Synthesis of novel antiproliferative hybrid bis-(3-indolyl)methane phosphonate derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.09.011

An efficient synthetic methodology for the preparation of phosphorus substituted bis-(3-indolyl)methane through a double nucleophilic addition of indole derivatives to an in situ generated α-iminophosphonate is reported. In addition, bis-(3-indolyl)methane substrates showed in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines A549 (carcinomic human alveolar basal epithelial cell) and SKOV03 (human ovarian carcinoma).

Full text of DOI:10.1016/j.ejmech.2018.09.011

Accepted Manuscript
Synthesis of novel antiproliferative hybrid bis-(3-indolyl)methane phosphonate
derivatives
Aitor Maestro, Endika Martín-Encinas, Concepción Alonso, Edorta Martinez de
Marigorta, Gloria Rubiales, Javier Vicario, Francisco Palacios
PII:
S0223-5234(18)30783-9
10.1016/j.ejmech.2018.09.011
EJMECH 10722
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2018
Revised Date: 4 September 2018
Accepted Date: 4 September 2018
Please cite this article as: A. Maestro, E. Martín-Encinas, Concepció. Alonso, E. Martinez de Marigorta,
G. Rubiales, J. Vicario, F. Palacios, Synthesis of novel antiproliferative hybrid bis-(3-indolyl)methane
phosphonate derivatives, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.011.
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ACCEPTED MANUSCRIPT
Synthesis of novel antiproliferative hybrid bis-(3-indolyl)methane phosphonate derivatives
Aitor Maestro, Endika Martín-Encinas, Concepción Alonso, Edorta Martinez de Marigorta,
Gloria Rubiales, Javier Vicario,* and Francisco Palacios*
*Departamento de Química Orgánica I, Centro de Investigación y Estudios Avanzados “Lucio
Lascaray”- Facultad de Farmacia, University of the Basque Country, UPV/EHU Paseo de la
Universidad 7, 01006 Vitoria-Gasteiz, SPAIN. Fax: (+34)-945-013049.
E mail: javier.vicario@ehu.eus / francisco.palacios@ehu.eus
Keywords: Indole, bis-(3-indolyl)methanes (BIMs), Phosphonate, Antiproliferative effect.
Abstract:
An efficient synthetic methodology for the preparation of phosphorus substituted bis-(3-
indolyl)methane through a double nucleophilic addition of indole derivatives to an in situ
generated α-iminophosphonate is reported. In addition, bis-(3-indolyl)methane substrates
showed in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines A549
(carcinomic human alveolar basal epithelial cell) and SKOV03 (human ovarian carcinoma).
1. Introduction:
Due to the notable grown in the life expectancy during the last decades, cancer has become one
of the leading causes of death worldwide [1]. The World Health Organization (WHO) reports
8.8 million people died of cancer globally in 2015, being the most common cause of cancer
death the cancer of lung with 1,69 million (19,4%) of deaths [2]. Cancer treatment comprises, in
most of the cases, a combination of surgery and chemotherapy [3] and here is where Drug
Discovery can play a crucial role into this area. There is still a serious need to search for some
newer and safer anticancer agents and, therefore, the discovery of new active compounds and
the in vitro evaluation of their anticancer properties represents an important task in Medicinal
Chemistry, in order to improve our toolbox for the treatment of cancer.
Indole framework holds a very high affinity to multiple receptors and enzymes and,
accordingly, it is considered a privileged structure in many active medicine compounds for
human health and represents a promising scaffold for drug development [4]. In particular, bis-
indole family derivatives, are of extraordinary significance in Synthetic and Medicinal
Chemistry due to their wide occurrence in nature and their assorted biological activity [5].
Simple bis-(3-indolyl)methane (BIM) I and their derivatives (BIMs) II-VIII are nitrogen-

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containing heterocyclic compounds which structure can be found in many alkaloids isolated
from natural sources (Figure 1) [6]. Alkaloids such as arundine (I, R = H) [7], vidrindole (II, R
= Me) [8], antibiotic turbomycins B, C, D, E (III, IV; R = Ar)[9] or arsindoline B (V, R = nPr)
[10] are isolated from marine bacteria, while streptindole (VI, R = Me) [11] is found in human
feces. Cytotoxic barakacin (VII) has been isolated from a ruminal Pseudomonas [12] and the
antibacterial and anti-inflammatory dalesindole (VIII), a new alkaloid, was obtained by
mycosynthesis [13]. In addition, numerous BIMs and their analogues show a wide range
of biological and pharmacological activities [14], including growth inhibition of
numerous tumor types [15].
Figure 1. Structures of BIMs I-VIII and its phosphorated derivatives IX.
Among the innumerable amount of chemotherapeutic agents tested in vitro, we focused in
organophosphorus compounds [16], particularly phosphoric acid esters. It is well known that
structural modifications of active molecules involving the introduction of phosphorated
functionalities, very often results in increased or new activities [17]. Due to the stability of its P-
C bond and their chemical similitude to phosphate ester and anhydride metabolites, phosphonate
derivatives show an assorted biological activity and, consequently, they have found numerous
applications in medicine and agrochemistry [18]. Organophosphorus derivatives are interesting
compounds from a biological point of view, since it is known that phosphorus substituents may
affect the reactivity of heterocycles and regulate important biological functions [19]. Likewise,
the development of new strategies for the preparation of aminophosphonates [20], phosphinated
[21] or phosphorylated azaheterocycles [22] implies the incorporation of organophosphorus
functionalities in simple synthons.

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The hybrid anticancer drug approach is an innovative synthetic strategy for the discovery of new
biologically active hybrid molecules [23]. It is believed that the presence of two or more
pharmacophores in a single unit not only synergizes their biological effect but also upsurge their
ability to inhibit more than one biological target. Recently, the molecular hybrid approach has
resulted in several novel chemical entities with improved anticancer activity and selectivity with
reduced side effects [24].
With these considerations in mind, we believed that the development of new hybrid molecules
[25], such as phosphonate functionalized bis-indole derivatives IX (Figure 1), incorporating a
phosphonate group in the BIMs structure (arundine, I, figure 1) may be privileged scaffolds for
pharmaceuticals [26] and may improve the antiproliferative cytotoxic properties with respect to
other biologically active structures. This represents an interesting challenge, due to the potential
interest of these molecules not only in synthetic but also in medicinal chemistry. As far as we
know, not only the synthesis but also the study of the biological activity of BIMs containing a
phosphorated moiety have not been described.
2. Results and discussion:
2.1. Chemistry
The most straightforward method for the preparation of BIM derivatives, reported by Fischer in
the late 19th century [27], is the acid catalyzed Friedel-Crafts reaction between aldehydes 1 and
indoles 2. After the first addition of electron rich indole 2 to electrophilic aldehyde 1, the
intermediate indole-3-carbinol 3 yields azafulvene 4 by means of elimination of a molecule of
water. This new electrophilic species 4 undergoes a second acid catalyzed nucleophilic addition
of indole 2 leading to the formation of BIM derivatives 5 [28] (Scheme 1).
R
N
N
R
R
H
H
O
2
2
OH
H+ (cat)
H+ (cat)
R
N
N
HN
NH
H2O
H
1
3
4
5
Scheme 1. Conventional synthesis of BIM derivatives 5.
Following this approach, simply by using phosphorylated aldehydes 1 (R = P(O)OR2) as
starting substrates, our target molecules may be obtained in a single step. However, the use of
formylphosphonates should be avoided since it suffers of numerous drawbacks. First, they are
extremely moisture sensitive, affording readily acetal derivatives and, moreover, they have to be
prepared by oxidation of diazomethylphosphonates using unstable Murray reagent [29]. For
those reasons, we thought that α-phosphorylated aldimines would be much more suitable
substrates for this reaction. Although less electrophilic, this aldimine substrates show slightly
higher stability to moisture and, more importantly, they can be easily prepared by a formal

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oxidation from α-aminophosphonates in a two-step protocol that comprises halogenation
followed by β-elimination of hydrogen halide [30].
For our synthetic approach, we chose N-trityl substituted α-aminophosphonates 6 as starting
substrates. Those substrates are readily obtained by a hydrophosphorylation reaction of
dialkylphosphites and imines and, due to the bulkiness expected in the nitrogen atom, we
envisioned the elimination of tritylamine to be favored in order to drive the process to the
formation of azafulvene intermediate 4 (Scheme 1). We were delighted to discover that the
radical bromination of α-aminophosphonate 6 using N-bromosuccinimide as bromine source in
carbon tetrachloride led to the direct formation of a clean solution of α-iminophosphonate 7 by a
subsequent spontaneous β-elimination of hydrogen bromide (Scheme 2). The generated
succinimide together with the remaining N-bromosuccinimide are insoluble in carbon
tetrachloride and they can be quickly removed by a simple filtration under inert atmosphere, in
order to prevent degradation of α-iminophosphonate 7.
Scheme 2. Synthesis of bis-(3-indolyl)methyl phosphonate derivatives 9-13.
Next, as we expected, the reaction of α-iminophosphonates 7 with two equivalents of indole
derivative 8 in the presence of a catalytic amount of a phosphoric acid catalyst afforded the
target BIM derivatives 9-13 in good yields (Chart 1, Scheme 2).
The reaction using simple indole 8a (R2 = R = H) was initially applied to the preparation of
different phosphonates such as methylphosphonate 9 (R1 = Me, Table 1, Entry 1),
ethylphosphonate 10 (R1 = Et, Table 1, Entry 2), iso-propylphosphonate 11a (R1 = iPr, Table 1,
t
Entry 3), tert-butylphosphonate 12a (R1 = Bu, Table 1, Entry 7) and benzylphosphonate 13a
(R1 = CH2Ph = Bn, Table 1, Entry 10). Moreover, this methodology allows a wide range of
electron-donating and electron-withdrawing substituents at the heteroaromatic substrate.
According to this, the reaction was successfully extended to several indole substrates bearing
strong (Table 1, Entries 15-17) and weak electron donating substituents (Table 1, Entries 6, 9,
18-20) and to 5- and 6-fluorine substituted indoles (Table 1, Entries 4-5, 8, 11-12). Finally, even
some deactivated trifluoromethyl substituted [31] indoles were used with success in the reaction
(Table 1, Entries 13-14).

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Table 1. BIM derivatives 9-13 obtained
Entry Comp. R1 R2
Yield (%)[a]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Me
Et
H
H
H
59
61
60
69
67
62
68
69
72
55
65
67
54
53
57
61
9
10
iPr
11a
11b
11c
11d
12a
12b
12c
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
iPr 6-F
iPr 5-F
iPr 5-Me
tBu
H
tBu 5-F
tBu 5-Me
Bn
Bn 6-F
Bn 5-F
Bn 5-CF3
Bn 6-CF3
Bn 5-OMe
Bn 6-OMe
H
17
18
19
20
Bn 5,6-OMe 58
Bn 6-Me
Bn 5-Me
Bn 2-Me
64
65
63
[a] Yield after purification
In order to extend the diversity of substituents in our substrates, the acidic hydrolysis of
dimethylphosphonate 12a was performed using an aqueous solution of hydrochloric acid in
refluxing tetrahydrofurane (THF). Work up of the reaction afforded phosphonic acid derivative
14 in very good yield (Scheme 3).
Scheme 3. Hydrolysis of dimethylphosphonate 12a and N-methylation of 11d and 12c.
For the same purpose, the alkylation of indolic nitrogen of iso-propyl and tert-butyl
phosphonate derivatives 11d and 12c was performed in the presence of methyl iodide in
dimethylformamide, using sodium hydride as a base to yield bis-(3-N-methylindolyl)
derivatives 15-16 in almost quantitative yields (Scheme 3). It should be noted that the direct
synthesis of N-methylated BIM derivatives 15-16 using our reported protocol with N-

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methylindole (8 R=Me, Scheme 2) is not feasible, which may suggest a key role of NH group in
the phosphoric acid catalyzed nucleophilic addition of indole species to α-iminophosphonates.
This methodology represents an easy protocol for the synthesis of BIM derivatives that avoids
the use of unstable formylphosphonates and, as far as we know, this strategy is the first example
of the preparation of BIMs with a phosphorus containing moiety.
Next, with this collection of promising structures in our hands, we considered the study of their
biological activity. Therefore, the behavior of prepared compounds as antiproliferative agents
was investigated.
2.2. Biological results
In vitro cytotoxicity of our novel BIM derivatives was evaluated by testing their
antiproliferative activities against two human cancer cell lines: A549 (carcinomic human
alveolar basal epithelial cell) and SKOV03 (human ovarian carcinoma). Cell counting kit
(CCK-8) assay was used in order to evaluate growth inhibition. Cell proliferation inhibitory
activities of the bis-indol derivatives 9-16 and chemotherapeutic doxorubicin are shown in
Table 2 as IC50 values. Moreover, MRC-5 non-malignant lung fibroblasts were tested for
studying selective toxicity [32] and in contrast, none of the synthesized phosphorated
compounds or doxorubicin exhibited any toxicity toward MRC-5 cells.
Regarding the effect of the substitution at the phosphonate ester group in BIM derivatives 9-16
into their cytotoxicity against SKOV03 cell line in vitro, it was evidenced that the presence of a
bulky group, in general, resulted in an increased activity. Considering only substrates holding
non-substituted indole moieties, while IC50 values higher than 50 µM were observed for methyl
or ethyl esters 9 and 10 (Table 2, entries 1, 2), those values were dropped to 11.43±0.83 µM for
iso-propyl ester 11a (entry 3) and, interestingly, lower values of 1.06±0.30 µM and 1.26±0.13
µM were obtained for tert-butyl and benzyl esters 12a and 13a respectively (entries 7, 10).
Although practically the same pattern was observed for A549 cell line, in this case the best
result was observed for iso-propyl ester 11a with IC50 values of 1.06±0.30 µM (entry 3). In
contrast to this tendency, phosphonic acid derivative 14 did not show any cytotoxicity in any of
the cell lines tested. Moreover, simple bis-(3-indolyl)methane Arundine (I, Figure 1) did not
show cytotoxic activity in vitro against A549 cell line.33

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Table 2. Antiproliferative activity of BIM derivatives 9-16 against lung and ovarian cancer cell
lines.
IC50 ovarian
(µM)[a] (SKOV03)
>50
IC50 lung
(µM)[a] (A549)
>50
Entry Comp.
R1
R2
R3
1
Me
Et
H
H
H
6-F
5-F
5-Me
H
5-F
5-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9
2
>50
10.48±1.01
1.06±0.30
0.85±0.00
0.58±0.09
0.41±0.13
15.90±0.74
11.30±0.08
0.06±0.04
10.53±0.39
0.22±0.15
0.63±0.16
6.10±0.23
8.90±0.92
1.05±0.58
7.45±0.35
(≈10)[b]
10
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
iPr
iPr
iPr
iPr
tBu
tBu
tBu
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
11.43±0.83
6.30±0.24
3.52±0.37
7.78±0.47
1.06±0.30
0.65±0.05
9.01±0.58
1.26±0.13
7.78±0.47
7.18±1.20
5.98±0.32
11.99±1.60
6.77±1.03
(>50)[b]
11a
11b
11c
11d
12a
12b
12c
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
14
6-F
5-F
5-CF3
6-CF3
5-OMe
6-OMe
5,6-OMe
6-Me
5-Me
2-Me
H
>50
4.90±0.36
0.06±0.02
>50
7.16±0.89
0.93±0.13
10.79±2.52
>50
>50
>50
>50
iPr
tBu
-
5-Me
5-Me
-
Me 11.91±1.84
Me >50
15
16
0.00184±0.00022
0.48±0.017
Doxorubicin
-
[a] Concentration corresponding to 50% growth inhibition. [b] Approximate value due to
systematic error in experimental.
With the purpose to improve further the activity of the synthesized BIM substrates, next we
studied the effect of the substitution at the indole ring into their cytotoxicity against both cell
lines. Although generally, the effect of fluorine on the biological activity of organic compounds
is rather difficult to predict, it is well known that the introduction of fluorine substituents in
bioactive molecules very often leads to increased activities [34]. For this reason, first we tested
the in vitro cytotoxicity of fluorine substituted BIM derivatives against SKOV03 and A549 cell
lines. In the case of SKOV03 cell line, high cytotoxicity is observed for 6- and 5-fluoro
substituted indole moieties in iso-propyl esters 11b, 11c, tert-butyl esters 12b and benzyl ester
derivatives 13b and 13c (entries 4, 5, 8, 11 and 12). Alternatively, in the case of A549 cell lines,
substitution of indole ring with fluorine atoms has a higher cytotoxic effect since IC50 values of
0.85±0.007 µM and 0.58±0.09 µM were revealed for iso-propyl ester derivatives 11b and 11c

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(entries 4, 5), for tert-butyl ester derivative 12b with an IC50 value of 11.30±0.08 µM (entry 8)
and the values for benzyl ester derivatives 13b and 13c dropped to 0.22±0.15 µM and 0.63±.16
µM (entries 11,12). Surprisingly, the introduction of a trifluoromethyl electron withdrawing
group in the indole ring had a low effect into the cytotoxicity of BIMs either on SKOV03 cell
line nor when they were tested in A549 cell line (entries 13, 14).
Methoxy group is a strong electron donating group, when it is present in aromatic rings, that it is
known to be a widespread motif in drugs and natural products. For that reason, next we studied
the introduction of methoxy groups in the skeleton of indole ring in the benzyl ester BIM
derivatives. Respect to the antiproliferative activity in vitro in SKOV03 cell line, while 5-
methoxy substituted BIM substrate 13f showed an IC50 value of 6.77±1.03µM (entry 15),
substitution in the 6-position of the indole ring was affected with a complete loss of the
cytotoxic properties for 6-methoxy and 5,6-dimethoxy BIMs 13g and 13h (entries 16, 17).
Whereas, when the cytotoxicity of those methoxy-substituted BIM substrates was tested in
A549 cell line, a slight increase in the activity was observed for 5-methoxy and 6-methoxy
substituted BIMPs 13f and 13g with IC50 values of 1.05±0.58 µM and 7.45±0.35 µM.
The introduction of methyl groups into a bioactive structure makes it more lipophilic [35]. In
general, the methyl group in the indole ring of our BIM substrates 11d, 12c, 13i-k (entries 6, 9,
18-20) showed interesting cytotoxic effects and substitution with methyl group in 5-possition
had a very significant impact into the cytotoxicity of BIM derivatives. For instance, tert-butyl
ester derivative 12c showed an IC50 value of 9.01±0.58 µM in SKOV03 cell line and an
improved value of 0.06±0.04 µM was obtained in A549 cell line (entry 9). Consistently, benzyl
ester derivative 13j with a methyl group in 5-position showed an improved cytotoxicity in the
nanomolar range when tested against the A549 cell line, with an IC50 value of 0.06±0.02 µM
and a significant effect in the SKOV03 cell line with an IC50 value of 0.93±0.13 µM (entry 19).
Finally, in order to determine the importance of NH group in indole moiety, N-methylindole
derivatives 15 and 16 were tested as antiproliferative agents. In this case, an increase of IC50
values to 11.91±1.84 µM and 11.41±2.00 µM was observed in SKOV03 cell line for iso-propyl
ester derivative 15 and an absolute lack of toxicity in both cell lines was observed for tert-butyl
ester derivative 16. This result suggests a significant role of NH group in the cytotoxic activity
of BIMs.
In conclusion, the new synthetic methodology proves to be very efficient for the preparation for
the first time of BIMs containing a phosphonate substituent and, in addition, the use of
undesirable formylphosphonates is avoided. This strategy allows the possibility of assorted
structural diversity in the resultant scaffold depending on the starting phosphonate and
commercial indole substrates. Moreover, obtained BIMs showed in vitro cytotoxicity inhibiting
the growth of human tumor cell lines A549 (carcinomic human alveolar basal epithelial cell)
and SKOV03 (human ovarian carcinoma) and none of the synthesized phosphorated compounds

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exhibited any toxicity toward MRC-5 non-malignant lung fibroblasts. Substrates 13j and 12c
presented very promising IC50 values of about 0.06 µM.
3. Experimental section
3.1 Chemistry
3.1.1. General experimental information
Solvents for extraction and chromatography were technical grade. All solvents used in reactions
were freshly distilled from appropriate drying agents before use. All other reagents were
recrystallized or distilled as necessary. All reactions were performed under an atmosphere of dry
nitrogen. Analytical TLC was performed with silica gel 60 F254 plates. Visualization was
1
13
31
19
accomplished by UV light. H, C, P and F NMR spectra were recorded on a Varian Unity
Plus (at 300 MHz, 75 MHz, 120 MHz and 282 MHz respectively) and on a Bruker Avance 400
1
13
(at 400 MHz for H, and 100 MHz for C). Chemical shifts (δ) are reported in ppm relative to
residual CHCl3 (δ = 7.26 ppm for H and δ = 77.16 ppm for 13C NMR) and using phosphoric
1
acid (50 %) as external reference (δ = 0.0 ppm) for 31P NMR spectra. Coupling constants (J) are
reported in Hertz. Data for 1H NMR spectra are reported as follows: chemical shift, multiplicity,
coupling constant, integration). Multiplicity abbreviations are as follows: s = singlet, d =
13
doublet, t = triplet, q = quartet, m = multiplet). C NMR peak assignments were supported by
Distortionless Enhanced Polarization Transfer (DEPT). High resolution mass spectra (HRMS)
were obtained by positive-ion electrospray ionization (ESI). Data are reported in the form m/z
(intensity relative to base = 100). Infrared spectra (IR) were taken in a Nicolet iS10 Termo
Scientific spectrometer as neat solids. Peaks are reported in cm–1.
3.1.2. Compounds Purity Analysis
All synthesized compounds were analyzed by HPLC to determine their purity. The analyses
were performed on Agilent 1260 infinity HPLC system (C-18 column, Hypersil, BDS, 5µm, 0.4
mm×25 mm) at room temperature. All the tested compounds were dissolved in
dichloromethane, and 5µL of the sample was loaded onto the column. Ethanol and heptane were
used as mobile phase, and the flow rate was set at 1.0 mL/min. The maximal absorbance at the
range of 190−400 nm was used as the detection wavelength. The purity of all the tested BIMP
substrates 9-16 is >95%, which meets the purity requirement by the Journal.
3.1.3. Experimental procedure and characterization data for compounds 6 and 9-16.
General procedure for the synthesis of starting α-aminophosphonates 6:
According to the previously described procedure [36], aqueous formaldehyde (37 %, 0.81 mL,
10 mmol) was added to a solution of tritylamine (1.30 g, 5 mmol) in toluene (20 mL) and the
reaction mixture was stirred for 20 h at room temperature. Then, water was removed using a
Dean-Stark and the corresponding hydrogen phosphate (6 mmol) and triethylamine (69.6 µL,
0.5 mmol) were added. The reaction was stirred under reflux in toluene overnight and the
volatiles were distilled off at reduced pressure to yield the crude product as clear liquids which
can be crystallized from toluene-pentane.
Dimethyl ((tritylamino)methyl)phosphonate 6a.[36] The general procedure was followed
affording 1.81 g (95%) of 6a as a white solid. M.p. (Toluene-pentane) = 200-204 ºC (Literature
1
3
[36] 210-211°C (CHCl3-MeOH)). H NMR (300 MHz, CDCl3): δ 7.48 (d, JHH = 7.7 Hz, 6H),
2
7.34 – 7.26 (m, 6H), 7.26 – 7.17 (m, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 2.54 (d, JPH = 13.9 Hz,
2H), 2.05 (broad s, 1H, NH). 13C {1H} NMR (75 MHz, CDCl3): δ 144.8 (Cquat), 128.6 (CH),

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3
2
128.2 (CH), 126.7 (CH), 71.6 (d, JPC = 19.1 Hz, Cquat), 53.1 (d, JPC = 6.6 Hz, CH3), 39.3 (d,
1JPC = 160.1 Hz, CH2). 31P NMR (120 MHz, CDCl3) δ 30.9.
Diethyl ((tritylamino)methyl)phosphonate 6b.[36] The general procedure was followed
affording 1.88 g (92%) of 6b as a white solid. M.p. (Toluene-pentane) = 116-117 °C (Literature
3
[36] 115-117 °C (CHCl3-MeOH)). 1H NMR (300 MHz, CDCl3): δ 7.49 (d, JHH = 8.0 Hz, 6H),
2
7.30 (m, 6H), 7.21 (m, 3H), 4.38 – 4.05 (m, 4H), 2.52 (d, JPH = 13.8 Hz, 2H), 2.04 (broad s,
3
13
1H, NH), 1.38 (t, JHH = 7.0 Hz, 6H). C {1H} NMR (75 MHz, CDCl3) δ 145.0 (Cquat), 128.7
(CH), 128.1 (CH), 126.7 (CH), 71.5 (d, 3JPC = 19.1 Hz, Cquat), 62.3 (d, 2JPC = 6.6 Hz, CH2), 39.9
(d, 1JPC = 160.2 Hz, CH2), 16.7 (d, 3JPC = 5.9 Hz, CH3). 31P NMR (120 MHz, CDCl3): δ 28.4.
Di-iso-propyl ((tritylamino)methyl)phosphonate 6c. The general procedure was followed
1
affording 1.99 g (91%) of 6c as a white solid. M.p. (Toluene-pentane) = 74-76 °C. H NMR
3
(300 MHz, CDCl3): δ 7.50 (d, JHH = 7.6 Hz, 6H), 7.37 – 7.26 (m, 6H), 7.26 – 7.16 (m, 3H),
2
3
4.86 – 4.70 (m, 2H), 2.46 (d, JPH = 14.0 Hz, 2H), 2.06 (broad s, 1H), 1.37 (d, JHH = 6.2 Hz,
12H). C {1H} NMR (75 MHz, CDCl3): δ 145.1 (Cquat), 128.7 (CH), 128.1 (CH), 126.6 (CH),
13
71.5 (d, 3JPC = 19.2 Hz, Cquat), 70.8 (d, 2JPC = 6.8 Hz, CH), 40.7 (d, 1JPC = 161.3 Hz, CH2), 24.3
(d, 3JPC = 5.3 Hz, CH3), 24.2 (d, 3JPC = 6.3 Hz, CH3). 31P NMR (120 MHz, CDCl3): δ 26.4.
Di-tert-butyl ((tritylamino)methyl)phosphonate 6d. The general procedure was followed
affording 2.26 g (97%) of 6d as a colorless liquid. 1H NMR (300 MHz, CDCl3): δ 7.49 (d, 3JHH
3
3
3
= 7.7 Hz, 6H), 7.31 – 7.23 (m, 6H), 7.17 (t, JHH = 7.2 Hz, 3H), 2.35 (dd, JPH = 13.5, JHH 8.1
Hz, 2H), 1.95 (broad s, 1H), 1.49 (s, 18H). 13C {1H} NMR (75 MHz, CDCl3): δ 145.5 (Cquat),
2
3
128.8 (CH), 128.0 (CH), 126.5 (CH), 82.2 (d, JPC = 8.8 Hz, Cquat), 71.4 (d, JPC = 18.8 Hz,
Cquat), 43.1 (d, JPC = 163.2 Hz, CH2), 30.6 (d, JPC = 4.0 Hz, CH3). 31P NMR (120 MHz,
1
3
CDCl3): δ 19.3.
Dibenzyl ((tritylamino)methyl)phosphonate 6e. The general procedure was followed
1
affording 2.53 g (95%) of 6e as a white solid. M.p. (Toluene-pentane) = 104-105 °C. H NMR
2
(300 MHz, CDCl3): δ 7.55 – 7.02 (m, 25H), 5.37 – 4.88 (m, 4H), 3.00 (m, 1H), 2.51 (d, JPH
=
13.5 Hz, 2H). 13C {1H} NMR (75 MHz, CDCl3): δ 144.9 (Cquat), 136.5 (d, 3JPC = 5.9 Hz, Cquat),
128.8 – 128.5 (CH), 128.1 (CH), 126.7 (CH), 71.5 (d, 3JPC = 19.4 Hz, Cquat), 67.9 (d, 2JPC = 6.7
Hz, CH2), 40.3 (d, 1JPC = 160.5 Hz, CH2). 31P NMR (120 MHz, CDCl3): δ 29.4.
General procedure for the synthesis of BIM substrates 9-13:
N-Bromosuccinimide (178 mg, 1 mmol) was added on a solution of corresponding
((tritylamino)methyl)phosphonate (1 mmol) in CCl4 (3 mL) The mixture was stirred in quartz
flask under UV light until observing the disappearance of starting aminomethylphosphonate by
31P-NMR. Then, the corresponding indole (2 mmol) and phosphoric acid catalyst (0.1 mmol) in
CH2Cl2 (10 mL) were added at room temperature and the reaction mixture was stirred for 36 h
at room temperature. After that, 20 mL of water were added and the organic phase was extracted
with CH2Cl2 (2x10 mL) and dried of in MgSO4. The volatiles were distilled off at reduced
pressure to yield the crude product, which was purified by column chromatography (AcOEt /
Hexanes).
Dimethyl (di(1H-indol-3-yl)methyl)phosphonate 9. The general procedure was followed
1
affording 209 mg (59%) of 9 as a white solid. M.p. (Et2O) = 239-240°C. H NMR (400 MHz,
MeOH-d4): δ 7.61 (dt, 3JHH = 8.0 Hz, 4JHH = 1.0 Hz, 2H), 7.39 (d, 3JHH = 2.7 Hz, 2H), 7.34 (dt,
3JHH = 8.1 Hz, 4JHH = 1.0 Hz, 2H), 7.08 (ddd, 3JHH = 8.1 Hz, 3JHH = 7.0 Hz, 4JHH = 1.1 Hz, 2H),
3
2
7.00 (ddd, 3JHH = 8.0Hz , JHH = 7.0 Hz, 4JHH = 1.0 Hz, 2H), 5.16 (d, JPH = 25.3 Hz, 1H), 3.52
3
13
2
(d, JPH = 10.6 Hz, 6H). C {1H} NMR (75 MHz, MeOH-d4): δ 137.8 (Cquat), 128.3 (d, JPC
=
3
8.7 Hz, Cquat), 125.4 (d, JPC = 6.4 Hz, CH), 122.5 (CH), 120.0 (CH), 119.6 (CH), 112.3 (CH),
111.2 (d, JPC = 6.1 Hz, Cquat), 53.9 (d, JPC = 7.4 Hz, CH3), 32.8 (d, JPC = 143.7 Hz, CH). P
3
2
1
31

ACCEPTED MANUSCRIPT
NMR (120 MHz, CDCl3:) δ 30.0. FTIR (neat) νmax : 3378 (N-H), 3177 (C-HAr), 1209 (P=O),
1059 (P-O-C). HRMS (Q-TOF) m/z calcd for C19H19N2O3P 354,1133, found 354,1144.
Diethyl (di(1H-indol-3-yl)methyl)phosphonate 10. The general procedure was followed
affording 233 mg (61%) of 10 as a white solid. M.p. (Et2O) = 144-146 °C. 1H NMR (300 MHz,
3
CDCl3): δ 8.65 (s, 2H), 7.65 (d, JHH = 7.8 Hz, 2H), 7.39 – 7.20 (m, 4H), 7.20 – 6.93 (m, 4H),
2
3
5.07 (d, JPH = 24.9 Hz, 1H), 4.04 – 3.84 (m, 2H), 3.84 – 3.61 (m, 2H), 1.04 (t, JHH = 7.0 Hz,
6H). 13C {1H} NMR (75 MHz, CDCl3): δ 136.1 (Cquat), 127.1 (d, 2JPC = 8.6 Hz, Cquat), 124.6 (d,
3JPC = 6.2 Hz, CH), 121.9 (CH), 119.4 (CH), 119.1 (CH), 111.4 (CH), 111.2 (d, 3JPC = 5.8 Hz,
Cquat), 62.8 (d, 2JPC = 7.3 Hz, CH2), 32.1 (d, 1JPC = 143.6 Hz, CH), 16.4 (d, 3JPC = 5.7 Hz, CH3).
31P NMR (120 MHz, CDCl3) δ 28.1. FTIR (neat) νmax : 3406 (N-H), 3248 (C-HAr), 1206 (P=O),
1076 (P-O-C). HRMS (Q-TOF) m/z calcd for C21H23N2O3P 382,1446, found 382,1456.
Di-iso-propyl (di(1H-indol-3-yl)methyl)phosphonate 11a. The general procedure was
followed affording 246 mg (60%) of 10 as a white solid. M.p. (Et2O) = 162-164 °C. 1H NMR
(400 MHz, CDCl3): δ 8.96 (s, 2H), 7.68 (d, 3JHH = 7.7 Hz, 2H), 7.31 – 7.21 (m, 4H), 7.15 – 7.04
(m, 4H), 5.04 (d, 2JPH = 25.2 Hz, 1H), 4.58 – 4.27 (m, 2H), 1.20 (d, 3JHH = 6.2 Hz, 6H), 0.72 (d,
3JHH = 6.2 Hz, 6H). 13C {1H} NMR (100 MHz, CDCl3): δ 136.0 (Cquat), 128.4 (CH), 128.1 (CH),
127.3 (d, 2JPC = 8.7 Hz, Cquat), 124.6 (d, 3JPC = 6.0 Hz, CH), 121.6 (CH), 119.1 (CH), 111.2 (d,
3JPC = 5.6 Hz, Cquat), 71.5 (d, 2JPC = 7.8 Hz, CH), 32.4 (d, 1JPC = 145.5 Hz, CH), 24.3 (d, 3JPC
3.2 Hz, CH3), 23.2 (d, JPC = 5.3 Hz, CH3). P NMR (120 MHz, CDCl3): δ 26.3. FTIR (neat)
νmax : 3404 (N-H), 3180 (C-HAr), 1216 (P=O), 1099 (P-O-C). HRMS (Q-TOF) m/z calcd for
C23H27N2O3P 410,1759, found 410,1778.
=
3
31
Di-iso-propyl (bis(6-fluoro-1H-indol-3-yl)methyl)phosphonate 11b. The general procedure
was followed affording 322 mg (69%) of 10 as a pale brown solid. M.p. (Et2O) = 85-86 °C. 1H
NMR (400 MHz, CDCl3): δ 8.89 (s, 2H), 7.53 (dd, 3JHH = 8.8 Hz, 4JFH = 5.3 Hz, 2H), 7.29-7.26
3
4
3
3
(m, 2H), 7.00 (dd, JFH = 9.7 Hz, JHH =2.3 Hz, 2H), 6.81 (ddd, JFH = 9.6 Hz, JHH = 8.8 Hz,
4JHH = 2.3 Hz, 2H), 4.91 (d, JPH = 25.4 Hz, 1H), 4.55 – 4.43 (m, 2H), 1.22 (d, JHH = 6.1 Hz,
2
3
6H), 0.77 (d, JHH = 6.2 Hz, 6H). C {1H} NMR (100 MHz, CDCl3): δ 160.0 (d, JFC = 237.3
3
13
1
3
3
5
Hz, 2x Cquat), 136.08 (d, JFC = 12.7 Hz, Cquat), 124.56 (dd, JPC = 6.4 Hz, JFC = 3.57 Hz, CH),
2
3
3
124.00 (d, JPC = 8.5 Hz, Cquat), 120.0 (d, JFC = 10.2 Hz, CH), 111.6 (d, JPC = 5.5 Hz, Cquat),
108.1 (d, 2JFC = 24.6 Hz, CH), 97.6 (d, 2JFC = 26.0 Hz, CH), 71.6 (d, 2JPC = 7.8 Hz, CH), 32.8 (d,
1JPC = 146.1 Hz, CH), 24.4 (d, 3JPC = 3.3 Hz, CH3), 23.4 (d, 3JPC = 5.3 Hz, CH3). 31P NMR (120
MHz, CDCl3): δ 24.9. 19F NMR (282 MHz, CDCl3): δ -121.9. FTIR (neat) νmax : 3398 (N-H),
3216 (C-HAr), 1218 (P=O), 1100 (P-O-C). HRMS (Q-TOF) m/z calcd for C31H25F2N2O3P
446,1571, found 466,1573.
Di-iso-propyl (bis(5-fluoro-1H-indol-3-yl)methyl)phosphonate 11c. The general procedure
was followed affording 299 mg (67%) of 10 as a pale brown solid. M.p. (Et2O) = 88-89 °C
3
(dec.). 1H NMR (400 MHz, CDCl3): δ 9.11 (d, JHH = 1.7 Hz, 2H), 7.30 – 7.24 (m, 4H,), 7.15
3
4
3
3
4
(dd, JHH = 8.8 Hz, JFH = 4.4 Hz, 2H), 6.85 (td, JFH = JHH = 9.1 Hz, JHH = 2.5 Hz, 2H), 4.82
(d, 2JPH = 25.3 Hz, 1H), 4.57 – 4.46 (m, 2H), 1.22 (d, 3JHH = 6.2 Hz, 6H), 0.79 (d, 3JHH = 6.2 Hz,
6H). 13C {1H} NMR (100 MHz, CDCl3): δ 157.9 (d, 1JFC = 234.3 Hz, Cquat), 132.7 (Cquat), 127.7
(m, Cquat),126.1 (d, 3JPC = 6.0 Hz, CH), 112.0 (d, 3JFC = 9.5 Hz, CH), 111.7 (m, Cquat), 110.5 (d,
2JFC = 26.3 Hz, CH), 104.3 (d, 2JFC = 23.8 Hz, CH), 71.7 (d, 2JPC = 7.7 Hz, CH), 33.0 (d, 1JPC
=
145.9 Hz, CH), 24.3 (d, 3JPC = 3.3 Hz, CH3), 23.4 (d, 3JPC = 5.1 Hz, CH3). 31P NMR (120 MHz,
CDCl3): δ 24.8. 19F NMR (282 MHz, CDCl3): δ -125.1. FTIR (neat) νmax : 3419 (N-H), 3213 (C-
HAr), 1218 (P=O), 1101 (P-O-C). HRMS (Q-TOF) m/z calcd for C31H25F2N2O3P 446,1571,
found 446,1573.
Di-iso-propyl (bis(5-methyl-1H-indol-3-yl)methyl)phosphonate 11d. The general procedure
was followed affording 272 mg (62%) of 10 as a brown solid. M.p. (Et2O) = 92-94 °C (dec.). 1H

ACCEPTED MANUSCRIPT
3
NMR (300 MHz, CDCl3): δ 8.34 (s, 2H), 7.48 (s, 2H), 7.34-7.29 (m, 2H), 7.18 (d, JHH = 8.3
3
2
Hz, 2H), 6.96 (dd, JHH = 8.1, 1.1 Hz, 2H), 4.94 (d, JPH = 25.2 Hz, 1H), 4.57 – 4.42 (m, 2H),
2.42 (s, 6H), 1.22 (d, 3JHH = 6.2 Hz, 6H), 0.77 (d, 3JHH = 6.2 Hz, 6H). 13C {1H} NMR (75 MHz,
2
3
CDCl3): δ 134.5 (Cquat), 128.4 (Cquat), 127.7 (d, JPC = 8.5 Hz, Cquat), 124.5 (d, JPC = 5.9 Hz,
CH), 123.5 (CH), 119.1 (CH), 111.5 (d, 3JPC = 5.6 Hz, Cquat), 110.8 (CH), 71.2 (d, 2JPC = 7.7 Hz,
CH), 32.6 (d, 1JPC = 145.3 Hz, CH), 24.5 (d, JPC = 2.8 Hz, CH3), 23.4 (d, 3JPC = 5.3 Hz, CH3),
3
31
21.7 (CH3). P NMR (120 MHz, CDCl3) δ 25.5. FTIR (neat) νmax : 3401 (N-H), 3245 (C-HAr),
1380 (C-HMe), 1218 (P=O), 1097 (P-O-C). HRMS (Q-TOF) m/z calcd for C31H25F2N2O3P
438,2072, found 438,2067.
Di-tert-butyl (di(1H-indol-3-yl)methyl)phosphonate 12a. The general procedure was
followed affording 298 mg (68%) of 12a as a white solid. M.p. (Et2O) = 162-164 °C (dec.). 1H
NMR (300 MHz, CDCl3): δ 8.66 (s, 2H), 7.66 (d, 3JHH = 7.8 Hz, 2H), 7.39 – 7.28 (m, 4H), 7.18
– 6.98 (m, 4H), 4.95 (d, 2JPH = 25.6 Hz, 1H), 1.20 (s, 18H). 13C {1H} NMR (75 MHz, CDCl3): δ
2
3
136.0 (Cquat), 127.8 (d, JPC = 8.4 Hz, Cquat), 124.6 (d, JPC = 6.1 Hz, CH), 121.7 (CH), 119.5
3
2
(CH), 119.2 (CH), 112.6 (d, JPC = 5.6 Hz, Cquat), 111.2 (CH), 82.7 (d, JPC = 10.5 Hz, Cquat),
35.1 (d, JPC = 149.4 Hz, CH), 30.3 (d, JPC = 3.8 Hz, CH3). 31P NMR (120 MHz, CDCl3): δ
18.7. FTIR (neat) νmax : 3416 (N-H), 3219 (C-HAr), 1224 (P=O), 1168 (P-O-C). HRMS (Q-TOF)
m/z calcd for C25H31N2O3P 438,2072, found 438,2090.
1
3
Di-tert-butyl (bis(5-fluoro-1H-indol-3-yl)methyl)phosphonate 12b. The general procedure
was followed affording 327 mg (69%) of 12b as a pale brown solid. M.p. (Et2O) = 145-147 °C
(dec.). 1H NMR (300 MHz, Acetone-d6): δ 10.39 (s, 2H), 7.60 (t, 3JHH = 2.4 Hz, 2H,), 7.42 (dd,
3JFH = 10.3, 4JHH = 2.5 Hz, 2H), 7.34 (dd, 3JHH = 8.8 Hz, 4JFH = 4.5 Hz, 2H), 6.83 (td, 3JFH = 3JHH
4
2
= 9.1 Hz, JHH = 2.5 Hz, 2H), 4.88 (d, JPH = 25.7 Hz, 1H), 1.28 (s, 18H). 13C {1H} NMR (75
1
2
MHz, Acetone-d6): δ 158.2 (d, JFC = 231.1 Hz, Cquat), 133.9 (Cquat), 129.1 (d, JPC = 10.1 Hz,
3
5
3
Cquat), 127.5 (dd, JPC = 6.3 Hz, JFC = 3.2 Hz, CH), 113.2 (m, Cquat), 112.9 (d, JFC = 9.6 Hz,
CH), 110.0 (d, 2JFC = 26.4 Hz, CH), 105.1 (d, 2JFC = 23.9 Hz, CH), 82.5 (d, 2JPC = 9.9 Hz, Cquat),
36.9 (d, 1JPC = 149.7 Hz, CH), 30.6 (d, 3JPC = 3.8 Hz, CH3). 31P NMR (120 MHz, Acetone-d6): δ
19
18.9. F NMR (282 MHz, Acetone-d6): δ -127.5. FTIR (neat) νmax : 3412 (N-H), 1217 (P=O),
1103 (P-O-C). HRMS (Q-TOF) m/z calcd for C25H29F2N2O3P 474,1884, found 474,1886.
Di-tert-butyl (bis(5-methyl-1H-indol-3-yl)methyl)phosphonate 12c. The general procedure
was followed affording 336 mg (72%) of 12c as a white solid. M.p. (Et2O) = 194-195 °C (dec.).
3
1H NMR (300 MHz, Acetone-d6): δ 10.25 (s, 2H), 7.59 – 7.52 (m, 2H), 7.47 (t, JHH = 2.7 Hz,
3
3
3
2
2H), 7.27 (d, JHH = 8.2 Hz, 2H), 6.92 (dd, JHH = 8.3 Hz, JHH = 1.5 Hz, 2H), 4.96 (d, JPH
=
26.0 Hz, 1H), 2.41 (s, 6H), 1.27 (s, 18H). 13C {1H} NMR (75 MHz, Acetone-d6): δ 135.5 (Cquat),
2
3
128.9 (d, JPC = 8.0 Hz, Cquat), 127.80 (Cquat), 125.6 (d, JPC = 6.2 Hz, CH), 123.4 (CH), 119.7
(CH), 112.4 (d, 3JPC = 5.7 Hz, Cquat), 111.7 (CH), 82.4 (d, 2JPC = 10.3 Hz, Cquat), 36.1 (d, 1JPC
=
3
31
149.7 Hz, CH), 30.5 (d, JPC = 3.8 Hz, CH3), 21.8 (CH3). P NMR (120 MHz, Acetone-d6): δ
19.5. FTIR (neat) νmax: 3407 (N-H), 1442 (C-HMe), 1227 (P=O), 1106 (P-O-C) cm–1. HRMS (Q-
TOF) m/z calcd for C31H25F2N2O3P 466,2385, found 466,2392.
Dibenzyl (di(1H-indol-3-yl)methyl)phosphonate 13a. The general procedure was followed
affording 273 mg (55%) of 13a as a white solid. M.p. (Et2O) = 131-132 °C (dec.). 1H NMR (400
3
3
MHz, CDCl3): δ 8.24 (s, 2H), 7.64 (d, JHH = 8.0 Hz, 2H), 7.41 (t, JHH = 2.4 Hz, 2H), 7.34 (d,
3JHH = 8.1 Hz, 2H), 7.25 – 7.14 (m, 8H), 7.10 – 7.01 (m, 6H), 5.15 (d, 2JPH = 25.2 Hz, 1H), 4.88
(dd, 2JHH = 11.8 Hz, 3JPH = 7.3 Hz, 2H), 4.62 (dd, 2JHH = 11.8 Hz, 3JPH = 8.7 Hz, 2H). 13C {1H}
3
NMR (100 MHz, CDCl3): δ 136.6 (d, JPC = 5.8 Hz, Cquat), 136.1 (Cquat), 128.4 (CH), 128.2
2
3
(CH), 128.0 (CH), 127.1 (d, JPC = 8.9 Hz, Cquat), 124.4 (d, JPC = 6.4 Hz, CH), 122.2 (CH),
3
2
119.7 (CH), 119.3 (CH), 111.5 (d, JPC = 5.4 Hz, Cquat), 111.3 (CH), 68.2 (d, JPC = 7.3 Hz,
CH2), 32.6 (d, JPC = 143.5 Hz, CH). 31P NMR (120 MHz, CDCl3): δ 28.9. FTIR (neat) νmax
1
:

ACCEPTED MANUSCRIPT
3399 (N-H), 3172 (C-HAr), 1218 (P=O), 1092 (P-O-C). HRMS (Q-TOF) m/z calcd for
C31H27N2O3P 506,1759, found 506,1758.
Dibenzyl (bis(6-fluoro-1H-indol-3-yl)methyl)phosphonate 13b. The general procedure was
followed affording 352 mg (65%) of 13b as a pale orange solid. M.p. (Et2O) = 60-62 °C (dec.).
1H NMR (300 MHz, CDCl3): δ 9.00 (s, 2H), 7.47 (dd, 3JFH = 8.6 Hz, 3JHH =5.3 Hz, 2H), 7.30 –
7.11 (m, 8H,), 7.01 (d, 3JHH = 7.5 Hz, 4H), 6.94 (d, 3JHH = 9.6 Hz, 2H), 6.83-6.73 (m, 2H), 5.06
(d, 2JPH = 25.1 Hz, 1H), 4.84 (dd, 2JHH = 11.6 Hz, 3JPH = 7.4 Hz, 2H), 4.63 (dd, 2JHH = 11.6 Hz,
3JPH = 9.1 Hz, 2H). 13C {1H} NMR (75 MHz, CDCl3): δ 160.0 (d, 1JFC = 237.4 Hz, Cquat), 136.1
(d, 3JPC = 8.5 Hz, Cquat), 136.0 (d, 3JFC = 9.8 Hz, Cquat), 128.5 (m, CH), 128.3 (m, CH), 127.9 (m,
CH), 124.9 (dd, 3JPC = 6.5 Hz, 5JFC = 3.3 Hz, CH), 123.5 (d, 3JPC = 8.4 Hz, Cquat), 119.8 (d, 3JPC
3
2
2
= 10.0 Hz, CH), 110.6 (d, JPC = 6.0 Hz, Cquat), 108.3 (d, JFC = 24.5 Hz, CH), 97.8 (d, JFC
=
25.8 Hz, CH), 68.4 (d, 2JPC = 7.4 Hz, CH2), 32.7 (d, 1JPC = 143.3 Hz, CH). 31P NMR (120 MHz,
CDCl3): δ 27.6. 19F NMR (282 MHz, CDCl3): δ -121.6. FTIR (neat) νmax: 3431 (N-H), 1224
(P=O), 1098 (P-O-C). HRMS (Q-TOF) m/z calcd for C31H25F2N2O3P 542,1571, found
542,1561.
Dibenzyl (bis(5-fluoro-1H-indol-3-yl)methyl)phosphonate 13c. The general procedure was
followed affording 363 mg (67%) of 13c as a pale orange solid. M.p. (Et2O) = 87-89 °C (dec.).
1H NMR (300 MHz, CDCl3): δ 8.84 (s, 2H), 7.54 – 7.04 (m, 12H), 6.96 (d, 3JHH = 6.9 Hz, 4H),
2
2
3
6.90-6.79 (m, 2H), 4.91 (d, JPH = 25.0 Hz, 1H), 4.73 (dd, JHH = 11.3 Hz, JPH = 7.4 Hz, 2H),
4.51 (m, 2H). 13C {1H} NMR (75 MHz, CDCl3): δ 157.9 (d, JFC = 234.6 Hz, Cquat), 143.8
1
(Cquat), 135.9 (d, 3JPC = 5.9 Hz, Cquat), 132.5 (Cquat), 128.5 (CH), 128.4 (CH), 127.9 (CH), 127.2
(m, Cquat), 126.6 (d, 3JPC = 5.9 Hz, CH), 112.3 (d, 3JFC = 9.2 Hz, CH), 110.5 (d, 2JFC = 26.6 Hz,
CH), 103.8 (d, 2JFC = 23.7 Hz, CH), 68.4 (d, 2JPC = 7.4 Hz, CH2), 32.4 (d, 1JPC = 143.3 Hz, CH).
31P NMR (120 MHz, CDCl3): δ 27.0. 19F NMR (282 MHz, CDCl3): δ -124.8. FTIR (neat) νmax
:
3432 (N-H), 1212 (P=O), 1105 (P-O-C). HRMS (Q-TOF) m/z calcd for C31H25F2N2O3P
542,1571, found 542,1561.
Dibenzyl (bis(5-(trifluoromethyl)-1H-indol-3-yl)methyl)phosphonate 13d. The general
procedure was followed affording 347 mg (54%) of 13d as a pale orange solid. M.p. (Et2O) =
107-108 °C (dec.). 1H NMR (400 MHz, CDCl3): δ 9.27 (s, 2H), 7.91 (s, 2H), 7.40 – 7.13 (m,
12H), 7.00 (d, 3JHH = 6.9 Hz, 4H), 5.07 (d, 2JPH = 24.9 Hz, 1H), 4.87 (dd, 2JHH = 11.4 Hz, 3JPH
=
8.2 Hz, 2H), 4.71 – 4.63 (m, 2H). 13C {1H} NMR (100 MHz, CDCl3): δ 137.5 (Cquat), 135.7 (d,
3JPC = 5.4 Hz, Cquat), 128.3 (CH), 128.1 (CH), 128.0 (CH) , 126.3 (d, 3JPC = 6.4 Hz, CH), 126.1
(d, 2JPC = 8.0 Hz, Cquat), 125.4 (q, 1JFC = 271.5 Hz, Cquat), 122.1 (q, 2JFC = 31.8 Hz, Cquat), 119.0
(q, 3JFC = 3.3 Hz, CH), 116.8 (q, 3JFC = 4.5 Hz, CH), 111.9 (CH), 111.2 (d, 3JPC = 6.0 Hz, Cquat),
2
1
68.7 (d, JPC = 7.5 Hz, CH2), 32.6 (d, JPC = 142.4 Hz, CH). 31P NMR (120 MHz, CDCl3): δ
26.5. 19F NMR (282 MHz, CDCl3): δ -60.6. FTIR (neat) νmax: 3423 (N-H), 1256 (P=O), 1101
(P-O-C). HRMS (Q-TOF) m/z calcd for C33H25F6N2O3P 642,1507, found 642,1487.
Dibenzyl (bis(6-(trifluoromethyl)-1H-indol-3-yl)methyl)phosphonate 13e. The general
procedure was followed affording 340 mg (53%) of 13e as a pale orange solid. M.p. (Et2O) =
96-97 °C (dec.). 1H NMR (400 MHz, CDCl3): δ 9.30 (s, 2H), 7.63 – 7.59 (m, 4H), 7.35 – 7.22
·
2
(m, 4H), 7.21 – 7.13 (m, 6H), 7.03 – 6.97 (d, JHH = 6.7 Hz, 4H), 5.11 (d, JPH = 25.2 Hz, 1H),
4.87 (dd, JHH = 11.7 Hz, JPH = 7.7 Hz, 2H), 4.71 (dd, JHH = 11.7 Hz, JPH = 9.3 Hz, 2H). 13C
2
3
2
3
{1H} NMR (100 MHz, CDCl3): δ 135.8 (d, JPC = 5.7 Hz, Cquat), 135.0 (Cquat), 129.0 (d, JPC
=
3
2
9.3 Hz, Cquat), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.1 (d, 3JPC = 6.2 Hz, CH), 125.3 (q, 1JFC
2
3
= 271.6 Hz, Cquat), 124.3 (q, JFC = 31.9 Hz, Cquat), 119.5 (CH), 116.4 (q, JFC = 3.5 Hz, CH),
110.9 (d, JPC = 5.8 Hz, Cquat), 109.2 (q, JFC = 4.4 Hz, CH), 68.6 (d, JPC = 7.5 Hz, CH2), 32.6
(d, 1JPC = 143.9 Hz, CH). 31P NMR (120 MHz, CDCl3) δ 26.9. 19F NMR (282 MHz, CDCl3) δ -
3
3
2

ACCEPTED MANUSCRIPT
61.0. FTIR (neat) νmax: 3419 (N-H), 1260 (P=O), 1097 (P-O-C) cm–1. HRMS (Q-TOF) m/z
calcd for C33H25F6N2O3P 642,1507, found 642,1496.
Dibenzyl (bis(5-methoxy-1H-indol-3-yl)methyl)phosphonate 13f. The general procedure was
followed affording 323 mg (57%) of 13f as a brown solid. M.p. (Et2O) = 140-142 °C (dec.). 1H
NMR (400 MHz, CDCl3): δ 8.32 (s, 2H), 7.41 – 7.34 (m, 2H), 7.24 – 7.10 (m, 8H), 7.08 – 6.96
(m, 6H), 6.82 (dd, 3JHH = 8.7 Hz, 4JHH = 2.5 Hz, 2H), 5.03 (d, 2JPH = 25.1 Hz, 1H), 4.86 (dd, 2JHH
3
2
3
= 11.6 Hz, JPH = 7.0 Hz, 2H), 4.60 (dd, JHH = 11.6 Hz, JPH = 8.4 Hz, 2H), 3.70 (s, 6H). 13C
{1H} NMR (75 MHz, CDCl3): δ 154.1 (Cquat), 136.4 (d, 3JPC = 6.3 Hz, Cquat), 131.3 (Cquat), 128.4
(CH), 128.2 (CH), 128.0 (CH), 127.5 (d, JPC = 8.4 Hz, Cquat), 125.4 (d, JPC = 6.1 Hz, CH),
2
3
3
2
112.4 (CH), 112.2 (CH), 110.7 (d, JPC = 5.8 Hz, Cquat), 101.0 (CH), 68.3 (d, JPC = 7.3 Hz,
CH2), 55.9 (CH3), 32.1 (d, JPC = 142.7 Hz, CH). 31P NMR (120 MHz, CDCl3): δ 27.7. FTIR
1
(neat) νmax: 3425 (N-H), 3189 (C-HAr), 1452 (C-HOMe), 1218 (P=O). HRMS (Q-TOF) m/z calcd
for C33H31N2O5P 566,1971, found 566,1965.
Dibenzyl (bis(6-methoxy-1H-indol-3-yl)methyl)phosphonate 13g. The general procedure was
followed affording 345 mg (61%) of 13g as a brown solid. M.p. (Et2O) = 109-110 °C (dec.). 1H
NMR (400 MHz, CDCl3): δ 8.37 (s, 2H), 7.47 (d, 3JHH = 9.28 Hz, 2H), 7.29 – 7.26 (m, 4H), 7.21
– 7.17 (m, 4H), 7.03 – 6.98 (m, 4H), 6.72 – 6.68 (m, 4H), 5.04 (d, 2JPH = 25.1 Hz, 1H), 4.82 (dd,
3
2
3
2JHH = 11.7 Hz, JPH = 7.1 Hz, 2H), 4.56 (dd, JHH = 11.7 Hz, JPH = 8.6 Hz, 2H), 3.75 (s, 6H).
13C {1H} NMR (101 MHz, CDCl3): δ 156.5 (Cquat), 136.8 (Cquat), 136.4 (d, 3JPC = 6.0 Hz, Cquat),
3
2
128.4 (CH), 128.1 (CH), 128.0 (CH), 123.5 (d, JPC = 6.6 Hz, CH), 121.5 (d, JPC = 8.7 Hz,
C
quat), 119.7 (CH), 110.8 (d, 3JPC = 6.1 Hz, Cquat), 109.7 (CH), 94.7 (CH), 68.2 (d, 2JPC = 7.3 Hz,
1
31
CH2), 55.7 (CH3), 32.7 (d, JPC = 142.7 Hz, CHP). P NMR (120 MHz, CDCl3): δ 27.9. FTIR
(neat) νmax: 3422 (N-H), 3245 (C-HAr), 1448 (C-HOMe), 1194 (P=O), 1059 (P-O-C). HRMS (Q-
TOF) m/z calcd for C33H31N2O5P 566,1971, found 566,1968.
Dibenzyl (bis(5,6-dimethoxy-1H-indol-3-yl)methyl)phosphonate 13h. The general procedure
was followed affording 363 mg (58%) of 13h as a brown solid. M.p. (Et2O) = 134-136 °C
(dec.). 1H NMR (400 MHz, DMSO-d6): δ 10.66 (s, 2H), 7.37 – 7.29 (m, 2H), 7.27 – 7.20 (m,
2
2
4H), 7.14 – 7.04 (m, 8H), 6.86 (s, 2H), 5.19 (d, JPH = 25.3 Hz, 1H), 4.87 (dd, JHH = 12.1 Hz,
3JPH = 6.5 Hz, 2H), 4.70 (dd, 2JHH = 12.1 Hz, 3JPH = 7.8 Hz, 2H), 3.73 (s, 6H), 3.65 (s, 6H). 13C
{1H} NMR (75 MHz, CDCl3): δ 147.1 (Cquat), 145.0 (Cquat), 136.2 (d, 3JPC = 6.4 Hz, Cquat), 130.2
3
2
(Cquat), 128.6 (CH), 128.4 (CH), 127.9 (CH), 123.7 (d, JPC = 5.9 Hz, CH), 119.7 (d, JPC = 7.9
3
2
Hz, Cquat), 110.0 (d, JPC = 6.0 Hz, Cquat), 100.6 (CH), 94.7 (CH), 68.3 (d, JPC = 7.3 Hz, CH2),
56.3 (CH3), 56.1 (CH3), 32.6 (d, JPC = 142.2 Hz, CH). 31P NMR (120 MHz, CDCl3): δ 27.4.
1
FTIR (neat) νmax: 3401 (N-H), 1445(C-HOMe), 1203 (P=O), 1150 (P-O-C). HRMS (Q-TOF) m/z
calcd for C35H35N2O7P 626,2182, found 626,2168.
Dibenzyl (bis(6-methyl-1H-indol-3-yl)methyl)phosphonate 13i. The general procedure was
followed affording 342 mg (64%) of 13i as a pale brown solid. M.p. (Et2O) = 81-83 °C (dec.).
3
1H NMR (300 MHz, CDCl3): δ 8.23 (s, 2H), 7.49 (d, JHH = 8.1 Hz, 2H), 7.29 – 7.13 (m, 8H),
3
7.10 – 6.98 (m, 6H), 6.87 (d, JHH = 8.1 Hz, 2H), 5.09 (d, 2JPH = 25.1 Hz, 1H), 4.86 (dd, 2JHH
=
11.7 Hz, 3JPH = 7.1 Hz, 2H), 4.60 (dd, 2JHH = 11.7 Hz, 3JPH = 8.6 Hz, 2H), 2.42 (s, 6H). 13C {1H}
NMR (75 MHz, CDCl3): δ 136.6 (Cquat), 131.8 (Cquat), 128.4 (CH), 128.1 (CH), 128.0 (CH),
2
3
125.0 (d, JPC = 8.8 Hz, Cquat), 123.9 (d, JPC = 6.3 Hz, CH), 121.4 (CH), 118.9 (CH), 111.3
(CH), 111.0 (d, 3JPC = 5.8 Hz, Cquat), 68.2 (d, 2JPC = 6.9 Hz, CH2), 32.7 (d, 1JPC = 142.8 Hz, CH),
21.8 (CH3). 31P NMR (120 MHz, CDCl3): δ 27.9. FTIR (neat) νmax: 3430 (N-H), 1454 (C-HMe),
1222 (P=O). HRMS (Q-TOF) m/z calcd for C33H31N2O3P 534,2072, found 534,2072.
Dibenzyl (bis(5-methyl-1H-indol-3-yl)methyl)phosphonate 13j. The general procedure was
followed affording 347 mg (65%) of 13j as a white solid. M.p. (Et2O) = 185-187 °C (dec.). 1H
NMR (300 MHz, CDCl3): δ 8.43 (s, 2H), 7.43 (s, 2H), 7.32 – 7.27 (m, 2H), 7.25 – 7.13 (m, 8H),

ACCEPTED MANUSCRIPT
7.06 – 6.94 (m, 6H), 5.11 (d, 2JPH = 25.1 Hz, 1H), 4.89 (dd, 2JHH = 11.7 Hz, 3JPH = 7.0 Hz, 2H),
4.59 (dd, 2JHH = 11.7 Hz, 3JPH = 8.1 Hz, 2H), 2.38 (s, 6H). 13C {1H} NMR (75 MHz, CDCl3): δ
136.6 (d, 3JPC = 6.3 Hz, Cquat), 134.5 (Cquat), 128.7 (Cquat), 128.4 (CH), 128.1 (CH), 127.97 (CH),
2
3
127.30 (d, JPC = 8.4 Hz, Cquat), 124.81 (d, JPC = 6.2 Hz, CH), 123.72 (CH), 118.84 (CH),
3
2
1
111.05 (CH), 110.52 (d, JPC = 5.9 Hz, Cquat), 68.20 (d, JPC = 7.2 Hz, CH2), 32.47 (d, JPC
=
31
142.7 Hz, CH), 21.65 (CH3). P NMR (120 MHz, CDCl3): δ 28.0. FTIR (neat) νmax: 3431 (N-
H), 1451 (C-HMe), 1221 (P=O). HRMS (Q-TOF) m/z calcd for C33H31N2O3P 534,2072, found
534,2068.
Dibenzyl (bis(2-methyl-1H-indol-3-yl)methyl)phosphonate 13k. The general procedure was
followed affording 337 mg (63%) of 13k as a pale brown solid. M.p. (Et2O) = 212-213 °C
(dec.). 1H NMR (300 MHz, CDCl3): δ 7.87 (m, 4H), 7.32 – 7.13 (m, 8H), 7.12 – 6.97 (m, 8H),
2
2
3
5.11 (d, JPH = 30.0 Hz, 1H), 4.85 (dd, JHH = 11.6 Hz, JPH = 7.5 Hz, 2H), 4.72-4.61 (m, 2H),
2.19 (s, 3H). 13C {1H} NMR (75 MHz, CDCl3): δ 136.6 (d, JPC = 5.9 Hz, Cquat), 134.9 (Cquat),
3
3
2
133.6 (d, JPC = 10.1 Hz, Cquat), 128.6 (d, JPC = 6.7 Hz, Cquat), 128.4 (CH), 128.2 (CH), 128.1
(CH), 120.9 (CH), 119.55 (CH), 119.5 (CH), 110.3 (CH), 106.6 (d, 3JPC = 3.6 Hz, Cquat), 67.9 (d,
2JPC = 7.3 Hz, CH2), 33.2 (d, 1JPC = 146.2 Hz, CH), 12.8 (CH3). 31P NMR (120 MHz, CDCl3): δ
28.4. FTIR (neat) νmax: 3396 (N-H), 1451 (C-HMe), 1227 (P=O). HRMS (Q-TOF) m/z calcd for
C33H31N2O3P 534,2072, found 534,2073.
Synthesis
of
(di(1H-indol-3-yl)methyl)phosphonic
acid
14.
Di-tert-butyl
((tritylamino)methyl)phosphonate (438.5 mg, 1 mmol) was diluted in THF (3 mL) and HCl 2M
(3 mL) were added. The reaction mixture was stirred at room temperature until the starting
material was consumed (monitored by TLC). The mixture was diluted in CH2Cl2 and the
combined organic phases were collected, dried with anhydrous MgSO4 and concentrated at
reduced pressure to yield the crude product which was crystallized in CHCl3-MeOH to afford
323 mg (99%) as a pink solid. M.p. (CHCl3-MeOH) = 217-219 °C (dec.). 1H NMR (400 MHz,
3
3
3
MeOH-d4): δ 7.52 (d, JHH = 7.9 Hz, 2H), 7.33 (d, JHH = 2.2 Hz, 2H), 7.26 (d, JHH = 8.1 Hz,
1H), 7.00 (ddd, 3JHH = 8.1 Hz, 3JHH =7.1 Hz, 4JHH = 1.1 Hz, 2H), 6.90 (ddd, 3JHH = 7.9 Hz, 3JHH
= 7.1 Hz, 4JHH =1.0 Hz, 2H), 4.95 (d, 1JPH = 24.8 Hz, 1H). 13C {1H} NMR (75 MHz, MeOH-d4):
2
3
δ 137.8 (Cquat), 128.8 (d, JPC = 8.5 Hz, Cquat), 125.1 (d, JPC = 5.9 Hz, CH), 122.2 (CH), 119.9
(CH), 119.6 (CH), 113.2 (d, 3JPC = 5.0 Hz, Cquat), 112.1 (CH), 34.7 (d, 1JPC = 141.5 Hz, CH). 31P
NMR (120 MHz, CDCl3): δ 26.4. FTIR (neat) νmax: 3431 (N-H), 1216 (P=O). HRMS (Q-TOF)
m/z calcd for C17H15N2O3P 326,0820, found 326,0811.
General procedure for the synthesis N-methylated substrates 15, 16:
The corresponding dialkyl bis(1H-indol-3-yl)methyl phosphonate (0.5 mmol) was diluted in
freshly distilled DMF (3 mL) under nitrogen atmosphere and the reaction mixture was cooled to
0ºC. NaH (36.0 mg, 1.5 mmol) were added and the reaction was stirred at room temperature for
1h. Then, the reaction was cooled to 0ºC, methyl iodide (0.12 mL, 2 mmol) was added and
stirring was continued at room temperature for 4h. The reaction mixture was quenched with
saturated NH4Cl solution (15 mL) and diluted in 10 mL of Et2O. DMF was removed by multiple
washing with saturated NH4Cl solution (10x15 mL). The organic phase was dried of in MgSO4
and concentrated at reduced pressure to yield the pure product in quantitative yield.
Di-iso-propyl (bis(1,5-dimethyl-1H-indol-3-yl)methyl)phosphonate 15. The general
procedure was followed affording 231 mg (99%) of 15 as a pale brown solid. M.p. (Et2O) = 78-
80 °C (dec.). 1H NMR (400 MHz, CDCl3): δ 7.50 – 7.46 (m, 2H), 7.34 – 7.22 (m, 2H), 7.14 (d,
3
4
2
3JHH = 8.3 Hz, 2H), 7.00 (dd, JHH = 8.3 Hz, JHH = 1.6 Hz, 2H), 4.91 (d, JPH = 25.2 Hz, 1H),
4.55 – 4.44 (m, 2H), 3.70 (s, 6H), 2.44 (s, 6H), 1.22 (d, 3JHH = 6.2 Hz, 6H), 0.78 (d, 3JHH = 6.2
Hz, 6H). 13C {1H} NMR (100 MHz, CDCl3): δ 135.3 (Cquat), 128.8 (d, 3JPC = 6.1 Hz, CH), 128.1
(d, JPC = 8.7 Hz, Cquat), 128.0 (Cquat), 123.2 (CH), 119.3 (CH), 110.2 (d, 32JPC = 5.4 Hz, Cquat),
2

ACCEPTED MANUSCRIPT
108.8 (CH), 71.1 (d, 2JPC = 7.5 Hz, CH), 33.0 (CH3), 32.4 (d, 1JPC = 145.2 Hz, CH), 24.5 (d, 3JPC
3
31
= 2.7 Hz, CH3), 23.4 (d, JPC = 5.5 Hz, CH3), 21.7 (CH3). P NMR (120 MHz, CDCl3) δ 25.5.
FTIR (neat) νmax: 1448 (C-HMe), 1222 (P=O), 1098 (P-O-C). HRMS (Q-TOF) m/z calcd for
C31H25F2N2O3P 466,2385, found 466,2390.
Di-tert-butyl (bis(1,5-dimethyl-1H-indol-3-yl)methyl)phosphonate 16. The general
procedure was followed affording 245 mg (99%) of 16 as a pale brown solid. M.p. (Et2O) = 82-
85 °C (dec.). 1H NMR (300 MHz, CDCl3): δ 7.45 (s, 2H, 2x indol-H2), 7.28 – 7.26 (m, 2H,
3
3
4
2xCHAr), 7.14 (d, JHH = 8.3 Hz, 2H, 2xCHAr), 7.00 (dd, JHH = 8.3 Hz, JHH = 1.3 Hz, 2H, 2x
indol-H6), 4.84 (d, 2JPH = 25.5 Hz, 1H, CHP), 3.71 (s, 6H, 2xNCH3), 2.44 (s, 6H, 2xCH3), 1.18
(s, 18H, 6xCH3). 13C {1H} NMR (75 MHz, CDCl3): δ 135.2 (Cquat), 129.1 (d, JPC = 6.0 Hz,
3
CH), 128.44 (d, 2JPC = 8.5 Hz, Cquat), 127.8 (Cquat), 123.0 (CH), 119.3 (CH), 111.1 (d, 3JPC = 5.4
Hz, Cquat), 108.7 (CH), 82.3 (d, 2JPC = 10.5 Hz, Cquat), 34.7 (d, 1JPC = 149.4 Hz, CH), 32.9 (CH3),
30.3 (d, 3JPC = 3.8 Hz, CH3), 21.7 (CH3). 31P NMR (120 MHz, CDCl3) δ 18.1. FTIR (neat) νmax
:
1452 (C-HMe), 1227 (P=O), 1101 (P-O-C). HRMS (Q-TOF) m/z calcd for C29H39N2O3P
494,2698, found 494,2709.
3.2 Biology
3.2.1. Materials
Reagents and solvents were used as purchased without further purification. All stock solutions
of the investigated compounds were prepared by dissolving the powered materials in
appropriate amounts of DMSO. The final concentration of DMSO never exceeded 10% (v/v) in
reactions. The stock solution was stored at 5ºC until it was used.
3.2.2. Cytotoxicity assays.
Cells were cultured according to the supplier´s instructions. Cells were seeded in 96-well plates
at a density of 2-4 x 103 cells per well and incubated overnight in 0.1 mL of media supplied
with 10% Fetal Bovine Serum (Lonza) in 5% CO2 incubator at 37 ºC. On day 2, drugs were
added and samples were incubated for 48 hours. After treatment, 10 µL of cell counting kit-8
was added into each well for additional 2 hours incubation at 37 ºC. The absorbance of each
well was determined by an Automatic Elisa Reader System at 450 nm wavelength.
Camptothecin was purchased from Sigma-Aldrich and used as positive control.
4. Acknowledgments
Financial support by Ministerio de Economía, Industria y Competividad (MINECO, CTQ-2015-
67871R) and Gobierno Vasco (GV, IT 992-16) is gratefully acknowledged. We also thank
SGIker (UPV/EHU) technical support for NMR spectra (MINECO, GV/EJ, and European
Social Found).
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A double nucleophilic addition of indole derivatives to an in situ generated α-iminophosphonate
yields very efficiently phosphorus substituted bis-(3-indolyl)methane derivatives.
Phosphorated bis-(3-indolyl)methane derivatives are hybrid substrates, holding indole and
phosphonate moieties, and they showed in vitro cytotoxicity, inhibiting the growth of carcinoma
human tumor cell lines A549 and SKOV03.
Substrates 13j and 12c derived from 5-methylindole show very good IC50 values of about 0.06
µM.