_8.1.13.0P).Anhydrous NL_8.1.0.0 (2, 0.20 g; 0.534
mmol) was dissolved in dry and amine-free dimethylformamide (6 ml). After addition of dry
K2CO3 (0.193 g; 1.4 mmol) and stirring of the suspension for 30 min at room temperature,
all-transfarnesyl bromide (0.18 ml; 0.6 mmol) was added dropwise under N2 atmosphere.
Stirring was continued for 24 h. The salt was filtered off and washed three times. The
filtrate and washings were evaporated to give a yellowish oil which was again co-
evaporated from CH2Cl2 to remove residual DMF (water bath below 35 °C). The raw
material was then further dried in high vacuo overnight. Separation ofthe products was
performed by silica gel chromatography (SiO260, column: 5.5 x 17 cm; CH2Cl2/MeOH 95:5;
v/v; 0.3 bar). The contents of two zones was isolated, and the solvent was evaporated.
The faster migrating compound formed a colorless foam in high vacuo(3b, NL_8.1.13.0),
the slower migrating compound formed a yellowish oil (4b, NL_8.1.13.0P).
NL_8.1.13.0(3b): 0.120 g, 0.2 mmol, 37.4 %; diastereoisomeric mixture.
TLC(SiO260, CH2Cl2/MeOH 95:5; v/v):Rf, 0.48. UV (MeOH):λmax 305.8 nm (ε = 10.500 mol-
1 cm-1). logPOW(ALOGPS 3.01): 3.61 ± 0.74. 1H-NMR (500.13 MHz, DMSO-d6), 8.16 (s, H-
C(5)(1R+1S)); 7.29 (s, br, 2H, NH2); 5.29-5.25 (m, 3H, H-C(10’’), H-C(6’’), HO-(5’),
(1R+1S)); 5.18-5.17 (m, 2H, H-C(2’), H-C(2’’), (1R+1S)); 5.08 (d, 3J(H-C(1’),H-C(2’)) = 4.5,
3
3
H-C(1’)(1R+1S)); 5.01 (t, J(H2-C(1’’),H-C(2’’)) = 6.5, H2-C(1’’)); 4.86 (dd, J(H-C(3’),H-
C(2’)) = 7.0, 3J(H-C(3’),H-C(4’)) = 4.0, H-C(3’)(1R+1S)); 4.09-4.05 (m, 3H,
CH2(ester)(1R+1S), H-C(4’)(1R)); 4.02 (ψq, 3J(CH2(ester),CH3(ester))
=
5.0,
3
(CH2(ester)(1S)); 3.54-3.42 (m, 2H, H2-C(5’)); 2.46 (t, J(CH2C=O,CH2(acetal)) = 7.5,
CH2C=O(1R+1S)); 2.07-1.83 (m, 10H, H2-C(4’’), H2-C(5’’); H2-C(8’’); H2-C(9’’); CH2(acetal),
(1R+1S)); 1.77 (s, 3H, H3-C(13‘‘)(1R+1S)); 1.61 (s, 3H, H3-C(14‘‘)(1R+1S)); 1.52 (s, 3H,
H3-C(12‘‘)(1R+1S)); 1.49 (s, 3H, H3-C(15‘‘)(1R+1S)); 1.29 (s, 3H, CH3(acetal)(1R)); 1.20 (t,
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
28
3J(CH3(ester)(1R),CH2(ester)(1R))
=
7.0,
CH3(ester)(1R));
1.14
(t,
3J(CH3(ester)(1S),CH2(ester)(1S)) = 7.0, CH3(ester)(1S)).13C-NMR (125.76 MHz, DMSO-
d6), 172.50 (C=O(ester)(1R)); 172.39 (C=O(ester)(1S)); 151.41 (C(5)(1R+1S)); 150.83
(C(7)(1R+1S)); 141.19 (C(3)(1S)); 141.08 (C(3)(1R)); 140.89 (C(3a)(1R+1S)); 139.35
(C(3’’)(1R+1S)); 134.88 (C(7’’)(1R+1S)); 130.44 (C(11’’)(1R+1S)); 124.00 (C(6’’)(1R+1S));
123.28 (C(10’’)(1R+1S)); 121.30 (C(7a)(1R+1S)); 119.62 (C(2’’)(1R+1S)); 114.00
(C(acetal)(1S)); 113.93 (C(acetal)(1R)); 85.24 (C(1’)(1S)); 84.89 (C(1’)(1R)); 83.74
(C(4’)(1S)); 82.99 (C(4’)(1R)); 82.30 (C(2’)(1R)); 79.97 (C(3’)(1S)); 79.38 (C(3’)(1R));
61.90 (C(5’)(1S)); 61.66 (C(5’)(1R)); 59.75 (CH2(ester)(1S)); 59.69 (CH2(ester)(1R)); 38.99
(C(1’’)(1R+1S)), 38.69 (C(4’’)(1R+1S)); 38.38 (C(8’’)(1R+1S)); 33.61 (CH2C=O(1R)); 33.77
(CH2C=O(1S)); 29.00 (CH2(acetal)(1S)); 28.22 (CH2(acetal)(1R)); 26.04 (C(5’’)(1R+1S));
25.56 (C(9’’)(1R+1S)); 25.32 (C(12’’)(1R+1S)); 25.11 (Me(acetal)(1S)); 23.61
(Me(acetal)(1R)); 17.38 (C(15’’)(1R+1S)); 16.29 (C(14’’)(1R+1S)); 15.62 (C(13’’)(1R+1S));
13.95 (Me(ester)(1R)); 13.96 (Me(ester)(1S)). ESI-MS: 598.38 [M+H]+; calculated: 597.75.
Anal calc. forC32H47N5O6 (597.75) * 0.5 DMF: C, 63.00; H, 8.02; N, 12.15. Found: C,
63.25; H, 7.84; N, 12.33.
NL_8.1.13.0P(4b):0.0485 g, 0.0747 mmol, 14 %; diasteroisomeric mixture. TLC
(SiO260, CH2Cl2/MeOH 95:5; v/v):Rf, 0.54. UV (MeOH):λmax 343.0 nm (ε = 8.100 mol-1cm-
1
1). logPOW(ALOGPS 3.01): 3.05 ± 0.74. H-NMR (500.13 MHz, DMSO-d6), 8.98 (s, H-
3
C(10)); 8.40 (s, H-C(5)); 5.44-5.37 (m, 4H, H2-C(1‘‘), H2-C(11)); 5.31 (dd, J(H-C(2’),H-
3
3
C(1’)) = 4.5, J(H-C(2’),H-C(3’)) = 6.5, H-C(2’)); 5.19 (t, J(HO-C(5’),H2-C(5’)) = 6.0, HO-
3
C(5’)); 5.13 (d, J(H-C(1‘),H-C(2‘)) = 4.5, H-C(1‘)); 5.01-5.00 (m, 2H, H-C(10‘‘), H-C(6‘‘));
4.87 (dd, 3J(H-C(3‘),H-C(2‘)) = 7.0, 3J(H-C(3‘),H-C(4‘)) = 3.0, H-C(3‘)); 4.10-4.05 (m, 3H, H-
3
C(4‘), CH2(ester)(1R)); 4.02 (q, J(CH2(ester),Me(ester)) = 7.5; CH2(ester)(1S)); 3.54-
2
3
3
3.43(m, J(H‘-CH2(5‘),H‘‘-CH2(5‘)) = -12.0, J(HO-(5‘),CH2-C(5‘)) = 5.0, HO-C(5‘)); J(H-
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201900012
Chemistry & Biodiversity
29
3
C(4‘),H2-C(5‘)) = 5.0, H2-C(5‘)); 3.25 (s, Me(12)); 2.47 (t, J(CH2(C=O),CH2(acetal)) = 7.5,
CH2(C=O)); 2.07-1.83 (m, 10H, CH2(4‘‘), CH2(5‘‘), CH2(8‘‘), CH2(9‘‘), CH2(acetal)); 1.78 (s,
3H, Me(13‘‘)(1R)); 1.77 (s, 3H, Me(13‘‘)(1S)); 1.62 (s, 3H, Me(14‘‘)(1S)); 1.60 (s, 3H,
Me(14‘‘)(1R)); 1.51 (s, 3H, Me(12‘‘)); 1.50 (s, 3H, Me(15‘‘)); 1.30 (s, 3H, Me(acetal)(1R));
3
3
1.20 (t, J(Me(ester),CH2(ester)) = 7.5, Me(ester)(1R)); 1.14 (t, J(Me(ester),CH2(ester)) =
7.5, Me(ester)(1S)).13C-NMR (125.76 MHz, DMSO-d6), 172.53 (C=O(ester)); 157.18
(C(10)); 154.51 (C(9)); 151.06 (C(5)); 141.25 (C(3)); 140.86 (C(3a)); 138.31 (C(3‘‘));
134.52 (C(7‘‘)); 130.46 (C(11‘‘)); 125.82 (C(2’’)); 123.98 (C(6‘‘)); 123.41 (C(10’’)); 120.43
(C(7a)); 114.05 (C(acetal)); 84.93 (C(1’)); 83.00 (C(4’)); 82.28 (C(2’)); 79.34 (C(3’)); 61.72
(C(5’)); 59.76 (CH2(ester)); 48.88 (C(11)); 40.85 (C(12)); 39.04 (C(1’’)); 39.00 (C(4’’));
38.64 (C(8’’)); 33.59 (CH2C=O); 28.23 (CH2(acetal)); 26.06 (C(5’’)); 25.57 (C(9’’)); 25.32
(C(12’’)); 23.65 (Me(acetal)); 17.37 (C(15’’)); 16.30 (C(14’’)); 15.64 (C(13’’)); 13.97
(Me(ester)). ESI-MS: 653.50 [M+H+H2]+;calculated:649.28. Anal. calc. forC36H51N5O6
(649.28) * 2 MeOH: C, 63.93; H, 8.33; N, 10.79. Found: C, 63.92; H, 8.06; N, 10.89.
3-{4-[7-Amino-1-(3-methyl-but-2-enyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl]-6-
hydroxymethyl-2-methyl-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}-propionic acid ethyl ester
(3a, NL_8.1.11.0) andEthyl-3-{4-Hydroxymethyl-2-methyl-6-[8-methyl-1-(3-methyl-but-2-
enyl)-1,6-dihydro-1,2,4,5a,9-pentaaza-cyclopenta[a]naphthalen-3-yl]-tetrahydro-furo[3,4-
d][1,3] dioxol-2-yl}-propionate (4a, NL_8.1.11.0P). Anhydrous NL_8.1.0.0 (2, 0.2 g; 0.534
mmol) was dissolved in dry and amine-free dimethylformamide (DMF, 6 ml). To this
solution dry K2CO3 (0.24 g; 1.7 mmol) was added, and the suspension was stirred for 30
min at ambient temperature. Then, isopentenyl bromide (0.2 mL; 1.5 mmol) was added
dropwise under N2 atmosphere, and the resulting mixture was stirred for 24 h at room
temperature. The salt was filtered off and washed three times with CH2Cl2. The filtrate and
washings were combined and evaporated on a rotary evaporator. The resulting yellowish
This article is protected by copyright. All rights reserved.