Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents

Article abstract of DOI:10.1016/j.bmc.2019.01.016

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential i

Full text of DOI:10.1016/j.bmc.2019.01.016

Accepted Manuscript
Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and
antitubercular agents
Charlotte Courtens, Martijn Risseeuw, Guy Caljon, Louis Maes, Anandi Martin,
Serge Van Calenbergh
PII:
S0968-0896(18)31953-9
https://doi.org/10.1016/j.bmc.2019.01.016
BMC 14708
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 November 2018
3 January 2019
13 January 2019
Please cite this article as: Courtens, C., Risseeuw, M., Caljon, G., Maes, L., Martin, A., Van Calenbergh, S., Amino
acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.016
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Amino acid based prodrugs of a fosmidomycin
surrogate as antimalarial and antitubercular agents
a
a
b
b
c
Charlotte Courtens , Martijn Risseeuw , Guy Caljon , Louis Maes , Anandi Martin and Serge
a,*
Van Calenbergh
a
Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000
Ghent, Belgium
b
Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp,
Universiteitsplein 1 (S7), B-2610 Wilrijk, Belgium
c
Medical Microbiology, Institute of Experimental and Clinical Research, Université catholique
de Louvain, Avenue Hippocrate 55, B-1200 Woluwe-Saint-Lambert, Belgium
KEYWORDS fosmidomycin, prodrugs, non-mevalonate pathway, isoprenoid biosynthesis,
malaria, tuberculosis
ABSTRACT Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-D-
xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first
committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in
Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity,
fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is
inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall.
Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues.
We report the application of two amino acid based prodrug approaches on a fosmidomycin
1

surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in
vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs
display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei
malaria mouse model. These results indicate good in vivo antiplasmodial potential.
INTRODUCTION
Despite considerable international efforts, malaria and tuberculosis (TB) control and
elimination remain major challenges. According to the World Health Organization (WHO), 216
million malaria cases and 445,000 malaria-related deaths were reported worldwide in 2016, of
which 90% occurred in Africa. Malaria incidence has decreased significantly since 2010. Since
1
2
014, however, the number of malaria cases is steadily increasing. TB is the world’s deadliest
infectious disease. In 2016, more than 10 million cases of TB and almost 2 million TB-related
deaths, of which approximately 400,000 among people co-infected with HIV, were reported. TB
mortality rates have dropped by 37% between 2010 and 2016. However, the proportion of
multidrug-resistant (MDR) TB cases is steadily increasing. Approximately 5% of active TB
2
,3
cases are MDR, of which 6% are extensively drug-resistant (XDR). In 2009, the first totally
4
drug-resistant (TDR) strains have been detected in India. Due to the rising problem of acquired
resistance against all currently available antimalarial and antitubercular agents, there is a
pressing need for novel therapies. Over the past forty years, only one new synthetic antimalarial
5
(
tafenoquine) has been launched on the market. Plenty of research is being conducted on
antitubercular drugs, recently resulting in the approval of bedaquiline (2012) and delamanid
(2014). However, no new first-line drug has been approved since the introduction of the current
6
short-course chemotherapy in the 1970s. Compounds with a completely novel mechanism of
action (MOA) are preferably pursued, as resistance against these compounds is expected to
2

develop more slowly in comparison with compounds acting on previously addressed targets or
pathways.
An interesting pathway in this respect is the non-mevalonate isoprenoid biosynthesis pathway,
also known as the methylerythritolphosphate (MEP) or 1-deoxyxylulose 5-phosphate (DXP)
7
,8
pathway. This pathway is essential in a number of pathogens, including Plasmodium parasites,
the causative agents of malaria, and Mycobacterium tuberculosis (Mtb), the causative agent of
TB. Humans synthesize the essential isoprenoid building blocks via the evolutionarily distinct
mevalonate pathway. Since the two pathways utilize non-homologous enzymes, selective
9
interference is possible. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the most
extensively studied enzyme of the non-mevalonate pathway and catalyzes the first committed
7,10–12
reaction step.
Fosmidomycin (1, Figure 1) and FR900098 (2, Figure 1) are natural
1
3
antibiotics and potent inhibitors of DXR.
Figure 1. Structural formulae of fosmidomycin, FR900098, their reverse analogues and α-aryl
derivatives.
3

Significant research has been devoted to expanding the structure-activity relationship (SAR) of
these phosphonate antibiotics. This has also resulted in a number of analogues with improved
1
0,12,14,15
DXR inhibitory activity, including the α-aryl derivatives 5-7 (Figure 1).
Fosmidomycin (1,
Figure 1) is a highly hydrophilic compound, mainly due to its phosphonate functionality, which
is charged at physiological pH. As a result, fosmidomycin exhibits suboptimal pharmacokinetic
(PK) properties including moderate oral bioavailability (20-40%), a short plasma half-life (< 2h)
1
6,17
and high rates of parasite recrudescence after monotherapy.
The poor permeation by passive
diffusion of fosmidomycin additionally results in a lack of activity against pathogens not actively
importing the drug, including Mtb. In an attempt to improve these properties, a number of
prodrugs of fosmidomycin (analogues) have been reported and have been shown to possess
1
0,12,18
enhanced in vitro antimalarial and antitubercular activity.
A selection of representative
1
9–23
prodrug derivatives is shown in Figure 2.
4

Figure 2. In vitro antiplasmodial and antitubercular activities of representative prodrug
derivatives of fosmidomycin analogues.
This article reports on the synthesis, in vitro and in vivo antimalarial, and in vitro
antitubercular activity, of two prodrug series of the fosmidomycin surrogate 4 and the α-aryl
analog 7 (Figure 1): the phosphonodiamidates and tyrosine-derived esters (Figure 3). Both series
are inspired by phosphonate and phosphate prodrug strategies successfully applied in the
2
4,25
nucleoside field.
has been shown to increase cellular uptake of antivirals.
valid in Mtb, as it enables cellular uptake of acyclic nucleoside phosphonates (ANPs), resulting
The phosphonodiamidate prodrug approach, developed by McGuigan et al.,
2
6,27
This approach has also proven to be
2
8
in highly active antituberculosis agents. The putative cleavage mechanism of these
phosphonodiamidates relies on two enzymatic steps, catalyzed by carboxypeptidase and
2
4
phosphoramidase. The tyrosine prodrug approach, developed by McKenna and coworkers, has
been shown to lead to significantly enhanced oral bioavailability of ANPs. Release of the
2
9
prodrug promoiety is mediated by enzymatic or chemical hydrolysis. We hypothesized that
corresponding prodrugs of 4 and 7 (Figure 1) could exhibit enhanced in vitro antimalarial and
antitubercular activity as a result of increased cellular uptake.
Figure 3. Target compounds: phosphonodiamidate and tyrosine ester series.
5

RESULTS AND DISCUSSION
Synthesis. The synthesis of the phosphonamidates started from 4-bromo-1-butene 9 (Scheme
1
). Substitution with diethyl phosphite yielded diethylphosphonate 10. TMSBr-mediated
deprotection, followed by chlorination with oxalyl chloride provided phosphonic dichloride 12.
Treatment with the appropriate amino acid (AA) derivatives (For AA structures, see Table 1)
yielded the symmetric phosphonamides 13a-k. Hydroboration-oxidation with 9-BBN produced
primary alcohols 14a-k, which were subsequently converted to carboxylic acids 15a-k using
TEMPO-BAIB oxidation. EDC-coupling with benzyl-protected hydroxylamine 16 gave
protected hydroxamates 18a-j. Catalytic hydrogenolysis afforded the corresponding free
hydroxamates 19a-j. For the synthesis of the benzyl L-leucine diamidate 19k orthogonal
protection of the hydroxamate was required. Therefore, carboxylic acid 15k was EDC coupled
with TBS-protected hydroxylamine 17 .
a
Scheme 1. Synthesis of NH-linked amino acid based prodrugs
a
Reagents and conditions: (a) (i) (EtO) PH, NaH, THF, 0 °C; (ii) 4-bromo-1-butene 9 (78%);
2
(
b) (i) TMSBr, DCM; (ii) H O, THF; (c) oxalyl chloride, DMF, DCM, 50 °C; (d) AA alkyl ester
2
HCl, DIPEA, pyridine, DCM (35-80%); e) (i) 9-BBN, THF; (ii) SPB.H O, H O (24-86%); (f)
2
2
TEMPO, BAIB, NaHCO , MeCN/H O (34-84%); (g) EDC.HCl, HOBt, BnONHMe (16) or
3
2
TBSONHMe (17), Et N, DCM (33-91%); (h) H , Pd/C, solvent (50%-quantitative).
3
2
6

Interestingly, during the coupling of 15k with 17, the silyl group was unexpectedly removed in
situ, immediately yielding final compound 19k in an acceptable yield of 33%. For the synthesis
of L-alanine ethyl ester prodrug derivative 28, hydroboration of the terminal alkene intermediate
was attempted using a range of different reaction conditions, but failed to produce the desired
product. Therefore, an alternative synthesis route was envisaged (Scheme 2).
a
Scheme 2. Synthesis of NH-linked L-alanine based prodrug
a
Reagents and conditions: (a) (EtO) P, reflux, 71%; (b) (i) TMSBr, DCM; (ii) H O, THF; (c)
3
2
oxalyl chloride, DMF, DCM, 50 °C; (d) L-Ala OEt.HCl, DIPEA, pyridine, DCM (37% over 3
steps); (e) H , Pd/C, EtOH (89%); (f) TEMPO, BAIB, NaHCO , MeCN/H O (53%); (g)
2
3
2
EDC.HCl, HOBt, BnONHMe (16), Et N, DCM (79%); (h) H , Pd/C, EtOH (91%).
3
2
The synthesis of 28 started from benzyl 4-bromobutyl ether 20. Arbuzov reaction with triethyl
phosphite yielded phosphonate 21. TMSBr-mediated deprotection followed by chlorination with
oxalyl chloride produced phosphonic dichloride 23. Treatment with L-alanine ethyl ester yielded
phosphonamide 24, which was converted into the primary alcohol 25 upon hydrogenation.
TEMPO-BAIB oxidation and EDC coupling of the resulting carboxylic acid 26 with benzyl-
protected hydroxylamine 16 gave protected hydroxamate 27. Debenzylation by hydrogenation
7

afforded final compound 28. Tyrosine based prodrugs 37a and 37b (Table 1) were synthesized as
2
2
described in our previous work using a cross metathesis synthetic approach (Scheme 3).
a
Scheme 3. Synthesis of O-linked L-tyrosine based prodrugs
a
Reagents and conditions: (a) triethyl phosphite, 150 °C (97%); (b) (i) TMSBr, DCM; (ii) H O,
2
THF; (c) oxalyl chloride, DMF, DCM, 45 °C; (d) N-acetyl L-tyrosine OEt or N-Cbz L-tyrosine
OEt, DIPEA, pyridine, DCM (60−68% over 3 steps); (e) Hoveyda−Grubbs second generation
catalyst, toluene, 70 °C (67%); (f) NiCl ·6H O, NaBH , THF (50−65%); (g) H , Pd/C, EtOH
2
2
4
2
(55−90%).
In addition, two prodrug promoieties were selected for the synthesis of prodrugs of the α-3,4-
dichlorophenyl analog 7 (Figure 1). Their synthesis started from 3,4-dichlorobenzyl bromide 38
(Scheme 4). Arbuzov reaction with triethyl phosphite yielded diethylphosphonate 39. Alkylation
with allyl bromide in the presence of n-butyllithium afforded 40. Transformation into the
corresponding phosphonamides 43a-b was performed using the same reaction sequence as
before (40 →4 1 →4 2 →4 3). Hydroboration with BH , followed by TEMPO-BAIB oxidation gave
3
carboxylic acids 45a-b. Attempts to couple the carboxylic acids 45a-b with TBDPS-protected
hydroxylamine 46 using a number of different coupling agents, including EDC, HATU, CDI and
8

a
Scheme 4. Synthesis of prodrugs of α-3,4-dichlorophenyl analog 7
a
Reagents and conditions: (a) (OEt) P, reflux, 92%; (b) (i) n-BuLi, THF, ˗78 °C; (ii) allyl
3
bromide (70%); (c) (i) TMSBr, DCM; (ii) H O, THF; (d) oxalyl chloride, DMF, DCM, 50 °C;
2
(
e) AA alkyl ester HCl, DIPEA, pyridine, DCM (43–98% over 3 steps); (f) (i) BH .THF, THF;
3
(
ii) SPB.H O, H O (35-80%); (g) TEMPO, BAIB, NaHCO , MeCN/H O (66-79%); (h) (i) 4-
2
2
3
2
methylmorpholine, ethyl chloroformate, ˗20 °C; (ii) TBDPSONHMe (46) (23-36%).
PyBOP, failed to yield acceptable amounts of desired products. Eventually, the desired
3
0
compounds 47a-b were obtained via the mixed anhydride method using ethyl chloroformate.
Also in this case, the silyl protecting groups were removed in situ to yield the corresponding free
hydroxamic acids 47a-b.
Biological Evaluation. Table 1 summarizes the in vitro inhibitory activity on the growth of
M. tuberculosis (H37Rv) and asexual blood stages of P. falciparum (Pf-K1). The cytotoxicity of
all compounds was assessed using human fibroblasts (MRC-5). Of the phosphonamidate
prodrugs evaluated against P. falciparum, only L-lysine derivative 19e displayed submicromolar
activity. All other phosphonamidates displayed antiplasmodial activity comparable or inferior to
fosmidomycin. Furthermore, derivatives 19i-k suffered from MRC-5 cell toxicity. Interestingly,
9

tyrosine esters 37a and 37b displayed better antiplasmodial activity than fosmidomycin. Both
9e
1
a
Table 1. In vitro biological evaluation of amino acid based prodrugs
Pf-K1
Ester
H37Rv
MRC-5
Compound
1
Amino acid (AA)
2
R
(
R )
IC [µM]
MIC [µM] [µM]
5
0
31
19
-
-
-
1.73
>500
>64
phosphonodiamidate series
1
1
1
1
1
1
1
1
1
1
1
2
4
9a
9b
9c
9d
9e
9f
L-Val
L-Phe
L-Pro
L-iLe
L-Lys
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Ala
L-Ala
Et
H
12.7
>50
>50
>50
>50
>50
>50
50
>64
Et
H
4.71 (±0.05)
10.2 (±8.2)
2.00
>64
Et
H
>64
Et
H
>64
Et
H
0.96 (±0.35)
1.76
>64
Me
Et
H
>64
9g
9h
9i
H
3.91 (±2.85)
4.97
>64
iPr
Pentyl
cHx
Bn
Et
H
>50
>50
>50
>50
20
>64
H
2.37
7.46
7.65
7.94 (±3.22)
>64
9j
H
4.56
9k
8
H
2.02 (±0.89)
5.06 (±0.54)
>36.6
H
7a
Et
3,4-dichlorophenyl
>50
>64
tyrosine ester series
7a
N-Acetyl L-Tyr
3
Et
H
0.31
>50
>64
1
0

3
4
7b
7b
L-Tyr
Et
Et
H
0.23
>50
>50
>64
N-Acetyl L-Tyr
3,4-dichlorophenyl
>41.1
36.9 (±5.5)
a
values for which standard deviations are given are the calculated mean values of at least two
measurement results
and 37b are positively charged at physiological pH, which does not seem to hamper transport
into the infected red blood cells (RBCs). Ethyl ester L-leucine derivative 19g and ethyl ester L-
alanine derivative 28 displayed moderate antitubercular activities, with MIC values of 50 µM and
2
0 µM. All other compounds did not show any activity against M. tuberculosis, presumably as a
result of lack of uptake or efflux. This observation led us to also test the L-alanine and N-acetyl
L-tyrosine prodrug derivatives of α-3,4-dichlorophenyl analog 7, which compared to
fosmidomycin is more active against PfDXR (IC50 = 2.8 nM) and equally active against
14,15
MtbDXR (IC = 280 nM).
Surprisingly, however, compound 47a was inactive against M.
50
tuberculosis and the antiplasmodial activity of 47b was clearly inferior to that of derivative 37a.
These results are in marked contrast to the previously reported activities of prodrugs of the
2
0
corresponding α-3,4-difluorophenyl analog with antiplasmodial IC values up to 4 nM.
5
0
Compounds 8, 19e and 37a, belonging to different prodrug classes, were selected for
evaluation in a P. berghei malaria mouse model. The isopropyloxycarbonyloxymethyl (POC)
prodrug 8 (Figure 2) was previously reported by us to display excellent in vitro antiplasmodial
22
activity (Pf –K1 IC of 110 nM) . The compounds were dosed intraperitoneally (ip) at 50 mg/kg
50
for 5 consecutive days. Chloroquine (10 mg/kg for 5 days) was included as reference treatment.
Parasitemia was determined on days 4, 7, 11 and 14 on surviving animals using flow cytometry.
Percentage reduction of parasitemia and mean survival time (MST) of mice compared to vehicle-
treated infected controls is used as a measure for drug activity (Table 2). The results in Table 2
show that compound 19e failed to show relevant activity. On the other hand, 37a resulted in 82%
suppression of parasitemia at 4 days post infection (dpi), which dropped to 66% at 7 dpi and 50%
1
1

at 14 dpi. The mean survival time was 12.7 days. In line with its potent in vitro antiplasmodial
activity data, compound 8 was superior to the others, resulting in 99% suppression of
parasitaemia
Table 2. In vivo biological evaluation of selected prodrug derivatives in P. berghei infected
mice.
mean %
suppression
of infected
mean % infected RBC
RBC at 4
a
Treatment
ip for 5
dpi
(
b
MST
consecutive
days)
4
dpi
8.6
4dpi
7dpi
11dpi
14dpi
64.6
Vehicle
17.1
0.2
6/6
6/6
19.0
0.6
4/6
6/6
44.0
11.9
3/6
6/6
2/6
0/6
11.2
14.0
chloroquine
10 mg/kg)
9
(
1
9e
0
.6
17.1
0.2
6/6
6/6
6/6
30.1
0.04
6.5
2/6
6/6
6/6
34.7
3.7
2/6
6/6
3/6
0/6
4/6
2/6
8.7
(
50mg/kg)
8
9
9
2
16.4
32.6
15.3
12.7
(
(
50mg/kg)
37a
8
3.0
29.2
50mg/kg)
a
dpi = days post infection
MST = mean survival time
b
at 4 dpi, which dropped slightly to 92% at 7 dpi and 75% at 14 dpi. The overall MST was 15.3
days. These data demonstrate that the tyrosine ester prodrug 37a exhibits moderate in vivo
antiplasmodial activity, while POC-prodrug 8 compares favorably with the reference chloroquine
treatment. The weak in vivo activity of 19e is possibly the result of chemical instability,
metabolic instability and/or insufficient bioactivation.
1
2

CONCLUSION
In conclusion, we report here on the synthesis, antiplasmodial and antimycobacterial activity of
a series of 14 amino acid-based prodrug derivatives of fosmidomycin surrogate 4 (Figure 1) and
2
prodrug derivatives of the α-3,4-dichlorophenyl analog 7 (Figure 1). Ethyl L-alanine and ethyl
L-leucine diamidate derivatives resulted in moderate in vitro inhibitory activity of M.
tuberculosis (H37Rv) growth. These findings suggests that a phosphonamidate prodrug approach
may enable uptake of fosmidomycin surrogate 4 in mycobacterial cells. Furthermore, both L-
tyrosine-derived ester prodrug derivatives of 4 displayed potent in vitro antiplasmodial (Pf-K1)
activity. Preliminary in vivo experiments indicate that the promising in vitro activity of the POC-
prodrug 8 and N-acetyl L-tyrosine ester 37a is reflected in the P. berghei malaria mouse model,
while the L-lysine derivative 19e failed to show significant in vivo activity despite its promising
in vitro activity.
EXPERIMENTAL SECTION
General. All reactions were performed using oven dried round-bottomed flasks sealed with
rubber septa, unless otherwise stated. Reactions were magnetically stirred using teflon-coated stir
bars. Where appropriate, reactions were carried out in dry solvents and under an inert nitrogen
atmosphere. Dropwise addition of reagents or solutions was carried out using a syringe pump.
1
Yields refer to chromatographically and spectroscopically ( H NMR) pure homogeneous
materials. Reagents were purchased at the highest commercial quality and used without
additional purification, unless otherwise stated. Hexanes for flash column chromatography were
distilled prior to use. Reactions were monitored by thin layer chromatography (TLC) carried out
on precoated Macherey-Nagel® SIL G/UV254 plates using ultraviolet light (254 nm wave
length) as visualizing agent and either potassium permanganate or ceric ammonium molybdate
1
3

(CAM) as developing agents. Flash column chromatography was performed manually using
Grace Davisil® silica gel (40-60 µm particle size) or automatically using a Grace Reveleris X2
purification system with flash cartridges. NMR spectra were recorded at 25 °C on a Varian
1
Mercury-300 spectrometer. H NMR spectra were calibrated using either TMS as a reference
1
(
TMS: H NMR = 0.00) or residual undeuterated solvent as an internal reference (METHANOL:
1
13
H NMR = 3.31). C NMR spectra were calibrated using residual undeuterated solvent as an
1
3
13
internal reference (CHLOROFORM-d: C NMR = 77.16, METHANOL-d4: C NMR = 49.00).
3
1
In P NMR, signals are referenced to the CHLOROFORM-d lock resonance frequency
according to IUPAC referencing, with H PO set to 0.00 ppm. The following abbreviations or
3
4
combinations thereof were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q
quartet, m = multiplet, b = broad signal. Chemical shifts are expressed in ppm and coupling
=
constants are given in Hertz (Hz). Weak carbon signals were assigned using HSQC experiments.
High resolution mass spectra (HRMS) were recorded on a Waters LCT Premier XE TOF
equipped with an electrospray ionization (ESI-MS) interface and a modular Lockspray TM
interface. Samples were infused in a MeCN/water (1:1) + 0.1% formic acid mixture at a rate of
1
00 μL/min. LC-MS analyses were carried out on a Waters AutoPurification System equipped
with PDA and ESI-MS detection and using a Waters CORTECS C18 Column (4.6 × 100 mm,
.7 µm) and a water/acetonitrile/formic acid linear gradient system at a flow rate of 1.44 mL/min.
2
Preparative HPLC purifications were carried out on a Waters AutoPurification System equipped
with PDA and ESI-MS detection and using a Waters Xbridge C18 column (19 × 250 mm, 5µm)
and a water/acetonitrile/formic acid linear gradient system at a flow rate of 20 mL/min.
Materials. Final compound 8 and protected hydroxylamines 16 and 46 have been previously
22
reported by us. Hydroxylamine 17 was synthesized in the same way as 46.
1
4

General procedure 1 for TMSBr-mediated deprotection. Phosphonate ethyl ester (1 eq) was
dissolved in DCM (0.1 M) and cooled to 0 °C in an ice bath. TMSBr (5 eq) was added and the
3
1
solution was subsequently stirred for 10 min at 0 °C and 2 h at RT. P NMR confirmed
completion of the reaction, after which all volatiles were removed in vacuo. The crude oil was
dissolved in THF (0.3 M) and treated with H O (3 M). The reaction was stirred for 1 h at RT,
2
after which all volatiles were removed in vacuo. The residue was coevaporated three times with
toluene in order to remove all traces of water. The resulting crude material was dried overnight at
high vacuum and immediately used in the next reaction without further purification or
characterization.
General procedure 2 for phosphonic dichloride formation. A solution of crude phosphonic
acid (1 eq) in DCM (0.4 M) was heated to 50 °C. After addition of a catalytic amount of DMF,
3
1
oxalyl chloride (5 eq) was added dropwise over 30 minutes. After 3 h at 50 °C, P NMR
confirmed completion of the reaction, after which all volatiles were removed in vacuo. The
resulting crude material was immediately used in the next reaction without further purification or
characterization.
General procedure 3 for phosphonamide formation. A solution of crude phosphonic
dichloride (1 eq) in DCM (0.2 M) was cooled to 0 °C in an ice bath. DIPEA (6 eq) was added to
a solution of the corresponding amino acid (2.5 eq) in DCM (0.3 M). Anhydrous pyridine (1 eq)
was added to the first solution, followed by addition of the second to the first solution. The
reaction mixture was stirred overnight, slowly warming to RT. After overnight stirring, the
reaction mixture was further diluted with DCM and washed with 0.1 M HCl (aq. soln.) and
NaHCO (sat. aq. soln.). The organic layer was dried over Na SO , filtered and concentrated in
3
2
4
vacuo. The products were purified by column chromatography with appropriate eluents.
1
5

General procedure 4 for hydroboration-oxidation. 9-BBN in THF 0.5 M (4 eq) was added
to terminal alkene (1 eq). The resulting solution was allowed to stir overnight at RT, after which
H O (0.13 M) and SPB.H O (8 eq) were added. The reaction mixture was stirred vigorously at
2
2
RT for 3 h, after which LC-MS confirmed completion of the reaction. The reaction mixture was
filtered and concentrated in vacuo. The products were purified by column chromatography with
appropriate eluents.
General procedure 5 for TEMPO-BAIB oxidation. To a solution of primary alcohol (1 eq)
in MeCN (0.2 M) were added consecutively H O (0.2 M), TEMPO (0.3 eq), BAIB (3 eq) and
2
NaHCO (1.5 eq). The reaction was protected from light and stirred overnight at RT. The
3
reaction mixture was partially evaporated in vacuo in order to remove MeCN. The crude was
further diluted with H O, the pH of the reaction was subsequently lowered to pH 2 with 1 M HCl
2
(aq. soln.) and the reaction mixture was extracted three times with EtOAc. The combined organic
layers were dried over Na SO , filtered and concentrated in vacuo The products were purified by
2
4
column chromatography with appropriate eluents.
General procedure 6 for EDC-mediated coupling. EDC.HCl (1.3 eq) and HOBt (1 eq) were
added to a solution of acid (1 eq) in DCM (0.3 M). A solution of hydroxylamine (1.3 eq) and
Et N (4 eq) in DCM (0.6 M) was added to the first solution and the reaction was allowed to stir
3
overnight at RT. The reaction mixture was further diluted with DCM and washed with H O,
2
citric acid (1 M aq. soln.) and brine. The organic layer was dried over Na SO , filtered and
2
4
concentrated in vacuo. The products were purified by column chromatography with appropriate
eluents.
General procedure 7 for hydrogenation. Benzyl protected compounds (1 eq) were dissolved
in MeOH, EtOH, iPrOH or EtOAc (+ formic acid, 1 eq) (0.05 M). A catalytic amount of Pd/C
1
6

was added to the reaction mixture. The reaction was stirred for 30 minutes under a hydrogen
atmosphere, after which TLC analysis confirmed complete conversion of the starting material.
The reaction mixture was filtered and all volatiles were removed in vacuo. The products were
purified by column chromatography with appropriate eluents.
General procedure 8 for cross metathesis. Phosphonate esters (1 eq) and benzyl-protected
hydroxamate (4 eq) were dissolved in toluene (0.1 M). This solution was heated to 70 °C.
Grubbs Hoveyda 2 catalyst (0.05 eq) was dissolved in a small amount of toluene and a volume
corresponding to 0.01 eq of catalyst was added to the reaction mixture every hour. After
overnight stirring at 70 °C, all volatiles were removed in vacuo and the resulting crude was
purified by flash column chromatography with appropriate eluents.
General procedure 9 for nickel boride reduction. Unsaturated compounds (1 eq) were
dissolved in THF (0.1 M).The resulting solution was cooled to 0 °C in an ice bath and
NiCl .6H O (2 eq) and NaBH (4 eq) were subsequently added. After 1 h, HRMS confirmed
2
2
4
complete conversion of the starting material. The reaction was quenched with NH Cl (sat. aq.
4
soln.) and was vigorously stirred at RT for 2 h until a clear blue solution was obtained. The
reaction mixture was extracted three times with DCM. The organic layer was dried over Na SO ,
2
4
filtered and concentrated in vacuo. The products were purified by column chromatography with
appropriate eluents.
Diethyl but-3-en-1-ylphosphonate (10). A solution of diethyl phosphite (0.64 mL, 5.0 mmol)
in dry THF (5 mL) was cooled to 0°C in an ice bath. NaH (0.20 g, 5.0 mmol) was added
gradually and the reaction mixture was allowed to warm to RT. After 1 h, 4-bromo-1-butene
(
0.76 mL, 7.5 mmol) was added. The reaction was quenched with NH Cl (sat. aq. soln.) after
4
overnight stirring and extracted three times with DCM. The organic layer was dried over
1
7

Na SO , filtered and concentrated in vacuo. Column chromatography (DCM  4% MeOH in
2
4
1
DCM) yielded pure 10 as a colorless oil (78%). H NMR (300 MHz, CHLOROFORM-d) δppm
1
5
.26 (t, J = 7.1 Hz, 6 H), 1.69 - 1.83 (m, 2 H), 2.21 - 2.35 (m, 2 H), 3.94 - 4.14 (m, 4 H), 4.91 -
1
3
.04 (m, 2 H), 5.79 (ddt, J = 16.9, 10.4, 6.4 Hz, 1 H). C NMR (75MHz, CHLOROFORM-d) δ
3
1
2
2
ppm 16.39 (d, J = 5.8 Hz), 24.96 (d, J = 140.5 Hz), 26.46 (d, J = 4.6 Hz), 61.41 (d, J
C-P
C-P
C-P
C-P
3
31
=
6.9 Hz), 115.01, 137.17 (d, J = 18.4 Hz). P NMR (121 MHz, CHLOROFORM-d) δppm =
C-P
+
3
1.45. HRMS (ESI-MS): calculated for C H O P [M+H] : 193.0988, found: 193.0987.
8
18
3
But-3-en-1-ylphosphonic acid (11). Following general procedure 1, 10 (0.58 g, 3.0 mmol)
afforded 11 as a crude material, which was dried overnight at high vacuum and immediately used
in the next reaction without further purification or characterization.
But-3-en-1-ylphosphonic dichloride (12). Following general procedure 2, 11 (0.54 g, 4.0
mmol) afforded 12 as a crude material, which was immediately used in the next reaction without
further purification or characterization.
Diethyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(3-methylbutanoate)
(13a). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded 13a (71%) as a faint
1
yellow oil after purification by column chromatography (DCM  3% EtOH in DCM). H NMR
(300 MHz, CHLOROFORM-d) δ ppm 0.87 (dd, J = 9.5, 6.9 Hz, 6 H), 0.98 (dd, J = 6.8, 5.5 Hz,
6
3
1
H), 1.23 - 1.33 (m, 6 H), 1.75 - 1.89 (m, 2 H), 2.01 - 2.18 (m, 2 H), 2.30 - 2.45 (m, 2 H), 2.83 -
.00 (m, 2 H), 3.73 - 3.91 (m, 2 H), 4.09 - 4.28 (m, 4 H), 5.00 - 5.06 (m, 1 H), 5.10 (dq, J = 17.1,
1
3
.6 Hz, 1 H), 5.87 (ddt, J = 16.9, 10.3, 6.4 Hz, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ
2
1
3
ppm 14.23, 17.3, 17.5, 19.0, 27.1 (d, J = 3.5 Hz), 28.7 (d, J = 112.9 Hz), 32.0 (d, J = 4.6
C-P
C-P
C-P
3
3
3
Hz), 32.2 (d, J = 5.8 Hz), 57.7, 58.5, 61.0, 61.1, 115.4, 137.8 (d, J = 15.0 Hz), 173.9 (d, J
C-P
C-P
C-
1
8

3
31
=
2.3 Hz), 174.1 (d, J = 2.3 Hz). P NMR (121 MHz, CHLOROFORM-d) δ ppm 29.67.
P
C-P
+
HRMS (ESI-MS): calculated for C H N O P [M+H] : 391.2356, found: 391.2367.
18
36
2
5
Diethyl 2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(3-phenylpropanoate)
(13b). Following general procedure 3, crude 12 (0.35 g, 2.0 mmol) afforded 13b (44%) as a
1
yellow oil after purification by column chromatography (DCM  3.5% EtOH in DCM). H
NMR (300 MHz, CHLOROFORM-d) δ ppm 1.18 - 1.29 (m, 6 H), 1.41 - 1.61 (m, 2 H), 1.98 -
2
4
.17 (m, 2 H), 2.46 (dd, J = 13.0, 11.1 Hz, 1 H), 2.83 - 3.10 (m, 5 H), 4.02 - 4.30 (m, 6 H), 4.87 -
1
3
.98 (m, 2 H), 5.67 (ddt, J = 16.9, 10.4, 6.3 Hz, 1 H), 7.08 - 7.34 (m, 10 H). C NMR (75 MHz,
2
1
CHLOROFORM-d) δ ppm 14.2, 14.3, 26.9 (d, J = 4.6 Hz), 28.5 (d, J = 115.2 Hz), 40.8 (d,
C-P
C-P
3
3
JC-P = 4.6 Hz), 41.3 (d, J = 4.61 Hz), 53.9, 54.3, 61.41, 61.44, 115.3, 127.08, 127.11, 128.6,
C-P
3
3
31
1
29.7, 129.8, 136.4, 136.7, 137.6 (d, J = 16.1 Hz), 173.3, 173.5 (d, J = 2.3 Hz). P NMR
C-P
C-P
(
121 MHz, CHLOROFORM-d) δ ppm 28.75. HRMS (ESI-MS): calculated for C H N O P
2
6
36
2
5
+
[
M+H] : 487.2356, found: 487.2348.
Diethyl (but-3-en-1-ylphosphoryl)(S)-di-L-prolinate (13c). Following general procedure 3,
crude 12 (0.69 g, 4.0 mmol) afforded 13c (80%) as a yellow oil after purification by column
1
chromatography (toluene  70% acetone in toluene). H NMR (300 MHz, CHLOROFORM-d)
δ ppm 1.27 (t, J = 7.1 Hz, 6 H), 1.71 - 2.05 (m, 8 H), 2.08 - 2.34 (m, 2 H), 2.34 - 2.68 (m, 2 H),
3
4
.12 - 3.39 (m, 3 H), 3.53 (dq, J = 9.3, 6.6 Hz, 1 H), 4.10 – 4.21 (m, 4 H), 4.21 – 4.33 (m, 2 H),
1
3
.90 - 5.09 (m, 2 H), 5.86 (ddt, J = 16.9, 10.3, 6.3 Hz, 1 H). C NMR (75 MHz,
3
3
1
CHLOROFORM-d) δ ppm 14.2, 25.1 (d, J = 2.3 Hz), 25.2 (d, J = 2.3 Hz), 25.9 (d, J
=
C-P
C-P
C-P
2
3
3
1
12.9 Hz), 26.1 (d, J = 3.5 Hz), 31.6 (d, J = 5.8 Hz), 31.9 (d, J = 5.8 Hz), 46.1, 47.0 (d,
C-P
C-P
C-P
2
2
2
3
JC-P = 4.6 Hz), 59.1 (d, J = 5.8 Hz), 59.4 (d, J = 4.6 Hz), 60.8, 60.9, 114.6, 138.1 (d, J
=
C-P
C-P
C-P
1
9

31
1
8.4 Hz), 174.5, 174.6. P NMR (121 MHz, CHLOROFORM-d) δ ppm 30.09. HRMS (ESI-
+
MS): calculated for C H N O P [M+H] : 387.2043, found: 387.2050.
18
32
2
5
Diethyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S,3R,3'R)-bis(3-
methylpentanoate) (13d). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded
1
3d (35%) as a faint yellow foam after purification by column chromatography (DCM  2.5%
1
EtOH in DCM). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.84 - 1.01 (m, 12 H), 1.01 -
1
.23 (m, 2 H), 1.23 - 1.33 (m, 6 H), 1.33 - 1.52 (m, 2 H), 1.71 - 1.91 (m, 4 H), 2.27 - 2.44 (m, 2
H), 2.81 - 2.99 (m, 2 H), 3.86 (dddd, J = 17.6, 11.0, 9.7, 4.7 Hz, 2 H), 4.06 - 4.29 (m, 4 H), 4.98 -
1
3
5
.16 (m, 2 H), 5.86 (ddt, J = 16.9, 10.3, 6.4 Hz, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ
2
1
ppm 11.8, 11.9, 14.4, 15.57, 15.60, 24.9, 25.2, 27.2 (d, JC-P = 3.5 Hz), 28.8 (d, J = 111.7 Hz),
C-P
3
3
3
3
1
2
9.3 (d, J = 4.6 Hz), 39.5 (d, J = 4.6 Hz), 57.2, 578.0, 61.1, 61.2, 115.5, 137.9 (d, J
=
C-P
C-P
C-P
3
31
5.0 Hz), 173.9, 174.1 (d, J = 2.3 Hz). P NMR (121 MHz, CHLOROFORM-d) δ ppm
C-P
+
9.43. HRMS (ESI-MS): calculated for C H N O P [M+H] : 419.2669, found: 419.2678.
2
0
40
2
5
Diethyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-
bis(6{[(benzyloxy)carbonyl] amino} hexanoate) (13e). Following general procedure 3, crude
1
2 (1.0 g, 6.0 mmol) afforded 13e (59%) as a yellow oil after purification by column
1
chromatography (DCM  5% EtOH in DCM). H NMR (300 MHz, CHLOROFORM-d) δ ppm
1
3
.18 - 1.32 (m, 6 H), 1.32 - 1.66 (m, 10 H), 1.69 - 1.87 (m, 4 H), 2.22 – 2.41 (m, 2 H), 2.88 –
.06 (m, 2 H), 3.09 - 3.26 (m, 4 H), 3.81 – 4.01 (m, 2 H), 4.09 - 4.26 (m, 4 H), 4.97 – 5.05 (m, 2
1
3
H), 5.08 (s, 4 H), 5.23 – 5.42 (m, 2 H), 5.72 – 5.90 (m, 1 H), 7.28 - 7.38 (m, 10 H). C NMR (75
2
1
MHz, CHLOROFORM-d) δ ppm 14.3, 22.0, 22.2, 27.0 (d, JC-P = 3.5 Hz), 28.7 (d, J = 112.9
C-P
Hz), 28.9, 29.1, 33.9, 34.5, 40.6, 40.8, 52.3, 53.1, 61.4, 61.6, 66.6, 115.6, 128.1, 128.6, 136.9,
2
0

3
31
1
2
37.6 (d, J = 15.0 Hz), 156.7, 174.2, 174.3. P NMR (121 MHz, CHLOROFORM-d) δ ppm
C-P
+
9.43. HRMS (ESI-MS): calculated for C H N O P [M+H] : 717.3623, found: 717.3592.
3
6
54
4
9
Dimethyl 2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-methylpentanoate)
(13f). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded 13f (59%) as a
1
yellow foam after purification by column chromatography (DCM  3.5% MeOH in DCM). H
NMR (300 MHz, CHLOROFORM-d) δ ppm 0.89 - 0.98 (m, 12 H), 1.40 - 1.87 (m, 8 H), 2.25 -
2
1
.42 (m, 2 H), 2.80 (br s, 2 H), 3.72 (d, J = 3.2 Hz, 6 H), 3.90 - 4.03 (m, 2 H), 5.02 (dq, J = 10.2,
1
3
.4 Hz, 1 H), 5.09 (dq, J = 17.1, 1.6 Hz, 1 H), 5.85 (ddt, J = 16.9, 10.3, 6.4 Hz, 1 H). C NMR
2
(
75 MHz, CHLOROFORM-d) δ ppm 22.1, 22.2, 22.8, 22.9, 24.7, 24.8, 27.1 (d, J = 3.5 Hz),
C-P
1
3
2
1
8.6 (d, J = 112.9 Hz), 44.1, 44.2, 44.3, 51.3, 52.0, 52.2, 115.51, 137.8 (d, J = 16.1 Hz),
C-P
C-P
31
75.5, 175.56, 175.60. P NMR (121 MHz, CHLOROFORM-d) δ ppm 28.85. HRMS (ESI-
+
MS): calculated for C H N O P [M+H] : 391.2356, found: 391.2353.
18
36
2
5
Diethyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-methylpentanoate)
(13g). Following general procedure 3, crude 12 (0.86 g, 5.0 mmol) afforded 13g (62%) as a
1
colorless foam after purification by column chromatography (DCM  4.5% EtOH in DCM). H
NMR (300 MHz, CHLOROFORM-d) δ ppm 0.88 - 1.00 (m, 12 H), 1.28 (td, J = 7.1, 5.1 Hz, 6
H), 1.42 - 1.87 (m, 8 H), 2.25 - 2.43 (m, 2 H), 2.71 - 2.84 (m, 2 H), 3.88 – 4.01 (m, 2 H), 4.08 -
4
.26 (m, 4 H), 4.98 - 5.05 (m, 1 H), 5.09 (dq, J = 17.1, 1.6 Hz, 1 H), 5.85 (ddt, J = 17.0, 10.3, 6.4
1
3
Hz, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.3, 22.1, 22.2, 22.8, 22.9, 24.7, 24.8,
2
1
3
3
2
5
7.1 (d, J = 3.5 Hz), 28.7 (d, J = 112.9 Hz), 44.1 (d, J = 5.8 Hz), 44.3 (d, J = 4.6 Hz),
C-P
C-P
C-P
C-P
3
31
1.3, 52.1, 61.2, 61.3, 115.4, 137.8 (d, J
= 16.1 Hz), 175.0. P NMR (121 MHz,
C-P
+
CHLOROFORM-d) δ ppm 28.56. HRMS (ESI-MS): calculated for C H N O P [M+H] :
2
0
40
2
5
4
19.2669, found: 419.2659.
2
1

Diisopropyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-
methylpentanoate) (13h). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded
1
3h (56%) as a faint yellow foam after purification by column chromatography (DCM  4%
1
iPrOH in DCM). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.85 - 1.03 (m, 12 H), 1.19 -
1
3
.31 (m, 12 H), 1.39 - 1.63 (m, 4 H), 1.65 - 1.97 (m, 4 H), 2.23 - 2.45 (m, 2 H), 2.84 (br s, 2 H),
13
.90 (td, J = 8.5, 6.2 Hz, 2 H), 4.97 - 5.12 (m, 4 H), 5.85 (ddt, J = 17.0, 10.3, 6.4 Hz, 1 H). C
NMR (75 MHz, CHLOROFORM-d) δ ppm 21.8, 21.9, 22.1, 22.2, 22.7, 22.8, 24.66, 24.72, 27.1
2
1
3
3
(
d, J = 3.5 Hz), 28.7 (d, J = 112.9 Hz), 44.0 (d, J = 5.8 Hz), 44.3 (d, J = 5.8 Hz),
C-P
C-P
C-P
C-P
3
31
5
1.4, 52.2, 68.7, 68.9, 115.3, 137.8 (d, J = 15.0 Hz), 174.49, 174.52. P NMR (121 MHz,
C-P
+
CHLOROFORM-d) δ ppm 28.48. HRMS (ESI-MS): calculated for C H N O P [M+H] :
2
2
44
2
5
4
47.2982, found: 447.2963.
Dipentyl 2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-methylpentanoate)
13i). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded 13i (37%) as a
(
yellow oil after purification by column chromatography (petroleum ether  40% EtOAc in
1
petroleum ether). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.87 - 0.99 (m, 18 H), 1.28 -
1
.39 (m, 8 H), 1.45 - 1.84 (m, 12 H), 2.26 - 2.42 (m, 2 H), 2.79 (t, J = 11.2 Hz, 2 H), 3.87 - 4.03
(m, 2 H), 4.03 - 4.20 (m, 4 H), 4.98 - 5.04 (m, 1 H), 5.08 (dq, J = 17.1, 1.6 Hz, 1 H), 5.85 (ddt, J
13
=
16.9, 10.3, 6.4 Hz, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.1, 22.2, 22.3, 22.4,
2
1
3
2
2.8, 22.9, 24.7, 24.8, 27.1 (d, J = 3.5 Hz), 28.7 (d, J = 111.7 Hz), 28.1, 28.4, 44.2 (d, J
C-P
C-P
C-P
3
3
=
5.8 Hz), 44.4 (d, J = 5.8 Hz), 51.3, 52.1, 65.4, 65.5, 115.5, 137.8 (d, J = 15.0 Hz), 175.1
C-P
C-P
3
3
31
(
d, J = 2.3 Hz), 175.2 (d, J = 3.5 Hz). P NMR (121 MHz, CHLOROFORM-d) δ ppm
C-P
C-P
+
2
8.59. HRMS (ESI-MS): calculated for C H N O P [M+H] : 503.3608, found: 503.3595.
2
6
52
2
5
2
2

Dicyclohexyl
2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-
methylpentanoate) (13j). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded
1
3j (75%) as a yellow oil after purification by column chromatography (petroleum ether  60%
1
EtOAc in petroleum ether). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.86 - 1.02 (m, 12
H), 1.23 - 1.62 (m, 16 H), 1.66 - 1.88 (m, 12 H), 2.23 - 2.45 (m, 2 H), 2.70 - 2.88 (m, 2 H), 3.82 -
4
.03 (m, 2 H), 4.69 - 4.86 (m, 2 H), 5.01 (dq, J = 10.2, 1.4 Hz, 1 H), 5.08 (dq, J = 17.1, 1.6 Hz, 1
1
3
H), 5.84 (ddt, J = 17.0, 10.3, 6.4 Hz, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 22.2,
2
1
2
3
=
2.3, 22.7, 22.8, 23.8, 24.7, 24.8, 25.4, 27.1 (d, J = 3.5 Hz), 28.7 (d, J =111.7 Hz), 31.6,
C-P
C-P
3
3
3
1.7, 44.2 (d, J = 5.8 Hz), 44.4 (d, J = 5.8 Hz), 51.4, 52.2, 73.6, 73.7, 115.4, 137.8 (d, J
C-P
C-P
C-P
31
15.0 Hz), 174.4, 174.5. P NMR (121 MHz, CHLOROFORM-d) δ ppm 28.42. HRMS (ESI-
+
MS): calculated for C H N O P [M+H] : 527.3608, found: 527.3632.
28
52
2
5
Dibenzyl 2,2'-[(but-3-en-1-ylphosphoryl)bis(azanediyl)](2S,2'S)-bis(4-methylpentanoate)
(13k). Following general procedure 3, crude 12 (0.69 g, 4.0 mmol) afforded 13k (56%) as a
1
yellow oil after purification by column chromatography (toluene  75%EtOAc in toluene). H
NMR (300 MHz, CHLOROFORM-d) δ ppm 0.79 - 0.99 (m, 12 H), 1.34 - 1.81 (m, 8 H), 2.19 -
2
1
2
4
1
2
.39 (m, 2 H), 2.75 - 2.94 (m, 2 H), 3.93 - 4.06 (m, 2 H), 4.95 - 5.19 (m, 6 H), 5.78 (ddt, J =
1
3
6.9, 10.3, 6.4 Hz, 1 H), 7.29 - 7.36 (m, 10 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm
2
1
1.9, 22.0, 22.65, 22.73, 24.56, 24.59, 27.0 (d, J = 3.5 Hz), 28.5 (d, J = 112.9Hz), 43.8,
C-P
C-P
3.9, 44.0, 51.3, 52.0, 66.8, 67.0, 115.3, 128.28, 128.33, 128.5, 128.56, 128.63, 135.4, 135.5,
3
31
37.7 (d, J = 16.1 Hz), 174.67, 174.72. P NMR (121 MHz, CHLOROFORM-d) δ ppm
C-P
+
8.54. HRMS (ESI-MS): calculated for C H N O P [M+H] : 543.2982, found: 543.2967.
3
0
44
2
5
Diethyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(3-
methylbutanoate) (14a). Following general procedure 4, 13a (0.75 g, 1.9 mmol) afforded 14a
2
3

(86%) as a transparent wax after purification by column chromatography (EtOAc  8% EtOH in
1
EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.81 - 0.92 (m, 6 H), 0.92 - 1.02 (m, 6
H), 1.28 (app td, J = 7.1, 4.3 Hz, 6 H), 1.59 - 1.87 (m, 6 H), 2.00 - 2.17 (m, 2 H), 2.88 - 3.08 (m,
1
3
2
H), 3.64 (t, J = 5.7 Hz, 2 H), 3.72 - 3.87 (m, 2 H), 4.09 - 4.31 (m, 4 H). C NMR (75 MHz,
2
1
CHLOROFORM-d) δ ppm 14.3, 17.4, 17.6, 19.06, 19.09, 19.2 (d, J = 3.5 Hz), 28.8 (d, J
=
C-P
C-P
3
3
3
1
6
12.9 Hz), 32.1 (d, J = 5.8 Hz), 32.2 (d, J = 4.6 Hz), 33.2 (d, J = 15.0 Hz), 57.8, 58.6,
C-P
C-P
C-P
3
1
1.2, 61.4, 174.1. P NMR (121 MHz, CHLOROFORM-d) δ ppm 30.78. HRMS (ESI-MS):
+
calculated for C C N O P [M+H] : 409.2462, found: 409.2479.
18
38
2
6
Diethyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(3-
phenylpropanoate) (14b). Following general procedure 4, 13b (558 mg, 1.15 mmol) afforded
1
4b (83%) as a transparent oil after purification by column chromatography (toluene  10%
1
EtOH in toluene). H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.23 (app q, J = 7.1 Hz, 6 H),
1
.33 - 1.48 (m, 6 H), 1.86 - 2.07 (br s, 1 H), 2.46 (dd, J = 11.1, 11.0 Hz, 1 H), 2.79 - 3.19 (m, 5
13
H), 3.52 (t, J = 5.7 Hz, 2 H), 3.97 - 4.29 (m, 6 H), 7.07 - 7.12 (m, 2 H), 7.17 - 7.34 (m, 8 H). C
2
1
NMR (75 MHz, CHLOROFORM-d) δ ppm 14.2, 14.3, 19.0 (d, JC-P = 4.6 Hz), 28.5 (d, J
=
C-P
3
1
1
12.9 Hz), 33.2 (d, J = 15.0 Hz), 40.8, 41.2, 54.0, 54.3, 61.4, 61.5, 61.6, 127.1, 128.6, 129.8,
C-P
31
29.8, 136.5, 136.8. P NMR (121 MHz, CHLOROFORM-d) δ ppm 29.67. HRMS (ESI-MS):
+
calculated for C H N O P [M+H] : 505.2462, found: 505.2447.
26
38
2
6
Diethyl [(4-hydroxybutyl)phosphoryl](S)-di-L-prolinate (14c). Following general procedure
4
, 13c (985 mg, 2.55 mmol) afforded 14c (67%) as a transparent oil after purification by column
1
chromatography (EtOAc  20% EtOH in EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ
ppm 1.16 - 1.35 (m, 6 H), 1.57 - 1.81 (m, 6 H), 1.81 - 2.05 (m, 6 H), 2.09 - 2.26 (m, 2 H), 3.13 -
3
.39 (m, 3 H), 3.44 - 3.56 (m, 1 H), 3.62 (t, J = 5.8 Hz, 2 H), 4.11 - 4.20 (m, 4 H), 4.20 - 4.33 (m,
2
4

1
3
2
1
2
H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.3, 17.9 (d, J = 4.6 Hz), 25.7 (d, J
C-P
C-P
3
3
3
=
114.0 Hz), 25.2, 25.3, 31.6 (d, J = 5.8 Hz), 31.9 (d, J = 5.8 Hz), 33.5 (d, J = 13.8 Hz),
C-P
C-P
C-P
2
2
2
2
4
6
6.1 (d, J = 2.3 Hz), 47.1 (d, J = 4.6 Hz), 59.1 (d, J = 5.8 Hz), 59.5 (d, J = 4.6 Hz),
C-P
C-P
C-P
C-P
3
1
0.9, 61.0, 61.4, 174.5, 174.7. P NMR (121 MHz, CHLOROFORM-d) δ ppm 31.59. HRMS
+
(
ESI-MS): calculated for C H N O P [M+H] : 405.2149, found: 405.2152.
18
34
2
6
Diethyl2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S,3R,3'R)-bis(3-methyl
pentanoate) (14d). Following general procedure 4, 13d (603 mg, 1.44 mmol) afforded 14d
(61%) as a transparent oil after purification by column chromatography (EtOAc  8% EtOH in
1
EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.83 - 0.97 (m, 12 H), 1.03 - 1.22 (m, 2
H), 1.28 (td, J = 7.1, 4.3 Hz, 6 H), 1.33 - 1.50 (m, 2 H), 1.59 - 1.87 (m, 8 H), 2.86 - 3.04 (m, 2
1
3
H), 3.61 - 3.69 (m, 2 H), 3.77 - 3.92 (m, 2 H), 4.08 - 4.29 (m, 4 H). C NMR (75 MHz,
2
CHLOROFORM-d) δ ppm 11.8, 11.9, 14.3, 15.57, 15.60, 19.2 (d, JC-P = 3.5 Hz), 24.9, 25.1,
1
3
3
3
2
8.8 (d, J = 112.9 Hz), 33.3 (d, J = 15.0 Hz), 39.3 (d, J = 4.6 Hz), 39.4 (d, J = 4.6
C-P
C-P
C-P
C-P
3
1
Hz), 57.2, 58.0, 61.2, 61.5, 174.08, 174.12, 174.2. P NMR (121 MHz, CHLOROFORM-d) δ
+
ppm 30.43. HRMS (ESI-MS): calculated for C H N O P [M+H] : 437.2775, found: 437.2763.
2
0
42
2
6
Diethyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(6-{[(benzyloxy)
carbonyl]amino}hexanoate) (14e). Following general procedure 4, 13e (2.51 g, 3.50 mmol)
afforded 14e (63%) as a transparent oil after purification by column chromatography (petroleum
1
ether  acetone  10% EtOH in acetone). H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.25
(app tt, J = 7.1, 1.6 Hz, 6 H), 1.28 - 1.84 (m, 18 H), 2.86 (br s, 1 H), 3.00 - 3.26 (m, 6 H), 3.54 -
3
-
.63 (m, 2 H), 3.80 - 4.00 (m, 2 H), 4.04 - 4.25 (m, 4 H), 5.07 (s, 4 H), 5.37 - 5.60 (m, 2 H), 7.25
13
2
7.37 (m, 10 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.25, 14.28, 19.2 (d, J
=
C-P
1
3
3
4
.6 Hz), 22.2, 28.7 (d, J = 112.9 Hz), 29.0, 29.1, 33.1 (d, J = 15.0 Hz), 33.9 (d, J = 4.6
C-P
C-P
C-P
2
5

3
Hz), 34.4 (d, J = 4.6 Hz), 40.7, 40.8, 52.3, 53.1, 60.5, 61.4, 61.5, 66.5, 128.1, 128.5, 136.9,
C-P
31
1
56.7, 156.8, 174.3, 174.4. P NMR (121 MHz, CHLOROFORM-d) δ ppm 30.35. HRMS (ESI-
+
MS): calculated for C H N O P [M+H] : 735.3729, found: 735.3717.
36
56
4
10
Dimethyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-
methylpentanoate) (14f). Following general procedure 4, 13f (911 mg, 2.33 mmol) afforded 14f
(24%) as a transparent oil after purification by column chromatography (toluene  15% MeOH
1
in toluene). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.88 - 1.00 (m, 12 H), 1.42 - 1.80
(m, 12 H), 2.83 - 3.00 (m, 2 H), 3.64 (t, J = 5.7 Hz, 2 H), 3.72 (app d, J = 2.3 Hz, 6 H), 3.88 –
1
3
2
4
2
2
.01 (m, 2 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 19.1 (d, J = 3.5 Hz), 22.0,
C-P
1
3
3
2.1, 22.8, 22.9, 24.66, 24.72, 28.6 (d, J = 112.9 Hz), 33.2 (d, J = 15.0 Hz), 44.0 (d, J
=
C-P
C-P
C-P
3
31
.3 Hz), 44.1 (d, J = 2.3 Hz), 51.3, 52.0, 52.2, 61.4, 175.59, 175.62, 175.7. P NMR (121
C-P
+
MHz, CHLOROFORM-d) δ ppm 29.82. HRMS (ESI-MS): calculated for C H N O P [M+H] :
1
8
38
2
6
4
09.2462, found: 409.2480.
Diethyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-
methylpentanoate) (14g). Following general procedure 4, 13g (0.13 g, 0.30 mmol) afforded 14g
(76%) as a transparent oil after purification by column chromatography (toluene  10% EtOH
1
in toluene). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.90 - 0.98 (m, 12 H), 1.28 (td, J =
7
3
.1, 4.4 Hz, 6 H), 1.42 - 1.80 (m, 12 H), 2.88 - 3.11 (m, 2 H), 3.62 (t, J = 5.6 Hz, 2 H), 3.87 –
1
3
.98 (m, 2 H), 4.10 - 4.23 (m, 4 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.2, 19.1
2
1
3
(
d, J = 3.5 Hz), 22.0, 22.1, 22.7, 22.8, 24.6, 24.7, 28.6 (d, J = 112.9 Hz), 33.2 (d, J
=
C-P
C-P
C-P
3
1
1
5.0 Hz), 43.9, 44.0, 44.1, 51.2, 52.0, 61.2, 175.2. P NMR (121 MHz, CHLOROFORM-d) δ
+
ppm 29.57. HRMS (ESI-MS): calculated for C H N O P [M+H] : 437.2775, found: 437.2758.
2
0
42
2
6
2
6

Diisopropyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-
methylpentanoate) (14h). Following general procedure 4, 13h (991 mg, 2.22 mmol) afforded
1
4h (62%) as a transparent oil after purification by column chromatography (toluene  10%
1
iPrOH in toluene). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.88 - 1.00 (m, 12 H), 1.20 -
1
.30 (m, 12 H), 1.37 - 1.81 (m, 12 H), 2.78 - 2.94 (m, 2 H), 3.64 (t, J = 5.8 Hz, 2 H), 3.82 - 3.96
13
2
(
m, 2 H), 4.94 - 5.09 (m, 2 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 19.1 (d, J
=
C-P
1
3
3
.5 Hz), 21.8, 21.9, 22.1, 22.2, 22.8, 22.9, 24.7, 28.7 (d, J = 111.7 Hz), 33.2 (d, J = 15.0
C-P
C-P
3
3
Hz), 44.0 (d, J = 5.8 Hz), 44.2 (d, J = 5.8 Hz), 51.4, 52.2, 61.4, 68.86, 68.93, 174.60,
C-P
C-P
31
1
74.63, 174.79, 174.83. P NMR (121 MHz, CHLOROFORM-d) δ ppm 29.49. HRMS (ESI-
+
MS): calculated for C H N O P [M+H] : 465.3088, found: 465.3107.
22
46
2
6
Dipentyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-
methylpentanoate) (14i). Following general procedure 4, 13i (706 mg, 1.41 mmol) afforded 14i
(51%) as a transparent oil after purification by column chromatography (toluene  EtOAc 
1
3
1
4
0% acetone in EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.85 - 1.01 (m, 18 H),
.26 - 1.42 (m, 8 H), 1.42 - 1.79 (m, 16 H), 2.78 - 3.00 (m, 3 H), 3.64 (t, J = 5.6 Hz, 2 H), 3.84 -
1
3
.01 (m, 2 H), 4.01 - 4.17 (m, 4 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.0, 19.1
2
1
(
d, J = 4.6 Hz), 22.1, 22.2, 22.4, 22.7, 22.9, 24.7, 24.8, 28.7 (d, JC-P = 111.7 Hz), 28.08,
C-P
3
2
1
8.11, 28.3, 33.2 (d, J = 13.8 Hz), 44.1, 44.2, 44.3, 51.3, 52.1, 61.4, 65.4, 65.5, 175.18,
C-P
31
75.22, 175.3. P NMR (121 MHz, CHLOROFORM-d) δ ppm 29.49. HRMS (ESI-MS):
+
calculated for C H N O P [M+H] : 521.3714, found: 521.3690.
26
54
2
6
Dicyclohexyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-methyl
pentanoate) (14j). Following general procedure 4, 13j (1.41 g, 2.67 mmol) afforded 14j (70%)
as a transparent oil after purification by column chromatography (toluene  EtOAc  30%
2
7

1
acetone in EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.91 - 0.97 (m, 12 H), 1.25 -
1
.91 (m, 32 H), 2.75 - 2.92 (m, 2 H), 3.64 (t, J = 5.7 Hz, 2 H), 3.85 – 4.97 (m, 2 H), 4.70 - 4.84
1
3
2
(
m, 2 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 19.1 (d, J = 3.5 Hz), 22.2, 22.3,
C-P
1
3
2
2.8, 22.9, 23.8, 24.76, 24.80, 25.4, 28.7 (d, J = 111.7 Hz), 31.6, 31.7, 33.3 (d, J = 13.8
C-P
C-P
3
3
3
Hz), 44.2 (d, J = 5.8 Hz), 44.4 (d, J = 5.8 Hz), 51.4, 52.3, 61.5, 73.7, 73.8, 174.5 (d, J
=
C-P
C-P
C-P
3
31
3
.5 Hz), 174.7 (d, J = 2.3 Hz). P NMR (121 MHz, CHLOROFORM-d) δ ppm 29.38. HRMS
C-P
+
(
ESI-MS): calculated for C H N O P [M+H] : 545.3714, found: 545.3732.
28
54
2
6
Dibenzyl
2,2'-{[(4-hydroxybutyl)phosphoryl]bis(azanediyl)}(2S,2'S)-bis(4-
methylpentanoate) (14k). Following general procedure 4, 13k (1.26 g, 2.32 mmol) afforded
1
4k (61%) as a faint yellow oil after purification by column chromatography (EtOAc  40%
1
acetone in EtOAc). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.82 - 0.95 (m, 12 H), 1.33 -
1
3
.77 (m, 12 H), 2.85 - 2.98 (m, 1 H), 3.06 (dd, J = 14.1, 10.9 Hz, 1 H), 3.56 (t, J = 5.8 Hz, 2 H),
1
3
.98 (dtd, J = 10.8, 8.5, 8.5, 6.1 Hz, 2 H), 5.05 - 5.19 (m, 4 H), 7.29 - 7.37 (m, 10 H). C NMR
2
(
75 MHz, CHLOROFORM-d) δ ppm 19.1 (d, J = 4.6 Hz), 22.0, 22.1, 22.7, 22.8, 24.6, 28.6
C-P
1
3
(
d, J = 111.7 Hz), 33.1 (d, JC-P = 15.0 Hz), 43.8, 43.9, 51.3, 52.0, 61.2, 66.9, 67.0, 128.3,
C-P
3
3
31
1
28.4, 128.5, 128.6, 128.7, 135.4, 135.6, 174.8 (d, J = 3.5 Hz), 174.9 (d, J = 2.3 Hz). P
C-P
C-P
NMR (121 MHz, CHLOROFORM-d) δ ppm 29.73. HRMS (ESI-MS): calculated for
+
C H N O P [M+H] : 561.3088, found: 561.3093.
3
0
46
2
6
4
-(Bis{[(S)-1-ethoxy-3-methyl-1-oxobutan-2-yl]amino}phosphoryl)butanoic acid (15a).
Following general procedure 5, 14a (664 mg, 1.63 mmol) afforded 15a (81%) as an off-white
wax after purification by column chromatography (toluene  7.5% EtOH in toluene + 0.1%
1
acetic acid). H NMR (300 MHz, CHLOROFORM-d) δ ppm 0.81 - 0.93 (m, 6 H), 0.97 (t, J =
7
.0 Hz, 6 H), 1.23 - 1.33 (m, 6 H), 1.78 - 1.99 (m, 4 H), 2.01 - 2.18 (m, 2 H), 2.38 - 2.50 (m, 2
2
8

H), 3.10 - 3.23 (m, 1 H), 3.34 (dd, J = 13.7, 11.1 Hz, 1 H), 3.71 - 3.89 (m, 2 H), 4.08 - 4.28 (m, 4
1
3
2
H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.4, 17.4, 17.6, 18.6 (d, J = 3.5 Hz),
C-P
2
2
2
3
1
1
9.1, 28.3 (d, J = 112.9 Hz), 32.1 (d, J = 4.6 Hz), 32.2 (d, J = 5.8 Hz), 34.6 (d, J
=
C-P
C-P
C-P
C-P
3
3
31
5.0 Hz), 58.0, 58.6, 61.28, 61.33, 173.9 (d, J = 2.3 Hz), 174.1 (d, J = 2.3 Hz), 175.5. P
C-P
C-P
NMR (121 MHz, CHLOROFORM-d) δ ppm 31.84. HRMS (ESI-MS): calculated for
+
C H N O P [M+H] : 423.2255, found: 423.2274.
1
8
36
2
7
4
-(Bis{[(S)-1-ethoxy-1-oxo-3-phenylpropan-2-yl]amino}phosphoryl)butanoic acid (15b).
Following general procedure 5, 14b (0.15 g, 0.30 mmol) afforded 15b (66%) as a transparent oil
after purification by column chromatography (DCM  6% EtOH in DCM + 0.1% acetic acid).
1
H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.18 - 1.27 (m, 6 H), 1.30 - 1.64 (m, 4 H), 2.17
(br t, J = 6.3 Hz, 2 H), 2.52 - 2.64 (m, 1 H), 2.73 - 2.86 (m, 2 H), 3.02 (ddd, J = 18.6, 13.5, 5.3
Hz, 2 H), 3.54 (br t, J = 11.1 Hz, 1 H), 3.99 - 4.26 (m, 6 H), 7.06 - 7.11 (m, 2 H), 7.17 - 7.34 (m,
1
3
2
8
H), 10.31 (br s, 1 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.2, 14.3, 19.0 (d, J
C-P
1
3
=
4.6 Hz), 28.5 (d, J = 107.8 Hz), 33.2 (d, J = 15.0 Hz), 40.8, 41.2, 54.0, 54.3, 61.4, 61.5,
C-P
C-P
3
1
6
1.6, 127.1, 128.6, 129.75, 129.84, 136.5, 136.8. P NMR (121 MHz, CHLOROFORM-d) δ
+
ppm 31.01. HRMS (ESI-MS): calculated for C H N O P [M+H] : 519.2255, found: 519.2274.
2
6
36
2
7
4
-{Bis[(S)-2-(ethoxycarbonyl)pyrrolidin-1-yl]phosphoryl}butanoic acid (15c). Following
general procedure 5, 14c (0.37 g, 0.90 mmol) afforded 15c (84%) as a faint yellow oil after
purification by column chromatography (toluene  25% EtOH in toluene + 0.1% acetic acid).
1
H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.26 (t, J = 7.1 Hz, 6 H), 1.79 - 2.05 (m, 10 H),
2
.08 – 2.27 (m, 2 H), 2.32 – 2.53 (m, 2 H), 3.14 - 3.38 (m, 3 H), 3.46 – 3.59 (m, 1 H), 4.15 (qd, J
1
3
=
7.1, 1.8 Hz, 4 H), 4.25 - 4.34 (m, 2 H). C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.3,
2
3
1
3
1
7.8 (d, J = 3.5 Hz), 25.2 (d, J = 5.8 Hz), 25.25 (d, J = 114.0 Hz), 25.31 (d, J = 6.9
C-P
C-P
C-P
C-P
2
9