Synthesis of 2-guanidinyl pyridines and their trypsin inhibition and docking

Synthesis of 2-Guanidinyl Pyridines and their Trypsin Inhibition and Docking

a

b

c

Nahlah Ahmed Al-Hadhrami, Angelique Ladwig, Adeyemi Rahman, Isabel Rozas, J. Paul G. Malthouse and Paul

a

Evans *

^aSchool of Chemistry, Centre for Synthesis and Chemical Biology, University College Dublin, Dublin D04 N2E2, Ireland.

Email: paul.evans@ucd.ie

^bSchool of Chemistry, TBSI, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland.

^cSchool of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute,

University College Dublin, Dublin D04 N2E2, Ireland.

Abstract-A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These

compounds were evaluated in relation to their competitive inhibition of bovine pancreatic trypsin. Results

demonstrate that compounds in which the guanidinyl substituent can form an intramolecular hydrogen bond (IMHB)

with the pyridinyl nitrogen atom (6a-p) are better trypsin inhibitors than their counterparts (10-13) that are unable to

form an IMHB. Among the compounds 6a-p, examples containing a 5-halo substituent were, generally, found to be

better trypsin inhibitors. This trend was inversely related to electronegativity, thus, 1-(5-iodopyridin-2-yl)guanidinium

ion 6e (K

i

= 0.0151 mM) was the optimum inhibitor in the 5-halo series. Amongst the isomeric methyl substituted

compounds, 1-(3-methylpyridin-2-yl)guanidinium ion 6h demonstrated optimum levels of trypsin inhibition (K

i

=

0

.0140 mM). In order to rationalise the measured enzyme inhibition, selected compounds were docked with bovine

and human trypsin with a view to understanding active site occupancy and taken together with the K values the order

i

of inhibitory ability suggests that the 5-halo 2-guanidinyl pyridine inhibitors form a halogen bond with the catalytically

active serine hydroxy group.

Keywords: Serine protease; inhibitor; pyridin-2-yl guanidine; halogen bonding; hydrogen bonding; enzymology;

molecular docking.

1

. Introduction

1

Proteases comprise of a very large enzyme family which play organism essential roles. They catalyse the hydrolysis

of an amide/peptide bond within their substrate(s) and are, therefore, also widely referred to as peptidases and/or

hydrolytic enzymes. Different protease sub-sets are classified according to the catalytically active residue and the

serine proteases, relying on the nucleophilicity of the serine hydroxy group, are then further divided according to their

1,2

particular type of substrate. For example, trypsin, a well-documented and an archetypal member of the Clan PA

1

-3

group of serine proteases, preferentially digests substrates (e.g. 1) presenting a basic residue in the P

1

position (e.g.

lysine and arginine).⁴This residue, which is positively charged at physiological pH, interacts with the negatively

charged side chain carboxylate group of aspartate-189, located in the S site of trypsin (see Figure 1).

1-4

1

Figure 1. The catalytic triad in serine protease-based peptide (1) hydrolysis and the general guanidinium ion substrate mimic 2.

The selective inhibition of trypsin is of interest in relation to certain diseases, for example pancreatitis and conditions

5

associated with respiration. Furthermore, due to its availability, low cost and the fact that it is a representative

1

member of the Clan PA group of proteases, members of which have more widespread disease relevance (e.g.

6,7

thrombin, urokinase plasminogen activator (uPA), tryptase, the kallikreins, matripases and hepsin ), it represents a

8

useful model in order to identify compounds with more general serine protease action. Due to the negatively charged

S

1

region, designed competitive inhibitors of this type of enzyme often incorporate a guanidine, or an alternative basic

7

functional group (e.g. amine, amidine etc.) within their structures. One interesting series of compounds (e.g. 2)

includes a pyridine ring substituted by a guanidine, which with, or without additional substituents, has been evaluated

9

as a promising class of uPA inhibitor.¹⁰We were interested to investigate how the relative position of the basic

guanidine residue effected the degree of trypsin inhibition. For example, some of us have demonstrated that 2-

pyridinyl guanidinium ions form intramolecular hydrogen bonds (IMHBs) between the pyridinyl N-atom and a

9a

guanidinium ion proton, which serves to lock the conformation of the molecule. Additionally, we sought to gauge

the consequences introduction of additional substituents to this ring, and their relative position, have on their

inhibitory action. Specifically, assuming the electrostatic S

1

-P interaction anchors the inhibitor we wondered if further

1

substituents in the aromatic ring might be orientated towards the catalytically active serine residue thereby picking

up additional binding interactions. In relation to this, Barber and co-workers have demonstrated using molecular

docking that compounds of the type 2 bind to uPA in a manner such that any 5-substituent in the pyridine ring is

located in proximity to the catalytic serine residue - Ser-195.¹

0

2

. Results and Discussion

.1. Chemistry

A range of 2-amino substituted pyridines are commercially available, and, as shown in Scheme 1, using the well-

11

established mercury-based S-methyl isothiourea guanidinylation reaction, the corresponding bis-Boc protected

guanidines, 5a-5j, were prepared. Although this protocol reliably installed the 2-guanidine unit, the yields for this

reaction were generally modest. In the case of 2-amino-3-methylpyridine 3h optimisation was required and 5h was

only formed when the reaction mixture was heated to reflux in CHCl

3

(as opposed to the typical DCM, rt method), with

an excess of HgCl . We ascribe these difficulties to a combination of the low nucleophilicity of the conjugated amino

2

group and specifically in the case of 3h, additional steric hindrance adjacent to the reaction site. Removal of both

carbamate protecting groups with TFA proceeded smoothly to afford the corresponding guanidinium salts 6a-6j, which

could be precipitated in analytically pure form from ether. Despite use of an excess of TFA this procedure was found

to generate the mono-salts selectively (Scheme 1).

Scheme 1. Synthesis of 2-guanidinyl pyridine 6a and substituted analogues 6b-6j.

2

As shown in Schemes 2-4, non-commercially available 5-substitued 2-aminopyridines were also accessed and used to

12

make the corresponding guanidinium salts. For instance, as Shown in Scheme 2, a Suzuki-Miyaura reaction using 3d

gave 2-amino-5-phenylpyridine 3k which was then converted into guanidinium salt 6k via the intermediate bis-Boc

guanidine 5k (not shown).

Scheme 2. Suzuki-based synthesis of biaryl 6k.

Building upon the concept of introducing alternative units in the pyridine ring, 6-amino nicotinic acid, 3l, was converted

into methyl ester 3m, which was in turn used to prepare guanidinium ion 6m (Scheme 3). Similarly, 3l was employed

to prepare Weinreb amide 3n, which was also converted, in two steps, into guanidinium salt 6n.

Scheme 3. Synthesis of 5-carbonyl derivatised 2-guanidinium substituted pyridines 6m and 6m.

In this sequence the intermediate bis-Boc compound (5n) proved crystalline and a single crystal X-ray diffraction

structure was obtained (Figure 2).¹³Two points are notable in relation to this structure. Firstly, a hydrogen-bonding

network forming two 6-membered rings between the NH and CO groups in the protected guanidine unit are evident.

14

Secondly, reported X-ray structures for Weinreb amides are relatively rare and the anti-alignment of the amido

carbonyl group and the N-methoxy group is clearly apparent.

As shown in Scheme 4, Weinreb amide 5n was exploited as a means to forming alternative carbonyl substituted 2-

guanidinyl pyridines 6o-6p. Briefly, aldehyde 5o was formed using excess LiAlH

reasonable to good yield and without interference with the guanindine group (Scheme 4). Subsequent conversion of

o-5p into 6o-6p with TFA proved straightforward.

4

and similarly MeMgCl gave 5p, in

5

3

Scheme 4. Synthesis of 5-carbonyl derivatised 2-guanidinium substituted pyridines 6o-6p.

C17

C19

C18

C16

O5

C3

C15

C2

O6

N4

N3

N2

N5

C9

N3

C1

N4

C4

C3

C5

C4

N1

O1

O4

C10

C6 C2

N5

C11

C5

C1 N1

C13

C7

N2

C8

O2

O3

C14

C12

Figure 2. Single crystal X-ray crystallographic structures for compounds 5n and 10 (trifluoroacetate omitted). Hydrogen-bond

13

networks highlighted (thermal ellipsoids shown on the 50% probability level).

In order to explore the affect the structure of the heteroaromatic and guanidine entity has upon activities for this

series, the compounds depicted in Scheme 5 were also prepared. From commercially available 4-aminopyrimidine 7,

compound 10, in which the additional nitrogen atom takes the place of the previously described 5-substituents 6b-6g,

15

was prepared. The natural product pyraxinine (11) was prepared from 8. This compound bears a 3- as opposed to

-guanidinyl pyridine connection. Then aniline derivative 12 was prepared to probe the relevance of the

2

heteroaromatic nitrogen atom. In relation to the formation of 11 and 12, the dramatic impact of the conjugation of

the amino substituent can be seen, since in both the latter cases the yields for the installation of the guanidine were

significantly higher (83% and 90% respectively) than their 2-aminopyridine counterparts 3a-3n (17-70%).

4

Scheme 5. Synthesis of alternative aromatic guanidinium-substituted compounds 10-12, and amidine 13.

X-Ray crystallography was performed on both the bis-Boc pyrimidine intermediate and the final pyrimidine-based

guanidinium salt 10.¹³For the bis-Boc compound (see ESI) the structure closely resembled the previously depicted

Weinreb amide 5n, presenting the bis-6-membered hydrogen bond network. However, in the case of the salt 10, in

the solid state, rotation of the guanidinium ion occurred and an interaction between the proximal nitrogen atom and

the protonated guanidine unit is present (Figure 2). This may explain why when the Boc groups are removed, even

with excess TFA, the basic pyridine nitrogen atom is not protonated. With the objective of probing the relevance of

the guanidine unit, amidine: (E)-N'-(5-bromopyridin-2-yl)-N,N-dimethylformimidamide 13, was synthesised from 3d

using a modified method from the literature.¹⁶This gave amidine substituted pyridine, 13, in good yield.

2

.2. Trypsin inhibition

The K values for the inhibition of trypsin were determined by assessing the effect different concentrations of each

i

inhibitor have upon the trypsin catalysed hydrolysis of α-N-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA).

Briefly, using a reported method,¹⁷the range of synthetic 2-pyridinyl guanidine compounds (6a- 6p), and their

analogues (10-13), were studied at 25 °C and pH 7.2. The initial rate of the trypsin catalysed hydrolysis of the substrate

L-BAPA (where [S] was < 10 times the K

8

m

value) was followed by the measuring the release of 4-nitroaniline (Ɛ410

,800 M cm ). Solutions of different concentrations of each inhibitor were then added to the enzyme and the

consequent effect on the rate of 4-nitroaniline formation was determined. K values for competitive inhibition were

=

-

1

-1 17

i

determined from non-linear regression analysis of the plots of rate of product formation versus inhibitor

concentration, as described by Malthouse et al.¹⁷Representative examples of the data generated and analysed in this

manner are shown in Figure 3.

5

Figure 3. Inhibition of trypsin catalysed hydrolysis of 0.124 mM L-BAPA by compounds 6a-6f at pH 7.2 and 25 °C. All samples

dp

contained dimethyl sulfoxide (3.33%) and 0.1 M phosphate buffer. The solid lines were calculated using the equation _d_t

=

K

kcaꢀ[Eꢁ][Sꢁ]

i

v_o₍

and the fitted values of v

O

( v =

) and ꢂ . The fitted values of ꢂ are included in Figures 3A-F. The trypsin

o

ꢃ

[I]+ K )

Km

i

-

8

-1

-8

-1

concentration and fitted values of v were: A = 0.8 μM, 8.09 ± 0.12 x 10 Ms ; B = 0.42 μM, 3.79 ± 0.062 x 10 Ms ; C = 0.8 μM,

O

-

8

-1

-8

-1

-8

-1

-8

-

8

.26 ± 0.13 x 10 Ms ; D = 0.42 μM, 3.85 ± 0.09 x 10 Ms ; E = 0.42 μM, 4.40 ± 0.13 x 10 Ms ; F = 0.8 μM, 10.3 ± 0.09 x 10 Ms

1

.

Using the method described above the remaining guanidinium salts 6g-6p, and analogues 10-13, were studied and K

values calculated. As can be seen in Table 1, beginning with the monosubstituted pyridine derivative 6a a K of 0.083

i

mM was measured (Entry 1). This value was used then as a reference with the intention of comparing the effect

6

incorporation of different substituents, and their relative positions have on their trypsin inhibition. Firstly,

introduction of a 5-halo substituent was evaluated (Table 1, Entries 2-5). The 5-fluoro analogue 6b proved a less

potent inhibitor than 6a (K = 0.144 mM), whereas the 5-chloro, bromo and iodo analogues (6c-6e) were all more

i

potent inhibitors than 6a. The level of inhibition follows the trend of increasing van der Waals radius/decreasing

electronegativity. Moving to 6f, the 5-trifluoromethyl electron withdrawing substituent led to significantly reduced

levels of trypsin inhibition relative to compounds 6a-6e (Entry 6). In order to further explore the relationship between

the size of the substituents and their location within the pyridine ring, methyl substituted compounds 6g-6j were next

evaluated. Firstly, the effect altering the 5-iodo (6e) and 5-trifluoromethyl (6f) substituents to a methyl substituent in

6

g was studied. As shown in Entry 7, compound 6g proved a significantly worse trypsin inhibitor than 6e but was

comparatively a better inhibitor than 6f. Moving to the alternative mono-methyl substituted compounds 6h-6j, the

4

3

0

- and 6-methyl compounds (6i and 6j) proved similar in their level of inhibition to 6g (Entries 9 and 10). However, the

-methyl substituted compound, 6h, was a significantly better inhibitor with a measured K

.014 mM (Entry 8). This value is approximately the same as 5-iodo analogue 6e (Entry 5).

i

for trypsin inhibition of

Table 1. Inhibition of the trypsin cataysed hydrolysis of L-BAPA by pyridine-based inhibitors 6a-6p and analogues 10-13.

Entry

Compound

6a (R = H)

6b (R = 5-F)

6c (R = 5-Cl)

6d (R = 5-Br)

6e (R = 5-I)

K

i

(mM)

Entry

11

12

13

14

15

16

17

18

Compound

6k (R= 5-Ph)

K (mM)

0.955 ± 0.234

1.08 ± 0.05

2.55 ± 0.22

0.289 ± 0.016

5.04 ± 0.30

i

1

2

3

4

5

6

7

8

9

0.0832 ± 0.0038

0.144 ± 0.010

6m (R = 5-CO Me)

2

0.0347 ± 0.0021

0.0205 ± 0.0017

0.0151 ± 0.0015

0.218 ± 0.0067

0.104 ± 0.0093

0.0140 ±0.0014

0.119 ± 0.0054

0.0913 ± 0.0039

6n (R = 5-CONMeOMe)

6o (R = 5-CHO)

6p (R = 5-COMe)

10

11 (Pyraxinine)

12

6f (R = 5-CF

3

)

0.514 ± 0.029

0.950 ± 0.072

0.127 ± 0.013

>17.2

6g (R = 5-Me)

6h (R = 3-Me)

6i (R = 4-Me)

6j (R= 6-Me)

19

20^a

13

10

Benzamidine

0.032,¹⁸^a0.035¹⁸^b

a_V_a_l_u_e_s_f_r_o_m_l_i_t_e_r_a_t_u_r_e_R_e_f_.₁₈_[₍₃₀_°_C_,_p_H₌₇₎_,¹⁸^a(23 °C, pH = 7.5)¹⁸^b

Alternative 5-susbtituted compounds 6k-6p were next evaluated (Entries 11-15). The Suzuki-derived biaryl compound

6

1

k proved to be a poor inhibitor (Entry 11), as did the carbonyl-derivatives, ester 6m and Weinreb amide 6n (Entries

2 and 13). It should be noted that 6m was of interest since it was anticipated that this might act as a trypsin substrate

due to the possible orientation of the ester group towards the catalytically active serine residue. Following on with

this idea, aldehyde 6o and methyl ketone 6p were considered since it was felt that they may reversibly interact with

the catalytic serine hydroxy group of trypsin to form a hemiacetal/ketal, the formation of which might enhance

inhibition (Entries 14 and 15). However, the both 6o and 6p were weaker inhibitors than 5-Me (6g, Entry 7), suggesting

that the described hemiacetal/ketal formation is unlikely. In order to investigate the role the pyridine nitrogen atom

plays in the inhibition of trypsin, compounds 10-12 were prepared and compared with compound 6a. Pyrimidine 10

proved to have a K of 0.514 mM, approximately ten times less potent than 6a (Entry 16 versus Entry 1). As shown in

i

Entry 17, adjustment of the relative position of the guanidine group to the heteroaromatic nitrogen atom causes a

dramatic effect, and the natural product pyraxinine (11) only weakly inhibits the action of trypsin. Complete removal

of the nitrogen atom from the aromatic ring, in the case of 12, also leads to a drop in the level of inhibition versus the

comparable pyridine derivative, 6c. However, at a K of 0.127 mM for 12 (Entry 18), versus 0.035 mM for 6c (Entry 3),

i

this result was less dramatic than expected. Finally, the exchange of a guanidine group for a dimethyl amidine was

not well tolerated and 13 proved inactive at the highest concentration that could be used without compound

precipitation (Entry 19). In terms of a comparison, benzamidine, a frequently used serine protease inhibitor, has been

reported to inhibit trypsin in conditions closely related to those used currently with K values between 0.032 and 0.035

i

18

mM (Entry 20).

In the interest of determining whether trypsin catalyses the hydrolysis of the ester bond in 6m it was incubated at pH

1

3

6

.90 with 0.185 mM trypsin. Using C-NMR spectroscopy the inhibitor was observed to gradually hydrolyse over a 6

7

day period. Assuming the K

mM, then kcat is 8.9 x 10 s and kcat/K

m

= K

s

= K

i

= 1.08 mM then from the complete hydrolysis curve we estimate that, if K = 1.08

m

-

5

-1

-1 -1

m

is 0.082 M s . Using these values, we estimate that during our inhibition

assays <0.002% of the inhibitor will be hydrolysed confirming that our K

i

value will not be significantly effected by the

trypsin catalysed hydrolysis of 6m. Benzoyl alanine methyl ester has a much higher K of 171 mM since it does not

have a positively charged residue to bind in the S subsite of trypsin. However, its kcat value of 0.185 s is ~21,000

19

m

-

1

greater than the kcat value of 6m. These findings suggest that ester bond in 6m is not bound by trypsin in the optimal

position for hydrolytic catalysis.

Summarising the K results the significance of the 2-guanidinyl group for trypsin inhibition is evident (compounds 6

i

versus 13). The presence and position of the heteroatom in the aromatic ring relative to the guanidine is also

important (compounds 6 versus 11 and 12). These differences, we believe, demonstrate the importance of an

intramolecular hydrogen bond (IMHB) between the heteroaromatic N-atom and the guanidinium ion (see X-ray, Figure

2

and discussion below). Within the main compound series, characterised by compounds belonging to family 6, 5-

halogen substitution in general terms provides optimum inhibition. This is particularly marked with the 5-iodo

substituent (6e). The trend observed in relation to the halogen substituents themselves fits with increasing ionic

radius/van der Waals radius and decreasing electronegativity/halogen bond donor capacity. Since the relative size of

and iodo substituent and a methyl substituent is comparable based on the significantly lower level of inhibition for the

5

-Me substituted compound versus the 5-iodo compound (6g versus 6e) it is likely that enhanced inhibition for 6e is

20

due to halogen bonding in the serine protease binding pocket. Examples of enhanced halogen bonding in relation

to enzyme inhibition have been reported.²In terms of the methyl substituted compounds, the inhibition associated

with the 3-Me substituted compound 6h is noteworthy.

1

2

.3. Docking studies

In order to understand how the synthesized compounds interacted with trypsin a docking study was undertaken.

Compounds 6a-e, 6g, 6h, 6k, 6o, 10-12 were selected. Briefly, pyridino-2-guanidinium ion (6a) was taken as a reference

to assess the effect of the different substituents on the theoretical binding and inhibitory action. The four 5-halogen

derivatives (6b-e) were selected to study the effect on trypsin inhibitory activity of potential halogen bonds with the

target. The 5-methyl and 5-phenyl derivatives (6g and 6k) were chosen to test the impact the size of the 5-substituent

has. Compound 6h, presenting a 3-methyl group, was studied to attempt to understand why its measured K

than its 5-isomer (6g). Aldehyde 6o was selected to investigate why, on the basis of the measured K

i

was lower

value, the

i

electrophilic 5-CHO group did not appear to interact with the nucleophilic hydroxy group of the catalytic serine residue

Ser-195). Compounds 10-12 were chosen to study the benefit of the inclusion and position of the heterocyclic N-

(

atom. Amidine 13 was chosen in order to probe how the identity of the basic substituent effects trypsin binding.

Initially, each of the selected structures were optimised at a DFT level (using the M06-2X functional and the 6-

22

3

1+G(d,p) basis set) with the SMD solvation model for water as implemented in Gaussian16 (see ESI, Table S1). In

all optimised 2-pyridino (compounds 6a-e,g,h,k,o) and 4-pyrimidino (10) guanidines an intramolecular hydrogen bond

IMHB) is clearly established between a N-atom from the heterocycle and a N-H bond in the guanidinium cation (see

(

also the single crystal X-ray structure of 10 in Figure 2). This IMHB locks the guanidinium system in an almost co-planar

conformation to the heterocycle. In contrast, those molecules (11-13) where such an IMHB cannot be formed show a

quasi-perpendicular arrangement between the aromatic unit and the guanidinium cation. All calculated IMHB

distances, and guanidine surrounding dihedral angles in the isolated solvated ligands, are presented in Table S2 (see

23

ESI). Regarding the target, bovine pancreatic trypsin (PDB: 1CE5, resolution 1.9 Å ) was selected with the purpose of

paralleling the experimental inhibition experiments. It should also be noted that this structure includes a benzamidine

ligand, which takes into account conformational differences between the structure used for the docking and the

enzyme without a ligand. The benzamidine was removed before docking studies were undertaken. Additionally, it

was of interest to compare the binding of the same suite of ligands to a human brain trypsin-4 (PDB: 1H4W, resolution

24

1

.7 Å ). It should be noted that both bovine and human trypsin structures have a very high levels of homology at the

catalytic binding site (for an example, see ESI Figure S1). Next, with the help of the Autodock Vina program, the

8

optimised ligand structures were docked to both forms of trypsin in a rigid structure-flexible ligand approach. Upon

binding, the ligands suffer subtle structural modifications and the optimum poses found for each ligand-trypsin

complex are presented in Figure S2 (ESI). For instance, in the compounds where an IMHB is possible (pyridines 6 and

pyrimidine 10) the distances between the heterocyclic N-atom and guanidinium ion become slightly larger (up to 0.9

Å) as a consequence of a torsion (from 4 to 49°) around these two moieties. As a trend this distortion is smaller when

docking into the bovine trypsin catalytic site than its human counterpart (see Table S2, ESI). The incremental

lengthening of the IMHB distances upon complexation is extreme in the case of compound 6o where it was observed

that the molecule must twist and break the IMHB in order to optimally locate into the binding pocket orientating the

electrophilic carbonyl group with the hydroxy group from Ser-195. The penalty associated with the loss of the IMHB

could explain the unanticipated, poor trypsin inhibition observed for compound 6o. Interactions between the

guanidinium cations of all the ligands studied and similar residues in the binding site of both bovine and human trypsin

enzymes (Gly-219, Gly-226 and/or Asp-189) are observed (see ESI, Table S3). Again, as a trend the distances for the

bovine protein complexes are slightly shorter. An exception is observed for 6h (3-Me pyridine and the compound with

the lowest measured K

i

) which forms a bifurcated hydrogen bond (HB) between a H-atom of the guanidine and Gly-

2

19 and Asp-189 (a very short HB) in the bovine trypsin complex, whereas in the human trypsin complex two different

HBs are formed between different H-atoms in the guanidinium cation and Gly-219 and Gly-226. Whilst an electrostatic

interaction with Asp-189 is observed no additional hydrogen bonding to this residue was predicted. For those

compounds where no IMHB can be formed (compounds 11-13), a structural modification reducing the torsion between

aromatic ring and basic residue in the 2-position takes place upon binding to the trypsin enzymes studied. This change

in conformation involves an energy penalty. In this set of molecules, similar interactions between the guanidinium

cation and Gly-219, Gly-226 and/or Asp-189 are observed. The exception is compound 13, which does not have a

guanidinium system explaining why this is the worst inhibitor (see ESI, Table S3). Additionally, the poor levels of

inhibition for compounds 6k and 13 can be explained because they are too large to fit into the trypsin binding pocket:

they clash with residues Trp-215, Gly-216 and Ser-214 and, hence, protrude from the binding site. Supporting the

interactions of the guanidinium cations with the Gly-219, Gly-226 and/or Asp-189 residues that seem to drive the

binding, additional interactions with the ligand substituents are observed. In particular, halogen substituents in 5-

position favour the interaction with the binding pocket by means of a halogen bond with the O-atom from the hydroxy

…

20

group of Ser-195. It is appreciated that the shorter the X O distance the stronger the halogen bond and in the case

…

of compounds 6b-e this distance correlates with the experimental K

i

values [d(X O) = 0.07 ln(K

i

) + 1.99, R² = 0.978] in

the bovine trypsin model. In the case of compound 12, which also has a 5-chloro substituent, even though a halogen

…

bond can be observed (Cl O= 2.17 Å –bovine trypsin- and 2.29 Å –human trypsin) the lack of the IMHB orients the

guanidinium moiety sub-optimally compared to the pyridine analogue 6c. Representative binding poses for

compounds 6a, 6b and 6 h, with both bovine and human trypsin, are shown in Figure 4 (see ESI for similar depictions

of the additional compounds mentioned).

6a

9

6e

6h

Figure 4. Representative optimal binding poses using Autodock: compounds 6a, 6e and 6h in bovine (on the left - PDB: 1CE5) and

human trypsin-4 (on the right - PDB: 1H4W).

Summarising, Structure-Activity Relationships (SARs) have been established for the optimal inhibition of trypsin (both

bovine and human) using a docking approach. Firstly, a guanidinium cation is needed to anchor the binding of the

inhibitor to the trypsin active site. Secondly, a 2-pyridyl core is required to facilitate an IMHB with the guanidinium

system. This serves to optimally orientate the cation with the binding pocket. Thirdly, a halogen in 5-position is

beneficial because it enables a halogen bond interaction with the catalytically active serine residue in the binding

20,21

pocket.

Within this series, the polarisable 5-iodo substituent is the optimal group. Large substituents (i.e. phenyl)

in the 5-position are detrimental for the inhibitory activity. Finally, a methyl group in the 3-position of the pyridine

core is beneficial for trypsin inhibition, possibly because this forms extra van de Waals interactions with a hydrophobic

region within the binding pocket.

3

. Experimental

.1. General Directions

Reagents were obtained from commercial suppliers and were used without further purification (including bovine

pancreatic trypsin). Flash column chromatography was performed using flash silica 60 Å (230-400 mesh) 9385 supplied

by Merck. Thin-layer chromatography was performed on silica coated aluminium sheet (60 F254) supplied by Merck.

Compounds were visualized with UV light and aqueous potassium permanganate, followed by heating. Known

compounds that were synthesized using literature conditions are specified and referenced correspondingly. Melting

points were recorded on a Gallenkamp electrothermal melting point apparatus and are uncorrected. Infrared spectra

1

13

19

were recorded on a Bruker ALPHA FT-IR spectrometer. H, C and F NMR spectra were recorded on Varian Unity 500

MHz, 400 MHz and 300 MHz system spectrometers. Where necessary, two-dimensional correlation spectroscopy

(COSY, HSQC, HMBC etc.) was utilized to assign/confirm the compound structure. Chemical shifts are quoted in parts

per million (ppm) relative to the internal standard reference tetramethylsilane and/or residual protonated solvent.

Note, for the guanidinium salts (6a-6k, 6m-6n and 10-12) the trifluoroacetate counterion is not routinely detected in

1

0

13

the C-NMR spectra. In the case of 6p increased numbers of scans enabled identification of these signals (reported

below). High resolution mass spectra were carried out on a Waters/Micromass LCT ESI mass spectrometer with a

time-of-flight analyser. Elemental analysis was carried out at the facilities in the Centre for Synthesis and Chemical

Biology, UCD.

3

.2. General Method – Di-tert-Butyloxycarbamate Guanidine Synthesis: Using a procedure adapted from the

9-11

literature, mercury(II) chloride (0.82 g, 2.7 mmol, 1 eq.) was added to a solution of the amine (2.7 mmol, 1 eq.),

N,N-di-Boc-S-methylisothiourea 4 (0.87 g, 2.7 mmol, 1 eq.), triethylamine (1.1 mL, 7.9 mmol, 3 eq.) and DCM (27 mL)

at 0 °C. The mixture was stirred for 30 minutes at 0 °C, then warmed to room temperature and stirred until reaction

was complete (16 – 48 h) as judged by disappearance of starting material in TLC analysis [an alternative procedure for

2

-amino-3-methylpyridine, 3h, involved using 1.5 eq. of HgCl

to reflux]. The reaction mixture was diluted with EtOAc (20 mL) and filtered through Celite and washed with EtOAc

20 mL). The filtrate was then washed with water (30 mL) followed by brine (30 mL), dried over anhydrous MgSO and

concentrated. The crude products were purified by SiO column chromatography, eluting with the appropriate eluent.

2

, 1 eq. of the thiourea in CHCl

3

and heating this mixture

(

4

2

3

.2.1. 1-(5-Iodopyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5e): 2-Amino-5-iodoopyridine 3e (0.59 g, 2.7

mmol) gave 5e (0.50 mg, 41%) as a colourless crystalline solid after purification by flash column chromatography (c-

Hex/EtOAc 9:1). Mp = 124 – 126 °C; R = 0.25 (c-Hex/EtOAc 9:1); IR: max = 3229, 2981, 2926, 1715, 1633, 1560, 1362

): δ = 1.53 (s, 9H, CH ), 1.53 (s, 9H, CH ), 7.96 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.25 (d, J = 9.0

Hz, 1H, CH), 8.47 (d, J = 2.5 Hz, 1H, CH), 10.88 (brs, 1H, NH), 11.50 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

C), 153.8 (C), 152.9 (C), 149.8 (C), 146.0 (CH), 137.9 (CH), 118.0 (CH), 86.1 (C), 84.2 (C), 80.1 (C), 28.1 (CH ), 28.0 (CH

f

-

1 1

cm ; H-NMR (400 MHz, CDCl

3

13

3

): 163.0

(

3

)

+

ppm; HRMS (ES ) C16

H24IN

4

O

4

[M+H] calcd. 463.0842; found 463.0854.

3

.3. General Method - Guanidinium salt formation: A solution of TFA (25 eq.) in DCM (3 mL) was added to the di-

Boc-protected guanidine (1 eq.). The reaction mixture was stirred at room temperature for 2 h. Solvent and excess

TFA were removed in vacuo. Diethyl ether (5 mL) was added to the crude product to effect precipitation. The liquid

was then siphoned, affording the 2-guanidinyl pyridine derivative which was dried under reduced pressure.

3

.3.1. 1-(5-Iodopyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6e): Colourless solid (67 mg, 80 %). Mp = 188 – 189 °C;

-

1 1

IR: max = 3291, 3092, 2930, 1696, 1580, 1461, 1189 cm ; H-NMR (400 MH

Z

, DMSO-d ): δ = 6.91 (dd, J = 8.5, 1.0 Hz,

6

1

H, CH), 8.17 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.34 (brs, 4H, NH), 8.51 (dd, J = 2.5, 1.0 Hz, 1H, CH), 11.22 (brs, 1H, NH) ppm;

13

19

C-NMR (100 MHz, DMSO-d

MHz, DMSO-d ): δ = -73.84 (s) ppm; HRMS (ES ) C

calcd. C% 25.55, H% 2.14 and N% 14.90; found C% 29.59, H% 1.94 and N% 14.59.

6

): 155.4 (C), 152.5 (C), 151.6 (CH), 147.6 (CH), 116.0 (CH), 86.8 (C) ppm; F-NMR (376

+

6

H

8

IN [M+H] calcd. 262.9794; found 262.9789; Microanalysis for

4

C

8 8

H IF

3

N

4

O

2

3

.4. Trypsin inhibition general directions:

α-N-Benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA) and 4-nitrophenyl 4-guanidinobenzoate hydrochloride

(

NPGB) were obtained from Sigma-Aldrich. The NPGB solution and buffers were prepared as described by Chase and

Shaw.²Deionised water was used in all cases. Absorption spectroscopic measurements were carried out using a Cary

5

0 Conc UV/Visible spectrometer. A Lauda A100 water bath, with a thermostat was used to maintain a constant

temperature (25 °C) in the cuvette. The pH measurements were performed using a Schott Blue-line 17 combination

pH electrode attached to a Thermo-Orion pH meter model 420. The pH meter was calibrated using pH buffers at pH 4

and pH 7. Bovine pancreatic trypsin was obtained from Sigma-Aldrich as a crystallized and lyophilized powder.

Spectrophotometric analysis (A280) determined that the trypsin protein used was 86.2% by weight using Ɛ280 = 36,960

-

1

-1

M cm . The concentration of fully active enzyme (74.3 % of the protein) was determined by titration with NPGB as

described by Malthouse et al.¹⁷Briefly, using the reported method the range of synthetic 2-pyridinyl guanidine

17

°

compounds (6a- 6p, 10-13) were studied at 25 C in 3 mL cuvettes and 0.1 M buffers pH 7.1−7.3 (potassium phosphate),

and 3.3% (v/v) dimethyl sulfoxide. The initial rate of hydrolysis of L-BAPA at pH 7 was followed by the measuring the

-

1

-1

release of p-nitroaniline (Ɛ410 = 8,800 M cm ). Stock solutions of substrate and inhibitor were dissolved in dimethyl

1

sulfoxide and deuterated dimethyl sulfoxide respectively. K values for competitive inhibition were determined when

i

[S

o

]<<K

m

, using the equation below:

풅풑

=

풗 푲 ⁄([푰] ꢄ 푲 ) where 풗 = 풌 [푬 ][푺 ]⁄푲

풐

풊

풐

풄풂풕

풐

풎

풅풕

풅풑

-

1

Where _풅_풕is the rate of product formation over time (Ms ), 풗 is the intial rate of product formation In the absence

풐

of inhibitor, 푲 is the inhibitor’s dissociation constant, [푬 ] and [푺 ] are free enzyme and free substrate concentration,

풊

풐

respectively. 풌_풄_풂_풕is the catalytic rate constant and 푲 is the Michaelis-Menten constant. K

the experimental data (_풅_풕and [I]) to the equation above as described by Malthouse et al.¹⁷

i

was determined by fitting

풎

풅풑

3

.5. Trypsin docking:

The chosen bovine (1CE5) and human trypsin (1H4W) structures are available from the protein databank

(https://www.rcsb.org/). The selected inhibitor structures were then geometry optimised by DFT [M06-2x/6-

3

1+g(d,p)] using an aqueous solvation model. Using Autodock, the co-crystallised ligand (benzamidine) was used to

set the docking coordinates. It was then removed prior to the docking. The different ligand-protease poses were

ranked using in-built scoring functions and optimal poses were selected and visualised using PyMOL and Visual

Molecular Dynamics (VMD). For more information see the ESI.

Declaration of interest

We declare that we have no financial and personal relationships with other people or organisations that could

inappropriately influence our work.

Acknowledgements

We thank the University of Taibah and the Ministry of Higher Education, kingdom of Saudi Arabia for a research

+

scholarship (NAl-H). AL (Humboldt-Universität zu Berlin) thanks the Erasmus traineeship scheme. Thanks are given

to the Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities and we thank the Irish

Research Council for postgraduate support (AR). Dr. Helge Müller-Bunz and Dr Yannick Ortin are thanked for X-ray

crystallography and NMR spectroscopy, respectively. Áine Crehan, Cecilia Morgenstern and Lola Gbadebo (UCD) are

thanked for some preliminary synthetic work.

Abbreviations

uPA

IMHB

HB

urokinase plasminogen activator

intramolecular hydrogen bond

hydrogen bond

DCM

Boc

dichloromethane

tert-Butyloxycarbonyl

TFA

trifluoroacetic acid

rt

room temperature

Ac

acetyl

HATU

DMF

DMAc

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

dimethylformamide

dimethylacetamide

1

2

DMSO

L-BAPA

NPGB

DFT

dimethyl sulfoxide

α-N-benzoyl-L-arginine-p-nitroanilide (hydrochloride)

4-nitrophenyl 4-guanidinobenzoate (hydrochloride)

density functional theory

solvent model density

SMD

EtOAc

c-Hex

ethyl acetate

cyclohexane

R

f

retention factor

Mp

melting point

IR

infrared

NMR

HRMS

ES

nuclear magnetic resonance

high resolution mass spectrometry

electrospray (ionisation)

Supplemental data

Supplementary data to this article (including synthetic procedure, NMR spectra, X-ray structures and full details for

the docking study) can be found online at:

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2. (a) Bumagin, B.; Bykov, V. Reaction of Organoboron Compounds with Organic Halides in Aqueous Media, Catalyzed

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3. X-Ray crystal data. Compound 5n: CCDC 2000231; unit cell parameters: a = 22.5653(1); b = 22.5653(1); c =

.45288(5) P63/m. Compound 10: CCDC 2000232; unit cell parameters: a = 7.8644(2); b = 15.0637(3); c = 17.8158(3)

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2

1

4. For examples of X-ray crystal structures for Weinreb amide-containing compounds, see: (a) Zheng, X.; Donkor, I.

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synthesis of a new imidazole library: Direct conversion of N-methoxy-N-methylamides (Weinreb amides) to 3-keto and

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-formylindazoles, Tetrahedron 2007, 63, 419-428.

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5. Al Mourabit, A.; Pusset, M.; Chtourou, M.; Gaigne, C.; Ahond, A.; Poupat, C.; Potier, P. Pyraxinine, a Novel

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Inhibition of bovine -trypsin, human -thrombin and porcine pancreatic -kallikrein-B by 4’,6-diamidino-2-

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2

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2

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1

5

Graphical Abstract

Synthesis of 2-Guanidinyl Pyridines and their Trypsin Inhibition and Docking

Nahlah Ahmed Al-Hadhrami, Angelique Ladwig, Adeyemi Rahman, Isabel Rozas, J. Paul G. Malthouse and Paul Evans

R = 5-I

Graphical Abstract

Synthesis of 2-Guanidinyl Pyridines and their Trypsin Inhibition and Docking

Nahlah Ahmed Al-Hadhrami, Angelique Ladwig, Adeyemi Rahman, Isabel Rozas, J. Paul M. G. Malthouse and Paul Evans

R = 5-I

Declaration of interests

☒

The authors declare that they have no known competing financial interests or personal relationships that could

have appeared to influence the work reported in this paper.

1

6

☐

The authors declare the following financial interests/personal relationships which may be considered as potential

competing interests:

Paul Evans

Electronic Supporting Information

Synthesis of 2-Guanidinyl Pyridines and their Trypsin Inhibition

a

b

c

Nahlah Ahmed Al-Hadhrami, Angelique Ladwig, Adeyemi Rahman, Isabel Rozas, J. Paul G. Malthouse and Paul

a

Evans *

^aSchool of Chemistry, Centre for Synthesis and Chemical Biology, University College Dublin, Dublin D04 N2E2, Ireland.

Email: paul.evans@ucd.ie

^bSchool of Chemistry, TBSI, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland.

^cSchool of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute,

University College Dublin, Dublin D04 N2E2, Ireland.

Contents

Experimental details ……………………..……………………………………………………………………………………… S2-S15

1

13

19

H-, C and F-NMR spectra ………………………………………………………………………………………………… S16-S75

1

7

X-Raycrystallographic details ……………………………………………………………………………………………… S76-S78

Docking study details ………………………………………………………………………………………………………… S79-S103

1

8

Experimental details

General Directions

Reagents were obtained from commercial suppliers and were used without further purification (including bovine

pancreatic trypsin). Anhydrous THF was freshly distilled from sodium benzophenone ketyl radical under nitrogen.

Flash column chromatography was performed using flash silica 60 Å (230-400 mesh) 9385 supplied by Merck. Thin-

layer chromatography was performed on silica coated aluminium sheet (60 F254) supplied by Merck. Compounds were

visualized with UV light and aqueous potassium permanganate, followed by heating. Known compounds that were

synthesized using literature conditions are specified and referenced correspondingly. Melting points were recorded

on a Gallenkamp electrothermal melting point apparatus and are uncorrected. Infrared spectra were recorded on a

1

13

19

Bruker ALPHA FT-IR spectrometer. H, C and F NMR spectra were recorded on Varian Unity 500 MHz, 400 MHz and

00 MHz system spectrometers. Where necessary, two-dimensional correlation spectroscopy (COSY, HSQC, HMBC

3

etc.) was utilized to assign/confirm the compound structure. Chemical shifts are quoted in parts per million (ppm)

relative to the internal standard reference tetramethylsilane and/or residual protonated solvent. Note, for the

13

guanidinium salts (6a-6k, 6m-6n and 10-12) the trifluoroacetate counterion is not routinely detected in the C-NMR

spectra. In the case of 6p increased numbers of scans enabled identification of these signals (reported below). High

resolution mass spectra were carried out on a Waters/Micromass LCT ESI mass spectrometer with a time-of-flight

analyser. Elemental analysis was carried out at the facilities in the Centre for Synthesis and Chemical Biology, UCD.

General Method – Di-tert-Butyloxycarbamate Guanidine Synthesis: Using a procedure adapted from the literature,¹

mercury(II) chloride (0.82 g, 2.7 mmol, 1 eq.) was added to a solution of the amine (2.7 mmol, 1 eq.), N,N-di-Boc-S-

methylisothiourea 4 (0.87 g, 2.7 mmol, 1 eq.), triethylamine (1.1 mL, 7.9 mmol, 3 eq.) and DCM (27 mL) at 0 °C. The

mixture was stirred for 30 minutes at 0 °C, then warmed to room temperature and stirred until reaction was complete

(16 – 48 h) as judged by disappearance of starting material in TLC analysis [an alternative procedure for less reactive

amines (e.g. 2-amino-3-methylpyridine, 3h) involved using 1.5 eq. of HgCl , 1 eq. of the thiourea in CHCl and heating

2

3

this mixture to reflux]. The reaction mixture was diluted with EtOAc (20 mL) and filtered through Celite and washed

with EtOAc (20 mL). The filtrate was then washed with water (30 mL) followed by brine (30 mL), dried over anhydrous

MgSO

4

and concentrated. The crude products were purified by SiO column chromatography, eluting with the

2

appropriate eluent.

General Method - Guanidinium salt formation: A solution of TFA (25 eq.) in DCM (3 mL) was added to the di-Boc-

protected guanidine (1 eq.). The reaction mixture was stirred at room temperature for 2 h. Solvent and excess TFA

were removed in vacuo. Diethyl ether (5 mL) was added to the crude product to effect precipitation. The liquid was

then siphoned, affording the 2-guanidinyl pyridine derivative which was dried under reduced pressure.

1

(

-(Pyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5a):¹Following the general procedure 2-aminopyridine 3a

0.25 g, 2.7 mmol) was converted into 5a (0.45 g, 50 %) was isolated as a colourless crystalline solid after purification

o

1

by flash column chromatography (c-Hex/EtOAc 9:1). Mp = 132 – 134 °C (Lit. Mp = 138 C ); R

IR: max = 3254, 2979, 1725, 1621, 1561, 1479, 1398 cm ; H-NMR (400 MHz, CDCl

=

1

f

= 0.30 (c-Hex/EtOAc 9:1);

): δ = 1.53 (s, 18H, CH ), 7.01 (dd, J

7.5, 5.0 Hz, 1H, CH), 7.68 - 7.73 (m, 1H, CH), 8.29 (dd, J = 5.0, 2.0 Hz, 1H, CH), 8.37 (brs, 1H, CH), 10.89 (brs, 1H, NH),

-

1 1

3

13

1.53 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

19.8 (C), 116.1 (CH), 83.9 (C), 80.0 (C), 28.1 (CH

3

): 163.2 (C), 153.1 (C), 152.6 (C) 150.7 (C), 148.0 (CH), 138.2 (CH),

+

3

), 28.0 (CH

3

) ppm. HRMS (ES ) C16

H

25

N

4

O [M+H] calcd. 337.1876;

4

found 337.1860.

1

9

4

1

-(Pyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6a): Following the general procedure 6a (110 mg, 79%) was isolated

4

as a colourless solid. Mp = 134 – 136 °C (Lit. Mp = 159.1 - 161.2 °C ); IR: max = 3217, 3069, 3043, 1689, 1566, 1466,

): δ = 7.05 (d, J = 8.5 Hz, 1H, CH), 7.15 - 7.19 (m, 1H, CH), 7.84 - 7.89 (m, 1H,

CH), 8.30 (dd, J = 5.0, 2.0 Hz, 1H, CH), 8.42 (brs, 4H, NH), 11.20 (brs, 1H, NH) ppm; C-NMR (100 MHz, DMSO-d

-

1 1

1

179 cm ; H-NMR (400 MHz, DMSO-d

6

1

3

6

): 155.7

): δ = -73.99 (s) ppm;

calcd. C% 38.41, H% 3.63

19

(

C), 152.5 (C), 147.0 (CH), 140.0 (CH), 119.8 (C), 113.8 (CH) ppm; F-NMR (282 MHz, DMSO-d

6

+

HRMS (ES ) C

H N

6 9 4

[M+H] calcd. 137.0827; found 137.0824; Microanalysis for C

8

H

9

F

3

N

4

O

2

and N% 22.39; found C% 38.28, H% 3.44 and N% 22.04.

1

-(5-Fluoropyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5b): 2-Amino-5-flouropyridine 3b (0.30 g, 2.7 mmol)

gave 5b (0.50 g, 56%) as a colourless crystalline material isolated after purification by flash column chromatography

c-Hex/DCM 1:4). Mp = 137 - 139 °C; R = 0.50 (c-Hex/DCM 1:4); IR: max = 3249, 2997, 1719, 1650, 1577, 1472, 1393

): δ = 1.53 (s, 18H, CH ), 7.44 (ddd, J = 9.0, 7.5, 3.0 Hz, 1H, CH), 8.14 (d, J = 3.0 Hz, 1H,

CH), 8.41 (dd, J = 9.0, 4.0 Hz, 1H, CH), 10.89 (brs, 1H, NH), 11.50 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

(

f

-

1 1

cm ; H-NMR (400 MHz, CDCl

3

1

3

): 163.1

C), 156.5 (d, JC-F = 251.5 Hz, C), 152.9 (C), 152.7 (C), 146.7 (CH), 135.5 (d, JC-F = 25.5 Hz, CH), 124.9 (d, JC-F = 19.5 Hz,

1

2

(

3

19

CH), 117.0 (d, JC-F = 4.0 Hz, CH), 84.0 (C), 80.0 (C), 28.1 (CH

3

), 28.0 (CH

3

) ppm; F-NMR (376 MHz, CDCl ): δ = -132.61

[M+H] calcd. 355.1782; found 355.1779.

3

+

–

-132.66 (m) ppm; HRMS (ES ) C16

H24FN

4

O

4

1

–

4

-(5-Fluoropyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6b): Isolated as a colourless solid (150 mg, 84 %). Mp = 160

-

1 1

162 °C; IR: max = 3276, 3134, 3076, 1693, 1578, 1473, 1186 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 7.12 (dd, J = 9.0,

.0 Hz, 1H, CH), 7.85 (td, J = 8.5, 3.0 Hz, 1H, CH), 8.30 (brs, 4H, NH), 8.31 (d, J = 3.5 Hz, 1H, CH), 11.27 (brs, 1H, NH)

13

1

4

ppm; C-NMR (100 MHz, DMSO-d ): 159.6 (C), 156.4 (d, JC-F = 201.5 Hz, C), 155.0 (C), 148.8 (d, JC-F = 2.5 Hz, C), 134.3

6

2

3

19

(d, JC-F = 26.5 Hz, CH), 127.9 (d, JC-F = 21.0 Hz, CH), 115.4 (d, JC-F = 5.5 Hz, CH) ppm; F-NMR (376 MHz, DMSO-d

6

): δ =

[M+H] calcd. 155.0733; found 155.0735; Microanalysis for

calcd. C% 35.83, H% 3.01 and N% 20.89; found C% 35.82, H% 2.83 and N% 20.61.

+

-73.90 (s), -133.37 – -133.40 (m) ppm; HRMS (ES ) C

H FN

6 8 4

C

8

H

8

F

4

N

4

O

2

1

-(5-Chloropyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5c): 2-Amino-5-chloropyridine 3c (0.50 g, 2.7 mmol)

was used to generate 5c (0.66 g, 70%) which was isolated after purification by flash column chromatography (c-

1

Hex/EtOAc 9:1) as a colourless crystalline material. Mp = 124 – 126 °C (Lit. Mp = 124 °C ); R

f

= 0.65 (c-Hex/EtOAc 9:1);

), 1.54 (s, 9H,

), 7.66 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.24 (d, J = 2.5 Hz, 1H, CH), 8.40 (d, J = 9.0 Hz, 1H, CH), 10.92 (brs, 1H, NH), 11.50

-

1 1

IR: max = 3433, 2981, 1724, 1645, 1558, 1455, 1378 cm ; H-NMR (300 MHz, CDCl

CH

brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl ): 162.0 (C), 151.9 (C), 151.7 (C), 147.9 (C), 145.6 (CH), 136.7 (CH), 125.9

3

): δ = 1.53 (s, 9H, CH

3

13

(

3

2

0

+

23³⁵ClN

+

(C), 115.8 (CH), 83.2 (C), 79.1 (C), 27.1 (CH

3

), 27.0 (CH

3

) ppm; HRMS (ES ) C16

H

4

O Na [M+Na] calcd. 393.1306;

4

found 393.1312.

1

–

-(5-Chloropyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6c): Isolated as a colourless solid (60 mg, 86 %). Mp = 148

-

1 1

150 °C; IR: max = 3296, 3192, 3097, 1697, 1587, 1465, 1192 cm ; H-NMR (400 MHz, DMSO-d ): δ = 7.09 (d, J = 9.0

6

Hz, 1H, CH), 7.98 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.28 (brs, 4H, NH), 8.36 (d, J = 2.5 Hz, 1H, CH), 11.17 (brs, 1H, NH) ppm;

1

3

19

C-NMR (100 MHz, DMSO-d

6

): 155.3 (C), 151.0 (C), 145.4 (CH), 139.8 (CH), 126.0 (C), 115.3 (CH) ppm; F-NMR (376

+

35

+

MHz, DMSO-d ): δ = -73.75 (s) ppm; HRMS (ES ) C

6

H

6 8

ClN [M+H] calcd. 171.0437; found 171.0440; Microanalysis for

4

8 8

C H ClF

3 4

N O

2

calcd. C% 33.76, H% 2.83, N% 19.68 and Cl% 12.46; found C% 33.70, H% 2.70, N% 19.39 and Cl% 12.23.

1

-(5-Bromopyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5d): With 2-amino-5-bromopyridine 3d (0.47 g, 2.7

mmol) 5d (0.61 g, 56%) was isolated after purification by flash column chromatography (c-Hex/EtOAc 9:1) as a

1

colourless crystalline solid. Mp = 120 – 122 °C (Lit. Mp = 126 °C ); R

f

= 0.40 (c-Hex/EtOAc 9:1); IR: max = 3249, 2980,

), 7.79 (dd, J = 9.0, 2.5 Hz, 1H, CH),

-

1 1

1

8

714, 1631, 1593, 1403, 1367 cm ; H-NMR (400 MHz, CDCl

3

): δ = 1.53 (s, 18H, CH

3

13

.33 (d, J = 2.5 Hz, 1H, CH), 8.36 (d, J = 9.0 Hz, 1H, CH), 10.90 (brs, 1H, NH), 11.50 (brs, 1H, NH) ppm; C-NMR (100

MHz, CDCl

3

): 163.0 (C), 152.9 (C), 155.7 (C), 149.4 (C), 148.8 (CH), 140.5 (CH), 117.4 (C), 114.9 (CH), 84.2 (C), 80.1 (C),

+

79

+

2

8.1 (CH

3

), 28.0 (CH

3 4

) ppm; HRMS (ES ) C16H23 BrN O

4

Na [M+Na] calcd. 437.0800; found 437.0785.

4

1

(

-(5-Bromopyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6d): Colourless solid (170 mg, 90 %). Mp = 164 – 166 °C

4

-1 1

Lit. Mp = 154 - 157 °C ); IR: max = 3292, 3091, 2914, 1696, 1587, 1461, 1190 cm ; H-NMR (400 MHz, DMSO-d

6

): δ =

7

1

.03 (d, J = 9.0 Hz, 1H, CH), 8.08 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.40 (brs, 4H, NH), 8.42 (d, J = 2.5 Hz, 1H, CH), 11.42 (brs,

13

H, NH) ppm; C-NMR (100 MHz, DMSO-d ): 155.5 (C), 151.4 (C), 147.5 (CH), 142.4 (CH), 115.7 (C), 114.2 (CH) ppm;

6

1

9

+

79

+

F-NMR (376 MHz, DMSO-d

Microanalysis for C BrF

6

): δ = -73.94 (s) ppm; HRMS (ES ) C

H

6 8

BrN [M+H] calcd. 214.9932; found 214.9941;

4

H

8 8

3

4

N O

2

calcd. C% 29.20, H% 2.45 and N% 17.03; found C% 29.19, H% 2.23 and N% 16.75.

1

-(5-Iodopyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5e): 2-Amino-5-iodoopyridine 3e (0.59 g, 2.7 mmol)

gave 5e (0.50 mg, 41%) as a colourless crystalline solid after purification by flash column chromatography (c-Hex/EtOAc

-

1 1

9

(

8

:1). Mp = 124 – 126 °C; R

400 MHz, CDCl ): δ = 1.53 (s, 9H, CH

.47 (d, J = 2.5 Hz, 1H, CH), 10.88 (brs, 1H, NH), 11.50 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

f

= 0.25 (c-Hex/EtOAc 9:1); IR: max = 3229, 2981, 2926, 1715, 1633, 1560, 1362 cm ; H-NMR

3

), 1.53 (s, 9H, CH ), 7.96 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.25 (d, J = 9.0 Hz, 1H, CH),

3

13

3

): 163.0 (C), 153.8

), 28.0 (CH ) ppm;

(C), 152.9 (C), 149.8 (C), 146.0 (CH), 137.9 (CH), 118.0 (CH), 86.1 (C), 84.2 (C), 80.1 (C), 28.1 (CH

3

+

HRMS (ES ) C16

H24IN

4

O [M+H] calcd. 463.0842; found 463.0854.

4

2

1

=

8

-(5-Iodopyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6e): Colourless solid (67 mg, 80 %). Mp = 188 – 189 °C; IR: max

-

1 1

3291, 3092, 2930, 1696, 1580, 1461, 1189 cm ; H-NMR (400 MH

.17 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.34 (brs, 4H, NH), 8.51 (dd, J = 2.5, 1.0 Hz, 1H, CH), 11.22 (brs, 1H, NH) ppm; C-NMR

Z

, DMSO-d ): δ = 6.91 (dd, J = 8.5, 1.0 Hz, 1H, CH),

6

13

19

(100 MHz, DMSO-d ): 155.4 (C), 152.5 (C), 151.6 (CH), 147.6 (CH), 116.0 (CH), 86.8 (C) ppm; F-NMR (376 MHz, DMSO-

6

+

d

6

): δ = -73.84 (s) ppm; HRMS (ES ) C

H IN

6 8 4

[M+H] calcd. 262.9794; found 262.9789; Microanalysis for C

H IF

8 8 3

4

N O

2

calcd. C% 25.55, H% 2.14 and N% 14.90; found C% 29.59, H% 1.94 and N% 14.59.

1

-(5-Trifluoromethylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5f): With 2-amino-5-(trifluoromethyl)

pyridine 3j (0.44 g, 2.7 mmol) compound 5j (0.45 g, 42%) was isolated after purification by flash column

chromatography (c-Hex/EtOAc 98:2) as a colourless solid. Mp = 132 – 134 °C; R

f

=0.30 (c-Hex/EtOAc 98:2); IR: max

=

),

-

1 1

3

7

250, 2986, 2901, 1722, 1638, 1559, 1369, 1229 cm ; H-NMR (500 MHz, CDCl

.93 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.55 - 8.59 (m, 2H, CH), 11.10 (brs, 1H, NH), 11.51 (brs, 1H, NH) ppm; C-NMR (125

3

): δ = 1.54 (s, 9H, CH ), 1.55 (s, 9H, CH

3

13

3

MHz, CDCl

3

): 162.9 (C), 153.3 (C), 153.0 (C), 152.6 (C), 145.3 (q, JC-F = 4.5 Hz, CH), 135.5 (q, JC-F = 4.0 Hz, CH), 123.6

1

2

19

(q, JC-F = 271.0 Hz, CF

3

), 122.4 (q, JC-F = 32.0 Hz, C), 115.4 (CH), 84.4 (C), 80.4 (C), 28.1 (CH

3

), 28.0 (CH

3

) ppm; F-NMR

+

(470 MHz, CDCl

3

): δ = -61.85 (s) ppm; HRMS (ES ) C17

H

24

F

3

N

4

O

4

[M+H] calcd. 405.1750; found 405.1723.

1

-(5-(Trifluoromethyl)pyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6f): Obtained as a colourless solid (89 mg, 76 %)

-

1

following the general procedure. Mp = 198 – 199 °C; IR: max = 3295, 3081, 2900, 1665, 1613, 1580, 1322, 1083 cm ;

1

H-NMR (500 MHz, DMSO-d

6

): δ = 7.21 (d, J = 8.5 Hz, 1H, CH), 8.22 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.43 (brs, 4H, NH), 8.68

13

3

(s, 1H, CH), 11.44 (brs, 1H, NH) ppm; C-NMR (125 MHz, DMSO-d ): 155.4 (C), 155.3 (C), 144.7 (q, JC-F = 4.0 Hz, CH),

6

3

1

2

19

1

37.1 (q, JC-F = 3.0 Hz, CH), 124.0 (q, JC-F = 271.0 Hz, CF

3

), 120.6 (q, JC-F = 33.0 Hz, C), 114.1 (CH) ppm; F-NMR (376

+

MHz, DMSO-d

Microanalysis for C

6

): δ = -60.5 (s) and -73.83 (s) ppm. HRMS (ES ) C

7

H

F

8 3

N [M+H] calcd. 205.0701; found 203.0702;

4

9 8 6

H F N

4

O

2

calcd. C% 33.97, H% 2.53 and N% 17.61; found C% 33.90, H% 2.42 and N% 17.39.

1

-(5-Methylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5g): With 2-amino-5-methylpyridine 3g (0.29 g, 2.7

mmol) 5g (0.55 g, 61%) was prepared which was isolated after purification by flash column chromatography (c-

1

Hex/EtOAc 9:1) as a colourless crystalline solid. Mp = 130 – 132 °C (Lit. Mp = 139 °C ); R

f

= 0.15 (c-Hex/EtOAc 9:1); IR:

), 2.28 (s, 3H,

), 7.51 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.10 (d, J = 2.5 Hz, 1H, CH), 8.21-8.24 (m, 1H, CH), 10.81 (brs, 1H, NH), 11.53

-1 1



CH

(

max = 3251, 2979, 1716, 1645, 1552, 1476, 1377 cm ; H-NMR (400 MHz, CDCl

3

): δ = 1.53 (s, 18H, CH

3

13

brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

3

): 163.3 (C), 153.0 (C), 152.6 (C), 148.3 (C), 147.9 (CH), 138.6 (C), 129.2

+

C), 115.6 (CH), 83.6 (C), 79.8 (C), 28.2 (CH ), 28.0 (CH

3

), 17.8 (CH

3

) ppm; HRMS (ES ) C17

H

27

N

4

O [M+H] calcd. 351.2032;

4

found 351.2042.

2

1



8

-(5-Methylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6g): Colourless solid (165 mg, 92 %). Mp = 152 – 154 °C; IR:

-

1 1

max = 3276, 3105, 2926, 1695, 1573, 1479, 1187 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 2.25 (s, 3H, CH ), 6.94 (d, J =

3

.5 Hz, 1H, CH), 7.67 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.11 (d, J = 2.5 Hz, 1H, CH), 8.39 (brs, 4H, NH), 11.16 (brs, 1H, NH)

13

ppm; C-NMR (100 MHz, DMSO-d

6

): 155.7 (C), 150.3 (C), 146.4 (CH), 140.6 (CH), 129.0 (C), 113.3 (CH), 17.6 (CH

3

) ppm;

[M+H] calcd. 151.0984; found 151.0981;

calcd. C% 40.91, H% 4.20 and N% 21.21; found C% 40.52, H% 4.03 and N% 20.77.

19

+

F-NMR (367 MHz, DMSO-d

Microanalysis for C

6

): δ = -73.82 (s) ppm; HRMS (ES ) C

7 11

H N

4

9 11

H F

3

N

4

O

2

1

-(3-Methylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5h): With 2-amino-3-methyl pyridine 3h (1.00 g, 9.2

mmol) 5h (0.55 g, 17%) was isolated as a yellow viscous solid after purification by flash column chromatography (c-

1

Hex/EtOAc 6:1). R

f

= 0.25 (c-Hex/EtOAc 6:1); H-NMR: (300 MHz, CDCl

3

): ẟ= 1.52 (m, 18H, CH

3

), 2.40 (s, 3H, CH

3

), 6.97-

+

6

.99 (m, 1H, CH), 7.54 (d, J =7.5, 1H, CH), 8.16-8.19 (m, 1H, CH), 10.06 (s, 1H, NH), 13.23 (s, 1H, NH) ppm;* HRMS (ES )

+

*

13

17

C H

27

N

4

O [M+H] calcd. 351.2032; found 351.2030. Note, C-NMR spectrum broadened due to rotation on the NMR

4

timescale.

1

–

7

-(3-Methylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6h): Colourless crystalline material (0.22 g, 52%). Mp = 169

-

1 1

171 °C; IR: max = 3295, 2987, 2901, 1675, 1591, 1460, 1183 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 2.26 (s, 3H, CH ),

3

.00 - 7.15 (m, 1H, CH), 7.71 (d, J = 7.5 Hz, 1H, CH), 8.14 - 8.15 (m, 1H, CH), 7.02 – 7.15 (m, 4H, NH), 9.37 (brs, 1H, NH)

1

3

ppm. C NMR (100 MHz, DMSO-d

6

): 155.8 (C=N), 150.0 (C), 144.4 (CH), 140.7 (CH), 122.5 (C), 120.1 (CH), 17.0 (CH )

3

1

9

+

ppm. F NMR (376 MHz, DMSO-d

51.0978. Microanalysis for C

N% 20.96 and F% 21.42.

6

): δ = -73.69 (s, CF

3

) ppm. HRMS (ES ) C

7

H

11

N [M+H] calcd. 151.0984; found

4

1

9 11

H F

3

N

4

O

2

calcd. C% 40.91, H% 4.20, N% 21.21 and F% 21.57; found C% 40.59, H% 4.08,

1

-(4-Methylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5i): 2-Amino-4-methylpyridine 3i (0.30 g, 2.7 mmol)

gave 5i (0.30 g, 32%) as a colourless crystalline solid after purification by flash column chromatography (c-Hex/EtOAc

-

1 1

9

:1). Mp = 158 – 160 °C; R

f

= 0.20 (c-Hex/EtOAc 9:1); IR: max = 3200, 2974, 2901, 1720, 1649, 1569, 1385 cm ; H-NMR

), 2.38 (s, 3H, CH ), 6.83 (d, J = 5.0 Hz, 1H, CH), 8.13-8.16 (m, 2H, CH), 10.85 (brs,

H, NH), 11.55 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl ): 163.2 (C), 153.1 (C), 152.6 (C), 150.6 (C), 149.5 (C), 147.6

), 28.0 (CH

(400 MHz, CDCl ): δ = 1.54 (s, 18H, CH

3

13

1

3

+

(CH), 121.1 (CH), 116.4 (CH), 83.8 (C), 79.9 (C), 28.1 (CH

3

), 21.5 (CH

3

) ppm; HRMS (ES ) C17

27

H N

4

O [M+H]

4

calcd. 351.2032; found 351.2029.

2

3

1

-

6

-(4-Methylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6i): Isolated as a colourless solid (110 mg, 86 %). Mp = 122

-

1 1

124 C. IR: max = 3389, 3088, 2987, 1683, 1545, 1465, 1206 cm . H NMR (400 MH

.83 - 6.84 (m, 1H, CH), 8.15 (d, J = 5.5 Hz, 1H, CH), 8.39 (brs, 4H, NH), 11.01 (brs, 1H, NH) ppm; C-NMR (100 MHz,

Z

, DMSO-d

6

): δ = 2.31 (s, 3H, CH ),

3

13

19

DMSO-d

C

6

): 155.8 (C), 152.5 (C), 151.1 (CH), 146.7 (CH), 121.1 (C), 113.6 (CH), 21.1 (CH

3

) ppm; F-NMR (376 MHz,

[M+H] calcd. 151.0984; found 151.0985; Microanalysis for

calcd. C% 40.91, H% 4.20, N% 21.21 and F% 21.57; found C% 40.89, H% 4.08, N% 21.00 and F% 21.14.

+

6

): δ = -73.84 (s) ppm; HRMS (ES ) C

H N

7 11 4

H F

9 11 3

4

N O

2

1

-(6-Methylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5j): Using 2-amino-6-methylpyridine 3j (0.30 g, 2.7

mmol), 5j (0.25 g, 27%) was isolated as a colourless crystalline material after purification via silica gel chromatography

(

1

c-Hex/EtOAc 98:2). Mp = 98 – 100 °C; R

f

= 0.20 (c-Hex/EtOAc 98:2); IR: max = 3273, 2972, 2901, 1726, 1646, 1576,

-

1 1

367 cm ; H-NMR (400 MHz, CDCl

3

): δ = 1.53 (s, 9H, CH ), 1.54 (s, 9H, CH ), 2.46 (s, 3H, CH ), 6.87 (d, J = 7.5 Hz, 1H,

3

13

CH), 7.58 (t, J = 7.5 Hz, 1H, CH), 8.18 (brs, 1H, CH), 10.74 (brs, 1H, NH), 11.56 (brs, 1H, NH) ppm; C-NMR (100 MHz,

CDCl ): 163.4 (C), 157.0 (C), 153.1 (C), 152.5 (C), 149.9 (C), 138.4.6 (CH), 119.3 (CH), 112.9 (CH), 83.7 (C), 79.8 (C), 28.1

3

+

(CH

3

), 28.0 (CH

3 3

), 24.0 (CH ) ppm. HRMS (ES ) C17

H N

27 4

O [M+H] calcd. 351.2032; found 351.2042.

4

1



8

-(6-Methylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6j): Colourless solid (80 mg, 80 %). Mp = 127 – 129 °C; IR:

-

1 1

max = 3398, 3300, 2988, 1682, 1563, 1455, 1125 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 2.44 (s, 3H, CH ), 6.84 (d, J =

3

.0 Hz, 1H, CH), 7.02 (d, J = 7.5 Hz, 1H, CH), 7.74 (dd, J = 8.0, 7.5 Hz, 1H, CH), 8.39 (brs, 4H, NH), 11.08 (brs, 1H, NH)

1

3

ppm; C-NMR (100 MHz, DMSO-d

6

): 156.3 (C), 155.7 (C), 151.9 (C), 140.2 (CH), 119.0 (CH), 110.5 (CH), 24.0 (CH

3

) ppm;

19

+

F-NMR (376 MHz, DMSO-d

6

): δ = -73.88 (s) ppm; HRMS (ES ) C

H N

7 11 4

[M+H] calcd. 151.0984; found 151.0990;

Microanalysis for C

and F% 21.27.

9 11 3

H F N

4

O

2

calcd. C% 40.91, H% 4.20, N% 21.21 and F% 21.57; found C% 40.77, H% 3.98, N% 20.81

2

-Amino-5-phenylpyridine (3k): A mixture of 2-amino-5-bromopyridine 3d (0.35 g, 2.0 mmol, 1 eq.), phenylboronic

acid (0.34 g, 2.8 mmol, 1.4 eq.), K

and MeCN mixture (5: 15 mL) was heated at 90 C for 16 h. The reaction mixture was allowed to cool, diluted with

2

CO

3

(0.41 g, 2.9 mmol, 1.5 eq.), and Pd(OAc)

2

(45 mg, 0.2 mmol, 0.1 eq.) in a H O

2

o

EtOAc, washed with brine, and then the organic phase was dried over anhydrous Na

2

SO , filtered and concentrated.

4

The residue was purified by flash column chromatography on silica gel (c-Hex/EtOAc 4:1) to give 3k (0.20 g, 59%) as a

2

yellow crystalline material. Mp = 127 – 128 °C (Lit. Mp = 133 °C ); R

f

= 0.10 (c-Hex; EtOAc 4:1); IR: max = 3383, 3326,

): δ = 4.49 (brs, 2H, NH ), 6.59 (d, J = 8.5 Hz, 1H, CH),

.29 - 7.34 (m, 1H, CH), 7.40 - 7.44 (m, 2H, CH), 7.49 - 7.52 (m, 2H, CH), 7.68 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.33 (brs, 1H,

-

1 1

3

7

139, 2944, 1693, 1656, 1269, 1116 cm ; H-NMR (400 MH

Z

, CDCl

3

2

4

1

3

CH) ppm; C-NMR (100 MHz, CDCl

3

): 157.6 (C), 146.4 (CH), 138.3 (C), 136.6 (CH), 128.9 (C), 126.9 (C), 126.3 (C), 108.5

+

(

CH) ppm; LRMS (ES ) C11

H N

11 2

[M+H] found 170.60.

1

-(5-Phenylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5k): 2-Amino-5-phenyl pyridine 3k (0.18 g, 1.1

mmol) gave the title compound (100 mg, 25 %) which was isolated as a colourless crystalline solid after purification by

flash column chromatography (c-Hex/EtOAc 98:2). Mp = 100 – 102 °C; R = 0.10 (c-Hex/EtOAc 98:2); IR: max = 3674,

): δ = 1.55 (s, 18H, CH ), 7.35 - 7.40 (m, 1H, CH),

.44 - 7.47 (m, 2H, CH), 7.55 - 7.57 (m, 2H, CH), 7.93 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.45 (brs, 1H, CH), 8.54 (d, J = 2.5 Hz,

f

-

1 1

2

7

1

(

2

986, 2901, 1714, 1628, 1552, 1380 cm ; H-NMR (400 MHz, CDCl

3

13

H, CH), 10.96 (brs, 1H, NH), 11.56 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

C), 146.2 (CH), 137.6 (C), 136.6 (CH), 132.9 (C), 129.1 (C), 127.8 (C), 126.8 (C), 115.9 (CH), 84.0 (C), 80.0 (C), 28.2 (CH

3

): 163.2 (C), 153.1 (C), 152.7 (C) 149.8

),

3

+

8.1 (CH

3

) ppm; HRMS (ES ) C22

29

H N

4

O [M+H] calcd. 413.2189; found 413.2185.

4

1

-(5-Phenylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6k): Treatment of 1-(5-phenyl pyridin-2-yl)-2,3-di(tert-

butoxycarbonyl) guanidine (75 mg, 0.18 mmol) using the general procedure gave 6k (45 mg, 76 %) which was isolated

-

1 1

as a colourless solid. Mp = 158 – 160 °C; IR: max = 3251, 3138, 2987, 1697, 1562, 1473, 1186 cm ; H-NMR (400 MHz,

DMSO-d ): δ = 7.13 (dd, J = 8.5, 1.0 Hz, 1H, CH), 7.39 – 7.41 (m, 1H, CH), 7.45 - 7.50 (m, 2H, CH), 7.67 (m, 2H, CH), 8.17

6

13

(

dd, J = 8.5, 2.5 Hz, 1H, CH), 8.32 (brs, 4H, NH), 8.60 (dd, J = 2.5, 1.0 Hz, 1H, CH), 11.05 (brs, 1H, NH) ppm; C-NMR

(

1

2

100 MHz, DMSO-d ): 155.5 (C), 151.6 (C), 144.8 (CH), 138.1 (CH), 136.6 (C), 131.8 (C), 129.6 (C), 128.5 (C), 127.0 (C),

6

19

+

13.9 (CH) ppm; F-NMR (376 MHz, DMSO-d

13.1134.

6

): δ = -73.72 (s) ppm; HRMS (ES ) C12

H

13

N [M+H] calcd. 213.1140; found

4

3

6

-Amino-nicotinic acid methyl ester (3m): To a stirred solution of 6-amino-nicotinic acid 3l (2.00 g, 14.5 mmol, 1 eq.)

o

in MeOH (16 mL, 27 eq.) was added concentrated H

at 80 C overnight. The mixture was cooled to room temperature and neutralized with saturated NaHCO

2

SO

4

(0.56 mL, 0.75eq.) at 0 C. The reaction mixture was heated

o

3

solution. The

precipitated solid was filtered and dried under vacuum to give 3m (1.7 g, 77%) as a yellow solid. Mp = 128 – 130 °C

3

-1 1

(

Lit. Mp = 154 – 156 °C ); IR: max = 3317, 3138, 2958, 2927, 1725, 1648, 1120 cm ; H-NMR (300 MHz, CDCl

s, 3H, CH ), 4.96 (brs, 2H, NH ), 6.47 (dd, J = 8.5, 1.0 Hz, CH), 8.01 (dd, J = 8.5, 2.5 Hz, CH), 8.73 (d, J = 2.5 Hz, 1H, CH)

): 166.2 (C), 161.1 (C), 151.5 (CH), 138.9 (CH), 116.4 (C), 107.4 (CH), 51.8 (CH ) ppm;

[M+H] found 153.30.

3

): δ = 3.88

3

2

13

ppm; C-NMR (100 MHz, DMSO-d

6

3

+

LRMS (ES ) C

7 9 2

H N O

2

5

Methyl 6-(2,3-di-(tert-butoxycarbonyl)guanidino)nicotinate (5m): Using methyl ester 3m (0.40 g, 2.7 mmol) the title

compound (0.55 g, 52%) was isolated after purification via silica gel chromatography (c-Hex/EtOAc 9:1) as a colourless

crystalline solid. Mp = 138 – 140 °C; R

f

= 0.20 (c-Hex/EtOAc 9:1); IR: max = 3254, 2976, 2900, 1720, 1633, 1580, 1370

): δ = 1.54 (s, 18H, CH ), 3.93 (s, 3H, CH ), 8.31 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.51 (d, J =

.5 Hz, 1H, CH), 8.91 (dd, J = 2.5, 1.0 Hz, 1H, CH), 11.11 (brs, 1H, NH), 11.51 (brs, 1H, NH) ppm; C-NMR (100 MHz,

-

1 1

cm ; H-NMR (400 MHz, CDCl

3

13

8

CDCl ): 165.5 (C), 163.0 (C), 153.7 (C), 152.9 (C), 152.6 (C), 150.0 (CH), 139.6 (CH), 121.9 (C), 114.9 (CH), 84.4 (C), 80.4

3

+

(C), 52.2 (CH

3 3

) 28.1 (CH ), 28.0 (CH

3

) ppm; HRMS (ES ) C18

H

27

N

4

O [M+H] calcd. 395.1931; found 395.1915.

6

Methyl 6-guanidinonicotinate 2,2,2-trifluoroacetate (6m): Isolated (165 mg, 95 %) as a colourless solid. Mp = 142 –

-

1 1

1

7

1

44 °C; IR: max = 3271, 3077, 1714, 1605, 1556, 1489, 1183 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 3.85 (s, 3H, CH

3

),

.13 (dd, J = 8.5, 1.0 Hz, 1H, CH), 8.30 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.41 (brs, 4H, NH), 8.82 (d, J = 2.5, 1.0 Hz, 1H, CH),

1.31 (brs, 1H, NH) ppm; C-NMR (100 MHz, DMSO-d ): 165.0 (C), 155.5 (C), 148.8 (C), 140.3 (CH), 127.1 (C), 121.4 (C),

13

6

19

+

13.7 (CH), 52.8 (CH

3

) ppm; F-NMR (376 MHz, DMSO-d

6

): δ = -73.74 (s) ppm; HRMS (ES ) C

calcd. C% 38.97, H% 3.60 and N% 18.18; found C% 38.95,

H N

8 11 4

O [M+H] calcd.

2

95.0882; found 195.0881; Microanalysis for C10

H F

11 3

4

N O

4

H% 3.49 and N% 17.95.

6

-Amino-N-methoxy-N-methylnicotinamide (3n): To a slurry of 6-aminonicotinic acid 3l (0.90 g, 6.5 mmol) in DMF (15

*

mL) was added HATU (2.48 g, 6.5 mmol), DIPEA (2.5 mL, 14.1 mmol) and N,O-dimethylhydroxylamine hydrochloride

(

0.65 g, 6.7 mmol), and the mixture was stirred at room temperature overnight. The mixture was concentrated in

vacuo. The resulting solution was partitioned with ethyl acetate and water (30 mL: 10 mL) and the resultant aqueous

layer was extracted with EtOAc (5 x 15 mL). The combined organic layers were dried over anhydrous MgSO , filtered

and concentrated to give a yellow thick oil which purified via silica gel flash chromatography (EtOAc) to provide the

amide 3n (1.07 g, 92 %) as a pale yellow crystalline solid. Mp = 90 – 92 °C; R = 0.30 (EtOAc); IR: max = 3362, 3169,

): δ = 3.35 (s, 3H, CH ), 3.58 (s, 3H, CH ), 4.82 (brs, 2H,

), 6.48 (d, J = 9.0 Hz, 1H, CH), 7.90 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.58 (d, J = 2.5 Hz, 1H, CH) ppm; C-NMR (100 MHz,

4

f

-

1 1

2

917, 1728, 1653, 1460, 1377 cm ; H-NMR (300 MH

NH

CDCl

Z

, CDCl

3

1

3

2

+

3

): 167.6 (C), 159.6 (C), 149.8 (CH), 139.0 (CH), 119.6 (C), 107.3 (CH), 60.9 (CH

3

), 33.5 (CH

3

) ppm; HRMS (ES )

+

*

C

8 12 3

H N O

2

[M+H] calcd. 182.0930; found 182.0933. [ Care: severe allergic reactions have been occasionally reported]

1

-(5-(Methoxy(methyl)carbamoyl) pyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5n): Following the general

procedure, Weinreb amide 3n (0.25 g, 1.35 mmol) gave 5n (0.36 g, 63%) which was isolated after purification via silica

gel flash chromatography (c-Hex/EtOAc 3:1) as a colourless solid. Mp = 114 – 115 °C; R = 0.20 (c-Hex/EtOAc 3:1); IR:

, CDCl ): δ = 1.54 (s, 18H, CH ),

), 8.13 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.45 (d, J = 8.5 Hz, 1H, CH), 8.72 (d, J = 2.5 Hz, 1H,

f

-

1 1



max = 3246, 2974, 2800, 1732, 1645, 1574, 1411, 1231, 1131 cm ; H-NMR (300 MH

Z

3

.38 (s, 3H, CH ), 3.55 (s, 3H, CH

3

13

CH), 11.05 (brs, 1H, NH), 11.52 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl ): 167.0 (C), 162.9 (C), 153.0 (C), 152.6 (C),

3

2

6

1

52.0 (C), 148.7 (CH), 139.0 (CH), 125.4 (C), 114.6 (CH), 84.2 (C), 80.3 (C), 61.2 (CH

3

), 33.2 (CH

3

), 28.1 (CH

3

), 28.0 (CH )

3

+

ppm; HRMS (ES ) C19

H

29

N

5

6

O Na [M+Na] calcd. 446.2016; found 446.2013.

6

-Guanidino-N-methoxy-N-methylnicotinamide 2,2,2-trifluoroacetate (6n): Following the general procedure, 6n (31

mg, 77 %) was isolated as a colourless solid. Mp = 123 -125 °C; IR: max = 3419, 3321, 3144, 2990, 2835, 1701, 1595,

-

1 1

1

(

CDCl

463, 1132 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 3.27 (s, 3H, CH

3

), 3.56 (s, 3H, CH

3

), 7.10 (d, J = 8.5 Hz, 1H, CH), 8.11

13

dd, J = 8.5, 2.0 Hz, 1H, CH), 8.40 (s, 4H, NH), 8.57 (d, J = 2.0 Hz, 1H, CH), 11.29 (s, 1H, NH) ppm; C-NMR (100 MHz,

): 166.3 (C), 155.5 (C), 153.5 (C), 147.0 (CH), 139.8 (CH), 125.6 (C), 113.0 (CH), 61.4 (CH

NMR (376 MHz, DMSO-d

19

3

), 28.0 (CH

3

) ppm; F-

+

6

): δ = -74.47 (s) ppm; HRMS (ES ) C

9 14

H N

5

O

2

[M+H] calcd. 224.1142; found 224.1140.

1

(

-(5-Formylpyridin-2-yl)-2,3-di-(tert-butoxycarbonyl)guanidine (5o): LiAlH

8 mL) at -78C, under nitrogen. Weinreb amide 5n (0.30 g, 0.7 mmol) in THF (3 mL) was added and the mixture stirred

for 90 minutes. Saturated NH Cl (10 mL) was cautiously added dropwise at -78 C. EtOAc (10 mL) was then added and

4

(0.11g, 2.8 mmol) was stirred in dry THF

4

the solution was allowed to warm to room temperature and stirred for 30 minutes. The organic layer was separated

and the aqueous layer was re-extracted with EtOAc (3 x 10 mL). The combined organic layers were successively washed

with 0.5 M HCl (2 x 10 mL), saturated NaHCO

Solvent was removed in vacuo, then the crude product was purified via silica gel chromatography (c-Hex/EtOAc 6:1)

to provide 5o (0.22 g, 88 %) as a pale yellow crystalline solid. Mp = 110 – 112 °C; R = 0.25 (c-Hex/EtOAc 6:1); IR: max

3230, 2980, 2934, 1792, 1702, 1635, 1578, 1395, 1119 cm ; H-NMR (300 MHz, CDCl ): δ = 1.55 (s, 9H, CH ), 1.54 (s,

H, CH ), 8.15 - 8.20 (m, 1H, CH), 8.62 (d, J = 8.5 Hz, 1H, CH), 8.76 (d, J = 2.0 Hz, 1H, CH), 10.00 (s, 1H, CHO), 11.22 (brs,

H, NH), 11.52 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

3

(2 x 10 mL), brine (10 mL) and dried over anhydrous MgSO

4

and filtered.

f

-

1 1

=

9

1

3

13

3

): 189.6 (C), 162.9 (C), 154.8 (C), 152.7 (C), 151.5 (CH),

+

48.7 (C), 138.4 (CH), 128.1 (C), 115.7 (CH), 84.5 (C), 80.6 (C), 28.1 (CH

87.1644; found 387.1629.

3

) ppm. HRMS (ES ) C17

H N

24 4

O Na[M+Na] calcd.

5

1

- (5-Formylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6o): Following the general procedure 6o (80 mg, 80 %) was

isolated as a beige solid. Mp = 132 – 134 °C; IR: max = 3304, 3086, 2923, 2857, 2794, 1664, 1602, 1580, 1482, 1199 cm^-

1

;

H-NMR (400 MHz, DMSO-d ): δ = 7.17 (d, J = 8.5 Hz, 1H, CH), 8.25 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.42 (brs, 4H, NH),

6

13

8

1

.82 (d, J = 2.5 Hz, 1H, CH), 9.98 (s, 1H, CHO), 11.32 (brs, 1H, NH) ppm; C-NMR (125 MHz, DMSO-d

60.0 (q, JC-F = 34.0 Hz, CF

6

): 191.2 (CHO),

2

19

3

), 156.1 (C), 155.5 (C), 151.4 (CH), 138.7 (CH), 127.9 (C), 114.1 (CH) ppm; F-NMR (376 MHz,

+

DMSO-d

6

): δ = -73.71 (s) ppm; HRMS (ES ) C

7

H

9

N O [M+H] calcd. 165.0776; found 165.0782; Microanalysis for

4

9 9

C H F

3

N

4

O

2

3. 0.5 H O calcd. C% 37.64, H% 3.51 and N% 19.51; found C% 37.78, H% 3.34 and N% 19.74.

2

7

1

-(5-Acetylpyridin-2-yl)-2,3-di(tert-butoxycarbonyl)guanidine (5p): Di-Boc-protected guanidine 5n (0.20 g, 0.48

mmol) was dissolved in dry THF (25 mL) under a nitrogen atmosphere. This solution was cooled to -78 C for 5 minutes,

then 3.0 M MeMgCl in THF (1.6 mL, 4.8 mmol) was added slowly at -78 °C. This solution was left to stir for 1 hr at -78

°

C, it was then allowed to warm to rt and stirred for 30 minutes. When the reaction was complete, the solution was

washed with NH Cl (sat.) (25 mL). The organic layer was washed with brine (50 mL), dried over anhydrous MgSO and

concentrated in vacuo. The crude product was purified via silica gel chromatography (c-Hex/EtOAc 4:1) to provide 5p

4

(

3

0.10 g, 67%) as a colourless crystalline material. Mp = 270 °C (decomposed); R

f

= 0.20 (c-Hex/EtOAc 4:1); IR: max

): δ = 1.53 (s, 9H, CH ), 1.54 (s, 9H, CH ), 2.58

), 8.26 (dd, J = 9.0, 2.5 Hz, 1H, CH), 8.54 (d, J = 9.0 Hz, 1H, CH), 8.87 (dd, J = 2.5, 1.0 Hz, 1H, CH), 11.13 (brs,

=

-

1 1

242, 2986, 1722, 1686, 1628, 1570, 1153 cm ; H-NMR (300 MHz, CDCl

3

(s, 3H, CH

3

13

1

H, NH), 11.52 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl ): 195.7 (C), 162.9 (C), 153.8 (C), 152.9 (C), 152.6 (C), 149.2

3

+

(CH), 138.2 (CH), 128.7 (C), 115.1 (CH), 84.4 (C), 80.3 (C), 28.1 (CH

3 3

), 28.0 (CH ) ppm; HRMS (ES ) C18

H

26

N

4

O Na[M+Na]

5

calcd. 401.1801; found 401.1785.

1

7

1

-(5-Acetylpyridin-2-yl)guanidine 2,2,2-trifluoroacetate (6p): Isolated as a colourless solid (35mg, 72 %). Mp = 142 –

-

1 1

44 °C; IR: max = 3255, 3072, 1710, 1681, 1530, 1478, 1206 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 2.57 (s, 3H, CH

3

),

.14 (d, J = 8.5 Hz, 1H, CH), 8.32 (dd, J = 8.5, 2.5 Hz, 1H, CH), 8.53 (brs, 4H, NH), 8.87 (d, J = 2.5 Hz, 1H, CH), 11.55 (brs,

13

H, NH) ppm; C-NMR (100 MHz, DMSO-d

6

): 196.1 (CHO), 155.6 (C), 155.3 (C), 148.6 (CH), 139.2 (CH), 128.3 (C), 113.5

(

CF

CH) 27.1 (CH

3

) ppm; note, increased scans led to identification of the trifluoroacetate signals: 117.4 (q, J = 297.5 Hz,

19

+

3

), 160.5 (q, J = 32.4 Hz, CO

2 6

) ppm; F-NMR (376 MHz, DMSO-d ): δ = -73.85 (s) ppm; HRMS (ES ) C

8 11

H N

4

O [M+H]

calcd. 179.0933; found 179.0066; Microanalysis for C10

H

11

F

3

N

4

O

3

calcd. C% 41.10, H% 3.79 and N% 19.17; found C%

4

0.95, H% 3.66 and N% 18.89.

1

-Pyrimidine-2,3-di-(tert-butoxycarbonyl)guanidine: Using 4-aminopyrimidine 7 (0.26 g, 2.7 mmol) the title

compound (0.40 g, 45 %) was isolated as a colourless crystalline solid after purification via silica gel chromatography

(

1

c-Hex/EtOAc 9:1). Mp = 160 °C (decomposed); R

f

= 0.15 (c-Hex/EtOAc 9:1); IR: max = 3217, 2998, 1772, 1635, 1553,

-

1 1

411, 1386 cm ; H-NMR (400 MHz, CDCl

3

): δ = 1.53 (s, 9H, CH ), 1.54 (s, 9H, CH ), 8.43 (d, J = 6.0 Hz, 1H, CH), 8.63 (d,

3

13

J = 6.0 Hz, 1H, CH), 8.86 (d, J = 1.0 Hz, 1H, CH), 10.99 (brs, 1H, NH), 11.47 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

3

):

1

62.7 (C), 158.2 (C), 156.6 (CH), 153.1 (CH), 152.5 (C), 112.1 (CH), 84.6 (C), 80.6 (C), 28.1 (CH ), 28.0 (CH ) ppm; HRMS

3

+

(

ES ) C15

H

24

N

5

O [M+H] calcd. 338.1828; found 338.1819.

4

2

8

1

-(Pyrimidin-4-yl)guanidine 2,2,2-trifluoroacetate (10): Compound 10 (75 mg, 75 %) was isolated as a beige solid

following the general procedure outlined above. Mp = 163 – 165 °C; IR: max = 3279, 3068, 2990, 1704, 1587, 1472,

-

1 1

1

175 cm ; H-NMR (400 MHz, DMSO-d

6

): δ = 7.08 (dd, J = 5.5, 1.5Hz, 1H, CH), 8.62 (brs, 4H, NH), 8.66 (d, J = 5.5 Hz, 1H,

13

CH), 8.88 (brs, 1H, CH), 11.70 (brs, 1H, NH) ppm; C-NMR (100 MHz, DMSO-d

C), 110.5 (CH) ppm; F-NMR (376 MHz, DMSO-d

6

): 158.7 (C), 158.4 (CH), 157.4 (CH), 155.6

19

+

(

6

): δ = -74.00 (s) ppm; HRMS (ES ) C

5

H

8

N [M+H] calcd 138.0780;

5

found 138.0778; Microanalysis for C

7 8 3

H F N

5

O

2

calcd. C% 33.47, H% 3.21, N% 27.88 and F% 22.69; found C% 32.71, H%

3

.04, N% 26.37 and F% 23.07.

5

1

-(Pyridin-3-yl)-2,3-di(tert-butoxycarbonyl)guanidine: Using 3-aminopyridine 8 (0.25 g, 2.7 mmol) and the general

procedure outlined above the title compound (0.75 g, 83 %) was isolated as a colourless crystalline solid after

5

purification via silica gel chromatography (c-Hex/EtOAc 4:1). Mp = 132 – 134 °C (Lit. Mp = 136 °C ); R

f

= 0.25 (c-

-

1 1

Hex/EtOAc 4:1); IR: max = 3147, 2980, 2930, 1724, 1642, 1561, 1329 cm ; H-NMR (400 MHz, CDCl

CH ), 1.55 (s, 9H, CH ), 7.28 - 7.30 (m, 1H, CH), 8.21 (ddd, J = 8.3, 2.5, 1.5 Hz, 1H, CH), 8.35 (dd, J = 8.3, 1.5 Hz, 1H, CH),

.67 (d, J = 2.5 Hz, 1H, CH), 10.40 (brs, 1H, NH), 11.61 (brs, 1H, NH) ppm; C-NMR (100 MHz, CDCl

C), 153.3 (C), 145.6 (CH), 143.3 (CH), 133.8 (C), 129.5 (CH), 123.5 (CH), 84.2 (C), 80.0 (C), 28.2 (CH

3

): δ = 1.51 (s, 9H,

3

13

8

(

3

): 163.2 (C), 153.7

), 28.0 (CH ) ppm;

3

+

HRMS (ES ) C16

H

25

N

4

O [M+H] calcd. 337.1876; found 337.1864.

4

1

-(Pyridin-3-yl)guanidine 2,2,2-trifluoroacetate [pyraxinine·TFA] (11): Compound 11 (60 mg, 66%) was isolated as a

-

1 1

colourless solid. Mp = 118 – 120 °C; IR: max = 3328, 3130, 2987, 2901, 1681, 1577, 1463, 1183 cm ; H-NMR (400 MHz,

DMSO-d

6

): δ = 7.45 (dd, J = 8.0, 4.5 Hz, 1H, CH), 7.66 - 7.69 (m, 5H, CH, NH), 8.47 (brs, 2H, CH), 10.05 (brs, 1H, NH)

13

19

ppm; C-NMR (100 MHz, DMSO-d

MHz, DMSO-d ): δ = -73.82 (s) ppm; HRMS (ES ) C

6

): 156.6 (C), 147.8 (CH), 146.5 (CH), 133.0 (CH,C), 124.8 (CH) ppm; F-NMR (376

+

6

H

9

N O [M+H] calcd. 137.0827; found 137.0821; Microanalysis for

4

8 9

C H F

3

N

4

O

2

calcd. C% 38.41, H% 3.63, N% 22.39 and F% 22.78; found C% 38.20, H% 3.43, N% 22.00 and F% 22.54.

6

1

-(4-Chlorophenyl)-2,3-di(tert-butoxycarbonyl)guanidine: 4-Chloroaniline (0.34 g, 2.7 mmol) was converted to the

title compound (0.89 g, 90 %) using the general method outlined above. This was isolated as a colourless solid after

purification via silica gel chromatography (c-Hex/EtOAc 98:2). Mp = 138 - 140 °C; R = 0.10 (c-Hex/EtOAc 98:2); IR: max

): δ = 1.51 (s, 9H, CH ), 1.54 (s, 9H, CH ),

.26 - 7.30 (m, 2H, CH), 7.55 - 7.59 (m, 2H, CH), 10.35 (brs, 1H, NH), 11.62 (brs, 1H, NH) ppm; C-NMR (100 MHz,

f

-

1 1

=

7

3145, 2975, 1717, 1633, 1561, 1455, 1335 cm ; H-NMR (400 MHz, CDCl

3

1

3

CDCl

3

): 163.1 (C), 153.4 (C), 153.3 (C), 135.5 (C), 129.8 (C), 128.9 (CH), 123.4 (CH), 84.0 (C), 79.8 (C), 28.1 (CH

3

), 28.0

+

35

+

(

CH ) ppm; HRMS (ES ) C17

3

H25 ClN

3

O [M+H] calcd. 370.1534; found 370.1517.

4

2

9

Article Doi

Synthesis of 2-guanidinyl pyridines and their trypsin inhibition and docking

DOI: 10.1016/j.bmc.2020.115612

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2020.115612

Full text of DOI:10.1016/j.bmc.2020.115612

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