Design and synthesis of 15-deoxyspergualin-biotin conjugates as novel binding probes for target protein screening

Full text of DOI:10.1038/ja.2016.32

The Journal of Antibiotics (2016), 1–5
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&
NOTE
Design and synthesis of 15-deoxyspergualin–biotin
conjugates as novel binding probes for target protein
screening
Masahiko Morioka1,2, Kuniki Kato1 and Kazuo Umezawa3
The Journal of Antibiotics advance online publication, 23 March 2016; doi:10.1038/ja.2016.32
Spergualin 1 (Figure 1) was isolated in 1981 from the culture filtrate of
In the present research, we designed and synthesized 15-deoxy-
Bacillus laterosporus as a novel anticancer antibiotic, and it inhibited spergualin–biotin conjugates, BDSG-S and BDSG-L, having shorter
the growth of chick embryo fibroblasts that had been transformed by and longer spacers in which the non-essential hydroxyl group was
Rous sarcoma virus.1,2 It was totally synthesized in 1985.3 The replaced by an aminoalkyl group.
An efficient synthesis of ( )-DSG and an enantioselective synthesis
of ( − )-15-DSG have been reported.13,14 Presently, DSG is supplied by
an industrial manufacturing method as the pharmaceutical product.15
From the study of biological activity of DSG and its structure− activity
relationship,12 we deduced that a compound having the N-biotinoyl-
4-aminoalkyl group instead of the hydroxyl group at position 11 of
DSG would neither lose its immunosuppressive activity nor compro-
mise its binding with the target protein. Based on this idea, we carried
out a synthesis of BDSG 4 and 5 (Figure 2).
BDSG 4 was synthesized from known N,N′-(bis-Cbz-guanidino)
heptanoic acid 6 prepared from N,N′-bis(Cbz)-S-methylisothiourea16
and 7-aminoheptanoic acid. First, coupling of compound 6 with
commercially available Nε-Boc-L-lysine allyl ester 712 using the usual
antitumor effect of spergualin was considered to involve potentiation
of cytotoxic T-lymphocytes.4 Spergualin showed not only anticancer
activity against mouse leukemia tumors but also potent immunosup-
pressive activity in mice.5 In the course of a structure–activity
relationship study, 15-deoxyspergualin (DSG; Figure 1) 2 was dis-
covered in 1982,6 and it showed ~ 10 times more potent than
spergualin in the therapeutic activity. DSG showed six times prolonga-
tion of survival in L1210-bearing mice with 0.20 mg kg− 1, whereas
spergualin showed four times prolongation with 3.1 mg kg− 1.6 DSG
showed immunosuppressive activity in mice7 and dogs.8 DSG was first
evaluated as an anticancer agent and its clinical trial was carried out.
Later, it was also found to be a potent immunosuppressive agent in
clinical studies.
Studies on the target protein of DSG showed that heat-shock WSC/HOAt (water-soluble carbodiimide/1-hydroxy-7-azabenzotria-
protein 70 (HSP70) would interact with DSG.9 Furthermore, DSG was zole) methodology provided the desired 8, which was subjected to
also found to bind to HSP90. Both HSP70 and HSP90 possess the cleavage of the allyl group using Pd(PPh3)4 and morpholine17 to
same EEVD motif, and this motif was considered to be the binding provide the corresponding carboxylic acid 9 in quantitative yield, as
site.10 However, although DSG binds to the EEVD motif of HSP70, shown in Scheme 1.
Next, N1, N5-bis-Cbz-spermidine 14 was synthesized as shown in
Scheme 2. Condensation of spermidine 10 with formaldehyde in water
followed by protection of the primary amine of the intermediate
compound with 2-(Boc-oxyimino)-2-phenylacetonitrile (BocON)
yielded tetrahydropyrimidine 11 protected with tert-butoxycarbonyl
group (Boc) group at primary amino group in 28% yield in two steps.
Subsequently, the resultant compound 11 was reacted with benzyl
DSG did not affect the chaperone function of HSP70.11 Nadler et al.9
prepared an affinity resin by coupling the terminal amino group of the
spermidine moiety of 11-methoxy-15-deoxyspergualin (Figure 1) 3, a
stable derivative of DSG, to a sepharose resin. Using this affinity
column, HSP70 was selectively trapped from the lysate of human
T-cell leukemia Jurkat cells. However, modification of the primary
amino group of 3 resulted in a loss of its immunosuppressive effect.12
Therefore, although DSG is known to bind to HSP70 and HSP90, hydrogen malonate and pyridine to give N10-Boc-protected spermi-
these target proteins cannot explain the mechanism of the immuno- dine 12. Then, N1 primary amino group and N5 secondary amino
suppressive effect of DSG. Thus, the essential target molecule remains group of 12 were protected with benzyloxylcarbonyl chloride to give
unclear.
N1, N5, N10 tri-functionalized spermidine 13 (25% yield in two steps).
1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan; 2R&D, Product and Service Development, CXS Corporation,
Yokohama, Japan and 3Department of Molecular Target Medicine, Aichi Medical University School of Medicine, Aichi, Japan
Correspondence: Professor K Umezawa, Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute, Aichi
480-1195, Japan.
E-mail: umezawa@aichi-med-u.ac.jp
Received 27 November 2015; revised 4 February 2016; accepted 12 February 2016

DSG as binding probes–biotin conjugates
M Morioka et al
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Next, the Boc group of N10 of 12 was removed under acidic conditions RAW264.7. The 40% inhibitory concentration (IC40) of DSG was
~3 μg ml− 1, whereas BDSG-S was ~ 30 μg ml− 1 to inhibit the growth
to give N1, N5-bis-Cbz-spermidine 14 in almost quantitative yield.
Synthesis of the target compound BDSG 4 was accomplished as of J774.1 cells. Then, we have used BDSG-S for the screening of
molecular target in J774 cells. Using BDSG-S, we have determined an
RNA-binding protein as a candidate target protein. Binding of BDSG-
S to PCBP-2 was confirmed by preparing the recombinant protein for
the in vitro binding and by the deletion mutant experiment. The result
of target screening will be published elsewhere.
shown in Scheme 3. The coupling reaction between carboxylic acid 9
and bis-Cbz-spermidine 14 was achieved smoothly to afford com-
pound 15 in 88% yield using the WSC/HOAt methodology. Then, the
Boc group of the amine at position 16 of 15 was removed under acidic
conditions to afford amine 16, which was subsequently reacted with
NHS-Biotin 17 in the presence of 4-dimethylaminopyridine (DMAP)
in pyridine to afford the biotinylated compound 18 in 77% yield in
two steps. Finally, complete deprotection of four Cbz groups of 18 was
accomplished using hydrogenolysis over palladium hydroxide under a
hydrogen atmosphere to give the target compound 4 (BDSG-S) in
58% yield and 5 (BDSG-L) in 57% yield (Scheme 3). The structures of
biotinylated agents for BDSG-S (17) and BDSG-L (19) as well as
BDSG-L (5) are shown in Figure 3.
We have prepared compound 4 (BDSG-S) and 5 (BDSG-L) as
biotin conjugates of DSG with linker. 15-DSG was reported to show
anticancer and immunosuppressive activities in vivo. However, its
cellular activity has been poorly understood so far. Therefore, it was
difficult for us to compare the biological activity of 15-DSG and
BDSGs. However, we found that 15-DSG efficiently inhibited the
growth of mouse macrophage-like leukemia cell lines J774.1 and
EXPERIMENTAL PROCEDURE
General synthetic procedures
1H-NMR (400 MHz) and 13C-NMR (100.5 MHz) spectra were obtained at an
ambient temperature using an AVANCE 400 Bruker spectrometer (Hamburg,
Germany) in dimethyl sulfoxide (DMSO)-d6, CDCl3 or D2O. Chemical shifts
are reported in δ (p.p.m.) referenced to internal tetramethylsilane (0.00 p.p.m.),
residual CHCl3 (7.26 p.p.m.) or DMSO (2.50 p.p.m.) for 1H NMR and
chloroform-d1 (77.16 p.p.m.) for 13C NMR, and the coupling constants (J)
are expressed in Hz. Mass spectra were measured on Thermo Scientic
(Waltham, MA, USA) LTQ ORBITRAP XL mass spectrometer. For column
chromatography, silica gel (MORITEX Purif-Pack SI 30 μm) or NH silica gel
(MORITEX Purif-Pack NH 60 μm) was used. Optical rotations were observed
with JASCO P-1020 polarimeter (Tokyo, Japan).
(S)-6-((Boc)amino)-2- [7-(N,N′-Bis(Cbz)guanidinoheptanamido)]
hexanoic acid (9)
To a solution of compound 6 (455 mg, 1 mmol) and Nε-Boc-L-lysine allyl ester
7 (387 mg, 1.2 mmol) in DMF (5 ml) were added the following, at room
temperature: WSC·HCl (210 mg, 1.1 mmol), HOAt (150 mg, 1.1 mmol) and
DIPEA (0.70 ml, 4.0 mmol). The resultant mixture was stirred at room
temperature for 21 h, the reaction mixture was added to 0.1N HCl aqueous
solution (10 ml) and then extracted with EtOAc three times. The combined
organic layer was washed with brine, dried over anhydrous MgSO4 and
concentrated under reduced pressure to give a viscous residue, which was used
in the next reaction without further purification. After reacting the residue with
Pd(PPh3)4 (116 mg, 0.10 mmol) and morpholine (870 μl, 10 mmol) in THF
(3 ml) at room temperature for 4 h, the reaction mixture was added to 0.1N HCl
aqueous solution (10 ml) and extracted with t-BuOMe three times. The combined
organic layer was washed with brine, dried over anhydrous MgSO4 and
concentrated under reduced pressure. The crude product was purified by silica
gel column chromatography (n-hexane/AcOEt =1:1) to provide 9 (720 mg, yield:
1
Figure 1 Structures of spergualin and its derivatives.
quant.) as an amorphous solid. H-NMR (DMSO-d6), δ 12.40 (br-s, 1H), 11.59
Figure 2 Molecular design of biotinylated 15-deoxyspergualins with short (BDSG-S) and long (BDSG-L) spacers.
Scheme 1 Reagents and conditions: (a) water-soluble carbodiimide (WSC), 1-hydroxy-7-azabenzotriazole (HOAt), N,N-diisopropylethylamine (DIPEA), N,N-
dimethylformamide (DMF), room temperature (rt), 21 h; (b) Pd(PPh3)4, morphorine, tetrahydrofuran (THF), rt, 2.5 h.
The Journal of Antibiotics

DSG as binding probes–biotin conjugates
M Morioka et al
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Scheme 2 Reagents and conditions: (a) HCHO, H2O, room temperature (rt), 72 h, and then 2-(Boc-oxyimino)-2-phenylacetonitrile (BocON), THF, 0 °C, 0.5 h;
(b) HOOCCH2COOBn, pyridine-EtOH, reflux, 2 h, and then CbzCl, pyridine, rt, 20 h; (c) 4N HCl-dioxane, rt, 1 h.
Scheme 3 Reagents and conditions: (a) water-soluble carbodiimide (WSC), 1-hydroxy-7-azabenzotriazole (HOAt), N,N-diisopropylethylamine (DIPEA), N,N-
dimethylformamide (DMF), room temperature (rt), 19 h, 88%; (b) 4N HCl-dioxane, rt, 1 h, 87%; (c) cat. 4-dimethylaminopyridine (DMAP), pyridine, 17, rt,
17 h, 33%: (d) H2, Pd(OH)2, MeOH, rt, 5 h, 58%.
(s, 1H), 8.39 (dd-like, 1H), 7.90 (d, 1H, J=5.8 Hz), 7.45–7.25 (complex, 10H), Benzyl (3-(Cbz)amino)propyl-(4-(Boc)aminobutyl)-carbamate (13)
5.21, 5.03 (each s, each 2H), 4.20–4.08 (m, 1H), 3.40–3.25 (complex, 2H),
To a solution of compound 11 (2.00 g, 7.77 mmol) in EtOH (100 ml)
2.95–2.80 (complex, 2H), 2.15–2.05 (complex, 2H), 1.72–1.40 (complex, 6H), were added pyridine (2.5 ml) and monobenzyl malonate (6.0 g, 31 mmol)
1.38 (s, 9H), 1.40–0.85 (complex, 8H); HRMS (CI) calcd. for C35H49N5O9 [M]+ under ice-cold conditions and the resulting mixture was stirred at 80 °C for 2 h.
= 684.3603. Found: 684.3612.; [α]24 +1.03° (c 0.96, MeOH).
The reaction mixture was concentrated under reduced pressure and the
residue was roughly purified by NH-silica gel column chromatography
(n-hexane/AcOEt= 1:1) to give a slurry (2.25 g), which was used in the next
D
reaction without further purification. To
a solution of the previously
tert-Butyl 4-(tetrahydropyrimidin-1(2H)-yl)butylcarbamate (11)
To a solution of spermidine 10 (25 g, 172 mmol) in water (200 ml) was added a
37% formaldehyde aqueous solution (14.2 ml, 189 mmol), and the resultant
mixture was stirred at room temperature for 3 days. The reaction mixture was
lyophilized to give the cyclic compound (23.2 g, yield: 86%), which was used in
the next reaction without further purification. 1H-NMR (CDCl3), δ 3.38 (br-s,
2H), 2.81 (t, 2H, J = 5.2 Hz), 2.71 (t, 2H, J = 6.9 Hz), 2.57 (br-s, 2H), 2.25 (t,
2H, J = 6.9 Hz), 1.7–1.4 (complex, 8H).
To a solution of previously obtained crude cyclic compound (5.79 g,
36.8 mmol) in THF (100 ml) was added BocON (7.3 g, 29.5 mmol) under
ice-cold conditions and the resultant mixture was allowed to stand for 30 min.
After addition of aqueous solution of 0.5 N sodium hydroxide (200 ml), the
reaction mixture was extracted with Et2O three times. The combined organic
obtained slurry in pyridine (20 ml) was added CbzCl (3.3 ml, 23.3 mmol)
under ice-cold conditions and the resultant mixture was allowed to stand at
room temperature for 17 h. The reaction mixture was concentrated under
reduced pressure and the residue was purified by NH-silica gel column
chromatography (n-hexane/AcOEt = 1:1) to give compound 13 (1.00 g, yield:
25% in two steps). 1H-NMR (DMSO-d6) δ 7.40–7.25 (complex, 10H), 7.25
(br-s, 1H), 6.80 (br-s, 1H), 5.06 (s, 2H), 5.01 (s, 2H), 3.25–3.10 (complex, 4H),
2.98 (q-like, 2H), 2.89 (br-s, 2H), 1.70–1.58 (complex, 2H), 1.5–1.25 (complex,
4H), 1.36 (s, 9H); HRMS (CI) calcd for C28H39N3O6 [M]+ = 514.2911. Found:
514.2914.
extracts were washed with brine, dried over anhydrous MgSO4, filtered and the Benzyl (4-aminobutyl)-(3-(Cbz)aminopropyl)-carbamate (14)
filtrate was concentrated under reduced pressure to give a slurry (8.06 g). The Compound 13 (300 mg, 0.584 mmol) was treated with 4N HCl-dioxane (1:3,
crude product was purified by NH-silica gel column chromatography 5.0 ml) at room temperature for 1 h and concentrated under reduced pressure
(n-hexane/AcOEt= 1:1) to give compound 11 (2.40 g, yield: 32%) as an to give diCbz-protected spermidine 14 (262 mg, yield: quant.), which was used
1
amorphous solid. 1H-NMR (CDCl3), δ 5.87 (br-s, 1H), 3.37 (br-s, 2H), 3.11 to the next reaction without further purification. H-NMR (DMSO-d6) δ 7.85
(t-like, 2H, J = 4.6 Hz), 2.82 (br-s, 2H), 2.57 (br-s, 2H), 2.23 (t, 2H, J =6.7 Hz), (br-s, 2H), 7.40–7.25 (complex, 10H), 5.06, (s, 2H), 5.00 (s, 2H), 3.28–3.14
1.63 (dt-like, 2H), 1.55–1.50 (complex, 4H), 1.45 (s, 9H); 13C-NMR (CDCl3), δ (complex, 4H), 2.99 (q-like, 2H), 2.76 (br-s, 2H), 1.75–1.60 (complex, 2H),
156.11, 69.89, 55.28, 53.15, 45.24, 40.66, 28.29, 28.16, 27.10, 24.55; HRMS (CI) 1.60–1.45 (complex, 4H); HRMS (CI) calcd for C23H31N3O4 [M]+ = 414.2387.
calcd for C13H27N3O2 [M]+ = 258.2176. Found: 258.2178.
Found: 414.2386.
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DSG as binding probes–biotin conjugates
M Morioka et al
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Figure 3 Biotinylated agents for biotinylated 15-deoxyspergualins with short (BDSG-S) (17) and long (BDSG-L) (19) spacers as well as BDSG-L (5).
(S)-N-(6-(Boc)amino-1-(4-(1,3-Bis(Cbz)aminopropylamino)
butylamino)-1-oxohexan-2-yl)-7-(N,N′-Bis(Cbz)guanidino)
heptanamide (15)
reverse phase silica gel chromatography (acetonitrile/water= 1:1) to give the
target compound, BDSG 4 (24.2 mg, yield: 58%). 1H-NMR (D2O), δ 4.65–4.58
(m, 1H), 4.44–4.35 (m, 1H), 4.18–4.08 (m, 1H), 3.33 (m, 1H, overlapped with
H2O), 3.26-2.93 (complex, 13H), 2.80–2.70 (m, 1H), 2.37–2.20 (complex, 4H),
2.18–2.00 (complex, 2H), 1.82–1.47 (complex, 16H), 1.45–1.27 (complex, 8H);
13C-NMR (D2O), δ 177.22, 176.56, 174.36, 165.28, 163.13, 156.62, 117.74,
114.84, 62.07, 60.21, 55.42, 54.12, 48.80, 47.29, 44.38, 41.04, 39.64, 38.85,
38.36, 35.41, 35.25, 30.54, 27.83, 27.75, 27.70, 27.64, 25.84, 25.51, 25.47, 25.14,
23.67, 22.84, 22.61; HRMS (CI) calcd for C31H60N10O4S [M]+ = 669.4598.
Found: 669.4597.
To a solution of compound 9 (720 mg, 1.0 mmol) in DMF (5.0 ml) under ice-
water conditions were added DIPEA (0.7 ml), diCbz-protected spermidine 14
(435 mg, 0.967 mmol), HOAt (177 mg, 1.3 mmol) and WSC·HCl (250 mg,
1.3 mmol) sequentially and the resultant mixture was stirred at room
temperature for 20 h. After addition of water (30 ml), the reaction mixture
was extracted with EtOAc and the organic layer was washed with brine, dried
over anhydrous MgSO4 and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography (n-hexane/AcOEt=
1:1) to give compound 15 (925 mg, yield: 88%) as an amorphous solid.
1H-NMR (DMSO-d6), δ 11.58 (s, 1H), 8.38 (dd-like, 1H), 7.90–7.78 (complex,
2H), 7.45–7.20 (complex, 20H), 6.73 (t, 1H, J = 4.4 Hz), 5.20 (s, 2H), 5.05 (s,
2H), 5.03 (s, 2H), 5.00 (s, 2H), 4.20–4.08 (m, 1H), 3.35–3.25 (complex, 2H),
3.25–3.10 (complex, 4H), 3.10–2.95 (complex, 4H), 2.90–2.75 (m, 1H),
2.15–1.00 (complex, 2H), 1.70–1.20 (complex, 20H), 1.38 (s, 9H); HRMS
N-((S)-1-amino-10,17,24-trioxo-28-(2-oxo-hexahydro-1H-thieno
[3,4-d]imidazol-4-yl)-4,9,16,23-tetraazaoctacosan-11-yl)-7-
guanidinoheptanamide (5)
In a similar procedure, the target compound 5 was prepared from 16 and NHS-
LC-Biotin 19 in 57% overall yield in three steps.1H-NMR (D2O), δ 4.64–4.56
(m, 1H), 4.44–4.37 (m, 1H), 4.15–4.08 (m, 1H), 3.35 (m, 1H, overlapped with
H2O), 3.35–3.05 (complex, 15H), 2.98 (dd, 1H, J = 4.9 and 13.0 Hz), 2.76
(d, 1H), 2.33–2.20 (complex, 6H), 2.22–2.02 (complex, 2H), 1.80–1.46
(complex, 22H), 1.45–1.26 (complex, 10H); 13C-NMR (D2O), δ 177.25,
177.66, 176.52, 174.33, 165.29, 156.63, 114.84, 62.06, 60.21, 55.39, 54.10,
48.80, 47.30, 44.48, 44.39, 41.05, 39.68, 39.01, 38.85, 38.37, 36.48, 35.65, 35.49,
35.26, 30.52, 27.98, 27.84, 27.77, 27.71, 27.67, 25.53, 25.48, 25.22, 25.13, 25.04,
23.68, 22.85, 22.58, 22.16; HRMS (CI) calcd for C37H71N11O5S [M]+
= 781.5360. Found: 782.5413.
(CI) calcd for C58H78N8O12 [M]+ = 1079.5811. Found: 1079.5814.; [α]24
− 4.24° (c 0.96, MeOH).
D
N-((S)-1-(4-(3-aminopropylamino)butylamino)-1-oxo-6-(5-(2-oxo-
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexan-2-
yl)-7-guanidinoheptanamide (4)
Compound 15 (925 mg, 0.857 mmol) was dissolved in 4N HCl-dioxane (1:3,
1.0 ml) and stirred at room temperature for 1 h. The solvent was removed
under reduced pressure to give the crude HCl salt of de-Boc compound 16
(760 mg), which was used in the next reaction without further purification. The
deblocked compound 16 was dissolved in 4 ml pyridine, from which 2 ml was
used. To the 2 ml pyridine solution under ice-bath conditions was added NHS-
Biotin 17 (83 mg, 0.242 mmol) and the resultant mixture was stirred at 0 °C for
17 h. The reaction mixture was concentrated under reduced pressure and the
obtained crude product was purified by reverse phase silica gel chromatography
(acetonitrile/water= 1:1) to give compound 18 (75 mg, yield: 31% from 17) as
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported, in part, by MEXT-Supported Program for the
Strategic Research Foundation at Private Universities, which is for Aichi
Medical University 2011-2015 (S1101027).
an amorphous solid. 1H-NMR(CDCl3),
δ 11.58 (s, 1H), 8.39 (t, 1H,
J = 3.8 Hz), 7.90–7.78 (complex, 2H), 7.72 (t, 1H, J = 4.1 Hz), 7.45–7.20
(complex, 20H), 6.42 (s, 1H), 6.36 (s, 1H), 5.30 (s, 2H), 5.05 (s, 2H), 5.03
(s, 2H), 5.00 (s, 2H), 4.29 (dd, 1H, J = 2.7 and 3.9 Hz), 4.20–4.00 (complex,
2H), 3.40–3.20 (complex, 2H), 3.20–3.10 (complex, 4H), 3.10–2.90 (complex,
7H), 2.85–2.72 (dd-like, 1H), 2.60–2.50 (m, 1H), 2.15–1.95 (complex, 4H),
1.70–1.10 (complex, 27H); HRMS (CI) calcd for C63H84N10O12S [M+H]+
=1205.6063. Found: 1205.6052.
Then, compound 18 was dissolved in methanol (1.0 ml) and hydrogenated
for 5 h in the presence of palladium hydroxide (5 mg) as a catalyst under a H2
atmosphere. After filtration of the mixture through a Celite pad, the filtrate was
concentrated under reduced pressure. The crude product was purified by
1
2
3
4
5
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