Revisiting aryl amidine synthesis using metal amide and/or ammonia gas: Novel molecules and their biological evaluation

Article abstract of DOI:10.1080/00397911.2017.1330959

Amidines, due to their unique biocompatibility and desirable physical characteristics, have been the functionality of choice as a scaffold for large number of drug synthesis. But still synthesis of amidines in the presence of other active functional groups or pharmacophore, remained a challenge. In this work, a simple and reliable protocol for conversion of nitrile-amide to unsubstituted amidine–amide is developed using metal amide and/or ammonia gas. The scope and efficiency of this synthetic strategy are demonstrated on several substrates which differ in functional groups will be discussed. In this process, 10 novel aryl amidines in good yields (upto 85%) were synthesized. Biological evaluation revealed that compound 4-(aminoiminomethyl)-N-(2-furanyl methyl) benzamide (IC₅₀ = 9 μM) and 4-(aminoiminomethyl)-N-(3-pyridinylmethyl) benzamide (73.36% growth inhibition) showed moderate efficacy for cancer cells.

Full text of DOI:10.1080/00397911.2017.1330959

Synthetic Communications
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Revisiting Aryl Amidine Synthesis Using Metal
Amide and/or Ammonia Gas: Novel Molecules and
Their Biological Evaluation
Ishani I. Sahay, Prasanna S. Ghalsasi, Mala Singh & Rasheedunnisa Begum
To cite this article: Ishani I. Sahay, Prasanna S. Ghalsasi, Mala Singh & Rasheedunnisa
Begum (2017): Revisiting Aryl Amidine Synthesis Using Metal Amide and/or Ammonia
Gas: Novel Molecules and Their Biological Evaluation, Synthetic Communications, DOI:
10.1080/00397911.2017.1330959
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Date: 19 May 2017, At: 21:23

Revisiting Aryl Amidine Synthesis using Metal Amide and/or Ammonia Gas: Novel
Molecules and their Biological Evaluation
Ishani I. Sahay1, Prasanna S. Ghalsasi1, Mala Singh1, Rasheedunnisa Begum2
1Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of
Baroda, Vadodara, India 2Department of Biochemistry, Faculty of Science, The Maharaja
Sayajirao University of Baroda, Vadodara, India
Corresponding Author Prasanna S. Ghalsasi E-mail: prasanna.ghalsasi@gmail.com
Abstract
Amidines, due to their unique biocompatibility and desirable physical characteristics,
have been the functionality of choice as a scaffold for large number of drug synthesis.
But still synthesis of amidines in presence of other active functional groups or
pharmacophore, remained a challenge. In this work, a simple and reliable protocol for
conversion of nitrile-amide to unsubstituted amidine-amide is developed using metal
amide and/or ammonia gas. The scope and efficiency of this synthetic strategy is
demonstrated on a number of substrates which differ in functional groups will be
discussed. In this process ten novel aryl amidines in good yields (upto 85%) were
synthesized. Biological evaluation revealed that compound 4-(aminoiminomethyl)-N-(2-
furanyl methyl) benzamide (IC50=9μM) and 4-(aminoiminomethyl)-N-(3-
pyridinylmethyl) benzamide (73.36% growth inhibition) showed moderate efficacy for
cancer cells.
1

Graphical Abstract
KEYWORDS: Amidine, amide, anti proliferative activity, single crystal, NCI
INTRODUCTION
Amidine functionality serves as synthon for the synthesis of variety of heterocyclic
compounds. [1] On the other hand amidines with their unique structural properties and
bio-compatibility are present in various active pharmaceuticals ingredients (API)
employed for antiviral, anti-inflamatory and anticancer[2]. This latter aspect will open up
new scope for amidines as a pharmacophore.
Syntheses of amidines have been explored by many scientists in the past. Out of the
various reagents available for the synthesis of amidine, single step conversion of nitrile to
amidine remained choice of synthetic chemists. In this method the nitrile functionality is
activated by the electron withdrawing groups or lewis acids [3,4] to give the desired
products.
Pinner reaction, to the best of our knowledge, happens to be the most commonly used
technique for the amidine synthesis from the parent nitrile compound.[5] Contemplating
2

the mechanism, first step being the cyanide activation using dry HCl gas in dry methanol,
leads to the formation of amidinates as shown in Scheme 1. Ammonia attack is the next
step leads to amidine, the desired product, in good yield. Key step in this process remains
the activation of nitrile group by HCl. This may lead to unwanted chemical reactions if
additional functional groups are present on substrate. This is what is observed in our case,
nitrile-amide system, where latter functional group started reacting prior to nitrile
acitivation leading to the formation of undesired products. Hence for our substrate there
is a need to find alternative methodology where nitrile group is activated prior to amide
linkage.
Further literature search resulted in few more alternative methods where scientist have
used different reagents/reaction conditions for the conversion of nitrile to amidine
(Scheme 2).[3,6,7] Cornell et al (1928) reported the synthesis of aliphatic and aromatic
amidines from their parent nitriles using liquefied ammonia gas with various metal
amides.[8] Criticality of this reaction remained with the difficulty in handling liquefied
ammonia gas in standard synthetic laboratory. Newbery et al (1947) used same reagent
(metal amides) but with a slightly milder conditions, using benzene as solvent at high
temperature.[9] The drawback of using this method was the solubility of the parent nitriles
in benzene and also the toxicity of the solvent.[10] Interestingly, today’s drug design
challenge revolves around presence of more than one functional group (pharmacophores)
on a single API.[11]
RESULTS AND DISCUSSIONS
3

Keeping above literature survey in mind, our efforts were focused on amidine synthesis
-
in presence of amide (R.CO.NH2) functionality, our substrates, using amide anion (NH2 ).
This process can be considered as a ‘direct’ amidine formation method, since it requires
-
no prior activation of nitrile functionality. Now, NH2 can be generated in-situ as well as
ex-situ means. Therefore we planned both these methods: Method 1- in-situ generation:
dry NH3(g) with sodium hydride in DMSO, and Method 2- ex-situ generation: Sodamide
in DMSO (Scheme 3). The role of sodium hydride in the Method-1 is to abstract proton
-
from ammonia and form NH2 (in-situ generation). Both these methods, when carried out
at room temperature (16-25°C), gave good yields with substantial reduction in the time of
the reaction as compared to literature. Interestingly bottle-neck for both these modified
method remains in the contact time of sodamide and nitrile functionality, which can be
controlled by tailoring the reaction conditions. Table 1 shows results of screening
different reaction conditions for both these methods on 4-(aminoiminomethyl)-
benzamide, a standard substrate with presence of amide functionality with nitrile. Thus,
standardized optimum conditions were employed to synthesis of ten new compounds
with amidine-amide linkages.
The synthesis of 4-(aminoiminomethyl)-benzamide, one of the standard lead compound,
was carried out using Scheme 3. To validate this synthetic procedures different
substituent’s in the form of halogens, heterocycles were introduced near amide
functionality. These structures are tabulated in table 1. While tailoring the structures
with different substituent, PAINS (Pan Assay Interference Compounds) were kept in
mind [12]
.
PAINS functionality doesn’t discriminate between target and non-target
4

moieties leading to plethora of side effects. We observed key step during the synthesis of
this series compounds remained in the formation of amidine from parent nitrile
functionality.
4-iodo-N-(4-methoxyphenyl) benzamide 5a was obtained from 4-iodobenzoic acid 4
(refer ESI for synthesis) in two stages, treatment with thionyl chloride in first step and
DMAP/R-NH2 [13–15] in second step (Scheme 2). For the later step, we employed variety
of bases triethyl amine (TEA), diethyl amine (DEA) and (dimethyl amino pyridine)
DMAP along with number of solvents such as dry ACN, MDC, CHCl3 and CCl4. The
best yields were obtained with MDC and DMAP. Traditionally, Iodo/nitrile exchange
employes NaCN or KCN but in our present strategy we preferred ‘green’ nitrile source in
the form of cuprous cyanide (CuCN). Good yield of 4-cyano-N-(4-methoxyphenyl)
benzamide (6a-j) were accessed by reacting 4-iodo-N-(4-methoxyphenyl) benzamide (5a-
j) with the requisite CuCN as the nitrile source in dry DMF(refer ESI for detailed
experimental procedure).
All the new compounds were characterized by FT-IR, 1HNMR, 13C NMR, Mass
spectrometry and micro analysis (Refer ESI for spectral details). Spectra analyses were
consistent with the assigned structures.
Figure 1 shows ORTEP diagram for single crystal of 7j. It crystallizes in triclinic crystal
system with P1 space group, with two molecules in an asymmetric unit. No prominent
hydrogen bonding is observed in spite of having presence of strong hydrogen bond donor
5

and hydrogen bond acceptor groups. Significant and expected short contact amongst two
neighboring amide bonds through NH…O bonding (3.247Å and 171.5°) is observed along
a-axis. Although this interaction is considered to be weak in nature but it is reported that
it plays an important role in the protein-drug binding.[16] This short-contact also helps in
maintaining planarity of two aromatic groups and hence pharmacophore. (CCDC no.
1432792, detailed structure information can be obtained from supporting information).
Cytotoxicity studies[17] on HeLa cell line was performed for all the new compounds. Cis-
platin was used as the reference drug. Percentage cell viability of synthesized compounds
on HeLa cell line at various concentrations was checked and then from that IC50 was
calculated (One way ANOVA (non-parametric test was carried out. P value=
0.0062(**)). Table 3 shows results of IC50 in µM concentrations.
All molecules were initially screened for anti-proliferative activity in silico by National
Cancer Institute (NCI), USA (Refer ESI for experimental procedure). Out of this,
compound 7b, 7c, 7d and 7g were further selected for actual screening anti-proliferative
activity at 10µM concentration. Graph 1 shows comparative study of compound 7b, 7c,
7d and 7g on selected human derived cell lines NCI-H522 (Non-Small cell lung cancer),
HCT-116 (Colon cancer), SF-539 (CNS cancer), OVCAR-8 (Ovarian cancer) and SN-12
(Renal cancer). 7g shows 73.36% growth inhibition in HCT-116 colon cancer cell line
(mean growth inhibition) at 10µM concentration.
6

Two heterocyclic structure containing derivatives of furan and picolyamine were found to
be most potent among all. Both compounds 7g and 7h have a heterocycle in conjugation
with NH side of amide linkage, but also have flexible -CH2 bridge. Thus, from anti
cancer activity perspective we can conclude that 4-(aminoiminomethyl)-N-(3-
pyridinylmethyl)benzamide (7g) and 4-(aminoiminomethyl)-N-(2-
furanylmethyl)benzamide (7h) can be investigated further for the development as new
leads.
EXPERIMENTAL
This includes synthesis and characterization of one of the new molecules. Experimental
procedures for the intermediates and rest of the derivatives are presented in supporting
information.
Materials And Methods
All the compounds were purified using column chromatography (2000- 400 mesh silica)
before characterization. TLC analysis was done using pre-coated silica on aluminum
sheets. Melting points were recorded in Thiele’s tube using paraffin oil and are
uncorrected. FT-IR (KBr pellets) spectra were recorded in the 4000-400 cm–1 range using
a Perkin-Elmer FT-IR spectrometer. The NMR spectra were obtained on a Bruker AV-III
400 MHz spectrometer using TMS as an internal standard. The chemical shifts were
reported in parts per million (ppm), coupling constants (J) were expressed in hertz (Hz)
and signals were described as singlet (s), doublet(d), triplet(t), broad (b) as well as
multiplet (m). The microanalysis was carried out using a Perkin-Elmer IA 2400 series
7

elemental analyzer. The mass spectra were recorded on Thermo scientific DSQ-II. All
chemicals and solvents were of commercial grade and were used without further
purification. Single crystal data was collected with Xcalibur, EoS, Gemini.
General Procedure For The Synthesis Of Compound 7
Method 1: Dry DMSO and NaH(60% suspension in oil, 0.100mg,0.0025 mol) were
stirred at 16-20°C for 15mins. Compound 6a (0.100g, 0.0037mol) was then added and
ammonia gas was purged in it till the completion of the reaction (monitored by TLC,
eluent, petroleum ether/ethyl acetate, 1/4, V/V), cooled the reaction. Then the water was
added slowly such that temperature of reaction should not exceed 30°C. Residue was
extracted with ethyl actetate (3x10ml) and purified by neutral alumina column
chromatography (eluent, petroleum ether/ ethyl acetate, 1/4, v/v) to afford compound 7 as
off white solid to pale yellow solid. Yield: 60-85%. Melting point: 200-202°C.
Method 2: Dry DMSO and compound 6a (0.100g, 0.0037mol) were stirred at 25°C for
5mins, added sodamide in it and stirred for 2-15mins at same temperature. Reaction was
monitored by TLC (eluent, petroleum ether/ethyl acetate, 1/4, v/v), cooled the reaction
and water was added slowly such that temperature of reaction should not exceed 30°C.
Residue was extracted with ethylacetate (3x10ml) and purified by neutral alumina
column chromatography (eluent: petroleum ether/ethylacetate, 1/4, v/v) to afford
compound 7 as off-white solid to pale yellow solid. Yield: 60-85%.
4-(Aminoiminomethyl)-N-(4-Methoxyphenyl) Benzamide (7a)
8

Following the above general procedure title compound was synthesized. Off-white solid.
Yield: 82%; M.P: 200-202°C ;IR (KBr) γ: 3336.77(amidine-NH), 2923 (w),
1681.31(C=O), 1647.57(C=N), 1534.10(NH), 1269.39(C-N), 1249.80(C-O), 1031.31(C-
O amide), 824.13(para substitution) cm-1; 1H NMR (400MHz,DMSO-d6) δ: 10.37(s,
NH), 8.04(d,2H, J=8 Hz), 7.99(d,2H,J=8 Hz),7.59(d,2H,J=8.8 Hz), 6.92(d,2H,J=8.8 Hz),
3.75(s,3H), 2.65(s,3H, amidine) ppm; 13C NMR (100MHz, DMSO-d6) δ: 198.9(C=NH),
165.3(C=O), 156.4(C-O), 139.1(C-C=O), 131.8(C-C=NH), 128.7(2C), 128.3(2C),
122.9(2C), 114.3(2C), 55.6(O-CH3) ppm; MS(m/z): (M+) 269.15; Micro Analysis: Anal.
Calc. for C15H15N3O2: C, 66.90; H, 5.61; N, 15.60 %; Found: C 67.20; H, 5.40; N, 15.82
%.
CONCLUSION
Conversion from nitrile to amidine can be achieved effectively in a single step and in the
presence of amide functionality using metal amide and/or ammonia gas. This method is
extended for the synthesis of ten new amidines-amide conjugates where strategy works
effectively in presence of heterocyclic functionality as well. Apart from nearing room
temperature most of the time yield observed in the modified reaction conditions clocks
above 70% for nitrile to amidine conversion. Our preliminary results confirmed that
present strategy, amidine-amide conjugates can act as anti-proliferative active
compounds, similar to observed in literature.[18] In short, this study paves a way to
synthesize not only novel amidines but also amidine-amide conjugates, a strategy for
future drug design.[19]
9

SUPPLEMENTARY DATA
Supplementary data (experimental procedures and full spectroscopic data for all new
compounds) associated with this article can be found, in the online version. CCDC
1432792 (7j) contains the supplementary crystallographic data for this paper. These data
can be obtained from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data request/cif.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENT
PSG thank Department of Science and Technology, New Delhi (SR/S1/IC-43/2009) for
financial support. IIS thanks JRF and SRF to DST-project and UGC-BSR fellowship. We
would like to thank Dr. Hemant Mande for single crystal X-ray study. The authors also
thank the DST-PURSE Single Crystal X-ray Diffraction facility at the Faculty of Science,
The Maharaja Sayajirao University of Baroda, Vadodara. The authors thank the National
Cancer Institute for evaluating compounds 7b, 7c, 7d and 7g in the NCI's Developmental
Therapeutics Program (DTP) in vitro cell line screening.
REFERENCES
[1]. (a) Chu, X. Q.; Cao, W. B.; Xu, X. P.; Ji, S. J. J. Org. Chem. 2017, 82, 1145-1154 ;
(b) Ma, B.; Wang, Y.; Peng, J.; Zhu, Q. J. Org. Chem. 2011, 76, 6362-6366 ;(c) Doise,
M.; Blondeau, D.; Sliwa, H. Synth. Commun. 1992, 22, 2891-2901.
10

[2]. (a)Yan, L.; Yan, C.; Qian, K.; Su, H.; Kofsky-Wofford, S. A.; Lee, W. C.; Zhao, X.;
Ho, M. C.; Ivanov, I.; Zheng, Y. G. J. Med. Chem. 2014, 57, 2611 ;(b) Wang, J.; Xu, F.;
Cai, T.; Shen, Q. Org. Lett. 2008, 10, 445-448 ;(c) Cortes-Salva, M.; Garvin, C.; Antilla,
J. C. J. Org. Chem. 2011, 76, 1456-1459.
[3]. Oxley, P.; Partridge, M. W.; Short, W. F. J. Chem. Soc. 1947, 209. Amidines. Part
VII, 1110-1116.
[4]. (a) Garigipati, R. S. Tet. Lett. 1990, 31, 1969-1972 ;(b) Grivas, J. C.; Taurins, A.
Can. J. Chem, 1961, 39, 761-764.
[5]. Pinner, A.; Klein, F. Eur. J. Inorg. Chem. 1877, 10, 1889-1897.
[6]. Boeré, R. T.; Oakley, R. T.; Reed, R. W. J. Organometallic Chem. 1987, 331, 161-
167.
[7]. Ullapu, P. R.; Ku, S. J.; Choi, Y. H.; Park, J. Y.; Han, S. Y.; Baek, D. J.; Lee, J. K.;
Pae, A. N.; Min, S. J.; Cho, Y. S. Bulletin of the Korean Chem. Soc. 2011, 32(spc8),
3063-3073.
[8]. (a) Cornell, E. F. J. Am. Chem. Soc. 1928, 50, 3311-3318 ;(b) Schaefer, F. C.;
Krapcho, A. P. JOC, 1962, 27, 1255-1258.
[9]. Newbery, G.; Webster, W. J. Chem. Soc. 1947, 738-742.
[10]. Nadrah, K.; Dolenc, M. S. Synlett, 2007, 8, 1257-1258.
[11]. (a)Yang, E. G.; Mustafa, N.; Tan, E. C.; Poulsen, A.; Ramanujulu, P. M.; Chng, W.
J.; Yen, J. J.; Dymock, B. W. J. Med. Chem. 2016, 59, 8233-8262 ;(b)Yang, S.Y. Drug
Discovery Today 2010, 15, 444-450.
[12]. Baell, J.; Walters, M. A. Nature 2014, 513, 481.
[13]. Vartale, S. P.; Pawar, Y. D.; Halikar, N. K. Heteroletters 2012, 2,71-78.
11

[14]. Hwang, S.; Choi, S. Y.; Lee, J. H.; Kim, S.; In, J.; Ha, S. K.; Lee, E; Kim, T. Y.;
Kim, S. Y.; Choi, S.; Kim, S. Bioorg Med. Chem. 2010, 18, 5602-5609.
[15]. Amin, K. M.; Gawad, N. M. A.; Rahman, D. E. A.; EI Ashry, M. K. Bioorg Chem.
2014, 52, 31-43.
[16]. Massorotti, A.; Aprile, S.; Mercalli, V.; Grosso, E. D.; Grosa, G.; Sorba, G.; Tron,
G. C. ChemMedChem. 2014, 9, 2497-2508.
[17]. Mosmann, T J. Immun. Methods, 1983, 65, 55-63.
[
18]. (a) Hu, H.; Owens, E. A.; Su, H.; Yan, L.; Levitz, A.; Zhao, X.; Henary, M.;
Zheng, Y. G. J. Med. Chem. 2015, 58, 1228 ;(b) Hart, P.; Thomas, D.; van
Ommeren, R.; Lakowski, T. M.; Frankel, A.; Martin, N. I. Med. Chem. Commun.
2012, 3, 1235-1244 ;(c) Spannhoff, A.; Heinke, R.; Bauer, I.; Trojer, P.; Metzger,
E.; Gust, R.; Schule, R.; Brosch, G.; Sippl, W.; Jung, M. J. Med. Chem. 2007, 50,
2319-2325 ;(d) Ragno, R.; Simeoni, S.; Castellano, S.; Vicidomini, C.; Mai, A.;
Caroli, A.; Tremontano, A.; Bonaccini, C.; Trojer, P.; Bauer, I.; Brosch, G. J. Med.
Chem. 2007, 50, 1241-1253
[19]. Medina-Franco, J. L.; Giulianotti, M. A.; Welmaker, G. S.; Houghten, R. A.
Drug Discovery Today 2013, 18, 495-501.
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Table 1 Investigation of solvents and reagents on our substrates
Reagentb
Entry
Solvent Yieldc %
1
Na, NH4Cl
MeOH NR
2
HCl(g), EtOH/NH3(g)
EtOH
NR
NR
2
NH2OH.HCl,TEA/NH3(g) EtOH
3
NH4Cl, Sid
MeOH NR
MeOH NR
4
EtOH.HCl/NH4Cl
NH3(g), NaH
NH3(g), NaH
NH3(g), NaH
NH4Cl, NaH
NH4Cl, NaH
NH3(g), NaH
NaNH2
5
THF
15
35
6
DMF
7
Toluene NR
Toluene NR
DMSO NR
DMSO 83
DMSO 85
8
9
10
11
*NR= No reaction, desired product is not obtained.
b
Reactions were performed based on the literature procedure.
c Yield of the isolated product after silica gel chromatography
d Reaction was performed in a sealed tube.
13

Table 2 List of derivatives of amidine-amide conjugates (7)
Entry Structure (R)
7a
Yield* Melting Point(°C)
82
85
74
200-202
230-232
204-205
7b
7c
7d
78
70
60
198-200
135-140
220-222
7e
7f
7g
65
156-158
7h
7i
75
83
144-146
170-172
14

7j
72
180-183
*Yields are reported for the final step f
15

Table 3 IC50 Values of the compounds 7a-j on HeLa cells
Compound 7a 7b 7c 7d 7e 7f 7g
7h 7i
7j
Cisplatin
IC50(µM)a >200 189 >200 153 >200 NDb >200 9 >200 NDc 28
a IC50 values are the mean of four independent determinations,
b IC50 value exceeded the mM concentration,
c Poor solubility
16

Scheme 1. Pinner Reaction condition
17

Scheme 2. Literature work for amidine synthesis
18

Scheme 3. Synthesis of target 4-(aminoiminomethyl)-benzamide derivatives
19

Figure 1. ORTEP diagram of 7j with 50 % probability
20

Graph 1. % Growth Inhibition on selected cell lines at 10µM concentration NCI-H522:
Non-Small cell lung cancer cell line; HCT-116: Colon cancer cell line; SF-539: CNS
cancer cell line; OVCAR-8: Ovarian cancer cell line; SN-12: Renal cancer cell line
21