Chemical Synthesis and Biological Effect on Xylem Formation of Xylemin and Its Analogues

Article abstract of DOI:10.1002/ejoc.202000322

Xylemin (6) and its designed structural analogues 18–23, N-(4-aminobutyl)alkylamines, were synthesized by 2-nitrobenzenesulfonamide (Ns) strategy. Investigation of the improved synthesis of 20–23 resulted in the development of one-step synthesis of these analogues from the commercially available corresponding ketones. Biological assessment of the synthetic molecules elucidated that xylemin (6) and the analogue N-(4-aminobutyl)cyclopentylamine (21) promoted the expression level of thermospermine synthase ACAULIS5 (ACL5) and enhanced xylem formation. In addition, xylemin (6) was found to significantly promote lateral root formation, whereas xylemin analogues 18–23 including 21 did not. These results indicate that the analogue 21 has the potential as a novel inhibitor of thermospermine synthesis to work specifically in xylem differentiation.

Full text of DOI:10.1002/ejoc.202000322

A Journal of
Accepted Article
Title: Chemical Synthesis and Biological Effect on Xylem Formation of
Xylemin and Its Analogues
Authors: Hiroyoshi Takamura, Hiroyasu Motose, Taichi Otsu, Shiori
Shinohara, Ryugo Kouno, Isao Kadota, and Taku Takahashi
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000322
Link to VoR: http://dx.doi.org/10.1002/ejoc.202000322
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10.1002/ejoc.202000322
European Journal of Organic Chemistry
FULL PAPER
Chemical Synthesis and Biological Effect on Xylem Formation of
Xylemin and Its Analogues
Hiroyoshi Takamura+,*[a] Hiroyasu Motose+,*[b] Taichi Otsu,[a] Shiori Shinohara,[b] Ryugo Kouno,[b] Isao
Kadota,[a] and Taku Takahashi[b]
Abstract: Xylemin (6) and its designed structural analogues 18–23,
N-(4-aminobutyl)alkylamines,
were
synthesized
by
2-
nitrobenzenesulfonamide (Ns) strategy. Investigation of the
improved synthesis of 20–23 resulted in the development of one-
step synthesis of these analogues from the commercially available
corresponding ketones. Biological assessment of the synthetic
molecules elucidated that xylemin (6) and the analogue N-(4-
aminobutyl)cyclopentylamine (21) promoted the expression level of
thermospermine synthase ACAULIS5 (ACL5) and enhanced xylem
formation. In addition, xylemin (6) was found to significantly promote
lateral root formation, whereas xylemin analogues 18–23 including
21 did not. These results indicate that the analogue 21 has the
potential as a novel inhibitor of thermospermine synthesis to work
specifically in xylem differentiation.
Scheme 1. Biosynthetic pathways of polyamines in plants.
Introduction
Thermospermine (5) is a structural isomer of spermine (4)
found in bacteria and plants.[4] Thermospermine (5) is generated
from spermidine (3) through adding an aminopropyl group by the
action of thermospermine synthase designated ACAULIS5
(ACL5).[5] Because ACL5 gene is well conserved in plant
kingdom from algae to angiosperms, thermospermine (5) is a
ubiquitous plant polyamine, whereas spermine (4) and SPMS
gene exist only in seed plants but not in algae, bryophytes, and
monilophytes.[4b] Thus, thermospermine (5) is a major polyamine
in plants. Thermospermine-deficient acl5 mutant of Arabidopsis
thaliana exhibits excess xylem formation, which results in severe
suppression of organ elongation.[6] Addition of exogenous
thermospermine significantly suppresses xylem differentiation
and recovers the defect of organ elongation in the acl5
mutant.[5b] These studies indicate that thermospermine (5) is a
unique plant growth regulator for limiting xylem differentiation
and promoting organ growth.
Polyamines are low-molecular cationic compounds existing in all
organisms. Polyamines regulate various biological processes
including RNA/DNA stabilization, transcription of RNA, protein
translation, and ion channel modulation through their binding
with nucleic acids and proteins.[1] Putrescine (2), spermidine (3),
and spermine (4) are commonly found in various species and
recognized as major polyamines (Scheme 1). In plants,
putrescine (2) is synthesized from L-arginine (1) through multiple
reaction steps.[2] Spermidine (3) and spermine (4) are
synthesized from putrescine (2) by sequential addition of an
aminopropyl group via the catalytic action of aminopropyl
transferases, spermidine synthase (SPDS) and spermine
synthase (SPMS), respectively. The aminopropyl group is
donated by decarboxylated S-adenosylmethionine. These
polyamines are involved in various physiological phenomena
during plant development and stress response, such as cell
death, alkaloid biosynthesis, and organ growth.[3]
Thermospermine (5) is not only a key regulator for plant
growth but also a powerful bioactive molecule to modulate plant
yield and biomass. However, its biological function has been
studied only in angiosperms, especially in Arabidopsis. To
investigate thermospermine function in various plants, we
decided to develop the inhibitor of thermospermine biosynthesis.
We previously reported chemical synthesis of the molecule to
inhibit thermospermine biosynthesis and named this biologically
active molecule xylemin.[7] Xylemin (6) is a spermidine analogue,
N-(4-aminobutyl)propylamine, which loses an amino group
required for the addition of the aminopropyl group in
thermospermine biosynthesis (Figure 1). Xylemin (6)
competitively inhibits biosynthesis of thermospermine (5) and
promotes xylem differentiation in various plants.[7] Therefore,
xylemin (6) is a novel chemical tool to analyze thermospermine
function and a useful biologically active compound, which can
manipulate xylem differentiation and organ elongation in plants
without any genetic modification. Xylem is a source of woody
[a]
Prof. Dr. H. Takamura, T. Otsu, Prof. Dr. I. Kadota
Department of Chemistry
Graduate School of Natural Science and Technology, Okayama
University
3-1-1 Tsushimanaka, Kita-ku, Okayama 700-8530 (Japan)
E-mail: takamura@cc.okayama-u.ac.jp
http://chem.okayama-u.ac.jp/english/staff/detail/hiroyoshi-
takamura.html
Prof. Dr. H. Motose, S. Shinohara, R. Kouno, Prof. Dr. T. Takahashi
Department of Biological Science
Graduate School of Natural Science and Technology, Okayama
University
3-1-1 Tsushimanaka, Kita-ku, Okayama 700-8530 (Japan)
E-mail: motose@cc.okayama-u.ac.jp
https://okayama.pure.elsevier.com/en/persons/hiroyasu-motose
These authors contributed equally to this work.
[b]
[+]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.202000322
European Journal of Organic Chemistry
FULL PAPER
biomass which is of enormous importance for industrial
applications including pulp and biofuel, therefore, xylemin (6)
could provide a novel and sustainable method to promote woody
biomass production with low environmental load. In this full
paper, we report the improved synthesis of xylemin (6) in
comparison with the previous synthetic scheme[7] and synthesis
of various xylemin analogues, and biological effect of their
synthetic products on xylem and root formation.
Synthesis of xylemin analogues 18–23
Because it was clarified that xylemin (6) works as the inhibitor of
thermospermine synthesis, we next decided to synthesize
xylemin structural analogues and investigate their structure–
activity relationship. Shirahata and co-workers designed N-(3-
aminopropyl)alkylamines as inhibitors of spermine synthase and
evaluated their activities.[13,14] As summarized in Figure 2, N-(3-
aminopropyl)butylamine (12) exhibited the inhibitory activity
against spermine synthase with an IC50 value of 0.42 μM.
Changing the alkyl chain moiety affected the activities. Thus, the
activity of N-(3-aminopropyl)pentylamine (13) was decreased to
an IC50 value of 4.2 μM and N-(3-aminopropyl)hexylamine (14)
was inactive. Shirahata’s group next evaluated the inhibitory
activities of cycloalkylamines 15–17 and found that N-(3-
Figure 1. Structure of xylemin (6).
aminopropyl)cyclopentylamine
aminopropyl)cyclohexylamine
(15),
N-(3-
N-(3-
(16),
and
Results and Discussion
aminopropyl)cycloheptylamine (17) were active with IC50 values
of 7.5 μM, 0.17 μM, and 1.1 μM, respectively. From these results,
it was proven that the number of carbons in the substituents on
nitrogen atom had influence on their inhibitory activities in both
alkylamines and cycloalkylamines. By using Shirahata’s results
described above as a reference, we designed and decided to
synthesize N-(4-aminobutyl)alkylamines 18–23 as xylemin
structural analogues (Figure 3).
Synthesis of xylemin (6)
We commenced the synthesis of xylemin (6) from commercially
available N-(4-aminobutyl)-2-nitrobenzenesulfonamide (7) as
shown in Scheme 2. Protection of the amino group of 7 with
Boc2O/Et3N afforded carbamate 8.[8] Selective alkylation of the 2-
nitrobenzenesulfonamide 8 was performed with 1-bromopropane
(9)/Cs2CO3/tetrabutylammonium iodide (TBAI) to provide
propylated product 10.[9] The 2-nitrobenzenesulfonyl (Ns)
protecting group of 10 was removed with PhSH/Cs2CO3 to give
amine 11, quantitatively. However, several experiments revealed
that this reaction has no reproducibility. We observed that this
reaction proceeded smoothly and thought that the problem might
result from the purification step by silica gel column
chromatography. Therefore, we carried out desulfonylation of 10
by using silica-supported 1-propanethiol (Si-Thiol)[10,11] to
simplify the purification procedure and the desired amine 11 was
obtained in 94% yield with reproducibility. Finally, the carbamate
[9,12]
11 was deprotected with SOCl2 in CH3OH/CH2Cl2
to furnish
xylemin (6). The synthetic xylemin (6) was found to promote
xylem differentiation by the inhibition of thermospermine
synthesis, as reported in our previous communication.[7]
Figure 2. Inhibitory activity of N-(3-aminopropyl)alkylamines against spermine
synthase.[13] IC50 = inhibitory concentration in 50% of test subjects.
Scheme 2. Synthesis of xylemin (6). Ns = 2-nitrobenzenesulfonyl, Boc = tert-
butoxycarbonyl, Et = ethyl, rt = room temperature, TBAI = tetrabutylammonium
iodide, quant = quantitative, Ph = phenyl.
Figure 3. Structures of xylemin (6) and its analogues 18–23.
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10.1002/ejoc.202000322
European Journal of Organic Chemistry
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Scheme 4. Unsuccessful results for the synthesis of cyclohexylamine 30.
N-(4-aminobutyl)butylamine
(18)
and
N-(4-
aminobutyl)pentylamine (19) were synthesized by the
transformation similar to that used for the synthesis of xylemin
(6). Thus, the butyl and pentyl substituents of amines 25 and 27
were introduced by Ns strategy (Scheme 3).[9] Subsequently, the
Ns and Boc moieties were removed by Si-Thiol[10,11] and
SOCl2/CH3OH,[9,12] respectively, to reach 18 and 19.
Scheme 3. Synthesis of xylemin analogues 18 (a) and 19 (b).
We
next
examined
the
synthesis
of
N-(4-
aminobutyl)cyclohexylamine (22). When the alkylation conditions
with bromocyclohexane (28)/Cs2CO3/TBAI, which were used for
the synthesis of 6, 18, and 19, were applied to the synthesis of
cyclohexylamine 30, the reaction did not proceed at all (Scheme
4a). Condensation of the sulfonamide 8 and cyclohexanol (29)
under Mitsunobu conditions[15] with diethyl azodicarboxylate
(DEAD)/Ph3P in toluene at reflux resulted in failure and the
starting material 8 was recovered (Scheme 4b). Therefore, we
tried to investigate other synthetic transformations toward the
Scheme 5. Synthesis of xylemin analogues 22 (a), 21 (b), 23 (c), and 20 (d).
synthesis of 22. First, treatment of cyclohexylamine (31) with
[16]
NsCl/NaHCO3
afforded
the
which reacted
corresponding
with 4-
Improved synthesis of xylemin analogues 20–23
nitrobenzenesulfonamide,[17]
Although we succeeded in the synthesis of xylemin analogues
20–23, these products were supplied from different amines 38,
34, 31, and 36 as starting materials, respectively, which resulted
in increasing the number of total steps for their synthesis.
Therefore, we next examined the alternative synthetic route for
obtaining 20–23 more concisely. We first surveyed the reaction
conditions of mono-reductive amination of cyclohexanone (40)
bromobutyronitrile (32) in the presence of Cs2CO3 and TBAI[9] to
provide the alkylated product 33 (Scheme 5a). Deprotection of
the nitrobenzenesulfonamide 33 with Si-Thiol[10,11] and
[17]
subsequent hydrogenation of the nitrile group with PtO2 were
performed
to
produce
the
desired
N-(4-
aminobutyl)cyclohexylamine (22).[13] Furthermore, the synthetic
sequence from 31 to 22 was successfully applied to amines 34,
36, and 38 to deliver the designed N-(4-aminobutyl)alkylamines
21,[17] 23, and 20 (Scheme 5b–d).
with
putrescine
(2)
for
delivering
N-(4-
aminobutyl)cyclohexylamine (22) in one-step as described in
Table 1. Treatment of 40 with 2 (2.0 equiv) in the presence of
Na2SO4 as dehydrating agent and AcOH followed by addition of
(a)
[18]
Cs2CO3, TBAI
CH3CN, reflux
Br
NaBH(OAc)3
gave trace amounts of 22 (Entry 1). When the
H
N
reductive amination of 40 with 2 (10 equiv) was carried out by
using NaBH3CN[19] to afford 22 in 22% yield (Entry 2).
Bhattacharyya et al. reported the reductive amination of
aldehydes and ketones utilizing Ti(OiPr)4–NaBH4 reagent
system.[20] These reaction conditions were successfully applied
to the synthesis of various secondary amines by Brunel and co-
28
29
Ns
Boc
Boc
N
H
(b)
8
DEAD, Ph3P
toluene, reflux
OH
H
N
N
30
Ns
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10.1002/ejoc.202000322
European Journal of Organic Chemistry
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workers in 2006.[21] When these reaction conditions were used in
our cases, thus, reaction of 40 with 2 (3.0 equiv) in the presence
of Ti(OiPr)4 in CH3OH at room temperature and subsequent
reduction with NaBH4 at –78 °C took place,[22] the desired
product 22 was produced in 68% yield (Entry 3). Changing the
reaction temperature from –78 °C to 0 °C in reduction step
increased the chemical yield of 22 to 84% yield (Entry 4). Having
succeeded in mono-reductive amination of cyclohexanone (40)
with putrescine (2) by the combination of Ti(OiPr)4 as a Lewis
acid and NaBH4 as a reducing reagent, we carried out one-step
synthesis of xylemin analogues 21,[17] 23, and 20 by the same
reaction conditions as shown in Scheme 6.
thermospermine. The expression level of ACL5 is feedback-
regulated by the amount of thermospermine (5); depletion of
thermospermine (5) leads to the increased expression of ACL5,
whereas the increase of thermospermine content causes a
reduction of ACL5 mRNA.[5b] The transcript level of ACL5 gene
was quantified in Arabidopsis wild-type seedlings treated with
xylemin (6), xylemin analogues 18–23, or thermospermine (5) by
reverse transcription-quantitative polymerase chain reaction
(RT-qPCR). As expected, the accumulation of ACL5 transcripts
was significantly increased in seedlings treated with xylemin (6)
(Figure 4). Interestingly, the expression of ACL5 was also
promoted to the similar level to that in the case of xylemin (6) by
adding the xylemin analogue 21, whereas it was not affected
when the other analogues were added to the culture medium.
These results have revealed that endogenous thermospermine
level is decreased by xylemin (6) and its analogue N-(4-
aminobutyl)cyclopentylamine (21). In Shirahata’s work about
Table 1. Mono-reductive amination of cyclohexanone (40) with putrescine (2).
inhibitors
of
spermine
synthase,
N-(3-
aminopropyl)cyclohexylamine (16) exhibited the inhibitory
activity with an IC50 value similar to that of N-(3-
aminopropyl)butylamine (12).[13,14] In our case of inhibitors of
thermospermine synthase, N-(4-aminobutyl)cyclopentylamine
(21) was active as well as xylemin (6), N-(4-
aminobutyl)propylamine. It is interesting that the expression
level of ACL5 is affected by the number of carbons on the
nitrogen atom, which has the similar tendency to that found in
inhibitors of spermine synthase.
Entry
Conditions[a]
Yield [%][b]
N.D.[c]
22
1
2
Na2SO4, AcOH, rt, then NaBH(OAc)3, rt
MS4Å, AcOH, CH3OH, reflux, then
NaBH3CN, rt
3
4
Ti(OiPr)4, CH3OH, rt, then NaBH4, –78 °C
Ti(OiPr)4, CH3OH, rt, then NaBH4, 0 °C
68
84
[a] Ac = acetyl, MS = molecular sieves, Pr = propyl. [b] Isolated yield. [c] N.D.
= not determined. Formation of trace amounts of 22 was observed.
Figure 4. Effects of xylemin (6), its analogues 18–23, and thermospermine (5)
on the expression of thermospermine synthase gene ACAULIS5 (ACL5). Wild
type seedlings of Arabidopsis thaliana were grown for 7 days in the liquid
medium without (Mock) or with 6, 18–23, or 5 at the concentration of 200 μM.
Expression of ACL5 was quantified by reverse transcription-quantitative
polymerase chain reaction (RT-qPCR). Expression of ACTIN8 (ACT8) was
used as a control. Columns and error bars indicate mean values and standard
deviations of n = 3, respectively. Asterisks show significant difference from the
value of Mock treatment (P < 0.01, t-test).
Scheme 6. One-step synthesis of xylemin analogues 21 (a), 23 (b), and 20 (c)
via mono-reductive amination.
Effect of xylemin (6) and its analogues 18–23 on the
expression of ACL5 gene
With xylemin (6) and its analogues 18–23 in hand, we next tried
to carry out the biological evaluation of these synthetic products.
First, for the quantitative evaluation of biological activity of the
synthetic products, we used expression level of the ACL5 gene
Effect of xylemin (6) and its analogues 18–23 on xylem
formation
Next, we analyzed effect of xylemin (6) and its analogues 18–23
on xylem differentiation because inhibition of thermospermine
synthesis by xylemin triggers excess xylem formation in
Arabidopsis.[7] Arabidopsis wild-type seedlings were grown
as
a sensitive indicator of the endogenous amount of
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Figure 6. Quantification of xylem differentiation. The width of xylem was
quantified in the hypocotyls of Arabidopsis wild type seedlings shown in Figure
5. Data were represented with box plots. The number of plants used for
quantification (n) in each treatment was shown under horizontal axis labels.
Asterisks show significant difference from the value of Mock treatment (P <
0.0003, t-test).
under the presence of xylemin (6) and xylemin analogues 18–23.
Xylem formation was observed under a microscope (Figure 5)
and quantification of xylem differentiation is depicted in Figure 6.
The addition of xylemin (6) and the xylemin analogue 21
remarkably promoted xylem formation, whereas the other
xylemin analogues did not affect the xylem differentiation. Thus,
N-(4-aminobutyl)cyclopentylamine
differentiation by inhibiting thermospermine biosynthesis as in
the case of xylemin (6).
(21)
promotes
xylem
Effect of xylemin (6) and its analogues 18–23 on lateral root
formation
Our previous study has shown that xylemin (6) promotes the
formation of lateral roots, whereas thermospermine suppresses
it.[7] Therefore, we examined effects of xylemin analogues 18–
23 on lateral root formation (Figure 7). Xylemin (6) significantly
promoted lateral root formation, whereas xylemin analogues 18–
23 including the analogue 21 did not. In consideration with the
similar effect of xylemin (6) and the analogue 21 on ACL5
expression and xylem formation, the physiological action of 6
and 21 may differ in lateral root formation.
Recently, crystal structure of thermospermine synthase of a
leguminous plant Medicago truncatula (MtACL5) has been
revealed.[23] The binding site of spermidine (3), a precursor of
thermospermine (5), is narrow and charged negatively. Acidic
amino acid residues of MtACL5 form hydrogen bonds with
amino groups of spermidine (3). Aromatic amino acid residues
generate hydrophobic interactions with 3. The protein structure
of MtACL5 is applicable to that of Arabidopsis thaliana ACL5,
because the amino acid sequences of ACL5 are highly
conserved in plants, especially within flowering plants including
Medicago and Arabidopsis.[2,4b,24] Thus, relatively compact alkyl
groups of xylemin (6) and the xylemin analogue 21 may possibly
be preferred for interactions with the active site of ACL5 protein.
Figure 7. Effects of xylemin (6), its analogues 18–23, and thermospermine (5)
on lateral root formation. Wild type seedlings of Arabidopsis thaliana were
grown for 7 days on the agar medium without (Mock) or with 6, 18–23, or 5 at
the concentration of 100 μM. Seedlings were observed under
a
stereomicroscope. The number of lateral roots per plant was measured. The
number of plants used for quantification (n) in each treatment was shown
under horizontal axis labels. Columns and error bars indicate mean values and
the standard errors, respectively. Single and double asterisks show significant
difference from the value of Mock treatment at the P level less than 0.03 and
0.003, respectively (t-test).
Figure 5. Effects of xylemin (6) and its analogues 18–23 on xylem
differentiation. Wild type seedlings of Arabidopsis thaliana were grown for 7
days in the liquid medium without (Mock) or with
6 or 18–23 at the
concentration of 200 μM. Hypocotyls were observed under a differential
interference contrast microscope. The region between white triangles indicates
xylem vessels, which exhibit brown color due to secondary cell wall thickening
and lignin deposition.
Conclusions
First, chemical synthesis of xylemin (6) and its designed
structural analogues 18–23 was examined. Xylemin (6) and N-
(4-aminobutyl)alkylamines 18–23 were successfully synthesized
by utilizing Ns strategy. In addition, improvement of the
synthesis of 20–23 was carried out to result in the synthesis of
these analogues in one-step via mono-reductive amination of
the corresponding ketones, respectively. This concise synthesis
by mono-reductive amination of ketones could be applied to the
synthesis of polyamines including other xylemin analogues. Next,
we evaluated the biological effect of synthetic molecules.
Xylemin (6) and the xylemin analogue 21, N-(4-
aminobutyl)cyclopentylamine, exhibited significant biological
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European Journal of Organic Chemistry
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HRMS (ESI–TOF) calcd for C15H23N3O6SNa [M + Na]+ 396.1205, found
396.1201.
activity to promote xylem formation. Enhanced transcript
accumulation of thermospermine synthase gene ACL5 by the
addition of
6 or 21 supports their inhibitory effects on
Sulfonamide 10: To a solution of sulfonamide 8 (1.02 g, 2.73 mmol) in
CH3CN (7.8 mL) were added Cs2CO3 (2.69 g, 8.26 mmol), TBAI (506 mg,
1.37 mmol), and 1-bromopropane (9, 0.30 mL, 3.28 mmol) at room
temperature. The mixture was stirred at 40 °C for 2 h. To the mixture was
added 1-bromopropane (9, 0.30 mL, 3.28 mmol) at room temperature.
The mixture was stirred at 40 °C for 1 h. The mixture was diluted with
EtOAc and washed with H2O. The aqueous phase was extracted with
EtOAc three times and the combined organic phase was washed with
brine and dried over Na2SO4. Concentration and column chromatography
(hexane/EtOAc = 4:1, 1:1) gave sulfonamide 10 (1.15 g, quant): yellow
oil; Rf = 0.52 (hexane/EtOAc = 1:1); IR (neat) 3412, 3094, 2971, 2934,
2876, 1699 cm–1; 1H NMR (400 MHz, CDCl3) δ 8.01–7.99 (m, 1 H), 7.68–
7.66 (m, 2 H), 7.61–7.59 (m, 1 H), 4.53 (brs, 1 H), 3.30 (t, J = 7.6 Hz, 2
H), 3.23 (t, J = 7.6 Hz, 2 H), 3.10 (q, J = 7.6 Hz, 2 H), 1.60–1.46 (m, 6 H),
1.44 (s, 9 H), 0.85 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ
155.9, 148.0, 133.7, 133.2, 131.4, 130.6, 124.0, 79.2, 49.1, 47.0, 40.0,
28.5, 27.3, 25.6, 21.5, 11.1; HRMS (ESI–TOF) calcd for C18H29N3O6SNa
[M + Na]+ 438.1675, found 438.1678.
biosynthesis of thermospermine. Other xylemin analogues (18–
20, 22, 23) did not affect xylem formation and transcript level of
ACL5. These differential effects may be due to larger alkyl
groups of the xylemin analogues 18–20, 22, and 23 than 6 and
21. Thus, the size and structure of the alkyl group are essential
for the activity to enhance xylem formation. Xylemin (6)
promoted lateral root formation, whereas the xylemin analogue
21 did not. This may reflect mild effect of 21 on xylem
differentiation. Lateral roots develop from pericycle cell layer,
which locates outside of vascular tissues. Pericycles in contact
with inner xylem region (xylem-pole pericycles) preferentially
generate lateral roots. Xylemin (6) enhances xylem formation,
which probably causes increased number of xylem-pole
pericycles and lateral roots, whereas xylem formation enhanced
by the xylemin analogue 21 is not enough to increase xylem-
pole pericycles. Thus, the analogue 21 could be utilized as a
novel inhibitor of thermospermine synthesis, which specifically
promotes xylem formation.
Deprotection of Sulfonamide 10 with PhSH: To
a solution of
sulfonamide 10 (11.3 mg, 27.2 μmol) in CH3CN (0.8 mL) were added
Cs2CO3 (44.3 mg, 0.136 mmol) and PhSH (10 μL, 0.101 mmol) at room
temperature. The mixture was stirred at the same temperature for 3 h.
The mixture was filtered through a Celite pad and washed with EtOAc.
Concentration and column chromatography (CH2Cl2, CH2Cl2/CH3OH =
5:1 including 2% Et3N) gave amine 11 (6.8 mg, quant): yellow oil; Rf =
0.15 (CH2Cl2/CH3OH = 5:1); IR (neat) 3340, 2962, 2932, 2873, 1697 cm–
1; 1H NMR (400 MHz, CD3OD) δ 3.04–3.03 (m, 2 H), 2.56–2.49 (m, 4 H),
1.54–1.48 (m, 6 H), 1.41 (s, 9 H), 0.95–0.89 (m, 3 H); 13C NMR (100 MHz,
CD3OD) δ 158.4, 79.7, 52.6, 50.3, 41.2, 28.8, 27.7, 23.5, 12.1; HRMS
(ESI–TOF) calcd for C12H27N2O2 [M + H]+ 231.2073, found 231.2072.
Experimental Section
General Methods: Reagents were used as received from commercial
suppliers unless otherwise indicated. All reactions were carried out under
an atmosphere of argon. Reaction solvents were purchased as
dehydrated solvents and stored with active molecular sieves 4Å under
argon prior to use for reactions. All solvents for work-up procedure were
used as received. Analytical thin-layer chromatography (TLC) was
performed with aluminium TLC plates (Merck TLC silica gel 60F254).
Column chromatography was performed with Fuji Silysia silica gel BW-
300 or Kanto Chemical silica gel 60N. Kugelrohr distillation was
performed with Shibata Glass Tube Oven GTO-250RS. IR spectra were
recorded on JASCO FT/IR-460 plus. 1H and 13C NMR spectra were
recorded on JEOL JNM-AL400 or Varian 400-MR. Chemical shifts in the
NMR spectra are reported in ppm with reference to the internal residual
Deprotection of Sulfonamide 10 with Si-Thiol: To a solution of
sulfonamide 10 (700 mg, 1.68 mmol) in CH3CN (17 mL) were added
Cs2CO3 (1.64 g, 5.04 mmol) and Si-Thiol (1.3 mmol/g, 5.24 g, 6.81 mmol)
at room temperature. The mixture was stirred at 80 °C for 5 h. The
mixture was filtered through a Celite pad and washed with EtOAc.
Concentration gave amine 11 (365 mg, 94%).
solvent (1H NMR, CDCl3 7.26 ppm, D2O 4.79 ppm, CD3OD 3.31 ppm; 13
C
NMR, CDCl3 77.0 ppm, CD3OD 49.0 ppm, (CD3)2SO 39.5 ppm). The
following abbreviations are used to designate the multiplicities: s = singlet,
t = triplet, q = quartet, quin = quintet, m = multiplet, br = broad. Coupling
constants (J) are in hertz. High resolution mass spectra were recorded
on Waters Micromass LCT (ESI–TOF–MS) or Bruker micOTOF II (ESI–
TOF–MS).
Xylemin (6): To a solution of carbamate 11 (92.2 mg, 0.400 mmol) in
CH3OH (2.6 mL) and CH2Cl2 (1.3 mL) was added SOCl2 (0.260 mL, 3.60
mmol) at 0 °C. The mixture was stirred at room temperature for 5 h.
Concentration gave xylemin (6, 131 mg, quant): yellow solid; IR (neat)
3422, 2930, 2778 cm–1 1H NMR (400 MHz, D2O) δ 3.13–3.02 (m, 6 H),
;
1.81–1.68 (m, 6 H), 1.00 (t, J = 7.6 Hz, 3 H); 13C NMR (100 MHz,
CD3CD) δ 50.6, 48.1, 40.1, 25.6, 24.3, 20.7, 11.3; HRMS (ESI–TOF)
calcd for C7H19N2 [M + H]+ 131.1548, found 131.1550.
Carbamate
8:
To
a
solution
of
N-(4-aminobutyl)-2-
nitrobenzenesulfonamide (7) (200 mg, 0.732 mmol) in CH2Cl2 (5.0 mL)
were added Et3N (0.12 mL, 0.878 mmol) and Boc2O (0.20 mL, 0.878
mmol) at room temperature. The mixture was stirred at the same
temperature for 2 h. The mixture was diluted with CH2Cl2 and washed
with 1N aqueous HCl. The aqueous phase was extracted with CH2Cl2
twice and the combined organic phase was washed with brine and dried
over Na2SO4. Concentration and column chromatography (hexane/EtOAc
= 2:1, 1:1) gave carbamate 8 (269 mg, 98%): yellow solid; Rf = 0.27
N-(4-Aminobutyl)butylamine (18): To a solution of sulfonamide 8 (30.6
mg, 81.9 μmol) in CH3CN (1.0 mL) were added Cs2CO3 (80.2 mg, 0.246
mmol), TBAI (15.1 mg, 41.0 μmol), and 1-bromobutane (24, 11 μL, 98.3
μmol) at room temperature. The mixture was stirred at 40 °C for 3 h. To
the mixture was added 1-bromobutane (24, 21 μL, 0.197 mmol) at room
temperature. The mixture was stirred at 40 °C for 2 h. The mixture was
diluted with EtOAc and washed with H2O. The aqueous phase was
extracted with EtOAc three times and the combined organic phase was
washed with brine and dried over Na2SO4. Concentration and short
column chromatography (hexane/EtOAc = 2:1) gave sulfonamide 25
(32.5 mg), which was used for the next step without further purification.
To a solution of sulfonamide 25 obtained above (32.5 mg) in CH3CN (1.0
mL) were added Cs2CO3 (77.0 mg, 0.236 mmol) and Si-Thiol (1.3 mmol/g,
(hexane/EtOAc = 1:1); IR (neat) 3294, 3085, 2980, 1703, 1676 cm–1 1H
;
NMR (400 MHz, CDCl3) δ 8.14–8.12 (m, 1 H), 7.87–7.85 (m, 1 H), 7.76–
7.73 (m, 2 H), 5.36 (brs, 1 H), 4.54 (brs, 1 H), 3.13–3.06 (m, 4 H), 1.61–
1.47 (m, 4 H), 1.42 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 156.0, 148.1,
133.6, 133.6, 132.8, 131.1, 125.4, 79.3, 43.4, 39.8, 28.4, 27.1, 26.8;
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000322
European Journal of Organic Chemistry
FULL PAPER
233 mg, 0.303 mmol) at room temperature. The mixture was stirred at
80 °C for 7 h. The mixture was filtered through a Celite pad and washed
with EtOAc. Concentration gave the corresponding amine (18.2 mg),
which was used for the next step without further purification. To a
solution of the carbamate obtained above (18.2 mg) in CH3OH (0.8 mL)
and CH2Cl2 (0.4 mL) was added SOCl2 (48 μL, 0.652 mmol) at 0 °C. The
mixture was stirred at room temperature for 5 h. Concentration gave N-
(4-aminobutyl)butylamine (18, 15.0 mg, quant in three steps): yellow
5 H), 1.44–1.26 (m, 4 H), 1.12–1.01 (m, 1 H); 13C NMR (100 MHz,
CDCl3) δ 148.0, 133.6, 133.5, 131.5, 130.7, 124.1, 118.9, 58.5, 42.7,
32.0, 27.6, 26.0, 25.2, 14.8; HRMS (ESI–TOF) calcd for C16H21N3O4SNa
[M + Na]+ 374.1151, found 374.1144.
N-(4-Aminobutyl)cyclohexylamine (22): To a solution of sulfonamide
33 (15.2 mg, 43.3 μmol) in CH3CN (1.4 mL) were added Cs2CO3 (44.1
mg, 0.135 mmol) and Si-Thiol (1.3 mmol/g, 133 mg, 0.173 mmol) at room
temperature. The mixture was stirred at 80 °C for 6 h. The mixture was
filtered through a Celite pad and washed with EtOAc. Concentration gave
the corresponding amine (6.7 mg), which was used for the next step
without further purification. A mixture of the nitrile obtained above (6.7
mg) and PtO2 (0.9 mg, 4.03 μmol) in EtOH (1.0 mL) and CHCl3 (0.1 mL)
was stirred for 20 h under a H2 atmosphere at room temperature. The
mixture was filtered and washed with CH3OH. Concentration gave N-(4-
aminobutyl)cyclohexylamine (22, 8.4 mg, quant in two steps).
solid; IR (neat) 3395, 2966 cm–1 1H NMR (400 MHz, D2O) δ 3.12–3.07
;
(m, 6 H), 1.81–1.79 (m, 4 H), 1.73–1.66 (m, 2 H), 1.43 (q, J = 6.8 Hz, 2
H), 0.96 (t, J = 6.8 Hz, 3 H); 13C NMR (100 MHz, CD3OD) δ 49.9, 48.2,
40.1, 29.2, 25.6, 24.3, 20.8, 13.9; HRMS (ESI–TOF) calcd for C8H21N2 [M
+ H]+ 145.1705, found 145.1710.
N-(4-Aminobutyl)pentylamine (19): To a solution of sulfonamide 8
(33.1 mg, 88.6 μmol) in CH3CN (1.0 mL) were added Cs2CO3 (86.7 mg,
0.266 mmol), TBAI (16.4 mg, 44.3 μmol), and 1-bromopentane (26, 13
μL, 0.106 mmol) at room temperature. The mixture was stirred at 40 °C
for 1 h. To the mixture was added 1-bromopentane (26, 39 μL, 0.315
mmol) at room temperature. The mixture was stirred at 60 °C for 1 h. The
mixture was diluted with EtOAc and washed with H2O. The aqueous
phase was extracted with EtOAc three times and the combined organic
phase was washed with brine and dried over Na2SO4. Concentration and
N-(4-Aminobutyl)cyclopentylamine
(21):
To
a
solution
of
cyclopentylamine (34, 20 μL, 0.202 mmol) in THF (2.0 mL) were added
NaHCO3 (67.9 mg, 0.808 mmol) and NsCl (89.5 mg, 0.404 mmol) at 0 °C.
The mixture was stirred at the same temperature for 10 min. The reaction
was quenched with 3N aqueous NaOH. The mixture was stirred at room
temperature for 30 min. The mixture was diluted with EtOAc, washed
with saturated 3N aqueous NaOH, 3N aqueous HCl, and brine, and then
dried over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 4:1) gave the corresponding sulfonamide (51.8 mg),
which was used for the next step without further purification. To a
solution of the sulfonamide obtained above (51.8 mg) in CH3CN (1.9 mL)
were added Cs2CO3 (188 mg, 0.576 mmol), TBAI (35.5 mg, 96.0 μmol),
and 4-bromobutyronitrile (32, 23 μL, 0.230 mmol) at room temperature.
The mixture was stirred at 60 °C for 20 min. To the mixture was added 4-
bromobutyronitrile (32, 46 μL, 0.460 mmol) at room temperature. The
mixture was stirred at 60 °C for 1 h. The mixture was diluted with EtOAc
and washed with H2O. The aqueous phase was extracted with EtOAc
twice and the combined organic phase was washed with brine and dried
over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 4:1, 2:1) gave nitrile 35 (56.5 mg), which was used for
the next step without further purification. To a solution of sulfonamide 35
obtained above (56.5 mg) in CH3CN (5.6 mL) were added Cs2CO3 (163
mg, 0.501 mmol) and Si-Thiol (1.3 mmol/g, 514 mg, 0.668 mmol) at room
temperature. The mixture was stirred at 80 °C for 6 h. The mixture was
filtered through a Celite pad and washed with EtOAc. Concentration gave
the corresponding amine (24.7 mg), which was used for the next step
without further purification. A mixture of the nitrile obtained above (24.7
mg) and PtO2 (3.6 mg, 15.7 μmol) in EtOH (3.1 mL) and CHCl3 (0.3 mL)
was stirred for 20 h under a H2 atmosphere at room temperature. The
mixture was filtered and washed with CH3OH. Concentration gave N-(4-
aminobutyl)cyclopentylamine (21, 35.5 mg, quant in four steps).
short column chromatography (hexane/EtOAc
= 4:1, 2:1) gave
sulfonamide 27 (33.9 mg), which was used for the next step without
further purification. To a solution of sulfonamide 27 obtained above (33.9
mg) in CH3CN (1.0 mL) were added Cs2CO3 (74.6 mg, 0.229 mmol) and
Si-Thiol (1.3 mmol/g, 235 mg, 0.306 mmol) at room temperature. The
mixture was stirred at 80 °C for 8 h. The mixture was filtered through a
Celite pad and washed with EtOAc. Concentration gave the
corresponding amine (20.5 mg), which was used for the next step without
further purification. To a solution of the carbamate obtained above (20.5
mg) in CH3OH (0.8 mL) and CH2Cl2 (0.4 mL) was added SOCl2 (52 μL,
0.713 mmol) at 0 °C. The mixture was stirred at room temperature for 5 h.
Concentration gave N-(4-aminobutyl)pentylamine (19, 20.4 mg, quant in
three steps): yellow solid; IR (neat) 3409, 2963 cm–1; 1H NMR (400 MHz,
D2O) δ 3.12–3.06 (m, 6 H), 1.81–1.68 (m, 6 H), 1.39–1.37 (m, 4 H), 0.95–
0.91 (m, 3 H); 13C NMR (100 MHz, CD3OD) δ 49.6, 48.2, 40.1, 29.7, 27.0,
25.7, 24.3, 23.2, 14.1; HRMS (ESI–TOF) calcd for C9H23N2 [M + H]+
160.1891, found 160.1886.
Nitrile 33: To a solution of cyclohexylamine (31, 0.10 mL, 0.877 mmol) in
THF (8.8 mL) were added NaHCO3 (298 mg, 3.58 mmol) and NsCl (400
mg, 1.80 mmol) at 0 °C. The mixture was stirred at the same temperature
for 10 min. The reaction was quenched with 3N aqueous NaOH. The
mixture was stirred at room temperature for 1 h. The mixture was diluted
with EtOAc, washed with saturated 3N aqueous NaOH, 3N aqueous HCl,
and brine, and then dried over Na2SO4. Concentration and short column
chromatography (hexane/EtOAc
=
4:1) gave the corresponding
sulfonamide (273 mg), which was used for the next step without further
purification. To a solution of the sulfonamide obtained above (273 mg) in
CH3CN (9.6 mL) were added Cs2CO3 (940 mg, 2.89 mmol), TBAI (178
mg, 0.482 mmol), and 4-bromobutyronitrile (32, 0.11 mL, 1.11 mmol) at
room temperature. The mixture was stirred at 60 °C for 2 h. To the
mixture was added 4-bromobutyronitrile (32, 0.22 mL, 2.22 mmol) at
room temperature. The mixture was stirred at 60 °C for 1 h. The mixture
was diluted with EtOAc and washed with H2O. The aqueous phase was
extracted with EtOAc twice and the combined organic phase was washed
with brine and dried over Na2SO4. Concentration and column
chromatography (hexane/EtOAc = 5:1, 2:1) gave nitrile 33 (340 mg,
quant in two steps): yellow oil; Rf = 0.44 (hexane/EtOAc = 1:1); IR (neat)
N-(4-Aminobutyl)cycloheptylamine
(23):
To
a
solution
of
cycloheptylamine (36, 25 μL, 0.197 mmol) in THF (2.0 mL) were added
NaHCO3 (65.2 mg, 0.776 mmol) and NsCl (87.3 mg, 0.394 mmol) at 0 °C.
The mixture was stirred at the same temperature for 10 min. The reaction
was quenched with 3N aqueous NaOH. The mixture was stirred at room
temperature for 30 min. The mixture was diluted with EtOAc, washed
with saturated 3N aqueous NaOH, 3N aqueous HCl, and brine, and then
dried over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 4:1) gave the corresponding sulfonamide (65.2 mg),
which was used for the next step without further purification. To a
solution of the sulfonamide obtained above (65.2 mg) in CH3CN (2.2 mL)
were added Cs2CO3 (214 mg, 0.657 mmol), TBAI (29.6 mg, 80.1 μmol),
and 4-bromobutyronitrile (32, 26 μL, 0.263 mmol) at room temperature.
The mixture was stirred at 60 °C for 1 h. To the mixture was added 4-
bromobutyronitrile (32, 52 μL, 0.526 mmol) at room temperature. The
3095, 2935, 2859, 2246, 1734 cm–1 1H NMR (400 MHz, CDCl3) δ 8.04–
;
8.01 (m, 1 H), 7.73–7.59 (m, 3 H), 3.72–3.65 (m, 1 H), 3.37 (t, J = 7.6 Hz,
2 H), 2.42 (t, J = 7.0 Hz, 2 H), 2.02 (quin, J = 7.0 Hz, 2 H), 1.80–1.60 (m,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000322
European Journal of Organic Chemistry
FULL PAPER
mixture was stirred at 60 °C for 1 h. The mixture was diluted with EtOAc
and washed with H2O. The aqueous phase was extracted with EtOAc
twice and the combined organic phase was washed with brine and dried
over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 2:1) gave nitrile 37 (78.4 mg), which was used for the
next step without further purification. To a solution of sulfonamide 37
obtained above (78.4 mg) in CH3CN (6.5 mL) were added Cs2CO3 (190
mg, 0.582 mmol) and Si-Thiol (1.3 mmol/g, 597 mg, 0.776 mmol) at room
temperature. The mixture was stirred at 80 °C for 6 h. The mixture was
filtered through a Celite pad and washed with EtOAc. Concentration gave
the corresponding amine (33.3 mg), which was used for the next step
without further purification. A mixture of the nitrile obtained above (33.3
mg) and PtO2 (4.2 mg, 18.5 μmol) in EtOH (3.7 mL) and CHCl3 (0.2 mL)
was stirred for 20 h under a H2 atmosphere at room temperature. The
mixture was filtered and washed with CH3OH. Concentration gave N-(4-
aminobutyl)cycloheptylamine (23, 44.1 mg, quant in four steps).
(m, 4 H), 2.46–2.39 (m, 1 H), 1.92–1.64 (m, 5 H), 1.53–1.47 (m, 4 H),
1.34–1.06 (m, 5 H); 13C NMR (100 MHz, (CD3)2SO) δ 55.7, 42.6, 37.9,
28.3, 24.7, 24.1, 23.9, 22.5; HRMS (ESI–TOF) calcd for C10H23N2 [M +
H]+ 171.1861, found 171.1859.
One-Step Synthesis of N-(4-Aminobutyl)cyclopentylamine (21) via
Mono-Reductive Amination: To a solution of cyclopentanone (41, 0.25
mL, 2.82 mmol) in CH3OH (18 mL) were added putrescine (2, 0.79 mL,
8.46 mmol) and Ti(OiPr)4 (1.1 mL, 3.67 mmol) at room temperature. The
mixture was stirred at the same temperature for 3 h. To the mixture was
added NaBH4 (106 mg, 2.82 mmol) at 0 °C. The mixture was stirred at
the same temperature for 30 min. The reaction was quenched with H2O.
The mixture was stirred at room temperature for 20 min. The mixture was
filtered through a Celite pad and washed with EtOAc. The mixture was
washed with 3N aqueous NaOH. The aqueous phase was extracted with
EtOAc three times and the combined organic phase was washed with
basic brine (pH = 11 by adding aqueous NaOH) and dried over Na2SO4.
Concentration and kugelrohr distillation (9 mmHg, 115–130 °C) gave N-
(4-aminobutyl)cyclopentylamine (21, 320 mg, 73%): colorless oil; IR
N-(4-Aminobutyl)-1-ethylpropylamine (20): To
a solution of 1-
ethylpropylamine (38, 25 μL, 0.215 mmol) in THF (2.2 mL) were added
NaHCO3 (72.2 mg, 0.860 mmol) and NsCl (95.3 mg, 0.430 mmol) at 0 °C.
The mixture was stirred at the same temperature for 20 min. The reaction
was quenched with 3N aqueous NaOH. The mixture was stirred at room
temperature for 1 h. The mixture was diluted with EtOAc, washed with
saturated 3N aqueous NaOH, 3N aqueous HCl, and brine, and then dried
over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 4:1) gave the corresponding sulfonamide (55.4 mg),
which was used for the next step without further purification. To a
solution of the sulfonamide obtained above (55.4 mg) in CH3CN (2.0 mL)
were added Cs2CO3 (217 mg, 0.666 mmol), TBAI (31.0 mg, 83.9 μmol),
and 4-bromobutyronitrile (32, 24 μL, 0.242 mmol) at room temperature.
The mixture was stirred at 60 °C for 2 h. To the mixture was added 4-
bromobutyronitrile (32, 48 μL, 0.484 mmol) at room temperature. The
mixture was stirred at 60 °C for 1 h. The mixture was diluted with EtOAc
and washed with H2O. The aqueous phase was extracted with EtOAc
three times and the combined organic phase was washed with brine and
dried over Na2SO4. Concentration and short column chromatography
(hexane/EtOAc = 2:1) gave nitrile 39 (66.3 mg), which was used for the
next step without further purification. To a solution of sulfonamide 39
obtained above (66.3 mg) in CH3CN (6.5 mL) were added Cs2CO3 (201
mg, 0.616 mmol) and Si-Thiol (1.3 mmol/g, 600 mg, 0.780 mmol) at room
temperature. The mixture was stirred at 80 °C for 7 h. The mixture was
filtered through a Celite pad and washed with EtOAc. Concentration gave
the corresponding amine (29.1 mg), which was used for the next step
without further purification. A mixture of the nitrile obtained above (29.1
mg) and PtO2 (4.3 mg, 18.9 μmol) in EtOH (3.8 mL) and CHCl3 (0.2 mL)
was stirred for 20 h under a H2 atmosphere at room temperature. The
mixture was filtered and washed with CH3OH. Concentration gave N-(4-
aminobutyl)-1-ethylpropylamine (20, 40.3 mg, quant in four steps).
(neat) 3370, 3279, 2950, 2860 cm–1 1H NMR (400 MHz, CD3OD) δ 3.04
;
(quin, J = 7.2 Hz, 1 H), 2.64 (t, J = 6.8 Hz, 2 H), 2.57 (t, J = 6.8 Hz, 2 H),
1.92–1.29 (m, 14 H); 13C NMR (100 MHz, (CD3)2SO) δ 58.1, 44.8, 37.9,
28.9, 24.0, 23.5, 22.5; HRMS (ESI–TOF) calcd for C9H21N2 [M + H]+
157.1705, found 157.1698.
One-Step Synthesis of N-(4-Aminobutyl)cycloheptylamine (23) via
Mono-Reductive Amination: To a solution of cycloheptanone (42, 0.30
mL, 2.54 mmol) in CH3OH (16 mL) were added putrescine (2, 0.71 mL,
7.62 mmol) and Ti(OiPr)4 (0.97 mL, 3.30 mmol) at room temperature. The
mixture was stirred at the same temperature for 3 h. To the mixture was
added NaBH4 (101 mg, 2.67 mmol) at 0 °C. The mixture was stirred at
the same temperature for 30 min. The reaction was quenched with H2O.
The mixture was stirred at room temperature for 20 min. The mixture was
filtered through a Celite pad and washed with EtOAc. The mixture was
washed with 3N aqueous NaOH. The aqueous phase was extracted with
EtOAc three times and the combined organic phase was washed with
basic brine (pH = 11 by adding aqueous NaOH) and dried over Na2SO4.
Concentration and kugelrohr distillation (9 mmHg, 150–165 °C) gave N-
(4-aminobutyl)cycloheptylamine (23, 428 mg, 92%): colorless oil; IR
(neat) 3362, 3282, 2923, 2853 cm–1; 1H NMR (400 MHz, CD3OD) δ 2.65–
2.55 (m, 5 H), 1.92–1.35 (m, 19 H); 13C NMR (100 MHz, (CD3)2SO) δ
57.9, 43.1, 37.9, 30.0, 29.0, 27.3, 27.1, 23.3; HRMS (ESI–TOF) calcd for
C11H25N2 [M + H]+ 185.2018, found 185.2019.
One-Step Synthesis of N-(4-Aminobutyl)-1-ethylpropylamine (20) via
Mono-Reductive Amination: To a solution of 3-pentanone (43, 0.30 mL,
2.86 mmol) in CH3OH (18 mL) were added putrescine (2, 0.80 mL, 8.58
mmol) and Ti(OiPr)4 (1.1 mL, 3.72 mmol) at room temperature. The
mixture was stirred at the same temperature for 3 h. To the mixture was
added NaBH4 (108 mg, 2.86 mmol) at 0 °C. The mixture was stirred at
the same temperature for 30 min. The reaction was quenched with H2O.
The mixture was stirred at room temperature for 20 min. The mixture was
filtered through a Celite pad and washed with EtOAc. The mixture was
washed with 3N aqueous NaOH. The aqueous phase was extracted with
EtOAc three times and the combined organic phase was washed with
basic brine (pH = 11 by adding aqueous NaOH) and dried over Na2SO4.
Concentration and kugelrohr distillation (9 mmHg, 110–125 °C) gave N-
(4-aminobutyl)-1-ethylpropylamine (20, 325 mg, 72%): colorless oil; IR
(neat) 3362, 3292, 2928, 2872 cm–1; 1H NMR (400 MHz, CD3OD) δ 2.67–
2.56 (m, 4 H), 2.38 (quin, J = 6.0 Hz, 1 H), 1.52–1.43 (m, 10 H), 0.92–
0.75 (m, 7 H); 13C NMR (100 MHz, (CD3)2SO) δ 59.0, 43.3, 37.9, 24.1,
22.7, 22.4, 21.3, 21.1, 9.0; HRMS (ESI–TOF) calcd for C9H23N2 [M + H]+
159.1861, found 159.1855.
One-Step Synthesis of N-(4-Aminobutyl)cyclohexylamine (22) via
Mono-Reductive Amination: To a solution of cyclohexanone (40, 0.25
mL, 2.42 mmol) in CH3OH (15 mL) were added putrescine (2, 0.68 mL,
7.26 mmol) and Ti(OiPr)4 (0.93 mL, 3.15 mmol) at room temperature. The
mixture was stirred at the same temperature for 3 h. To the mixture was
added NaBH4 (98.7 mg, 2.61 mmol) at 0 °C. The mixture was stirred at
the same temperature for 30 min. The reaction was quenched with H2O.
The mixture was stirred at room temperature for 20 min. The mixture was
filtered through a Celite pad and washed with EtOAc. The mixture was
washed with 3N aqueous NaOH. The aqueous phase was extracted with
EtOAc three times and the combined organic phase was washed with
basic brine (pH = 11 by adding aqueous NaOH) and dried over Na2SO4.
Concentration and kugelrohr distillation (9 mmHg, 140–155 °C) gave N-
(4-aminobutyl)cyclohexylamine (22, 346 mg, 84%): colorless oil; IR (neat)
3358, 3281, 2926, 2852 cm–1 1H NMR (400 MHz, CD3OD) δ 2.65–2.57
;
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000322
European Journal of Organic Chemistry
FULL PAPER
RT-qPCR: Wild type seedlings of Arabidopsis thaliana (Columbia
accession) were grown for 7 days in 670 µl of Murashige-Skoog liquid
medium without (Mock) or with xylemin, xylemin analogues, or
thermospermine at the concentration of 200 µM. Total RNA was isolated
by NucleoSpin® RNA Plant RNA isolation kit (TaKaRa) according to the
manufacture instruction. 0.5 µg of total RNA of each sample was reverse
transcribed to cDNA by ReverTra Ace reverse transcriptase (TOYOBO)
according to the manufacture protocol. Quantitative PCR was performed
in Thermal Cycler Dice Real Time System Lite (TaKaRa) using KOD
SYBR qPCR Mix (TOYOBO) and gene-specific primers (ACL5: 5’-
ACCGT TAACC AGCGA TGCTT T-3’ and 5’-CCGTT AACTC TCTCT
TTGAT TCTTC GATCC-3’, and ACT8: 5’-GTGAG CCAGA TCTTC
ATTCG TC-3’ and 5’-TCTCT TGCTC GTAGT CGACA G-3’). ACT8 was
used as a reference gene for normalization of transcript abundance. RT-
qPCR was conducted in three biological replicates.
[3]
a) T. Kusano, T. Berberich, C. Tateda, Y. Takahashi, Planta 2008, 228,
367–381; b) A. F. Tiburcio, T. Altabella, M. Bitrián, R. Alciázar, Planta
2014, 240, 1–18.
[4]
[5]
a) T. Oshima, J. Biol. Chem. 1979, 254, 8720–8722; b) A. Takano, J.
Kakehi, T. Takahashi, Plant Cell Physiol. 2012, 53, 606–616.
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Acknowledgements
We are grateful to Division of Instrumental Analysis, Okayama
University, for the HRMS and NMR measurements. We
appreciate Okayama Foundation for Science and Technology for
the financial support to H.M. This research was supported by
MEXT/JSPS KAKENHI Grant Numbers 17K05862 (H.T.),
16H01245 (T.T.), and 19K06724 (T.T.).
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Keywords: Amines • Biological activity • Chemical synthesis •
Reductive amination • Structure-activity relationships
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This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000322
European Journal of Organic Chemistry
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Xylemin and its six designed analogues were successfully synthesized. Evaluation of their biological effect on xylem revealed that a
xylemin analogue, N-(4-aminobutyl)cyclopentylamine, could be a novel inhibitor of thermospermine synthesis to enhance specifically
xylem formation.
This article is protected by copyright. All rights reserved.