Development of amidine-based sphingosine kinase 1 nanomolar inhibitors and reduction of sphingosine 1-phosphate in human leukemia cells

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pubs.acs.org/jmc

Development of Amidine-Based Sphingosine Kinase 1 Nanomolar

Inhibitors and Reduction of Sphingosine 1-Phosphate in Human

Leukemia Cells^†

Andrew J. Kennedy,*^,‡Thomas P. Mathews,^‡,§Yugesh Kharel,^§Saundra D. Field,^‡Morgan L. Moyer,^‡

James E. East,^‡Joseph D. Houck,^‡Kevin R. Lynch,^§and Timothy L. Macdonald^‡,§

^‡Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virginia 22904, United States

^§Department of Pharmacology, University of Virginia, 1340 Jeﬀerson Park Avenue, Charlottesville, Virginia 22908,

United States

S Supporting Information

b

ABSTRACT: Sphingosine 1-phosphate (S1P) is a bioactive

lipid that has been identiﬁed as an accelerant of cancer

progression. The sphingosine kinases (SphKs) are the sole

producers of S1P, and thus, SphK inhibitors may prove eﬀective

in cancer mitigation and chemosensitization. Of the two SphKs,

SphK1 overexpression has been observed in a myriad of cancer

cell lines and tissues and has been recognized as the presumptive

target over that of the poorly characterized SphK2. Herein, we

present the design and synthesis of amidine-based nanomolar

SphK1 subtype-selective inhibitors. A homology model of

SphK1, trained with this library of amidine inhibitors, was then

used to predict the activity of additional, more potent, inhibi-

tors. Lastly, select amidine inhibitors were validated in human leukemia U937 cells, where they signiﬁcantly reduced endogenous

S1P levels at nanomolar concentrations.

’ INTRODUCTION

signaling,²⁷prolactin expression,²⁸and lysophosphatidic acid

(LPA) signaling,²⁹which indicates SphK1 inhibitors may be

capable of counteracting a range of oncogene-accelerated can-

cers. SphK1 expression has also been shown to protect rapidly

dividing cells from hypoxia,³⁰autophagy,³¹and chemotherapy.³²

SphK1 siRNA has been shown to slow the rate of growth of

cancer cells that have SphK1 overexpression.^20,21,32,33Breast

cancer,¹²gastric cancer,¹⁵and glioblastoma^8,9patients with high

levels of SphK1 have shorter life expectancies. The relationship

between SphK1 and cell survival can be described as linear, with

increased S1P facilitating more aggressive and chemotherapeutic

resistant cells and decreased S1P leading to a buildup of

ceramide, its biosynthetic precursor, and ceramide dependent

apoptosis.³⁴Indeed, the sphingosine rheostat (Scheme 1) that

governs cell fate by controlling the ratio of S1P to ceramide could

be manipulated by applying the correct resistance at SphK1 with

small molecule inhibitors that “dial down” S1P concentrations.

To state that the less inducible SphK2 is simply the house-

keeping isoenzyme of SphK1 would be misleading. Unlike

SphK1, which is cytosolic and when phosphorylated translo-

cates to the inner leaﬂet of the cell membrane,³⁵SphK2 is

The scientiﬁc community has identiﬁed the sphingosine

kinases (SphKs) as potential therapeutic targets for broad cancer

mitigation and chemotherapeutic sensitization.^1,2The SphKs are

the sole producers of sphingosine 1-phosphate (S1P), which

regulates cell survival, proliferation, neovascularization, and

migration through ﬁve G-protein-coupled receptors (S1PR_1ꢀ5

)

and through other intracellular mechanisms.^3ꢀ7Up-regulation of

the SphK1, the ﬁrst of two SphK isoforms, is found in many

cancers (brain,^8,9bladder,¹⁰breast,^11,12colon,^13,14gastric,¹⁵head

and neck,^16,17leukemia,¹⁸non-Hodgkin lymphoma,¹⁹prostate,^20,21

skin,²²and squamous cell carcinoma,²³among others), and the

overproduction of S1P has been shown to aid angiogenesis,

tumorigenesis, and metastasis.

Because of its deregulation in cancer, SphK1 has been

implicated as a potential oncogene;^2,24however, no genetic

mutations have yet been identiﬁed, indicating that malignancies

may become dependent on SphK1 through a non-oncogene

addiction.²⁵This theory is appealing because of the central role

that S1P plays in the signal ampliﬁcation of other known

oncogenes. SphK1 expression and activation increase with

mitogenic signaling from growth factors for a range of receptor

tyrosine kinases²⁶(epidermal (EGF), vascular endothelial

(VEGF), platelet derived (PDGF), among others), estrogen

Received: January 10, 2011

Published: April 15, 2011

r

2011 American Chemical Society

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Scheme 1. Sphingosine Rheostat

Table 1. Previously Described Amidine-Based Sphingosine Kinase Inhibitors

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

.

predominately located on or in the organelles, such as the ER or

the nucleus.³⁶Because of this location, S1P produced by SphK2

in the interior of the cell is not eﬀectively positioned to enter into

the inside-out S1P receptor signaling pathway occurring at the

cell membrane and therefore does not have the same proliferative

eﬀects.³⁷Instead, S1P synthesized in the nucleus by SphK2 causes

histone deacetylase 1 and 2 (HDAC 1/2) inhibition, p21 gene

expression, and cytostasis.⁷SphK2 overexpression causes apopto-

sis, which is most likely due to its degradation by the proteasome

and release of a short proapoptotic BH3-domain present in SphK2

that is absent in SphK1.³⁸The relationship between SphK2 and

cell survival appears to be parabolic, where up-regulation leads to

its degradation and caspase-mediated apoptosis, moderate activity

leads to p21 expression and cell cycle arrest, and down-regulation

leads to reduced p21 expression and apoptosis or proliferation

depending on cell environment.¹

If SphK inhibitors are to be used to mitigate the presentation

of cancer or to retard chemotherapeutic resistance, the ques-

tion must be raised: Is it necessary to selectively inhibit one of

the SphKs or inhibit both enzymes together? The inducibility

of SphK1 by mitogenic factors is an indication of disease

causing deregulation; however, siRNA experiments demon-

strate that “knocking down” SphK2 is more eﬃcacious at

retarding cell growth in two glioblastoma cell lines.⁹It is

possible that the inhibitor subtype selectivity necessary for

eﬀective treatment may be cancer dependent, and our research

aim is to synthesize a spectrum of dual and selective SphK

inhibitors.

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and tested. Scheme 2 shows the individual head and tail

optimizations and subsequent partnership to generate com-

pound 38, which has K_I= 75 nM at SphK1 and is 80-fold

selective over SphK2. The library of inhibitors synthesized was

then used as a test set in the generation of a SphK1 homology

model derived from the solved structure of diacylglycerol kinase

β (DGKB).⁵¹Lastly, a virtual library of possible linkers was

docked into the SphK1 model and a class of heteroaromatic

compounds with six fewer rotatable bonds was generated and

synthesized. Biochemical evaluation led to the identiﬁcation of

the most potent inhibitors of SphK1 reported in the literature to

date.⁵²Oxazole 56, which has K_I= 47 nM at SphK1 and 180-fold

selectivity, and other amidine-based inhibitors described are

shown to signiﬁcantly reduce S1P concentrations in human

leukemia U937 cells at nanomolar concentrations.

’ RESULTS AND DISCUSSION

Tail Modifications. The tail region was defined to be every-

thing distal to the amidine beyond the amide bond (Scheme 2).

Three major modifications were made to the scaffold of com-

pound 2: aryl deletion, the substitution of terminal ethers, and

the substitution of terminal aromatics. The aryl deletion series

was synthesized in two steps from the commercially available

starting aliphatic amines and 1-cyanocyclopropanecarboxylic

acid. In the example shown in Scheme 3, tetradecylamine was

coupled using PyBOP to form the nitrile 3a and then trans-

formed under base catalyzed Pinner conditions⁵³to yield the

corresponding amidine 4a.

The ether tail derivatives were then examined, and terminal

steric bulk was built into the ether from the corresponding

alcohol. In the example synthesis shown in Scheme 4, benzyl

alcohol was coupled to 7-bromo-1-heptene using sodium hydride

in DMF to form ether 5a. The terminal oleﬁn was reduced to an

alkylborane in situ using 9-BBN and then introduced to Suzuki

conditions to be coupled with 1-bromo-4-nitrobenzene to form

the arylnitro 6a. On reduction to the aniline 7a with zinc dust and

amide coupling facilitated by PyBOP to form nitrile 8a, our

standard amidine formation led to the ﬁnal product 9a.

The non-ether aromatic tails were synthesized to compare the

solubility eﬀects of introducing an ether linkage in the middle of

the tail region. In the example synthesis shown in Scheme 5,

benzylmagnesium bromide was catalytically converted to its

organocuprate with cuprous chloride and coupled to 8-bromo-

1-octene to form alkene 10a. This oleﬁn was identical to that of

compound 5a, with the exception of the ether linkage being

substituted with a methylene, and was converted to its corre-

sponding ﬁnal product under similar chemical transformations.

The K_Ivalues of these tail derivatives were determined by a

[γ-³²P]ATP in vitro assay⁵²of SphK enzymatic activity and are

shown in Table 2. The most striking observation about the aryl

deletion series 4aꢀc was the lack of a potency response to

changes in tail length. Unlike the aryl-containing analogues

described in Figure 1, these saturated tails had a ﬂat SAR in the

low micromolar range but did maintain SphK1 selectivity in the

longer tailed 4b and 4c. It was hypothesized that these more

hydrophobic compounds had strong aﬃnities for the active site

but were so water insoluble that their active concentrations were

small because of aggregation. The more soluble ether tails

performed with a more consistent SAR, with the smaller terminal

phenyl-containing 9a being less active than the cyclohexyl 9c by

more than a log order (Table 2). The terminal cyclohexyl

Figure 1. Comparison between tail length and potency for the amide

orientations in compounds 1 and 2. Compound 1 derivatives are shown

in red. Compound 2 derivatives are shown in blue. SphK1 inhibition is

shown as solid lines. SphK2 inhibition is shown as dotted lines.

Over the past few years several SphK inhibitors have appeared

in the literature.¹A large portion of these are amino alcohol

sphingosine analogues that compete for the substrate binding

pocket;^39ꢀ44however, the ATP competitive SKI-II is one notable

exception.⁴⁵Indeed, sphingosine kinase inhibitors with micro-

molar K_Ivalues have been eﬀective in vivo in suppressing tumor

growth in xenograft models^39,41,46and inhibited inﬂammation

response in Crohn’s,⁴⁷inﬂammatory bowl,⁴⁸and sepsis⁴⁹disease

models. However, there is still a need for a library of potent SphK

inhibitors with a range of subtype selectivities that could eluci-

date the currently enigmatic diﬀerences between the SphKs in

cancer disease states.

Previous work has led to the generation of submicromolar dual

and selective SphK inhibitors 1 and 2, which were derivatives of

the initial hit compound (S)-N-(1-amino-1-iminopropan-2-yl)-

4-octylbenzamide hydrochloride (VPC94075) (Table 1).⁵⁰

These amidine-based lipids were selective for the SphKs; they

did not inhibit other lipid kinases, such as the diacylglycerol

kinases (DGKs), or protein kinases, such as protein kinase C

(PKC). They were excellent starting points for drug optimiza-

tion. The most interesting feature of the preliminary SAR was the

selectivity for SphK1 induced by the direction of the amide

functional group present in compounds 1 and 2. The amide-

controlled selectivity was dependent on tail length, with a

maximum eﬀect only observed in the longer tailed derivatives.

Potency and selectivity are aﬀected by tail length and amide

conﬁguration as described in Figure 1. Shorter tails (C8 and

C10) inhibit both SphK1 and SphK2 equally, but the maximum

potency tail length of C12 diﬀerentiates dual inhibition and

SphK1 selectivity based on amide direction before potencies

drop oﬀ at longer tail lengths.

These diﬀerences can be explained by the tail-binding region

of the substrate pocket of SphK1 being larger than that of SphK2,

which forces an altered binding position for the inhibitors and

causes a repulsive electrostatic interaction for the amide conﬁg-

uration in compound 2. To exploit this tail length and amide

derived selectivity, inhibitors with increased terminal steric bulk

and amide rigid analogues derived from proline were synthesized

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Scheme 2. Outline of Inhibitor Optimization

derivative 9c was synthesized to evaluate saturation compared to

the aromaticity of 9a, and the positive performance of 9c suggests

a preference for the larger and more hydrophobic terminal

cyclohexane. Adding further steric bulk in the adamantyl deriva-

tive 9e caused a loss of activity and selectivity, suggesting an

alternative binding conformation for such a large substituent.

Short and longer cyclohexyl-containing tails, 9b and 9d, respec-

tively, both performed more poorly than 9c, indicating that is was

the optimum length.

water solubility to CLogP = 3.61 versus CLogP = 4.00 for com-

pound 2.⁵⁴This added polar character allowed us to reconsider

the aryl deletion series, and compounds 19a and 19b were then

synthesized. Shown in Scheme 6 is the example synthesis of 19a.

Cyclohexylmethanol was coupled to 10-bromo-1-decene using

sodium hydride in DMF to form ether 15a. The terminal oleﬁn

was converted to the primary alcohol 16a under hydroboration/

oxidation conditions and then displaced to the primary azide 17a

through its mesylate. The azide 17a was reduced and ligated

using Staudinger conditions⁵⁵to form nitrile 18a, before being

converted to amidine 19a. Compound 19a proved to be both

Unfortunately, compound 9c did not yield the substantial

gains in potency or selectivity that were expected but did increase

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Scheme 3. Example Synthesis of an Aryl Deleted Tail Modiﬁcation

Scheme 4. Example Synthesis of an Ether Tail Modiﬁcation

Scheme 5. Example Synthesis of a Non-Ether Tail Modiﬁcation

more potent, with K_I= 110 nM, and 470-fold selective for SphK1

over SphK2. The reduction in terminal ring size to the cyclo-

pentyl 19b demonstrated that the steric bulk of the six-mem-

bered saturated ring of 19a was optimal for both potency and

selectivity (Table 2).

to 1-amino-1-cyclopropanecarbonitrile through its acid chloride.

Nitrile 22a was then converted to its amidine to form the desired

23a. The synthesis of the non-aryl inverted amide analogue

26 was relatively simple, starting with the Williamson ether

coupling of cyclohexylmethanol and 11-bromoundecenoic acid

(Scheme 8). The acid 24 was then coupled to 1-amino-1-

cyclopropanecarbonitrile with PyBOP to form nitrile 25 and

converted to the corresponding amidine 26.

The results from the amide inversion experiments demon-

strated that a cyclohexane at the tail terminus does itself increase

selectivity for SphK1, as shown in the diﬀerences in activity

between compounds 1 and 23a (Table 3). Again, substitution to

the smaller cyclopentane reduced activity and selectivity. It was

expected that a direct ether substitution in the tail of compound 1

would lead to reduced activity against both kinases equally

because of its increased solubility in water; however, compound

Having achieved the design of a compound that is 2 ¹/₂log

order selective for SphK1, our attention shifted to whether the

bulkier tail design had aided selectivity in an amide-dependent

manner. To test this relationship, the inverted amide derivatives

of compounds 9c and 19a were synthesized. The synthesis of the

aryl containing inverted amide is shown in Scheme 7. Starting

from the same terminal alkene used in the synthesis of 9c, the

reduction of 5c to its alkylborane and coupling under Suzuki

conditions to 4-bromobenzaldehyde gave the arylaldehyde 20a.

The aldehyde was then oxidized to benzoic acid 21a using

Pinnick oxidation conditions.⁵⁶The carboxylic acid was coupled

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Table 2. K_Ifor the Tail Modiﬁed Compounds

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

.

23c lost potency disproportionately leading to a slight degree of

SphK1 selectivity. The selectivity was due to the position of the

ether linkage along the tail, and compound 30 was synthesized

(Scheme SI-1 in Supporting Information) and evaluated to show

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Scheme 6. Example Synthesis of Ether Containing/Aryl Deletion Tail Modiﬁed Compounds

Scheme 7. Synthesis of 23a

Scheme 8. Synthesis of 26

no such change in selectivity compared to the saturated parent

compound 1.

selectivity. One explanation is that a saturated amide increases

potency and accentuates the eﬀect that an amide already has on

selectivity. On the other hand, a bulky substitution at the tail

terminus, such as a cyclohexane, increases potency and selectivity

regardless of amide orientation.

Head Group Modifications. An early examination of sub-

stitution R to the amidine showed that small substituents, such as

An important subtlety of the tail modiﬁcation data is that the

deletion of the aromatic ring present in 9c and replacement with

a three carbon saturated spacer as in 19a increased both potency

and selectivity (Table 2). However, the same conversion from

23a to 26 increased potency without such an obvious eﬀect on

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Table 3. K_Ifor Alternative Amide Conﬁgurations

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

Scheme 9. Synthesis of the R,R-Cyclobutyl Head Group Analogue 33

.

Scheme 10. Synthesis of the Proline-Based Rigid Analogue 36a

methyl and cyclopropyl, were tolerated well by the enzyme.⁵⁰It

was therefore desirable to test a bulkier cyclobutyl derivative;

however, a ring expansion to the cyclobutyl would affect the

angle of presentation of the amidine, possibly hindering its

function. More promising was a rigid analogue design that

restricted the dihedral angle between the position of the amide

and that of the amidine. Restricting a bond between such

functionally important groups should have an effect on selectivity

and potency. Derivatives of both enantiomers of proline pro-

vided a synthetically useful avenue to rigidity and would allow

freedom of rotation about the amidine while restricting rotation

of the amide.

The synthesis of the R,R-cyclobutyl analogue 33 began with

the conversion of cyclobutanone under Strecker conditions to

1-amino-1-cyclobutanecarbonitrile 31 (Scheme 9). Immediate

acylation with 4-dodecylbenzoyl chloride to form nitrile 32 and

conversion to its amidine gave compound 33. Next, the proline-

based rigid analogue syntheses began from the corresponding

asymmetric amino acid (Scheme 10). ^L-Proline was ﬁrst N-Boc

protected, before conversion of its carboxylic acid to the primary

amide and lastly dehydration of that amide to the nitrile in

compound 34a. The Boc group was then deprotected and

the free amine coupled using PyBOP to 4-dodecylbenzoic acid

to form compound 35a. The nitrile was then converted to its

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Table 4. K_Ifor Head Group Modiﬁcations

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

.

amidine, and the synthesis was repeated for D-proline to produce

both enantiomers.

The ether present in the tail increases its calculated water

solubility and in the case of 23c reduces activity versus its non-

ether counterpart 1. A synthesis was then undertaken to elim-

inate the ether from compound 38 to investigate the limit of such

solubility dependence. The synthesis of the non-ether 47 was

completed (Scheme SI-2), and it was determined that its lower

water solubility caused a decrease in activity (Table 4). The loss

of activity for 47 and other compounds with high ClogP suggests

an ideal ClogP of around 4.2.

In Silico Linker Screening. Crystal structures of kinases that

bear close sequence homology to the ATP binding domain of the

SphKs have been solved for YegS,^57,58a bacterial lipid kinase,

phosphofructokinase (PFK),^59,60and DGKB.⁵¹Of these struc-

tures, DGKB has the greatest overall sequence identity of 20% to

SphK1. Cases of such low sequence identity are often referred to

as “twilight zone” cases,⁶¹and a 28 amino acid sequence that

defines the substrate binding pocket of SphK1 has no meaningful

sequence homology. Modelers tread lightly in such situations,

and any conclusions drawn should be supported by experimental

data. However, the sequence homology between the two kinases

suggests that SphK1 shares the basic quaternary structure of a

β-sandwich in DGKB, connected to the ATP binding domain

through a hinge.

Table 4 shows the biological evaluation of the head group

analogues. As suspected, the ring expansion from cyclopropane

to the cyclobutane present in 33 worsened activity equally

against both SphKs. The proline analogues 36a,b yielded selec-

tivity as expected, with the (S)-conﬁguration derived from

L-proline being 24-fold more selective for SphK1, while the

(R)-enantiomer was slightly SphK2 selective with less potency.

With compound 36a being more potent and selective for

SphK1 than compound 1, a synthesis combining our best tail

derivatives with an (S)-proline head group was undertaken

(Scheme 11). The aryl 38 and non-aryl 40 were synthesized

and evaluated to have K_Iof 75 and 130 nM, respectively

(Table 4). In previous series there was an increase in activity

for the non-aryl over the arylamide substitution (Tables 2 and 3).

However, that relationship was for mononitrogen substitution on

the amide bonds, while the proline derivatives are dinitrogen

substituted. For the proline arylamides, A^1,3strain prohibits bond

rotation about the carbonyl carbonꢀaryl bond, eﬀectively rigi-

difying two bonds compared with compound 23a. The saturated

40, which is monosubstituted R to the carbonyl, has the ability to

freely rotate and has only one rigidiﬁed bond compared with

compound 26. The potency of the proline analogues is therefore

dependent on a substitution R to the amide carbonyl that inhibits

bond rotation, which prepays the cost of freezing that bond prior

to reaching the enzyme active site.

A homology model of SphK1 was generated (see Supporting

Information for details pertaining to model generation and

inhibitor docking) from the solved crystal structure of DGKB⁵¹

(Figure 2A). The current library of amidine inhibitors was

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Scheme 11. Combination of Tail and Head Group Modiﬁcations

Figure 2. SphK homology model: (A) crystal structure DGKB (R-helices = teal, β-sheets = lavender, ATP = yellow stick model, and Mg^2þ= white

sphere); (B) SphK1 homologue; (C) theoretical binding orientation of compound 38 with ATP and protein backbone; (D) 2D representation of the

amidine in compound 38 chelating ATP.

docked into the SphK1 model (Figure 2B) and illuminated an

interesting hypothesis of how the amidine may interact with the

enzyme. The model suggests that the amidine interacts directly

with ATP through a bidentate chelation of its γ phosphate

(Figure 2C and Figure 2D). This supports a mechanism of

inhibition where SphK ﬁrst binds ATP and the inhibitor, and the

amidine acts to stabilize the [SphK ATP I ] complex. Using the

test set of known amidine-based inhibitors enabled the virtual

screening of theoretical amidine inhibitors and a prediction of

their enzymatic activity.

Long unrestricted alkyl chains have a large number of rotatable

bonds, which add a large entropic cost when forced to lock into a

single binding conformation. Our most potent compounds have

between 11 and 15 rotatable bonds; thus, it was desirable to

reduce these large degrees a freedom by incorporating linker

regions that comprise as many ring structures as possible. The

SphK1 model suggests a tail binding region that mostly com-

prises hydrophobic surface area, indicating that this region of the

pocket acts as a hydrocarbon ruler designed for sphingosine

recognition. Therefore, without much possibility of polar inter-

action the ideal tail would be one that maximizes the energy

associated with ligand and pocket desolvation. Assuming the

binding positions of the amidine head group and the cyclohexyl

tail fragments were accurate, several hundred possible linkers

were created in silico, docked into the SphK1 homology model,

and scored (see Supporting Information). These potential linker

regions consisted of substituted benzenes, heteroaromatics, satu-

rated rings, fused rings, and alkyl spacers in varying order, and

scaﬀolds were chosen for both their predicted potencies and ease

of synthesis. Figure 3 shows the general scaﬀold picked as a proof

of principle for the linker region generation. It is a proline-based

rigid analogue series that includes a ﬁve-membered heterocycle

with an arylꢀaryl bond to another benzene that is meta-substi-

tuted by a two-carbon spacer to the terminal cyclohexane. The

presence of a centralized heterocycle was ideal for solubility

manipulation, and the synthesis of the X/Z imidazole, oxazole,

and thiazole was undertaken to demonstrate a solubility/activity

relationship. Figure 4 illustrates the linker generation process

3

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Figure 3. Generic scaﬀold for the linker design proof of principle.

where the docking conformation of compound 38 (Figure 4A)

was fragmented into an arylamide head group and a cyclohexyl tail

terminus (Figure 4B) and the in silico linker screening procedure

led to a theoretical aromatic tail derivative (Figure 4C).

The synthesis of imidazole 53 began with the hydroboration of

vinylcyclohexane and subsequent Suzuki coupling with 3-bro-

moacetophenone to form ketone 48 (Scheme 12). The ketone

was then R-brominated with molecular bromine and displaced by

the cesium salt of mono-tert-butyl protected terephthalic acid to

yield ester 50. Compound 50 was then cyclized in reﬂuxing

xylenes with ammonium acetate to produce imidazole 51, which

was deprotected and coupled to form nitrile 52. Standard Pinner

conditions then yielded the desired imidazole containing ami-

dine 53. The synthesis of oxazole 56 diverges from that of the

imidazole at compound 50, which is cyclized in AcOH with

ammonium acetate to yield the acid deprotected oxazole 54 in

one step (Scheme 13). Amide followed by amidine formation

then produced the oxazole containing amidine 56. Synthesis of

the thiazole required the conversion of the mono-tert-butyl

protected terephthalic acid to its terminal amide using isobutyl

chloroformate and ammonia in methanol (Scheme 14). This

terminal amide could then be transformed into the thioamide 57

using Lawesson’s reagent. Thioamide 57 was smoothly coupled

then cyclized with the R-bromoketone 49 to yield the thioazole

58. tert-Butyl deprotection, amide formation, and then amidine

synthesis produced the desired thioazole containing amidine 60.

The SphK1 model-predicted and in vitro-determined K_I

values for the heterocycle series are listed in Table 5. All three

heterocycles were predicted to geometrically ﬁt in the substrate

pocket, but the SphK1 model predicted a “Goldilocks” eﬀect

based on solubility, where the oxazole 56 with a ClogP of 4.24

should have the lowest K_Iof 30 nM. The imidazole 53 and the

thiazole 60 were predicted to have lesser potencies due to being

too polar and hydrophobic, respectively. On biological evalua-

tion the model performed quite well, yielding the correct order of

potency and predicting the actual K_I(47 nM) of the oxazole 56

within the 95% conﬁdence limits. Indeed, the imidazole was the

only compound of the three that had an experimentally deter-

mined K_Ioutside the 95% conﬁdence limit, and this is probably

due to the ratio of protonated versus neutral states. The pK_aof

the protonated imidazole ring is predicted to be around 7 in

water, and if one assumes that the charged species has K_I> 10

μM, then that ratio would proportionally reduce the activity of

compound 53. Comparing ClogP to reverse-phase HLPC reten-

tion time, which is a standard measure for comparing relative

water solubilities, validates this reasoning (Figure SI-3). The

retention times of the presented library of amidine containing

inhibitors correlates well with ClogP (R²= 0.71), and compound

53 is an outlier of this trend (see Supporting Information).

In Vitro Evaluation of Inhibitors in U937 Cells. To evaluate

how well these amidine-based inhibitors penetrate and reduce

endogenous S1P levels in living cells, U937 cells were pretreated

with compounds 1, 19a, 38, and 56 for 2 h (Table 6 and

Figure 4. Progression of linker design: (A) theoretical binding orienta-

tion of compound 38 chelating ATP (both drawn as stick models); (B)

deletion of aliphatic linker between the aryl amide and cyclohexyl groups

in 38; (C) generation of a new linker (56) with a reduced number of

rotatable bonds.

Figure 5). U937 cells are a human monoblastic leukemia cell

line, whose S1P levels have been reduced by micromolar con-

centrations of the known sphingosine kinase inhibitor dimethyl

sphingosine (DMS).^40,42The amidine-based inhibitors indeed

showed inhibition at concentrations near the K_I; all showed

significant S1P reduction at 100 nM. At 10 nM, lower than the K_I

of all the inhibitors, S1P reduction was still observed for

compounds 19a and 38. In other experiments (not shown), it

was determined that the decreased accumulation of S1P in U937

cells was the result of blockade of synthesis rather than increased

decay or export of S1P.

To compare these amidine based inhibitors to other known

sphingosine kinase inhibitors, compounds 9ab⁴⁴and SKI-II⁴⁵

were also examined in living U937 cells (Table 6 and Figure 5).

Compound9ab didnot cause S1Preductionat100 nM, whichwas

expected given its K_Ibeing 1.4 μM for SphK1 and 31 μM for

SphK2.⁵²However, at 1 μM, nearer to the K_Iof compound 9ab at

SphK1, a 40% reduction of S1P is observed. Comparing the K_I

values for 9ab versus those of the SphK1 selective compound 19a,

110 nM for SphK1 and 26 μM for SphK2 (Table 6), suggests that

the observed reduction in S1P levels for 19a is accomplished

through the inhibition of SphK1. SKI-II also ﬁts this trend with a

higher SphK1 K_Iof 12 μM,⁵²and no signiﬁcant S1P reduction was

observed until 10 μM concentration was applied.

A notable outlier in the series is the performance of oxazole 56

on whole cells. With the lowest K_Iin the series (47 nM for

SphK1), 56 should inhibit S1P production most successfully.

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Scheme 12. Synthesis of Imidazole 53

Scheme 13. Synthesis of Oxazole 56

Compound 56 does reduce S1P levels signiﬁcantly, along with

the other amidine inhibitors, at 100 nM, but fails to outperform

compounds 19a and 38 at 10 nM despite having the lowest K_I.

This recombinant enzyme versus living cell deviation in activity is

subtle and suggests diﬀerences in uptake or eﬄux. Interestingly,

S1P reduction in U937 cells by these amidine-based inhibitors

did not cause caspase-mediated apoptosis as previous reports

have demonstrated with other SphK inhibitors (data not

shown).^40,42However, a more thorough investigation beyond

the characterization of these inhibitors is needed to better

understand these diﬀerences in cytotoxicity.

rheostat illuminates the practicality of an anticancer strategy that

targets their activity.¹Described herein is the optimization and

SAR of amidine-based SphK1 subtype-selective inhibitors. The

library of inhibitors evaluated were used as a test set in the

generation of a SphK1 homology model from the crystal

structure of DGKB and used for the in silico design and synthesis

of nanomolar SphK1 inhibitors. These inhibitors were found to

signiﬁcantly lower endogenous S1P levels in human leukemia

U937 cells at 10 and 100 nM.

’ EXPERIMENTAL SECTION

Sphingosine Kinase Assay. Human SphK1 and mouse SphK2

cDNAs were used to generate mutant baculoviruses that encoded these

proteins. Infection of Sf9 insect cells with the viruses for 72 h resulted in

>1000-fold increases in SphK activity in 10000g supernatant fluid from

’ CONCLUSION

The role of the SphKs as the sole producers of S1P, a lipid

promoter for tumorigenesis and angiogenesis, in the sphingosine

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Scheme 14. Synthesis of Thiazole 60

Table 5. K_Ifor the Heterocycle Linker Series

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

.

homogenized cell pellets. The enzyme assay conditions were exactly as

described,⁵²except infected Sf9 cell extract containing 2ꢀ3 μg of protein

was used as a source of enzyme.

up in 2 mL of 3:1 methanol/chloroform mixture and transferred to a

capped glass vial. To this suspension was added 10 μL of internal

standard solution containing 1 μM C17 S1P (purchased from Avanti

Polar Lipids). The mixture was homogenized via sonication for 10 min

and immediately incubated at 48 °C for 16 h. After this time, the mixture

was cooled to ambient temperature and 200 μL of 1 M KOH in

methanol was added to the suspension. The samples were again

sonicated and incubated at 37 °C for an additional 2 h. After this time,

the samples were neutralized through the addition of 30 μL of glacial

acetic acid and transferred to 2 mL microcentrifuge tubes. Samples were

then centrifuge at 10000g for 10 min at 4 °C. The supernatant fluid was

collected in a separate glass vial, and the pellets were discarded. The

resulting solution was evaporated (to a solid) with a stream of nitrogen.

U937 Cell Culture Assay. U937 cells were grown according to a

previously described literature procedure.⁴⁰In general, cells were grown

in RPMI 1640 medium enriched with ^L-glutamine, 10% penicillin and

streptomycin, and 10% fetal bovine serum (FBS). Twenty-four hours

before dosing with SphK inhibitors, the medium was replaced with

medium containing 2% FBS. All cell cultures were grown at a stable

temperature of 37 °C, and the SphK inhibitors were dosed for 2 h.

S1P Extraction and LCMS Quantification. Extraction proto-

cols and LCMS procedures were adapted from a previously reported

study.⁶²Samples of pelleted cells (approximately 4 million) were taken

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Table 6. K_Ifor Inhibitors Used for S1P Inhibition in Living U937 Cells

^aK_I= [I]/(K⁰_M/K_Mꢀ 1). K_Mof sphingosine at SphK1 is 10 μM. K_Mof sphingosine at SphK2 is 5 μM. ^bSelectivity = (K_I/K_M)^SphK2/(K_I/K_M)^SphK1

.

(Supelco Discovery C18 column; 50 mm, 2.1 mm (length, i.d.); 5 μm

bead size). Mobile phase A consisted of water/methanol/formic acid

(79:20:1), and mobile phase B consisted of methanol/formic acid

(99:1). The run started with 100% A for 0.5 min. Solvent B was then

increased linearly for 5.1 min to 100% of the total solvent composition

and held at 100% for an additional 4.3 min. The column was ﬁnally re-

equilibrated to 100% for 0.1 min and held for an additional 1 min. The

following analytes (and fragmentation patterns) were monitored simul-

taneously for identiﬁcation. C17S1P (366.4, 250.4); S1P (380.4, 264.4).

General Synthetic Materials and Methods. All nonaqueous

reactions were carried out in oven-dried or flame-dried glassware under

an argon or nitrogen atmosphere with dry solvents and magnetic stirring,

unless otherwise stated. The argon and nitrogen were dried by passing

through a tube of Drierite. Anhydrous diethyl ether (Et₂O), chloroform

(CHCl₃), dimethylsulfoxide (DMSO), toluene (PhMe), dichloro-

methane (CH₂Cl₂), methanol (MeOH), ethanol (EtOH), tetrahydro-

furan (THF), and N,N-dimethylformamide (DMF) were purchased

from Aldrich or VMR Chemicals and used as received. THF was dried

over activated molecular sieves (4 Å) prior to use. All other reagents

were purchased from Acros Chemicals and Aldrich Chemicals. Except as

indicated otherwise, reactions were monitored by thin layer chromatog-

raphy (TLC) using 0.25 mm Whatman precoated silica gel plates. Flash

chromatography was performed with the indicated solvents and Dy-

namic Adsorbents silica gel (particle size 0.023ꢀ0.040 mm). Proton

(¹H) and carbon (¹³C) NMR spectra were recorded on a Varian

UnityInova 500/51 or Varian UnityInova 300/54 at 300 K unless

otherwise noted. Chemical shifts are reported in ppm (δ) values relative

to the solvent as follows: CDCl₃(δ 7.24 for proton and δ 77.0 for carbon

NMR), DMSO-d₆(δ 2.50 for proton and δ 39.5 for carbon NMR),

Figure 5. S1P concentrations in human leukemia U937 cells dosed with

SphK inhibitors. Error bars indicate 95% conﬁdence limits. Concentra-

tions are indicated by shading and patterns.

Immediately prior to LCMS analysis, the solid material was taken up in

300 μL of methanol and centrifuged at 12000g for 12 min at 4 °C. An

autosampler vial was loaded with 150 μL of the resulting supernatant for

LCMS analysis.

S1P analysis from cellular extracts was performed on an Applied

Biosystems 4000 QTrap LC/MS/MS instrument. Chromatographic

resolution of analytes was achieved with a Shimadzu LC-20AD system.

A binary solvent gradient with a ﬂow rate of 1 mL/min was used to

separate sphingolipid analytes by reverse phase chromatography

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CD₃OD (δ 3.31 for proton and δ 47.6 for carbon NMR). All high-

resolution mass spectrometry was carried out by the Mass Spectrometry

Laboratory in the School of Chemical Sciences at the University of

Illinois Urbana-Champagne (Urbana, IL).

TLC stains were as follows. For KMnO₄stain, the mixture was 3 g of

KMnO₄and 20 g of K₂CO₃in 300 mL water and 5 mL of 5% NaOH. For

Seebach’s dip, to a solution of 25 g of phosphomolybdic acid and 7.5 g

cerium(IV) sulfate in 479 mL water was added 25 mL of concentrated

sulfuric acid dropwise. For the ninhydrin stain, the mixture was 1.5 g of

ninhydrin in 5 mL of AcOH and 500 mL of 95% EtOH. All stains

required TLC development on a hot plate set to 80 °C.

General Procedure D: Suzuki Coupling. To a solution of

alkene (1.0 equiv) in THF (0.2 M) at room temperature was added a

0.5 M solution of 9-BBN in THF (2.0 equiv). The mixture was allowed

to stir until consumption of the alkene was evident by TLC analysis (4 h

unless otherwise stated). The mixture was then treated with 3 M K₃PO_4(aq)

,

and diluted with DMF (0.2 M relative to the starting alkene). The aryl

bromide and PdCl₂(dppf) were then sequentially added, and the mixture

was allowed to stir for 16 h. The mixture was diluted with EtOAc (200 ꢁ

the volume of DMF) and washed 3ꢁ with neat water (100 ꢁ the volume of

DMF). The organic layer was then dried with Na₂SO₄, evaporated to a dark

red oil, and immediately purified by flash chromatography.

General Procedure E: Arylnitro Reduction with Zinc Dust.

To a solution of an arylnitro (1.0 equiv) in AcOH (0.1 M) at room

temperature was added zinc dust (10 equiv) in one portion, and the

mixture was allowed to stir for 16 h. The mixture was then diluted with

EtOAc and filtered through Celite. The filtrate was evaporated to

dryness, coevaporated with PhMe to remove residual AcOH, and

immediately purified by flash chromatography.

General Procedure F: Copper Mediated Alkane Synthesis.

To a suspension of cuprous chloride (0.05 equiv) in Et₂O (1.0 M relative

to the alkyl halide) at ꢀ78 °C was added a 2.0 M solution of a Gignard

reagent (2 equiv) in THF followed by the addition of an alkyl halide (Br

or I) (1.0 equiv). The mixture was allowed to warm to room temperature

and held at that temperature until the starting alkene was judged

consumed by TLC analysis (4 h unless otherwise stated). The mixture

was then cooled to 0 °C and quenched with 1 N HCl (50 ꢁ the volume

of Et₂O) and extracted into EtOAc (50 ꢁ the volume of Et₂O). The

organic layer was then dried with Na₂SO₄, evaporated to a dark green oil,

and immediately purified by flash chromatography.

General Procedure G: Hydroboration/Oxidation. To a solu-

tion of alkene (1.0 equiv) in THF (1.0 M) at room temperature was

added a 0.5 M solution of 9-BBN in THF (2.0 equiv), and the mixture

was allowed to stir until consumption of the alkene was evident by TLC

analysis (4 h unless otherwise stated). The mixture was then cooled to

0 °C and then diluted with EtOH (10 equiv), 3 M NaOH_(aq)(1.0 equiv)

and slowly treated with 30% H₂O₂in water (1.4 equiv) sequentially. The

mixture was allowed to warm to room temperature and then was stirred

for 30 min before being quenched with 1 N HCl (equal to the total

volume of THF) and extracted into EtOAc (10 ꢁ the total volume of

THF). The organic layer was then dried with Na₂SO₄, evaporated to a

colorless oil, and immediately purified by flash chromatography.

General Procedure H: Alkyl Mesylate Formation. To a

solution of an alcohol (1.0 equiv) in CH₂Cl₂(0.3 M) at 0 °C was

added TEA (2.0 equiv) and then methanesulfonyl chloride (1.1 equiv).

The mixture was allowed to warm to room temperature and then was

heated to reflux for 30 min. The mixture was then cooled to room

temperature, quenched with saturated NaHCO₃(10 ꢁ the volume of

CH₂Cl₂), and extracted into CHCl₃(200 ꢁ the volume of CH₂Cl₂).

The organic layer was then dried with Na₂SO₄, evaporated to a yellow

oil, and immediately purified by flash chromatography.

Liquid Chromatography and Mass Spectrometry for Eva-

luation of Chemical Purity. All compounds submitted for biological

evaluation were determined to be >95% pure by LCMS evaluation

performed by the Mass Spectrometry Laboratory in the School of

Chemical Sciences at the University of Illinois Urbana-Champagne

(Urbana, IL). High performance liquid chromatographyꢀmass spectro-

metry (LCMS) was carried out using an Agilent 2.1 mm ꢁ 50 mm C-18

column and a Micromass Q-tof Ultima mass spectrometer. Mobile phase

A consisted of HPLC grade H₂O and 0.01% TFA. Mobile phase B

consisted of MeCN and 0.01% TFA. LCMS identification and purity

utilized a binary gradient starting with 90% A and 10% B and linearly

increasing to 100% B over the course of 6 min, followed by an isocratic

flow of 100% B for an additional 3 min. A flow rate of 0.5 mL/min was

maintained throughout the HPLC method. The purity of all products

was determined by integration of the total ion count (TIC) spectra and

integration of the ultraviolet (UV) spectra at 214 nm. Retention times

are abbreviated as t_R; mass to charge ratios are abbreviated as m/z.

General Procedure A: Conversion of Nitriles to Amidines.

To a solution of a nitrile (1.0 equiv) in MeOH (0.10 M) was added a

0.5 M solution of sodium methoxide in MeOH (0.50 equiv) at room

temperature. The mixture was then heated to 50 °C for 24 h. The

intermediate imidate was detectable by TLC; however, since it is in

equilibrium with the nitrile, full conversion does not occur. Ammonium

chloride (2.0 equiv) was then added in one portion at that temperature

and allowed to react until the imidate was completely consumed by TLC

analysis. The mixture was then cooled to room temperature and

evacuated to dryness to yield a crude solid. The solid was reconstituted

with CHCl₃and filtered through a fine glass fritted funnel in order to

remove excess ammonium chloride, and the filtrate was again evacuated

to dryness. The material was then recrystallized in Et₂O to yield the pure

amidine hydrochloride salt. The yields varied greatly depending upon

substrate because amidine formation is dependent upon the equilibrium

ratio between nitrile and imidate established under the sodium methox-

ide conditions.

General Procedure B: PyBOP Mediated Couplings of

Amines and Anilines to Carboxylic Acids. To a suspension

of an amine or aniline (1.0 equiv), carboxylic acid (1.0 equiv), and

PyBOP (1.1 equiv) in CH₂Cl₂at room temperature was added DIEA

(4.0 equiv). The mixture was allowed to stir for 4 h unless otherwise

stated. The mixture was then evaporated to dryness and immediately

purified by flash chromatography.

General Procedure C: Williamson Ether Synthesis. To a

solution of an alcohol (2 equiv) in DMF (0.3 M) at 0 °C was added 60%

sodium hydride dispersed in mineral oil (2.0 equiv) at 0 °C. Then the

mixture was allowed to warm to room temperature and then allowed to

react for 45 min. The alkyl bromide was then added in one portion, and

the mixture was stirred for 12 h. The reaction was quenched with

saturated NaHCO₃(100 ꢁ the volume of DMF) and extracted into

EtOAc (100 ꢁ the volume of DMF). The organic layer was washed

3ꢁ with neat water (100 ꢁ the volume of DMF), dried with Na₂

SO₄, evaporated to a yellow oil, and immediately purified by flash

chromatography.

General Procedure I: Alkylazide Formation. To a solution of

an alkyl mesylate (1.0 equiv) in DMF (0.3 M) at room temperature was

added sodium azide (1.1 equiv), and the mixture was heated to reflux for

12 h. The mixture was then cooled to room temperature, diluted with

EtOAc (100 ꢁ the volume of DMF), and washed 3ꢁ with saturated

NaHCO₃(100 ꢁ the volume of DMF). The organic layer was then dried

with Na₂SO₄, evaporated to a yellow oil, and immediately purified by

flash chromatography.

General Procedure J: Staudinger Reduction and Ligation.

To a solution of an alkylazide (1.0 equiv) and triphenylphosphine

(1.1 equiv) in PhMe (0.3 M) was added neat water (2.0 equiv), and the

mixture was heated to reflux for 30 min. The mixture was then cooled to

room temperature and treated with a solution of 1-cyano-1-cyclopane-

carboxylic acid (1.5 equiv), PyBOP (1.5 equiv), and TEA (2.0 equiv) in

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CH₂Cl₂(1.5 M relative to the carboxylic acid). The mixture was allowed

to stir for 4 h, then evacuated to a dark red oil, and purified by flash

chromatography.

LCMS: t_R= 4.72; m/z = 324.3. HRMS m/z calcd for C₁₉H₃₈N₃O

(M þ H), 324.3015; found, 324.3010.

1-Carbamimidoyl-N-hexadecylcyclopropanecarboxamide

Hydrochloride (4b). General procedure A was used to convert 3b

(320 mg, 1.00 mmol) to the title compound. Yield, 46%. White solid. ¹H

NMR (300 MHz, DMSO) δ 9.16 (s, 2H), 8.96 (s, 2H), 7.82 (s, 1H),

3.01 (d, J = 5.1 Hz, 2H), 1.51ꢀ0.97 (m, 30H), 0.83 (s, 3H). ¹³C NMR

(75 MHz, DMSO) δ 168.54, 167.92, 40.04, 31.98, 29.74, 29.56, 29.49,

29.40, 27.05, 22.79, 14.86, 14.65. LCMS: t_R= 5.22; m/z = 352.3. HRMS

m/z calcd for C₂₁H₄₂N₃O (M þ H), 352.3328; found, 352.3329.

1-Carbamimidoyl-N-octadecylcyclopropanecarboxamide

Hydrochloride (4c). General procedure A was used to convert 3c

(362 mg, 1.00 mmol) to the title compound. Yield, 32%. White solid. ¹H

NMR (300 MHz, DMSO) δ 9.07 (s, 2H), 8.95 (s, 2H), 7.89 (s, 1H),

3.05 (d, J = 5.1 Hz, 2H), 1.58ꢀ0.95 (m, 32H), 0.83 (s, 3H). ¹³C NMR

(75 MHz, DMSO) δ 168.64, 167.95, 40.09, 32.01, 29.78, 29.59, 29.54,

29.43, 27.12, 22.80, 14.92, 14.70. LCMS: t_R= 6.22; m/z = 380.4. HRMS

m/z calcd for C₂₃H₄₆N₃O (M þ H), 380.3641; found, 380.3633.

((Hept-6-en-1-yloxy)methyl)benzene (5a). General proce-

dure C was used to couple benzyl alcohol and 7-bromohept-1-ene

(1.00 mL, 6.52 mmol) to yield the title compound. Yield, 87%. Yellow

General Procedure K: Pinnick Oxidation. To a solution of an

aldehyde (1.0 equiv), NaH₂PO₄(8.0 equiv), and 2-methyl-2-butene

t

(10 equiv) in THF, water, and BuOH (4:4:1) (0.04 M) at room

temperature was added sodium chlorite (4 equiv), and the mixture was

allowed to stir for 1 h. The mixture was diluted with EtOAc (10 ꢁ the

volume of the reaction’s mixture of solvents) and washed 3ꢁ with 1 N

HCl (5 ꢁ the volume of the reaction’s mixture of solvents). The organic

layer was then dried with Na₂SO₄and evaportated to a white solid. No

further purification was necessary.

General Procedure L: Acid Chloride Formation. To a solu-

tion of a carboxylic acid (1.0 equiv) and DMF (0.05 equiv) in CH₂Cl₂

(0.1 M) at 0 °C was added oxalyl chloride (2.0 equiv) dropwise, and the

mixture was allowed to warm to room temperature. The mixture pro-

gresses to a yellow green color, and after 3 h the mixture was evaporated to

dryness and then immediately purified by flash chromatography.

General Procedure M: Acid Chloride and Amine Coupling.

To a solution of an acid chloride (1.0 equiv) in CH₂Cl₂(0.3 M) at room

temperature was added DIEA (4.0 equiv) followed by an amine HCl

salt (1.5 equiv), and the mixture was stirred for 12 h. The mixture

was then evaporated to dryness and immediately purified by flash

chromatography.

1

4

oil. R_f= 0.88 (10% EtOAc in hexanes, KMnO ). H NMR (300 MHz,

CDCl₃) δ 7.57ꢀ7.19 (m, 5H), 5.89 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H),

5.22ꢀ4.89 (m, 2H), 4.57 (s, 2H), 3.54 (t, J = 6.6 Hz, 2H), 2.14 (m, 2H),

1.82ꢀ1.61 (m, 2H), 1.59ꢀ1.40 (m, 4H). ¹³C NMR (75 MHz, CDCl₃)

δ 139.16, 139.01, 128.62, 127.86, 127.73, 114.65, 73.16, 70.66, 34.08,

29.97, 29.08, 26.04.

General Procedure N: Deprotection of N-Boc and O-^tBu

Ester Protecting Groups. To a solution of either a N-Boc or O-^tBu

protecting group (1.0 equiv) in CH₂Cl₂(0.2 M) at room temperature

was added TFA (0.2 M), and the mixture was reacted until judged

complete by TLC analysis (30 min unless otherwise stated). The

mixture was then evaporated to dryness and taken on crude.

1-Cyano-N-tetradecylcyclopropanecarboxamide (3a).

General procedure B was used to couple tetradecan-1-amine (213 mg,

1.00 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield the title

compound. Yield, 99%. White solid. R_f= 0.50 (20% EtOAc in hexanes,

KMnO₄). ¹H NMR (300 MHz, CDCl₃) δ 6.41 (s, 1H), 3.27 (dt, J = 6.5,

5.3 Hz, 2H), 1.65 (dd, J = 8.1, 4.4 Hz, 2H), 1.52 (t, J = 6.9 Hz, 2H), 1.45

(dd, J = 8.0, 4.4 Hz, 2H), 1.36ꢀ1.11 (m, 22H), 0.86 (t, J = 6.7 Hz, 3H).

¹³C NMR (75 MHz, CDCl₃) δ 165.22, 120.54, 40.89, 32.14, 29.87,

29.79, 29.71, 29.58, 29.45, 27.01, 22.91, 17.65, 14.34, 13.67.

1-Cyano-N-hexadecylcyclopropanecarboxamide (3b). Gen-

eral procedure B was used to couple hexadecan-1-amine (241 mg,

1.00 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield the title

compound. Yield, 99%. White solid. R_f= 0.52 (20% EtOAc in hexanes,

KMnO₄). ¹H NMR (500 MHz, CDCl₃) δ 6.37 (s, 1H), 3.28 (dt, J = 6.6,

5.3 Hz, 2H), 1.66 (dd, J = 8.1, 4.3 Hz, 2H), 1.58ꢀ1.49 (m, 2H), 1.47 (dd,

J = 8.1, 4.3 Hz, 2H), 1.39ꢀ0.99 (m, 24H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 164.99, 120.33, 40.68, 31.91, 29.67, 29.56,

29.48, 29.38, 29.22, 26.78, 22.68, 17.43, 14.12, 13.55.

1-Cyano-N-octadecylcyclopropanecarboxamide (3c). Gen-

eral procedure B was used to couple octadecan-1-amine (270 mg,

1.00 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield the title

compound. Yield, 99%. White solid. R_f= 0.55 (20% EtOAc in hexanes,

KMnO₄). ¹H NMR (500 MHz, CDCl₃) δ 6.44 (s, 1H), 3.26 (dt, J = 6.6,

5.3 Hz, 2H), 1.65 (dd, J = 8.1, 4.3 Hz, 2H), 1.52 (dt, J = 13.7, 6.9 Hz, 2H),

1.45 (dd, J = 8.1, 4.3 Hz, 2H), 1.26 (m, 26H), 0.86 (t, J = 6.9 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 164.96, 120.28, 40.64, 31.91, 29.68,

29.56, 29.48, 29.36, 29.22, 26.78, 22.67, 17.40, 14.11, 13.42.

((Hex-5-en-1-yloxy)methyl)cyclohexane (5b). General pro-

cedure C was used to couple cyclohexylmethanol and 6-bromohex-1-ene

(1.00 mL, 7.48 mmol) to yield the title compound. Yield, 70%. Clear and

colorless oil. R_f= 0.39 (3% EtOAc in hexanes, KMnO ). ¹H NMR

4

(300 MHz, CDCl₃) δ 5.76 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.10ꢀ4.58

(m, 2H), 3.35 (t, J = 6.4, 2H), 3.15 (d, J = 6.5 Hz, 2H), 2.23ꢀ0.80 (m, 2H),

1.87ꢀ1.05 (m, 13H), 0.87 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ

138.87, 114.59, 77.03, 71.03, 38.28, 33.80, 30.36, 29.42, 26.87, 26.10, 25.73.

((Hept-6-en-1-yloxy)methyl)cyclohexane (5c). General pro-

cedure C was used to couple cyclohexylmethanol and 7-bromohept-1-

ene (1.00 mL, 6.52 mmol) to yield the title compound. Yield, 70%. Clear

1

4

and colorless oil. R_f= 0.73 (5% EtOAc in hexanes, KMnO ). H NMR

(300 MHz, CDCl₃) δ 5.75 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.07ꢀ4.70

(m, 2H), 3.32 (t, J = 6.6 Hz, 2H), 3.14 (d, J = 6.6 Hz, 2H), 2.15ꢀ1.90

(m, 2H), 1.84ꢀ0.98 (m, 15H), 0.98ꢀ0.65 (m, 2H). ¹³C NMR

(75 MHz, CDCl₃) δ 139.04, 114.42, 77.03, 71.18, 38.27, 33.96, 30.37,

29.80, 28.98, 26.88, 26.10, 25.91.

((Oct-7-en-1-yloxy)methyl)cyclohexane (5d). General proce-

dure C was used to couple cyclohexylmethanol and 8-bromooct-1-ene

(1.00 mL, 5.98 mmol) to yield the title compound. Yield, 77%. Clear and

1

4

colorless oil. R_f= 0.43 (3% EtOAc in hexanes, KMnO ). H NMR

(300 MHz, CDCl₃) δ 5.77 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.13ꢀ4.71

(m, 2H), 3.35 (t, J = 6.6 Hz, 5H), 3.17 (d, J = 6.5 Hz, 2H), 2.12ꢀ1.91 (m,

2H), 1.86ꢀ1.01 (m, 17H), 0.98ꢀ0.74 (m, 2H). ¹³C NMR (75 MHz,

CDCl₃) δ 139.18, 114.36, 77.03, 71.26, 38.29, 33.96, 30.39, 29.92, 29.19,

29.09, 26.89, 26.27, 26.11.

1-((Hept-6-en-1-yloxy)methyl)adamantane (5e). General

procedure C was used to couple 1-adamantane-methanol and 7-bromo-

hept-1-ene (1.00 mL, 6.52 mmol) to yield the title compound. Yield,

4

85%. Clear and colorless oil. R_f= 0.54 (5% EtOAc in hexanes, KMnO ).

¹H NMR (300 MHz, CDCl₃) δ 5.79 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H),

5.09ꢀ4.77 (m, 2H), 3.36 (t, J = 6.5 Hz, 2H), 2.94 (s, 2H), 2.12ꢀ1.86 (m,

5H), 1.79ꢀ0.99 (m, 18H). ¹³C NMR (75 MHz, CDCl₃) δ 139.14,

114.44, 82.10, 71.78, 39.97, 37.49, 34.00, 29.68, 29.00, 28.54, 25.90.

((Hept-6-en-1-yloxy)methyl)cyclopentane (5f). General pro-

cedure C was used to couple cyclopentylmethanol and 7-bromohept-1-

ene (1.00 mL, 6.56 mmol) to yield the title compound. Yield, 79%. Clear

1-Carbamimidoyl-N-tetradecylcyclopropanecarboxamide

Hydrochloride (4a). General procedure A was used to convert 3a

(306 mg, 1.00 mmol) to the title compound. Yield, 55%. White solid. ¹H

NMR (300 MHz, DMSO) δ 9.11 (s, 2H), 8.97 (s, 2H), 7.80 (t, J =

5.3 Hz, 1H), 3.01 (dd, J = 13.1, 6.5 Hz, 2H), 1.65ꢀ0.96 (m, 28H), 0.83

(t, J = 6.5 Hz, 3H). ¹³C NMR (75 MHz, DMSO) δ 168.54, 167.92,

40.13, 31.98, 29.74, 29.56, 29.48, 29.40, 29.09, 27.05, 22.78, 14.86, 14.65.

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4

and colorless oil. R_f= 0.69 (5% EtOAc in hexanes, KMnO ). H NMR

(300 MHz, CDCl₃) δ 5.98ꢀ5.57 (m, 1H), 5.10ꢀ4.79 (m, 2H), 3.38 (t,

J = 6.6 Hz, 2H), 3.25 (d, J = 7.1, 2H), 2.24ꢀ1.95 (m, 3H), 1.88ꢀ1.01 (m,

14H). ¹³C NMR (75 MHz, CDCl₃) δ 139.13, 114.45, 75.76, 71.17,

39.68, 33.97, 29.94, 29.81, 28.99, 25.92, 25.61.

CDCl₃) δ 144.41, 139.03, 133.17, 129.42, 128.65, 127.94, 127.78,

115.48, 73.15, 70.80, 35.37, 32.09, 30.09, 29.69, 29.52, 26.48.

4-(6-(Cyclohexylmethoxy)hexyl)aniline (7b). General proce-

dure E was used to convert 6b (252 mg, 0.789 mmol) to the title

compound. Yield, 99%. Clear and colorless oil. R_f= 0.25 (20% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 6.98 (d, J = 8.1

Hz, 2H), 6.62 (d, J = 8.1, 2H), 3.56 (s, 2H), 3.40 (t, J = 6.2, 2H), 3.21 (d,

J = 6.5, 2H), 2.51 (t, J = 7.6 Hz, 2H), 1.93ꢀ1.04 (m, 17H), 1.04ꢀ0.73

(m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 144.33, 133.10, 129.36, 115.42,

77.07, 71.33, 38.30, 35.27, 32.04, 30.44, 29.95, 29.36, 26.96, 26.34, 26.17.

4-(7-(Cyclohexylmethoxy)heptyl)aniline (7c). General pro-

cedure E was used to convert 6c (663 g, 2.00 mmol) to the title

compound. Yield, 99%. Clear and colorless oil. R_f= 0.34 (20% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 6.99 (d, J = 8.1

Hz, 2H), 6.62 (d, J = 8.2 Hz, 2H), 3.58 (s, 2H), 3.40 (t, J = 6.6 Hz, 2H),

3.23 (d, J = 6.6 Hz, 2H), 2.62ꢀ2.41 (m, 2H), 1.97ꢀ1.04 (m, 19H),

1.06ꢀ0.83 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 144.36, 133.13,

129.37, 115.43, 77.10, 71.38, 38.32, 35.34, 32.07, 30.47, 30.02, 29.67,

29.50, 26.98, 26.44, 26.20.

4-(8-(Cyclohexylmethoxy)octyl)aniline (7d). General proce-

dure E was used to convert 6d (572 mg, 1.65 mmol) to the title

compound. Yield, 99%. Clear and colorless oil. R_f= 0.38 (20% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 6.98 (d, J = 8.2

Hz, 2H), 6.62 (d, J = 8.3 Hz, 2H), 3.66ꢀ3.43 (m, 2H), 3.39 (t, J = 6.7 Hz,

2H), 3.21 (d, J = 6.6 Hz, 2H), 2.59ꢀ2.39 (m, 2H), 1.93ꢀ1.02 (m, 21H),

1.06ꢀ0.80 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 144.26, 133.25,

129.35, 115.43, 77.08, 71.37, 38.29, 35.32, 32.07, 30.43, 29.99, 29.70,

29.48, 26.94, 26.43, 26.16.

4-Adamantan-1-ylmethoxy)heptyl)aniline (7e). General pro-

cedure E was used to convert 6e (263 mg, 0.683 mmol) to the title

compound. Yield, 99%. Clear and colorless oil. R_f= 0.31 (20% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 6.96 (d, J = 7.8

Hz, 2H), 6.62 (d, J = 7.1 Hz, 2H), 3.54 (s, 2H), 3.37 (t, J = 6.6 Hz, 2H),

2.95 (d, J = 5.1 Hz, 2H), 2.49 (t, J = 6.3 Hz, 2H), 1.96 (s, 3H), 1.85 ꢀ1.19

(m, 22H). ¹³C NMR (75 MHz, CDCl₃) δ 144.22, 133.30, 129.36, 115.44,

82.11, 71.96, 39.98, 37.49, 35.30, 32.02, 29.82, 29.62, 29.47, 28.54, 26.33.

N-(4-(7-(Benzyloxy)heptyl)phenyl)-1-cyanocyclopropane-

carboxamide (8a). General procedure B was used to couple 7a

(183 mg, 0.616 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield

the title compound. Yield, 96%. Clear and colorless oil. R_f= 0.47 (20%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.13

(s, 1H), 7.41 (d, J = 8.3 Hz, 2H), 7.38ꢀ7.24 (m, 5H), 7.16 (d, J = 8.3 Hz,

2H), 4.51 (s, 2H), 3.48 (t, J = 6.6 Hz, 2H), 2.68ꢀ2.42 (m, 2H), 1.78 (dd,

J = 8.2, 4.3 Hz, 2H), 1.70ꢀ1.52 (m, 6H), 1.46ꢀ1.26 (m, 6H). ¹³C NMR

(75 MHz, CDCl₃) δ 163.54, 140.25, 138.93, 134.78, 129.19, 128.59,

127.88, 127.72, 120.86, 120.31, 73.09, 70.69, 35.59, 31.61, 29.99, 29.56,

29.39, 26.39, 18.49, 14.34.

7-Butoxyhept-1-ene (5g). General procedure C was used to

couple butanol and 7-bromohept-1-ene (1.50 mL, 9.84 mmol) to yield

the title compound. Yield, 75%. Clear and colorless oil. R_f= 0.74 (10%

EtOAc in hexanes, KMnO₄). ¹H NMR (300 MHz, CDCl₃) δ 5.73 (ddt,

J = 16.9, 10.2, 6.7 Hz, 1H), 5.07ꢀ4.65 (m, 2H), 3.33 (m, 4H), 2.17ꢀ1.79

(m, 2H), 1.62ꢀ1.42 (m, 4H), 1.33 (m, 6H), 0.86 (t, J = 7.4 Hz, 3H). ¹³C

NMR (75 MHz, CDCl₃) δ 138.95, 114.38, 70.96, 70.75, 33.92, 32.05,

29.81, 28.95, 25.89, 19.53, 14.03.

1-(7-(Benzyloxy)heptyl)-4-nitrobenzene (6a). General pro-

cedure D was used to couple 5a (564 mg, 2.76 mmol) and 1-bromo-4-

nitrobenzene to yield the title compound. Yield, 61%. Yellow solid. R_f=

0.16 (5% EtOAc in hexanes, Seebach’s dip). ¹H NMR (500 MHz,

CDCl₃) δ 8.13 (d, J = 7.9 Hz, 2H), 7.45ꢀ7.19 (m, 7H), 4.51 (s, 2H),

3.48 (t, J = 6.4 Hz, 2H), 2.70 (t, J = 7.7 Hz, 2H), 1.61 (m, 4H), 1.49ꢀ1.17

(m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 150.79, 146.20, 138.67,

129.16, 128.36, 127.62, 127.50, 123.55, 72.88, 70.39, 35.83, 30.92, 29.74,

29.23, 29.11, 26.11.

1-(6-(Cyclohexylmethoxy)hexyl)-4-nitrobenzene (6b). Gen-

eral procedure D was used to couple 5b (1.02 g, 4.56 mmol) and

1-bromo-4-nitrobenzene to yield the title compound. Yield, 52%. Tan

oil. R_f= 0.56 (5% EtOAc in hexanes, Seebach’s dip). H NMR (300

MHz, CDCl₃) δ 8.15 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 3.34

(t, J = 6.5 Hz, 2H), 3.15 (d, J = 6.6 Hz, 2H), 2.80ꢀ2.58 (m, 2H),

1.86ꢀ1.00 (m, 17H), 1.00ꢀ0.77 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

δ 150.97, 146.39, 129.36, 123.74, 77.05, 71.10, 38.25, 35.99, 31.15,

30.38, 29.81, 29.20, 26.88, 26.21, 26.11.

1

1-(7-(Cyclohexylmethoxy)heptyl)-4-nitrobenzene (6c). Gen-

eral procedure D was used to couple 5c (951 mg, 4.52 mmol) and

1-bromo-4-nitrobenzene to yield the title compound. Yield, 63%.

1

Tan oil. R_f= 0.61 (5% EtOAc in hexanes, Seebach’s dip). H NMR

(300 MHz, CDCl₃) δ 8.07 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H),

3.33 (t, J = 6.5 Hz, 2H), 3.14 (d, J = 6.6 Hz, 2H), 2.75ꢀ2.53 (m, 2H),

1.84ꢀ0.96 (m, 19H), 0.95ꢀ0.70 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

δ 150.99, 146.36, 129.33, 123.70, 77.03, 71.18, 38.25, 36.02, 31.13,

30.37, 29.89, 29.43, 29.32, 26.88, 26.28, 26.10.

1-(8-(Cyclohexylmethoxy)octyl)-4-nitrobenzene (6d). Gen-

eral procedure D was used to couple 5d (1.02 g, 4.56 mmol) and

1-bromo-4-nitrobenzene to yield the title compound. Yield, 60%. Tan

oil. R_f= 0.64 (5% EtOAc in hexanes, Seebach’s dip). H NMR (300

MHz, CDCl₃) δ 8.12 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 3.36

(t, J = 6.6 Hz, 2H), 3.17 (d, J = 6.6 Hz, 2H), 2.79ꢀ2.57 (m, 2H), 1.85ꢀ

1.00 (m, 21H), 0.86 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 151.03,

146.41, 129.35, 123.76, 77.06, 71.26, 38.26, 36.07, 31.19, 30.39, 29.93,

29.56, 29.33, 26.89, 26.37, 26.11.

1-(((7-(4-Nitrophenyl)heptyl)oxy)methyl)adamantane (6e).

General procedure D was used to couple 5e (671 mg, 2.77 mmol) and

1-bromo-4-nitrobenzene to yield the title compound. Yield, 59%. Tan oil.

R_f= 0.74 (10% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz,

CDCl₃) δ 8.15 (d, J = 6.6 Hz, 2H), 7.32 (m, J = 6.6 Hz, 2H), 3.36 (t, J = 6.5

Hz, 2H), 2.93 (s, 2H), 2.81ꢀ2.58 (m, 2H), 2.06ꢀ1.87 (m, 3H),

1.87ꢀ1.09 (m, 22H). ¹³C NMR (75 MHz, CDCl₃) δ 151.01, 142.80,

132.25, 124.11, 82.13, 71.82, 39.98, 37.48, 36.09, 29.78, 29.46, 29.37,

28.54, 26.27.

4-(7-(Benzyloxy)heptyl)aniline (7a). General procedure E was

used to convert 6a (215 mg, 0.657 mmol) to the title compound. Yield,

99%. Tan oil. R_f= 0.27 (20% EtOAc in hexanes, Seebach’s dip). ¹H NMR

(300 MHz, CDCl₃) δ 7.51ꢀ7.26 (m, 5H), 7.02 (d, J = 5.3 Hz, 2H), 6.66

(m, J = 5.3 Hz, 2H), 4.57 (s, 2H), 3.52 (m, 4H), 2.70ꢀ2.42 (m, 2H),

1.80ꢀ1.53 (m, 4H), 1.42 (t, J = 8.6 Hz, 6H). ¹³C NMR (75 MHz,

1

1-Cyano-N-(4-(6-(cyclohexylmethoxy)hexyl)phenyl)cyclo-

propanecarboxamide (8b). General procedure B was used to

couple 7b (205 mg, 0.708 mmol) and 1-cyano-1-cyclopanecarboxylic

acid to yield the title compound. Yield, 92%. Clear and colorless oil. R_f=

1

0.48 (20% EtOAc in hexanes, Seebach’s dip). H NMR (300 MHz,

CDCl₃) δ 8.12 (s, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H),

3.36 (t, J = 6.5 Hz, 2H), 3.17 (d, J = 6.5 Hz, 2H), 2.67ꢀ2.25 (m, 2H),

1.90ꢀ1.02 (m, 21H), 1.02ꢀ0.52 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

δ 163.50, 140.17, 134.75, 129.15, 120.83, 120.27, 77.04, 71.22, 38.27,

35.52, 31.60, 30.40, 29.88, 29.26, 26.91, 26.27, 26.13, 18.47, 14.31.

1-Cyano-N-(4-(7-(cyclohexylmethoxy)heptyl)phenyl)cyclo-

propanecarboxamide (8c). General procedure B was used to

couple 7c (288 mg, 0.950 mmol) and 1-cyano-1-cyclopanecarboxylic

acid to yield the title compound. Yield, 93%. Clear and colorless oil. R_f=

1

0.49 (20% EtOAc in hexanes, Seebach’s dip). H NMR (300 MHz,

CDCl₃) δ 8.15 (s, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H),

3.35 (t, J = 6.6 Hz, 2H), 3.19 (t, J = 6.5 Hz, 2H), 2.55 (t, J = 7.6 Hz, 2H),

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ARTICLE

1.89ꢀ0.98 (m, 23H), 0.89 (dd, J = 21.8, 10.6 Hz, 2H). ¹³C NMR

(75 MHz, CDCl₃) δ 163.52, 140.17, 134.79, 129.11, 120.86, 120.25,

77.04, 71.27, 38.26, 35.57, 31.60, 30.40, 29.94, 29.55, 29.38, 26.91, 26.35,

26.13, 18.45, 14.29.

(126 MHz, DMSO) δ 168.19, 166.47, 138.44, 136.51, 128.62, 121.33,

76.20, 70.55, 37.98, 35.00, 31.39, 30.03, 29.62, 29.24, 28.98, 26.63, 26.14,

25.82, 15.16. LCMS: t_R= 4.86; m/z = 428.3. HRMS m/z calcd for

C₂₆H₄₂N₃O₂(M þ H), 428.3277; found, 428.3277.

1-Cyano-N-(4-(8-(cyclohexylmethoxy)octyl)phenyl)cyclo-

propanecarboxamide (8d). General procedure B was used to

couple 7d (160 mg, 0.504 mmol) and 1-cyano-1-cyclopanecarboxylic

acid to yield the title compound. Yield, 86%. Clear and colorless oil. R_f=

N-(4-(7-((Adamantan-1-ylmethoxy)heptyl)phenyl)-1-car-

bamimidoylcyclopropanecarboxamide Hydrochloride (9e).

General procedure A was used to convert 8e (79 mg, 0.176 mmol) to the

title compound. Yield, 22%. White solid. ¹H NMR (500 MHz, DMSO)

δ 9.56 (s, 1H), 9.17 (s, 2H), 8.88 (s, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.10

(d, J = 8.5 Hz, 2H), 3.28 (t, J = 6.4 Hz, 2H), 2.88 (s, 2H), 2.50 (m, 2H),

1.89 (m, 2H), 1.65 (d, J = 12.1 Hz, 3H), 1.60ꢀ1.25 (s, 26H). ¹³C NMR

(126 MHz, DMSO) δ 176.39, 168.07, 167.14, 166.50, 163.30, 138.48,

136.45, 134.79, 128.61, 121.35, 81.37, 71.06, 37.16, 34.94, 34.08, 31.37,

29.93, 29.44, 28.97, 28.04, 26.04, 15.18. LCMS: t_R= 5.65; m/z = 466.4.

HRMS m/z calcd for C₂₉H₄₅N₃O₂(M þ H), 466.3434; found, 466.3424.

Non-8-en-1-ylbenzene (10a). General procedure F was used to

couple benzylmagnesium bromide and 8-bromooct-1-ene (1.00 mL,

5.98 mmol) to yield the title compound. Yield, 84%. Clear and colorless

1

0.62 (20% EtOAc in hexanes, Seebach’s dip). H NMR (300 MHz,

CDCl₃) δ 8.04 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H),

3.36 (t, J = 6.6 Hz, 2H), 3.18 (d, J = 6.6 Hz, 2H), 2.73ꢀ2.31 (m, 2H),

1.89ꢀ1.02 (m, 25H), 1.02ꢀ0.68 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

δ 163.47, 140.31, 134.69, 129.17, 120.78, 120.28, 77.05, 71.31, 38.26,

35.58, 31.64, 30.40, 29.95, 29.64, 29.38, 26.91, 26.39, 26.13, 18.44, 14.31.

N-(4-(7-Adamantan-1-ylmethoxy)heptyl)phenyl)-1-cyano-

cyclopropanecarboxamide (8e). General procedure B was used to

couple 7e (83 mg, 0.233 mmol) and 1-cyano-1-cyclopanecarboxylic acid

to yield the title compound. Yield, 85%. Clear and colorless oil. R_f= 0.52

(20% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ

8.01 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 3.36 (t, J =

6.6 Hz, 2H), 2.94 (d, J = 6.7 Hz, 2H), 2.70ꢀ2.43 (m, 2H), 1.95 (s, 3H),

1.85ꢀ1.28 (m, 26H). ¹³C NMR (75 MHz, CDCl₃) δ 163.45, 140.38,

134.60, 129.23, 120.71, 120.32, 82.10, 71.90, 39.96, 37.47, 35.58, 34.30,

31.62, 29.79, 29.54, 29.39, 28.52, 26.30, 18.48, 14.34.

1

4

oil. R_f= 0.75 (hexanes, KMnO ). H NMR (300 MHz, CDCl₃) δ

7.50ꢀ6.98 (m, 5H), 5.84 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.20ꢀ4.82

(m, 2H), 2.78ꢀ2.47 (m, 2H), 1.89 (dt, J = 7.0, 6.8 Hz, 2H), 1.64 (p, J =

7.0 Hz, 2H), 1.55ꢀ1.21 (m, 8H). ¹³C NMR (75 MHz, CDCl₃) δ 143.09,

139.38, 128.63, 128.45, 125.81, 114.67, 114.42, 36.26, 34.09, 31.80,

29.56, 29.35, 29.20.

N-(4-(7-(Benzyloxy)heptyl)phenyl)-1-carbamimidoylcyclo-

propanecarboxamide Hydrochloride (9a). General procedure A

was used to convert 8a (230 mg, 0.590 mmol) to the title compound.

Yield, 52%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.54 (s, 1H),

9.15 (s, 2H), 8.88 (s, 2H), 7.44 (d, J = 8.3 Hz, 2H), 7.37ꢀ7.20 (m, 5H),

7.10 (d, J = 8.3 Hz, 2H), 4.40 (s, 2H), 3.38 (t, J = 6.4 Hz, 2H), 2.49 (t, J =

7.0 Hz, 2H), 1.66ꢀ1.35 (m, 8H), 1.26 (m, 6H). ¹³C NMR (126 MHz,

CDCl₃) δ 172.78, 171.30, 143.91, 143.23, 141.18, 133.40, 132.57,

132.49, 126.14, 76.96, 74.74, 39.69, 36.13, 34.34, 33.81, 33.71, 30.84,

19.96. LCMS: t_R= 4.23; m/z = 408.3. HRMS m/z calcd for

C₂₅H₃₄N₃O₂(M þ H), 408.2651; found, 408.2646.

Oct-7-en-1-ylbenzene (10b). General procedure F was used to

couple benzylmagnesium bromide and 7-bromohept-1-ene (1.00 mL,

6.52 mmol) to yield the title compound. Yield, 84%. Clear and colorless

1

4

oil. R_f= 0.75 (hexanes, KMnO ). H NMR (300 MHz, CDCl₃) δ

7.65ꢀ7.15 (m, 5H), 5.97 (ddt, J = 16.9, 10.0, 6.7 Hz, 1H), 5.13 (m, 2H),

2.98ꢀ2.56 (m, 2H), 2.21 (dt, J = 6.9, 6.5 Hz, 2H), 1.97ꢀ1.67 (m, 2H),

1.67ꢀ1.28 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 143.17, 139.42,

128.76, 128.59, 125.95, 114.60, 36.37, 34.20, 31.88, 29.56, 29.41, 29.26.

1-Nitro-4-(9-phenylnonyl)benzene (11a). General procedure

D was used to couple 10a (971 mg, 4.80 mmol) and 1-bromo-4-

nitrobenzene to yield the title compound. Yield, 59%. Tan oil. R_f=

1-Carbamimidoyl-N-(4-(6-(cyclohexylmethoxy)hexyl)phe-

nyl)cyclopropanecarboxamide Hydrochloride (9b). General

procedure A was used to convert 8b (248 mg, 0.648 mmol) to the title

1

0.67 (10% EtOAc in hexanes, Seebach’s dip). H NMR (300 MHz,

CDCl₃) δ 8.18 (d, J = 8.6 Hz, 2H), 7.42ꢀ7.30 (m, 4H), 7.30ꢀ7.18 (m,

3H), 2.72 (m, 4H), 1.66 (m, 24.4 Hz, 4H), 1.39 (m, 10H). ¹³C NMR (75

MHz, CDCl₃) δ 151.12, 146.49, 143.12, 129.45, 128.70, 128.55, 125.90,

123.81, 36.33, 36.15, 31.87, 31.31, 29.82, 29.75, 29.66, 29.53.

1-Nitro-4-(8-phenyloctyl)benzene (11b). General procedure

D was used to couple 10b (1.00 g, 5.31 mmol) and 1-bromo-4-

nitrobenzene to yield the title compound. Yield, 71%. Tan oil. R_f= 0.49

(5% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ

8.16 (d, J = 8.8 Hz, 2H), 7.39ꢀ7.27 (m, 4H), 7.27ꢀ7.15 (m, 3H),

2.72ꢀ2.60 (m, 4H), 1.78ꢀ1.48 (m, 4H), 1.40 (d, J = 19.9 Hz, 8H). ¹³C

NMR (75 MHz, CDCl₃) δ 151.09, 146.46, 143.08, 129.42, 128.67,

128.52, 125.87, 123.81, 36.25, 36.12, 31.78, 31.26, 29.67, 29.63,

29.55, 29.45.

4-(9-Phenylnonyl)aniline (12a). General procedure E was used

to convert 11a (426 mg, 1.42 mmol) to the title compound. Yield, 99%.

Tan oil. R_f= 0.33 (20% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300

MHz, CDCl₃) δ 7.48ꢀ7.20 (m, 5H), 7.08 (d, J = 8.3 Hz, 2H), 6.74 (d,

J = 8.3 Hz, 2H), 3.58 (s, 2H), 2.87ꢀ2.66 (m, 2H), 2.66ꢀ2.53 (m, 2H),

1.86ꢀ1.57 (m, 4H), 1.42 (s, 10H). ¹³C NMR (75 MHz, CDCl₃) δ

144.38, 143.27, 133.31, 129.46, 128.76, 128.58, 125.91, 115.54, 36.36,

35.45, 32.21, 31.90, 29.91, 29.71, 29.65.

1

compound. Yield, 41%. White solid. H NMR (300 MHz, DMSO) δ

9.67 (s, 1H), 9.03 (s, 4H), 7.47 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz,

2H), 3.27 (t, J = 6.3 Hz, 2H), 3.10 (d, J = 6.4 Hz, 2H), 2.49 (t, J = 5.0 Hz,

2H), 1.40 (m, 21H), 0.85 (dd, J = 21.4, 11.0 Hz, 2H). ¹³C NMR (75

MHz, CDCl₃) δ 173.21, 171.45, 143.36, 141.52, 133.61, 126.28, 81.17,

75.51, 42.96, 39.92, 36.41, 35.01, 34.58, 33.81, 31.61, 30.96, 30.79, 20.13.

LCMS: t_R= 4.44; m/z = 400.3. HRMS m/z calcd for C₂₄H₃₈N₃O₂(M þ

H), 400.2964; found, 400.2958.

1-Carbamimidoyl-N-(4-(7-(cyclohexylmethoxy)heptyl)

phenyl)cyclopropanecarboxamide Hydrochloride (9c). Gen-

eral procedure A was used to convert 8c (352 mg, 0.888 mmol) to the

title compound. Yield, 43%. White solid. ¹H NMR (300 MHz, DMSO)

δ 9.71 (s, 1H), 9.12 (s, 4H), 7.47 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz,

2H), 3.27 (t, J = 6.4 Hz, 2H), 3.12 (d, J = 5.9 Hz, 2H), 2.49 (t, J = 7.4 Hz,

2H), 1.78ꢀ0.97 (m, 23H), 0.85 (dd, J = 21.6, 10.9 Hz, 2H). ¹³C NMR

(75 MHz, DMSO) δ 168.53, 166.67, 138.61, 136.79, 128.84, 121.51,

76.43, 70.77, 38.21, 35.19, 31.64, 30.27, 29.84, 29.32, 29.23, 26.86, 26.34,

26.05, 15.36. LCMS: t_R= 4.65; m/z = 414.3. HRMS m/z calcd for

C₂₅H₄₀N₃O₂(M þ H), 414.3121; found, 414.3111.

1-Carbamimidoyl-N-(4-(8-(cyclohexylmethoxy)octyl)phe-

nyl)cyclopropanecarboxamide Hydrochloride (9d). General

procedure A was used to convert 8d (178 mg, 0.434 mmol) to the title

4-(8-Phenyloctyl)aniline (12b). General procedure E was used

to convert 11b (572 mg, 1.65 mmol) to the title compound. Yield, 99%.

Tan oil. R_f= 0.31 (20% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300

MHz, CDCl₃) δ 7.52ꢀ7.20 (m, 5H), 7.11 (d, J = 8.3 Hz, 2H), 6.73 (d,

J = 8.3 Hz, 2H), 3.81ꢀ3.37 (m, 2H), 2.77 (m, 2H), 2.63 (m, 2H), 1.72

(m, 4H), 1.43 (d, J = 19.9 Hz, 8H). ¹³C NMR (75 MHz, CDCl₃) δ

1

compound. Yield, 50%. White solid. H NMR (500 MHz, DMSO)

δ 9.65 (s, 1H), 9.22 (s, 2H), 8.99 (s, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.11

(d, J = 8.3 Hz, 2H), 3.29 (t, J = 6.3 Hz, 2H), 3.11 (d, J = 6.4 Hz, 2H), 2.50

(t, J = 7.4 Hz, 2H), 1.78ꢀ0.99 (m, 25H), 0.99ꢀ0.74 (m, 2H). ¹³C NMR

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144.45, 143.29, 133.31, 129.50, 128.81, 128.63, 125.96, 115.58, 36.40,

35.49, 32.25, 31.95, 29.90, 29.75, 29.69.

3.42 (t, J = 6.7 Hz, 2H), 3.23 (t, J = 6.7 Hz, 2H), 3.04 (d, J = 6.6 Hz, 2H),

1.67ꢀ1.48 (m, 5H), 1.48ꢀ1.32 (m, 5H), 1.24ꢀ0.93 (m, 13H),

0.85ꢀ0.66 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 76.66, 70.97,

62.19, 37.84, 32.53, 30.00, 29.54, 29.49, 29.46, 29.38, 26.54, 26.03, 25.75.

10-(Cyclopentylmethoxy)decan-1-ol (16b). General proce-

dure G was used to convert 15b (902 mg, 3.78 mmol) to the title

compound. Yield, 85%. Clear and colorless oil. R_f= 0.65 (30% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 3.69 (s, 1H),

3.38 (t, J = 6.7 Hz, 2H), 3.24 (t, J = 5.9 Hz, 2H), 3.05 (d, J = 6.3 Hz, 2H),

2.14 (sept, J = 7.4 Hz, 1H), 1.96ꢀ0.49 (m, 24H).

1-Cyano-N-(4-(9-phenylnonyl)phenyl)cyclopropanecar-

boxamide (13a). General procedure B was used to couple 12a

(251 mg, 0.850 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield

the title compound. Yield, 94%. Clear and colorless oil. R_f= 0.56 (20%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.12

(s, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.37ꢀ7.09 (m, 7H), 2.62 (m, 4H), 1.80

(dd, J = 8.2, 4.4 Hz, 2H), 1.74ꢀ1.47 (m, 6H), 1.34 (s, 10H). ¹³C NMR

(75 MHz, CDCl₃) δ 163.53, 143.15, 140.34, 134.78, 129.22, 128.66,

128.49, 125.83, 120.84, 120.33, 36.26, 35.64, 31.79, 31.70, 29.76, 29.59,

29.48, 18.50, 14.37.

1-Cyano-N-(4-(8-phenyloctyl)phenyl)cyclopropanecar-

boxamide (13b). General procedure B was used to couple 12b

(485 mg, 1.72 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield

the title compound. Yield, 91%. Clear and colorless oil. R_f= 0.54 (20%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.19

(s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.39ꢀ7.28 (m, 2H), 7.28ꢀ7.12 (m,

5H), 2.64 (m, 4H), 1.81 (dd, J = 8.2, 4.4 Hz, 2H), 1.75ꢀ1.50 (m, 6H),

1.50ꢀ1.21 (m, 8H). ¹³C NMR (75 MHz, CDCl₃) δ 163.59, 143.14,

140.29, 134.85, 129.22, 128.68, 128.52, 125.86, 120.91, 120.35, 36.27,

35.65, 31.81, 31.72, 29.72, 29.60, 29.49, 18.52, 14.38.

(((10-Azidodecyl)oxy)methyl)cyclohexane (17a). General

procedure H was used to convert 16a (2.35 g, 8.69 mmol) to 10-(cyclo-

hexylmethoxy)decyl methanesulfonate. 98%. Clear and colorless oil. R_f=

1

0.26 (10% EtOAc in hexanes, Seebach’s dip). H NMR (500 MHz,

CDCl₃) δ 4.07 (t, J = 6.6 Hz, 2H), 3.23 (t, J = 6.5 Hz, 2H), 3.05 (d, J =

6.6, 2H), 2.86 (s, 3H), 1.69ꢀ1.48 (m, 7H), 1.48ꢀ1.35 (m, 3H),

1.35ꢀ0.94 (m, 15H), 0.86ꢀ0.68 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 76.61, 70.88, 70.15, 37.94, 36.99, 30.02, 29.62, 29.33, 29.30,

29.23, 28.98, 28.89, 26.56, 26.04, 25.78, 25.28. General procedure I was

then used to convert the alkyl mesylate (2.05 g, 5.88 mmol) to the title

compound. Yield, 94%. Clear and colorless oil. R_f= 0.93 (10% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (500 MHz, CDCl₃) δ 3.30 (t, J = 6.6

Hz, 2H), 3.17 (t, J = 7.0 Hz, 2H), 3.12 (d, J = 6.6 Hz, 2H), 1.74ꢀ1.56 (m,

5H), 1.56ꢀ1.40 (m, 5H), 1.35ꢀ1.00 (m, 15H), 0.94ꢀ0.76 (m, 2H). ¹³C

NMR (126 MHz, CDCl₃) δ 76.71, 70.98, 51.33, 38.03, 30.10, 29.69,

29.42, 29.37, 29.07, 28.78, 26.63, 26.12, 25.85.

(((10-Azidodecyl)oxy)methyl)cyclopentane (17b). General

procedure H was used to convert 16b (904 mg, 3.21 mmol) to

10-(cyclohexylmethoxy)decyl methanesulfonate. R_f= 0.22 (10% EtOAc

in hexanes, Seebach’s dip). The alkyl mesylate was not purified by flash

chromatography in the example and was taken onto the next step crude.

This method was later shown to be inferior to that of the synthesis of

17a. General procedure I was then used to convert the alkyl mesylate to

the title compound. Yield, 63% over two steps. Clear and colorless oil.

R_f= 0.93 (10% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz,

CDCl₃) δ 3.38 (t, J = 6.6 Hz, 2H), 3.29ꢀ3.14 (m, 4H), 2.24ꢀ1.94 (m,

1H), 1.93ꢀ0.55 (m, 24H).

1-Carbamimidoyl-N-(4-(9-phenylnonyl)phenyl)cyclopro-

panecarboxamide Hydrochloride (14a). General procedure A

was used to convert 13a (312 mg, 0.803 mmol) to the title compound.

Yield, 44%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.61 (s, 1H),

9.21 (s, 2H), 8.97 (s, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.29ꢀ

7.19 (m, 2H), 7.18ꢀ7.05 (m, 5H), 2.58ꢀ2.48 (m, 4H), 1.69ꢀ1.31 (m,

8H), 1.23 (s, 10H). ¹³C NMR (126 MHz, DMSO) δ 168.18, 166.48,

142.74, 138.45, 136.48, 128.68, 128.64, 126.01, 121.35, 35.59, 34.97,

31.44, 29.39, 29.26, 29.06, 29.00, 15.16. LCMS: t_R= 4.79; m/z = 406.3.

HRMS m/z calcd for C₂₆H₃₆N₃O (M þ H), 406.2858; found, 406.2852.

1-Carbamimidoyl-N-(4-(9-phenylnonyl)phenyl)cyclopro-

panecarboxamide Hydrochloride (14b). General procedure A

was used to convert 13b (560 mg, 1.50 mmol) to the title compound.

Yield, 39%. White solid. ¹H NMR (300 MHz, DMSO) δ 10.50 (s, 2H),

9.62 (s, 1H), 8.89 (s, 2H), 7.45 (d, J = 7.9 Hz, 2H), 7.17 (m, 7H),

2.71ꢀ2.24 (m, 4H), 1.92ꢀ0.92 (m, 14H). ¹³C NMR (75 MHz, DMSO)

δ 170.61, 168.67, 166.78, 142.98, 138.63, 136.80, 128.91, 128.87, 126.25,

123.40, 121.59, 35.82, 35.20, 31.67, 30.30, 29.49, 29.30, 29.24, 16.07,

15.23. LCMS: t_R= 4.51; m/z = 392.3. HRMS m/z calcd for C₂₅H₃₄N₃O

(M þ H), 392.2702; found, 392.2695.

1-Cyano-N-(10-(cyclohexylmethoxy)decyl)cyclopropane-

carboxamide (18a). General procedure J was used to couple 17a

(1.55 g, 5.25 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield the

title compound. Yield, 82%. Clear and colorless oil. R_f= 0.56 (20%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (500 MHz, CDCl₃) δ 6.59

(t, J = 5.4 Hz, 1H), 3.25 (t, J = 6.6 Hz, 2H), 3.14 (dt, J = 6.6, 5.4 Hz, 2H),

3.06 (d, J = 6.6 Hz, 2H), 1.67ꢀ1.35 (m, 12H), 1.33 (m, 2H), 1.26ꢀ0.95

(m, 15H), 0.79 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 164.86,

120.06, 76.64, 70.92, 40.48, 37.95, 30.04, 29.63, 29.38, 29.33, 29.26,

29.11, 26.68, 26.57, 26.06, 25.79, 17.22, 13.28.

1-Cyano-N-(10-(cyclopentylmethoxy)decyl)cyclopropane-

carboxamide (18b). General procedure J was used to couple 17b

(569 mg, 2.02 mmol) and 1-cyano-1-cyclopanecarboxylic acid to yield

the title compound. Yield, 84%. Clear and colorless oil. R_f= 0.54 (20%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 6.52

(s, 1H), 3.32 (t, J = 6.6 Hz, 2H), 3.19 (d, J = 7.1 Hz, 2H), 2.07 (sept, J =

7.4 Hz, 1H), 1.77ꢀ0.99 (m, 28H).

((Dec-9-en-1-yloxy)methyl)cyclohexane (15a). General pro-

cedure C was used to couple cyclohexylmethanol and 10-bromodec-

1-ene (4.35 mL, 21.7 mmol) to yield the title compound. Yield, 74%.

1

4

Clear and colorless oil. R_f= 0.86 (10% EtOAc in hexanes, KMnO ). H

NMR (300 MHz, CDCl₃) δ 5.74 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H),

5.13ꢀ4.74 (m, 2H), 3.36 (t, J = 6.6 Hz, 2H), 3.17 (d, J = 6.6 Hz, 2H),

2.02 (dt, J = 9.9, 4.0 Hz, 2H), 1.90ꢀ0.99 (m, 21H), 0.98ꢀ0.70 (m, 2H).

¹³C NMR (75 MHz, CDCl₃) δ 139.30, 114.31, 77.05, 71.32, 38.29,

34.03, 30.40, 29.97, 29.66, 29.30, 29.14, 26.90, 26.40, 26.12.

((Dec-9-en-1-yloxy)methyl)cyclopentane (15b). General pro-

cedure C was used to couple cyclopentylmethanol and 10-bromodec-

1-ene (1.00mL, 4.98 mmol) toyield thetitle compound. Yield, 76%. Clear

1

4

and colorless oil. R_f= 0.85 (10% EtOAc in hexanes, KMnO ). H NMR

(300 MHz, CDCl₃) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.17ꢀ4.75

(m, 2H), 3.39 (t, J = 6.7 Hz, 2H), 3.26 (d, J = 7.1 Hz, 2H), 2.15 (dp, J =

15.0, 7.4 Hz, 1H), 2.03 (m, 2H), 1.82ꢀ0.70 (m, 21H). ¹³C NMR (75

MHz, CDCl₃) δ 139.39, 114.32, 75.77, 71.29, 39.67, 34.04, 29.94, 29.83,

29.67, 29.31, 29.14, 26.40, 25.62.

1-Carbamimidoyl-N-(10-(cyclohexylmethoxy)decyl)cyclo-

propanecarboxamide Hydrochloride (19a). General procedure

A was used to convert 18a (1.56 g, 4.30 mmol) to the title compound.

Yield, 44%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.04 (s, 4H),

7.83 (t, J = 5.6 Hz, 1H), 3.28 (t, J = 6.5 Hz, 2H), 3.11 (d, J = 6.4 Hz, 2H),

3.02 (dt, J = 6.7, 5.6 Hz, 2H), 1.71ꢀ1.54 (m, 5H), 1.53ꢀ1.00 (m, 24H),

0.94ꢀ0.76 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 168.34, 167.68,

76.20, 70.56, 37.97, 30.03, 29.64, 29.47, 29.42, 29.32, 29.23, 26.82, 26.62,

26.17, 25.81, 14.62. LCMS: t_R= 4.29; m/z = 380.3. HRMS m/z calcd for

C₂₂H₄₂N₃O₂(M þ H), 380.3277; found, 380.3268.

10-(Cyclohexylmethoxy)decan-1-ol (16a). General proce-

dure G was used to convert 15a (4.00 g, 15.8 mmol) to the title

compound. Yield, 81%. Clear and colorless oil. R_f= 0.68 (30% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (500 MHz, CDCl₃) δ 3.64 (s, 1H),

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1-Carbamimidoyl-N-(10-(cyclohexylmethoxy)decyl)cyclo-

propanecarboxamide Hydrochloride (19b). General procedure

A was used to convert 18b (591 mg, 1.70 mmol) to the title compound.

Yield, 29%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.08 (s, 4H),

7.78 (t, J = 5.4 Hz, 1H), 3.34ꢀ3.28 (m, 2H), 3.18 (d, J = 7.0 Hz, 2H),

3.03 (td, J = 6.8, 5.4 Hz, 2H), 2.12ꢀ1.95 (m, 1H), 1.68ꢀ1.57 (m, 2H),

1.57ꢀ1.31 (m, 10H), 1.31ꢀ1.10 (m, 16H). ¹³C NMR (126 MHz,

DMSO) δ 168.15, 167.73, 74.92, 70.52, 39.37, 29.63, 29.54, 29.46,

29.41, 29.34, 29.22, 28.91, 26.81, 26.17, 25.42, 16.85, 14.62. LCMS: t_R=

4.08; m/z = 366.3. HRMS m/z calcd for C₂₁H₄₀N₃O₂(M þ H),

366.3121; found, 366.3111.

4-(7-(Cyclohexylmethoxy)heptyl)benzaldehyde (20a). Gen-

eral procedure D was used to couple 5c (1.08 g, 5.12 mmol) and

4-bromobenzaldehyde to yield the title compound. Yield, 77%. Clear

and colorless oil. R_f= 0.60 (10% EtOAc in hexanes, Seebach’s dip). ¹H

NMR (300 MHz, CDCl₃) δ 9.94 (t, J = 2.0 Hz, 1H), 7.76 (d, J = 8.2 Hz,

2H), 7.30 (d, J = 8.0 Hz, 2H), 3.34 (t, J = 6.6 Hz, 2H), 3.16 (d, J = 6.6 Hz,

2H), 2.74ꢀ2.52 (m, 2H), 1.84ꢀ1.03 (m, 19H), 0.87 (m, 2H). ¹³C NMR

(75 MHz, CDCl₃) δ 191.83, 150.40, 134.57, 129.96, 129.17, 76.94,

71.13, 38.22, 36.29, 31.16, 30.32, 29.88, 29.43, 29.35, 26.84, 26.27, 26.07.

4-(7-(Cyclopentylmethoxy)heptyl)benzaldehyde (20b). Gen-

eral procedure D was used to couple 5f (500 mg, 2.55 mmol) and

4-bromobenzaldehyde to yield the title compound. Yield, 77%. Clear

and colorless oil. R_f= 0.72 (10% EtOAc in hexanes, Seebach’s dip). ¹H

NMR (300 MHz, CDCl₃) δ 9.89 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.26

(d, J = 8.1 Hz, 2H), 3.33 (t, J = 6.6 Hz, 2H), 3.19 (d, J = 7.1 Hz, 2H),

2.68ꢀ2.49 (m, 2H), 2.08 (sept, J = 7.4 Hz, 1H), 1.77ꢀ1.37 (m, 10H),

1.28ꢀ1.06 (m, 8H). ¹³C NMR (75 MHz, CDCl₃) δ 191.97, 150.49,

134.59, 130.01, 129.22, 75.69, 71.13, 39.65, 36.34, 31.18, 29.88, 29.78,

29.45, 29.37, 26.29, 25.60.

4-(7-Butoxyheptyl)benzaldehyde (20c). General procedure D

was used to couple 5g (1.26 g, 7.39 mmol) and 4-bromobenzaldehyde to

yield the title compound. Yield, 89%. Clear and colorless oil. R_f= 0.48

(10% EtOAc in hexanes, Seebach’s dip). ¹H NMR (500 MHz, CDCl₃) δ

9.83 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 3.26 (m,

4H), 2.70ꢀ2.35 (m, 2H), 1.60ꢀ1.06 (m, 14H), 0.77 (t, J = 7.5 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 191.53, 150.12, 134.33, 129.69, 128.90,

70.68, 70.46, 36.03, 31.78, 30.90, 29.65, 29.18, 29.08, 26.02, 19.29, 13.84.

N-(1-Cyanocyclopropyl)-4-(7-(cyclohexylmethoxy)heptyl)

benzamide (22a). General procedure K was used to convert 20a

(1.25 g, 3.94 mmol) to the corresponding carboxylic acid 21a, which was

taken onto the next reaction crude. General procedure I was then used to

convert the crude carboxylic acid to its acid chloride and was used

immediately after purification. General procedure M was used to couple

the acid chloride and 1-amino-1-cyclopropanecarbonitrile to yield the

title compound. Yield, 57% over three steps. While solid. R_f= 0.52 (40%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 7.68

(dd, J = 8.3, 2.7 Hz, 2H), 7.34ꢀ7.12 (m, 2H), 6.97 (d, J = 20.0 Hz, 1H),

3.36 (td, J = 6.6, 2.9 Hz, 2H), 3.18 (dd, J = 6.6, 2.8 Hz, 2H), 2.63 (dd, J =

10.1, 5.2 Hz, 2H), 1.87ꢀ0.99 (m, 25H), 0.99ꢀ0.73 (m, 2H). ¹³C NMR

(75 MHz, CDCl₃) δ 168.63, 148.03, 130.24, 128.87, 127.66, 120.65,

77.06, 71.26, 38.22, 36.03, 31.30, 30.37, 29.90, 29.49, 29.37, 26.87, 26.31,

26.09, 21.11, 16.99.

N-(1-Cyanocyclopropyl)-4-(7-(cyclopentylmethoxy)hep-

tyl)benzamide (22b). General procedure K was used to convert 20b

(536 mg, 1.77 mmol) to is corresponding carboxylic acid 21b, which was

taken onto the next reaction crude. General procedure I was then used to

convert the crude carboxylic acid to its acid chloride and was used

immediately after purification. General procedure M was used to couple

the acid chloride and 1-amino-1-cyclopropanecarbonitrile to yield the

title compound. Yield, 61% over three steps. White solid. R_f= 0.48 (40%

EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 7.91

(s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 3.36 (t, J = 9.6

Hz, 2H), 3.22 (d, J = 7.2 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H), 2.18ꢀ2.00

(m, 1H), 1.80ꢀ0.98 (m, 22H). ¹³C NMR (75 MHz, CDCl₃) δ 168.68,

147.90, 130.29, 128.80, 127.71, 120.70, 75.76, 71.20, 39.63, 36.02, 31.29,

29.87, 29.81, 29.49, 29.36, 26.31, 25.59, 21.11, 16.92.

4-(7-Butoxyheptyl)-N-(1-cyanocyclopropyl)benzamide (22c).

General procedure K was used to convert 20c (1.40 g, 5.07 mmol) to the

corresponding carboxylic acid 21c, which was taken onto the next

reaction crude. General procedure I was then used to convert the crude

carboxylic acid to its acid chloride and was used immediately after

purification. General procedure M was used to couple the acid chloride

and 1-amino-1-cyclopropanecarbonitirle to yield the title compound.

Yield, 78% over three steps. White solid. R_f= 0.32 (40% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.20 (s, 1H),

7.73 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 3.57ꢀ3.13 (m, 4H),

2.53 (m, 2H), 1.64ꢀ1.42 (m, 8H), 1.42ꢀ1.13 (m, 10H), 0.86 (t, J = 7.3

Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 169.04, 147.95, 130.24, 128.83,

127.77, 120.80, 71.05, 70.83, 36.01, 32.02, 31.29, 29.92, 29.48, 29.36,

26.31, 21.15, 19.56, 16.87, 14.16.

N-(1-Carbamimidoylcyclopropyl)-4-(7-(cyclohexylmethoxy)

heptyl)benzamide Hydrochloride (23a). General procedure A

was used to convert 22a (890 mg, 2.25 mmol) to the title compound.

Yield, 71%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.12 (s, 1H),

8.86 (s, 2H), 8.57 (s, 2H), 7.82 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz,

2H), 3.28 (t, J = 6.4 Hz, 2H), 3.11 (d, J = 6.4 Hz, 2H), 2.60 (t, J = 7.5 Hz,

2H), 1.78ꢀ0.52 (m, 25H). ¹³C NMR (126 MHz, CDCl₃) δ 177.08,

172.73, 151.72, 135.97, 133.16, 133.08, 80.94, 75.27, 42.72, 40.10, 37.82,

35.92, 34.78, 34.34, 33.80, 33.69, 31.37, 30.85, 30.55, 23.17. LCMS: t_R=

4.36; m/z = 414.3. HRMS m/z calcd for C₂₅H₄₀N₃O₂(M þ H),

414.3121; found, 414.3111.

N-(1-Carbamimidoylcyclopropyl)-4-(7-(cyclopentylmethoxy)

heptyl)benzamide Hydrochloride (23b). General procedure A

was used to convert 22b (413 mg, 1.08 mmol) to the title compound.

Yield, 73%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.15 (s, 1H),

8.94 (s, 2H), 8.60 (s, 2H), 7.81 (d, J = 7.6 Hz, 2H), 7.26 (d, J = 7.6 Hz,

2H), 3.30 (t, J = 5.6 Hz, 2H), 3.17 (d, J = 7.0 Hz, 2H), 2.60 (t, J = 7.4 Hz,

2H), 2.13ꢀ1.94 (m, 1H), 1.75ꢀ1.01 (m, 22H). ¹³C NMR (126 MHz,

DMSO) δ 172.42, 167.98, 146.94, 131.23, 128.41, 128.35, 74.92, 70.49,

35.35, 33.06, 31.16, 29.59, 29.54, 29.05, 28.95, 26.10, 25.42, 18.41.

LCMS: t_R= 4.29; m/z = 400.3. HRMS m/z calcd for C₂₄H₃₈N₃O₂(M þ

H), 400.2964; found, 400.2957.

4-(7-Butoxyheptyl)-N-(1-carbamimidoylcyclopropyl)ben-

zamide Hydrochloride (23c). General procedure A was used to

convert 22c (791 mg, 2.22 mmol) to the title compound. Yield, 60%.

White solid. ¹H NMR (500 MHz, DMSO) δ 9.14 (s, 1H), 8.75 (s, 4H),

7.80 (d, J = 7.7 Hz, 2H), 7.26 (d, J = 7.3 Hz, 2H), 3.29 (m, 4H), 2.60 (t,

J = 7.4 Hz, 2H), 1.75ꢀ0.93 (m, 18H), 0.93ꢀ0.48 (m, 3H). ¹³C NMR

(126 MHz, DMSO) δ 172.38, 167.98, 146.95, 131.22, 128.41, 128.34,

70.33, 70.04, 35.35, 33.07, 31.79, 31.17, 29.64, 29.07, 28.94, 26.11, 19.35,

18.40, 14.23. LCMS: t_R= 4.08; m/z = 374.3. HRMS m/z calcd for

C₂₂H₃₆N₃O₂(M þ H), 374.2808; found, 374.2802.

11-(Cyclohexylmethoxy)undecanoic Acid (24). General pro-

cedure C was used to couple cyclohexylmethanol and 11-bromounde-

cenoic acid (1.00 g, 3.77 mmol), with TBAB (121 mg, 0.38 mmol, 0.1

equiv) being added along with the 11-bromoundecenoic acid in one

portion, to yield the title compound. Yield, 74%. Clear and colorless oil.

1

4

R_f= 0.80 (60% EtOAc in hexanes, KMnO ). H NMR (500 MHz,

CDCl₃) δ 6.46 (s, 1H), 3.30 (d, J = 6.6 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H),

1.72ꢀ0.95 (m, 27H), 0.93ꢀ0.65 (m, 2H). ¹³C NMR (126 MHz,

CDCl₃) δ 177.87, 76.69, 71.00, 40.15, 34.00, 30.03, 29.52, 29.45,

29.36, 29.32, 29.18, 29.02, 26.52, 25.78, 24.73.

N-(1-Cyanocyclopropyl)-11-(cyclohexylmethoxy)undecana-

mide (25). General procedure B was used to couple 24 (358 mg,

3.02 mmol) and 1-amino-1-cyclopropanecarbonitirle to yield the title

compound. Yield, 81%. White solid. R_f= 0.51 (20% EtOAc in hexanes,

1

Seebach’s dip). H NMR (500 MHz, CDCl₃) δ 7.23ꢀ7.13 (m, 1H),

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3.34 (t, J = 6.7 Hz, 2H), 3.16 (d, J = 6.6 Hz, 2H), 2.16 (t, J = 7.6 Hz, 2H),

1.78ꢀ0.99 (m, 31H), 0.86 (m, 2H). ¹³C NMR (126 MHz, CDCl₃)

δ 174.62, 120.27, 76.79, 71.07, 37.95, 35.86, 30.12, 29.68, 29.49, 29.42,

29.37, 29.28, 29.14, 26.62, 26.11, 25.83, 25.28, 16.58.

saturated NaHCO₃(10 ꢁ the volume of DMF), 3ꢁ with 1 N HCl (10 ꢁ

the volume of DMF), and once with brine (10 ꢁ the volume of DMF).

The organic layer was then dried with Na₂SO₄, evaporated to a yellow

oil, and immediately purified by flash chromatography to yield the title

compound. Yield, 98%. Clear and colorless oil. R_f= 0.37 (20% EtOAc in

N-(1-Carbamimidoylcyclopropyl)-11-(cyclohexylmethoxy)

undecanamide Hydrochloride (26). General procedure A was

used to convert 25 (791 mg, 2.22 mmol) to the title compound. Yield,

68%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.26ꢀ7.91 (m, 5H),

3.28 (t, J = 6.4 Hz, 2H), 3.10 (d, J = 6.4 Hz, 2H), 2.09 (t, J = 7.5 Hz, 2H),

1.72ꢀ0.64 (m, 33H). ¹³C NMR (126 MHz, DMSO) δ 174.53, 172.36,

76.19, 70.55, 37.96, 35.41, 32.38, 30.02, 29.63, 29.49, 29.44, 29.26, 29.12,

26.61, 26.15, 25.81, 24.92, 18.36. LCMS: t_R= 4.36; m/z = 380.3. HRMS

m/z calcd for C₂₂H₄₂N₃O₂(M þ H), 380.3277; found, 380.3271.

1-Aminocyclobutanecarbonitrile Hydrochloride (31). To a

solution of cyclobutanone (930 mg, 1.0 equiv) in 2 N NH₃/MeOH

(0.2 M) were added potassium cyanide (2.0 equiv) and ammonium

chloride (2.0 equiv). The reaction was run under ammonia gas (1 atm)

at room temperature for 17 h. The mixture was then evaporated and the

inorganic salts precipitated in CHCl₃and removed via filtration through

a fine frit. The solution was diluted in MeOH (100 mL), cooled to 0 °C,

and treated with 12.1 N HCl (2.0 equiv). The mixture was allowed to stir

for 5 min and then evaporated to dryness to yield the title compound.

1

hexanes, ninhydrin). H NMR (300 MHz, CDCl₃) δ 4.46ꢀ4.10 (m,

1H), 3.30 (dd, J = 13.9, 7.8 Hz, 1H), 3.16 (dd, J = 17.5, 8.1 Hz, 1H),

2.15ꢀ1.95 (m, 2H), 1.95ꢀ1.76 (m, 2H), 1.39ꢀ1.21 (m, 9H). ¹³C NMR

(75 MHz, CDCl₃) δ 153.01, 119.28, 81.08, 47.23, 45.79, 31.68, 30.84,

28.29, 24.73, 23.87.

(S)-1-(4-Dodecylbenzoyl)pyrrolidine-2-carbonitrile (35a).

General procedure N was use to deprotect 34 (400 mg, 2.05 mmol).

General procedure B was used to couple the deprotected 34 and

4-dodecylbenzoic acid to yield the title compound. Yield, 57%. White

solid. R_f= 0.75 (40% EtOAc in hexanes, Seebach’s dip). ¹H NMR (500

MHz, CDCl₃) δ 7.50 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H),

4.96ꢀ4.84 (m, 1H), 3.73ꢀ3.63 (m, 1H), 3.62ꢀ3.52 (m, 1H),

2.68ꢀ2.58 (m, 2H), 2.39ꢀ2.26 (m, 1H), 2.24ꢀ2.10 (m, 1H),

2.06ꢀ1.96 (m, 1H), 1.67ꢀ1.55 (m, 1H), 1.39ꢀ1.18 (m, 20H), 0.87

(t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.18, 146.28,

128.40, 127.61, 127.17, 118.73, 49.51, 46.87, 35.84, 31.91, 31.20, 30.30,

29.65, 29.56, 29.46, 29.34, 29.23, 25.58, 22.68, 14.12.

(S)-1-(4-Dodecylbenzoyl)pyrrolidine-2-carboximidamide

Hydrochloride (36a). General procedure A was used to convert 35a

(213 mg, 0.519 mmol) to the title compound. Yield, 64%. White solid.

¹H NMR (500 MHz, CD₃OD) δ 7.63 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0

Hz, 2H), 4.73 (t, J = 7.5 Hz, 1H), 3.87 (dd, J = 15.4, 9.0 Hz, 1H),

3.73ꢀ3.60 (m, 1H), 2.67 (t, J = 7.6 Hz, 2H), 2.54 (dd, J = 13.3, 6.6 Hz,

1H), 2.12ꢀ1.93 (m, 1H), 1.71ꢀ1.56 (m, 1H), 1.34ꢀ1.28 (m, 18H),

0.90 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 171.79,

146.52, 131.91, 128.06, 127.63, 58.94, 50.59, 35.36, 31.66, 31.14, 31.03,

29.33, 29.28, 29.15, 29.06, 28.88, 25.27, 22.32, 13.03. LCMS: t_R= 5.04;

m/z = 386.3. HRMS m/z calcd for C₂₄H₄₀N₃O (M þ H), 386.3171;

found, 386.3175.

(R)-1-(4-Dodecylbenzoyl)pyrrolidine-2-carbonitrile (35b).

General procedure N was use to deprotect ent-34a (275 mg, 1.40

mmol). General procedure B was used to couple the deprotected ent-

34a and 4-dodecylbenzoic acid to yield the title compound. Yield, 53%.

White solid. R_f= 0.75 (40% EtOAc in hexanes, Seebach’s dip). ¹H and

¹³C NMR data were identical to the data of 35a.

(R)-1-(4-Dodecylbenzoyl)pyrrolidine-2-carboximidamide

Hydrochloride (36b). General procedure A was used to convert 35b

(213 mg, 0.519 mmol) to the title compound. Yield, 73%. White solid.

¹H and ¹³C NMR data were identical to the data of 36a.

(S)-1-(4-(7-(Cyclohexylmethoxy)heptyl)benzoyl)pyrrolidine-

2-carbonitrile (37). General procedure N was use to deprotect 34

(179 mg, 0.911 mmol). General procedure B was used to couple the

deprotected 34 and 21a to yield the title compound. Yield, 61%. Clear

and colorless oil. R_f= 0.38 (40% EtOAc in hexanes, Seebach’s dip). ¹H

NMR (300 MHz, CDCl₃) δ 7.48 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.0 Hz,

2H), 4.99ꢀ4.68 (m, 1H), 3.72ꢀ3.44 (m, 2H), 3.35 (t, J = 6.6 Hz, 2H),

3.16 (d, J = 6.6 Hz, 2H), 2.59 (t, J = 9.7 Hz, 2H), 2.42ꢀ1.90 (m, 4H),

1.57 (m, 10H), 1.40ꢀ1.01 (m, 9H), 1.01ꢀ0.72 (m, 2H). ¹³C NMR (75

MHz, CDCl₃) δ 170.19, 146.40, 133.46, 128.66, 127.79, 118.72, 77.05,

71.25, 58.68, 49.97, 38.24, 36.02, 31.33, 31.12, 30.37, 29.92, 29.50, 29.36,

26.88, 26.31, 26.10.

(S)-1-(4-(7-(Cyclohexylmethoxy)heptyl)benzoyl)pyrrolidine-

2-carboximidamide Hydrochloride (38). General procedure A

was used to convert 37 (213 mg, 0.519 mmol) to the title compound.

Yield, 85%. White solid. ¹H NMR (500 MHz, DMSO) δ 9.11 (s, 2H),

8.85 (s, 2H), 7.61 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 7.7 Hz, 2H), 4.61 (t, J =

6.7 Hz, 1H), 3.81 (dd, J = 14.4, 7.4 Hz, 1H), 3.39 (m, 1H), 3.28 (t, J = 8.5,

2H), 3.09 (d, J = 6.5 Hz, 2H), 2.57 (m, 2H), 2.35ꢀ1.87 (m, 4H),

1.71ꢀ1.35 (m, 9H), 1.35ꢀ0.96 (m, 10H), 0.86 (m, 2H). ¹³C NMR (126

1

Yield, 60%. White solid. H NMR (300 MHz, CD₃OD) δ 2.73ꢀ2.47

(m, 2H), 2.29 (dt, J = 12.7, 8.8 Hz, 2H), 2.19ꢀ1.93 (m, 2H). ¹³C NMR

(75 MHz, CD₃OD) δ 123.66, 37.44, 35.80, 15.47.

N-(1-Cyanocyclobutyl)-4-dodecylbenzamide (32). General

procedure L was used to couple 31 (87 mg, 0.90 mmol) and 4-dode-

cylbenzoyl chloride to yield the title compound. Yield, 30%. Clear and

colorless oil. R_f= 0.48 (25% EtOAc in hexanes, Seebach’s dip). ¹H NMR

(300 MHz, CDCl₃) δ 8.44 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H),

7.26 (s, 1H), 3.71ꢀ3.52 (m, 2H), 3.44ꢀ3.33 (m, 2H), 3.21 (dd, J = 21.0,

9.6 Hz, 2H), 3.11ꢀ2.78 (m, 2H), 2.35 (m, 2H), 2.16ꢀ1.91 (m, 18H),

1.62 (t, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.71, 147.92,

130.05, 128.73, 127.21, 120.88, 48.00, 35.86, 34.14, 31.90, 31.16, 29.63,

29.56, 29.45, 29.34, 29.22, 22.68, 16.17, 14.13.

N-(1-Carbamimidoylcyclobutyl)-4-dodecylbenzamide (33).

General procedure A was used to convert 32 (100 mg, 0.27 mmol) to the

title compound. Yield, 7%. White solid. ¹H NMR (500 MHz, DMSO) δ

9.15 (s, 1H), 8.82 (s, 4H), 7.88 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz,

2H), 2.68ꢀ2.56 (m, 4H), 2.42 (dd, J = 17.6, 11.1 Hz, 2H), 2.10ꢀ1.96

(m, 1H), 1.96ꢀ1.82 (m, 1H), 1.55 (pent, J = 6.8 Hz, 2H), 1.25 (m,

18H), 0.85 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 172.70,

166.46, 146.63, 130.64, 128.08, 127.95, 57.31, 34.98, 31.32, 31.01, 30.80,

29.03, 28.85, 28.73, 28.58, 22.12, 15.05, 13.99. LCMS: t_R= 5.29; m/z =

386.3. HRMS m/z calcd for C₂₄H₄₀N₃O (M þ H), 386.3171; found,

386.3178.

(S)-tert-Butyl 2-Cyanopyrrolidine-1-carboxylate (34a). To

a solution of ^L-proline (600 mg, 5.21 mmol) in dioxane (7 mL) and 10%

Na₂CO_3(aq)(14 mL) was added Boc₂O (2.0 equiv) at room tempera-

ture, and the mixture was allowed to stir for 16 h. The mixture was

washed 2ꢁ with hexanes (100 mL), then acidified with 1 N HCl

(100 mL) and extracted 3ꢁ with EtOAc (200 mL). The EtOAc layer was

dried with Na₂SO₄and evaporated to a white solid. The crude white

solid was then dissolved in CH₂Cl₂(0.3 M) at 0 °C and treated with

TEA (3.0 equiv) and then isobutyl chloroformate (1.1 equiv). The

mixture turned turbid after the addition and was allowed to warm to

room temperature before slowly clearing over time. After 1 h at room

temperature, the mixture was treated with 2 M NH₃in MeOH

(2.0 equiv) and allowed to stir for 6 h. The mixture was evaporated to

a white solid and taken on crude. The crude solid was dissolved in DMF

(0.1 M) and 2,4,6-collidine (8 equiv) at 0 °C and then treated with

cyanuric chloride (3.15 equiv) and allowed to warm to room tempera-

ture. The mixture was allowed to stir for 12 h. The mixture was then

extracted with EtOAc (20 ꢁ the volume of DMF) and washed 3ꢁ with

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MHz, DMSO) δ 171.55, 169.93, 145.60, 133.21, 128.38, 128.30, 76.19,

70.53, 58.49, 50.66, 37.97, 35.39, 31.40, 31.13, 30.03, 29.59, 29.04, 26.62,

26.07, 25.81, 25.67. LCMS: t_R= 4.65; m/z = 428.3. HRMS m/z calcd for

C₂₆H₄₂N₃O₂(M þ H), 428.3277; found, 428.3274.

NMR (300 MHz, DMSO) δ 8.20ꢀ7.74 (m, 4H), 1.41ꢀ1.17 (m, 9H).

Rotomer ratio A/B = 1.95.

tert-Butyl (2-(3-(2-Cyclohexylethyl)phenyl)-2-oxoethyl)

terephthalate (50). A solution of 4-(tert-butoxycarbonyl)benzoic

acid (1.10 g, 4.95 mmol) and Cs₂CO₃(0.51 equiv) in EtOH (80 mL,

0.06 M) was sonicated for 6 min at room temperature. The solvent was

then evaporated and dried under vacuum for 20 min. The cesium

carboxylate salt was then dissolved in DMF (30 mL), and 49 (1.2 equiv)

was transferred by cannula in DMF (10 mL) at room temperature. The

mixture was allowed to stir for 12 h before being diluted with EtOAc

(400 mL) and washed 3ꢁ with neat water (100 mL). The organic layer

was then dried with Na₂SO₄, evaporated to a yellow oil, and immediately

purified by flash chromatography to yield the title compound. Yield,

99%. Tan oil. R_f= 0.43 (10% EtOAc in hexanes, Seebach’s dip). ¹H NMR

(300 MHz, CDCl₃) δ 8.11 (d, J = 6.8 Hz, 2H), 8.01 (d, J = 6.8 Hz, 2H),

7.73 (m, 2H), 7.47ꢀ7.04 (m, 2H), 5.55 (s, 2H), 2.62 (t, J = 6.9 Hz, 2H),

1.84ꢀ0.52 (m, 22H). ¹³C NMR (75 MHz, CDCl₃) δ 192.11, 165.50,

164.95, 144.34, 136.30, 134.28, 133.00, 129.95, 129.60, 128.98, 127.82,

125.37, 81.86, 67.01, 39.41, 37.49, 33.44, 33.30, 28.31, 26.83, 26.50.

tert-Butyl 4-(4-(3-(2-Cyclohexylethyl)phenyl)-1H-imida-

zol-2-yl)benzoate (51). To a round-bottom flask, fitted with a

deans-stark trap containing 4 Å molecular sieves, were added 50

(1.00 g, 2.22 mmol) and ammonium acetate (5.0 equiv). The mixture

was dissolved in xylenes (30 mL, 0.07 M). The mixture was then heated

to reflux for 7 h. The mixture was then cooled to room temperature,

evaporated to a black oil, and immediately purified by flash chromatog-

raphy to yield the title compound. Yield, 36%. Yellow solid. R_f= 0.05

(10% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ

8.00 (d, J = 8.4 Hz, 2H), 87.92 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.52 (d,

J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.32ꢀ7.15 (m, 1H), 7.05 (d, J = 7.6 Hz,

1H), 2.70ꢀ2.39 (m, 2H), 1.85ꢀ1.01 (m, 20H), 1.01ꢀ0.69 (m, 2H). ¹³C

NMR (75 MHz, CDCl₃) δ 165.90, 146.77, 144.07, 134.13, 131.60,

130.22, 128.88, 127.64, 125.44, 122.74, 81.65, 39.61, 37.65, 33.56, 33.48,

28.42, 26.91, 26.56.

(S)-1-(11-(Cyclohexylmethoxy)undecanoyl)pyrrolidine-2-

carbonitrile (39). General procedure N was use to deprotect 34

(371 mg, 1.89 mmol). General procedure B was used to couple the

deprotected 34 and 24 to yield the title compound. Yield, 84%. Clear and

colorless oil. R_f= 0.29 (40% EtOAc in hexanes, Seebach’s dip). ¹H NMR

(300 MHz, CDCl₃) δ 4.64 (d, J = 6.0 Hz, 1H), 3.53 (dd, J = 8.3, 7.1 Hz,

1H), 3.42ꢀ3.22 (m, 3H), 3.09 (d, J = 6.5 Hz, 2H), 2.44ꢀ1.84 (m, 6H),

1.78ꢀ1.35 (m, 6H), 1.35ꢀ0.96 (m, 19H), 0.81 (dd, J = 21.9, 10.6 Hz,

2H). ¹³C NMR (75 MHz, CDCl₃) δ 172.23, 114.29, 77.45, 71.23, 46.51,

46.38, 38.19, 34.59, 30.31, 30.22, 29.89, 29.68, 29.55, 29.44, 26.83, 26.31,

26.05, 25.30, 24.56.

(S)-1-(11-(Cyclohexylmethoxy)undecanoyl)pyrrolidine-2-

carboximidamide Hydrochloride (40). General procedure A was

used to convert 39 (433 mg, 1.15 mmol) to the title compound. Yield,

1

53%. White solid. H NMR (500 MHz, DMSO) δ 8.89 (s, 4H), 4.45

(dd, J = 9.0, 4.0 Hz, 1H), 3.68 (ddd, J = 9.3, 7.2, 4.8 Hz, 1H), 3.41 (dd, J =

16.6, 7.3 Hz, 1H), 3.34 (s, 1H), 3.27 (t, J = 8.6 Hz, 2H), 3.09 (d, J = 8.5

Hz, 2H), 2.40ꢀ2.07 (m, 4H), 2.04ꢀ0.98 (m, 27H), 0.93ꢀ0.68 (m, 2H).

¹³C NMR (126 MHz, DMSO) δ 172.43, 171.78, 76.20, 70.56, 57.22,

47.31, 37.97, 33.86, 31.25, 30.03, 29.47, 29.39, 29.30, 29.23, 26.62, 25.81,

24.72, 24.22. LCMS: t_R= 4.51; m/z = 394.3. HRMS m/z calcd for

C₂₃H₄₄N₃O₂(M þ H), 394.3434; found, 394.3425.

1-(3-(2-Cyclohexylethyl)phenyl)ethanone (48). To a 0.5 M

solution of 9-BBN (1.5 equiv) was added vinylcyclohexane (1.5 mL,

11.3 mmol, 1.5 equiv). The mixture was allowed to stir for 12 h. The

mixture was then treated with 3 N NaOH_(aq)(1.7 equiv), followed by

the solid additions of 3-bromoacetophenone (1.00 mL, 7.56 mmol,

1.0 equiv) and Pd(PPh₃)₄sequentially. The mixture was then heated to

reflux for 1 h, then cooled to room temperature and extracted 2ꢁ into

EtOAc (400 mL). The organic layer was then dried with Na₂SO₄,

evaporated to a black oil, and immediately purified by flash chromatog-

raphy to yield the title compound. Yield, 82%. Clear and colorless oil.

R_f= 0.58 (10% EtOAc in hexanes, Seebach’s dip). ¹H NMR (300 MHz,

CDCl₃) δ 7.85ꢀ7.58 (m, 2H), 7.42ꢀ7.07 (m, 2H), 2.59 (t, J = 7.3 Hz,

2H), 2.51 (s, 3H), 1.88ꢀ1.53 (m, 5H), 1.53ꢀ1.35 (m, 2H), 1.32ꢀ1.00

(m, 4H), 0.88 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 198.29, 143.87,

137.36, 133.36, 128.63, 128.19, 126.02, 39.53, 37.54, 33.46, 33.33, 26.85,

26.79, 26.52.

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)-1H-imidazol-2-yl)

benzoyl)pyrrolidine-2-carbonitrile (52). General procedure N

t

was used to deprotect the Bu ester on compound 51 (193 mg, 0.448

mmol) to yield the corresponding carboxylic acid. In a separate flask,

general procedure N was used to deprotect 34 (88 mg, 0.448 mmol).

General procedure B was used to couple the deprotected 51 and 34 to

yield the title compound. Yield, 84%. Yellow solid. R_f= 0.57 (EtOAc,

Seebach’s dip). ¹H NMR (300 MHz, DMSO) δ 12.81 (s, 1H),

8.22ꢀ7.90 (m, 2H), 7.90ꢀ7.34 (m, 5H), 7.26 (t, J = 6.8 Hz, 1H),

7.03 (d, J = 6.5 Hz, 1H), 4.88 (dd, J = 7.5, 5.2 Hz, 1H), 4.09 (ddd, J =

12.3, 7.6, 3.3 Hz, 1H), 3.98ꢀ3.40 (m, 3H), 3.08ꢀ2.66 (m, 2H),

2.66ꢀ2.51 (m, 2H), 2.39ꢀ0.56 (m, 13H). ¹³C NMR (75 MHz, DMSO)

δ 168.98, 145.55, 143.45, 142.40, 134.95, 133.13, 129.09, 128.77, 128.44,

125.29, 124.94, 122.55, 120.15, 115.64, 49.93, 47.44, 39.89, 37.49, 33.45,

31.92, 30.28, 26.89, 26.52, 25.84.

2-Bromo-1-(3-(2-cyclohexylethyl)phenyl)ethanone (49).

To a solution of 48 (2.14 g, 9.28 mmol) in CHCl₃(40 mL, 0.23 M)

at 40 °C was added Br₂(1.0 equiv) dropwise. The reaction was complete

immediately after the addition of the bromine, and the mixture was

cooled to room temperature and quenched with saturated NaHCO₃

(100 mL). The mixture was then extracted 3ꢁ with CHCl₃(100 mL).

The organic layer was then dried with Na₂SO₄, evaporated to a black oil,

and immediately purified by flash chromatography to yield the title

compound. Yield, 92%. Yellow oil. R_f= 0.71 (10% EtOAc in hexanes,

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)-1H-imidazol-2-

yl)benzoyl)pyrrolidine-2-carboximidamide Hydrochloride

(53). General procedure A was used to convert 52 (140 mg, 0.310

mmol) to the title compound. Yield, 81%. Yellow solid. ¹H NMR (500

MHz, DMSO) δ 12.85 (s, 1H), 9.07 (s, 2H), 8.74 (s, 2H), 8.07 (d, J = 8.2

Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.65 (m, 2H), 7.25 (t, J = 7.6 Hz, 1H),

7.03 (d, J = 7.2 Hz, 1H), 4.62 (t, J = 7.5 Hz, 1H), 3.93ꢀ3.70 (m, 1H),

3.47 (dd, J = 19.6, 12.3 Hz, 1H), 2.67ꢀ2.53 (m, 2H), 2.37 (dd, J = 14.8,

8.7 Hz, 1H), 2.02ꢀ0.92 (m, 14H). ¹³C NMR (126 MHz, DMSO)

δ 171.36, 169.60, 145.32, 143.20, 142.05, 134.89, 134.67, 132.81, 129.15,

128.91, 126.78, 124.71, 122.30, 120.07, 58.61, 50.66, 39.71, 37.25, 33.22,

31.42, 26.64, 26.28, 25.93. LCMS: t_R= 3.79; m/z = 470.3. HRMS m/z

calcd for C₂₉H₃₆N₅O (M þ H), 470.2920; found, 470.2916.

1

Seebach’s dip). H NMR (300 MHz, CDCl₃) δ 7.90ꢀ7.66 (m, 2H),

7.45ꢀ7.28 (m, 2H), 4.42 (s, 2H), 2.74ꢀ2.56 (m, 2H), 1.80ꢀ1.54 (m,

5H), 1.47 (dt, J = 10.4, 7.1 Hz, 2H), 1.32ꢀ1.01 (m, 4H), 0.89 (m, 2H).

¹³C NMR (75 MHz, CDCl₃) δ 191.54, 144.30, 134.30, 134.21, 129.04,

128.95, 126.56, 39.45, 37.51, 33.49, 33.32, 31.67, 26.89, 26.56.

4-(tert-Butoxycarbonyl)benzoic Acid. To a mixture of ter-

ephthalic acid (6.64 g, 40.0 mmol), Boc₂O (1.0 equiv), and DMAP

t

(0.25 equiv) were added BuOH (60 mL) and THF (20 mL). The

mixture was heated to reflux for 24 h. The mixture was then evaporated

to dryness and immediately purified by flash chromatography to yield

the title compound. Yield, 36%. White solid. R_f= 0.50 (5% MeOH

1

in CHCl₃, KMnO₄). Rotomer A: H NMR (300 MHz, DMSO) δ

4-(4-(3-(2-Cyclohexylethyl)phenyl)oxazol-2-yl)benzoic Acid

(54). To a round-bottom flask containing 50 (1.10 g, 2.44 mmol) and

8.20ꢀ7.74 (m, 4H), 1.54 (s, 9H), 1.41ꢀ1.17 (m, 2H). Rotomer B: ¹H

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Journal of Medicinal Chemistry

ARTICLE

1

ammonium acetate (5.0 equiv) was added AcOH (20 mL, 0.12 M). The

mixture was then heated to reflux for 16 h. The mixture was then cooled

to room temperature, evaporated to dryness, and immediately purified

by flash chromatography to yield the title compound. Yield, 25%. Yellow

solid. R_f= 0.58 (EtOAc, Seebach’s dip). Compound was crude by NMR

and taken onto the next step without further purification.

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)oxazol-2-yl)ben-

zoyl)pyrrolidine-2-carbonitrile (55). General procedure N was

used to deprotect 34 (88 mg, 0.448 mmol). General procedure B was

used to couple the deprotected 34 and crude 54 to yield the title

compound. Yield, 81%. Yellow solid. R_f= 0.51 (50% EtOAc in hexanes,

Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.15 (d, J = 8.0 Hz, 2H),

7.96 (s, 1H), 7.81ꢀ7.52 (m, 4H), 7.30 (dt, J = 6.6, 4.9 Hz, 1H), 7.14 (d,

J = 7.6 Hz, 1H), 5.03ꢀ4.69 (m, 1H), 3.91ꢀ3.26 (m, 2H), 2.77ꢀ2.43 (m,

2H), 2.43ꢀ0.62 (m, 17H). ¹³C NMR (75 MHz, CDCl₃) δ 169.25,

160.94, 144.13, 142.74, 136.60, 134.30, 130.90, 129.79, 128.93, 128.65,

128.28, 126.69, 125.81, 123.13, 118.81, 49.69, 47.18, 39.68, 37.67, 33.54,

30.48, 26.92, 26.57, 25.82.

hexanes, Seebach’s dip). H NMR (300 MHz, CDCl₃) δ 8.18ꢀ7.95

(m, 4H), 7.84 (s, 1H), 7.81ꢀ7.64 (m, 1H), 7.45 (s, 1H), 7.33 (t, J = 7.6

Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 2.67 (t, J = 8.6 Hz, 2H), 1.94ꢀ1.44 (m,

15H), 1.44ꢀ1.06 (m, 5H), 0.96 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

δ 166.61, 165.35, 157.22, 143.99, 137.37, 135.15, 134.43, 133.24, 130.52,

128.94, 126.92, 126.47, 124.04, 113.70, 81.48, 77.68, 39.74, 37.70, 33.60,

33.48, 28.43, 26.99, 26.65.

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)thiazol-2-yl)ben-

zoyl)pyrrolidine-2-carbonitrile (59). General procedure N was

used to deprotect the ^tBu ester on compound 58 (2.10 mg, 4.69 mmol)

to yield the corresponding carboxylic acid. In a separate flask, general

procedure N was used to deprotect 34 (919 mg, 4.69 mmol). General

procedure B was used to couple the deprotected 58 and 34 to yield the

title compound. Yield, 65%. Yellow solid. R_f= 0.36 (50% EtOAc in

hexanes, Seebach’s dip). ¹H NMR (300 MHz, CDCl₃) δ 8.02 (d, J =

8.1 Hz, 2H), 7.79 (s, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.5 Hz,

2H), 7.48 (s, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 7.7 Hz, 1H),

4.82 (s, 1H), 3.85ꢀ3.35 (m, 2H), 2.64 (t, J = 5.3 Hz, 2H), 2.40ꢀ1.81

(m, 4H), 1.81ꢀ1.39 (m, 7H), 1.39ꢀ1.02 (m, 4H), 1.02ꢀ0.74 (m,

2H). ¹³C NMR (75 MHz, CDCl₃) δ 169.21, 168.71, 166.42, 157.05,

144.05, 136.02, 134.33, 128.95, 128.71, 128.51, 126.69, 123.97,

118.92, 113.77, 49.74, 47.23, 39.72, 37.64, 33.55, 33.43, 30.42,

26.93, 26.59, 25.83.

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)thiazol-2-yl)ben-

zoyl)pyrrolidine-2-carboximidamide Hydrochloride (60).

General procedure A was used to convert 59 (682 mg, 1.50 mmol) to the

title compound. Yield, 74%. White solid. ¹H NMR (500 MHz, DMSO)

δ 9.23 (s, 2H), 9.02 (s, 2H), 8.23 (s, 1H), 8.15ꢀ8.00 (m, 2H),

7.94ꢀ7.60 (m, 4H), 7.37 (t, J = 6.5 Hz, 1H), 7.17 (d, J = 6.4 Hz, 1H),

4.66 (s, 1H), 3.84 (dd, J = 34.3, 15.3 Hz, 1H), 3.53ꢀ3.20 (m, 2H), 2.62

(t, J = 5.6 Hz, 2H), 2.53ꢀ2.09 (m, 2H), 2.08ꢀ1.31 (m, 9H), 1.31ꢀ0.96

(m, 4H), 0.89 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 176.16, 173.99,

171.05, 160.83, 148.44, 142.06, 139.74, 138.98, 133.98, 133.47, 131.24,

131.00, 128.82, 124.56, 120.56, 63.26, 55.32, 41.95, 37.93, 37.79, 36.20,

31.37, 31.00, 30.38. LCMS: t_R= 5.08; m/z = 487.3. HRMS m/z calcd for

C₂₉H₃₅N₄OS (M þ H), 487.2532; found, 487.2530.

(S)-1-(4-(4-(3-(2-Cyclohexylethyl)phenyl)oxazol-2-yl)ben-

zoyl)pyrrolidine-2-carboximidamide Hydrochloride (56).

General procedure A was used to convert 55 (169 mg, 0.373 mmol) to

1

the title compound. Yield, 75%. Yellow solid. H NMR (500 MHz,

DMSO) δ 9.23 (s, 2H), 9.03 (s, 2H), 8.78 (s, 1H), 8.12 (d, J = 8.4 Hz,

2H), 7.90 (d, J = 8.4 Hz, 2H), 7.73ꢀ7.60 (m, 2H), 7.34 (t, J = 7.6 Hz,

1H), 7.16 (d, J = 7.6 Hz, 1H), 4.67 (t, J = 7.5 Hz, 1H), 3.99ꢀ3.69 (m,

1H), 3.50ꢀ3.37 (m, 1H), 2.61 (t, J = 9.1 Hz, 2H), 2.44ꢀ2.30 (m, 1H),

2.01ꢀ1.79 (m, 3H), 1.74 (d, J = 13.0 Hz, 2H), 1.69ꢀ1.54 (m, 3H),

1.54ꢀ1.41 (m, 2H), 1.30ꢀ1.00 (m, 4H), 1.00ꢀ0.75 (m, 2H). ¹³C NMR

(126 MHz, DMSO) δ 171.39, 169.19, 160.68, 143.73, 141.87, 137.70,

136.51, 130.93, 129.22, 129.15, 128.68, 126.16, 125.49, 123.13, 58.50,

50.57, 37.24, 33.18, 33.02, 31.46, 26.62, 26.25, 25.61. LCMS: t_R= 4.79;

m/z = 471.3. HRMS m/z calcd for C₂₉H₃₅N₄O₂(M þ H), 471.2760;

found, 471.2759.

tert-Butyl 4-Carbamothioylbenzoate (57). 4-(tert-Butoxycar-

bonyl)benzoic acid (3.24 g, 14.6 mmol) was then dissolved in CH₂Cl₂

(0.3 M) at 0 °C and treated with TEA (3.0 equiv) and then isobutyl

chloroformate (1.1 equiv). The mixture turned turbid after the addition

and was allowed to warm to room temperature before slowly clearing

over time. After 1 h at room temperature, the mixture was treated with

2 N NH₃in MeOH (2.0 equiv) and allowed to stir for 6 h. The mixture

was evaporated to a white solid and purified by flash chromatography to

yield the corresponding amide. Yield, 66%. White solid. R_f= 0.39 (60%

EtOAc in hexanes, UV). ¹H NMR (300 MHz, CDCl₃) δ 8.02 (d, J = 8.1

Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 6.48 (s, 2H), 1.59 (s, 9H). ¹³C NMR

(75 MHz, CDCl₃) δ 169.25, 165.10, 137.02, 135.22, 129.87, 127.47,

81.99, 28.35. The amide was then dissolved in THF (0.5 M) and treated

with Lawesson’s reagent (0.6 equiv) in one portion. The mixture was

allowed to stir for 3 h before being evaporated to dryness and purified by

flash chromatography to yield the title compound. Yield, 92%. Yellow/

green solid. R_f= 0.93 (50% EtOAc in hexanes, Seebach’s dip). ¹H NMR

(500 MHz, DMSO) δ 10.06 (s, 1H), 9.65 (s, 1H), 8.12ꢀ7.55 (m, 4H),

1.53 (s, 9H). ¹³C NMR (126 MHz, DMSO) δ 199.69, 164.80, 143.60,

133.51, 129.12, 127.88, 81.62, 28.18.

’ ASSOCIATED CONTENT

S

Supporting Information. Synthetic schemes for com-

b

pounds 30 and 47, tabulated results from the inhibitor docking

studies, the SphK1/DGKB sequence alignment, and NMR

spectra. This material is available free of charge via the Internet

at http://pubs.acs.org.

Accession Codes

^†PDB code for DGKB: 2QV7.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: 434-924-0595. Fax: 434-982-2302. E-mail: ajk3p@

virginia.edu.

tert-Butyl 4-(4-(3-(2-Cyclohexylethyl)phenyl)thiazol-2-yl)be-

nzoate (58). To a solution of 57 (1.95 g, 8.22 mmol) and KHCO₃

(1.1 equiv) in THF (45 mL) at ꢀ5 °C was added 49 (2.05 g, 6.63 mmol)

in THF (5 mL). The mixture was allowed to warm to room temperature.

The mixture was allowed to stir for 2 h and then cooled to ꢀ5 °C again

and treated with TEA (2.2 equiv) and TFAA (1.1 equiv). The mixture

was allowed to warm to room temperature and was stirred for 16 h. The

mixture was diluted with CHCl₃(400 mL) and quenched with water

(100 mL). The organic layer was then dried with Na₂SO₄, evaporated to

dryness, and immediately purified by flash chromatography to yield the

title compound. Yield, 80%. Yellow solid. R_f= 0.88 (10% EtOAc in

’ ACKNOWLEDGMENT

This work was supported by grants from the National

Institutes of Health (Grants R01 GM067958 to K.R.L. and

T.L.M.). The authors thank Jose L. Tomsig, Department of

Pharmacology, University of Virginia, for S1P/LCMS quantiﬁca-

tion. A.J.K. oﬀers special thanks to the Jeﬀerson Scholars

Foundation for ﬁnancial support.

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dx.doi.org/10.1021/jm2001053 |J. Med. Chem. 2011, 54, 3524–3548

Journal of Medicinal Chemistry

ARTICLE

’ ABBREVIATIONS USED

(14) Kawamori, T.; Kaneshiro, T.; Okumura, M.; Maalouf, S.;

Uﬂacker, A.; Bielawski, J.; Hannun, Y. A.; Obeid, L. M. Role for

sphingosine kinase 1 in colon carcinogenesis. FASEB J. 2009, 23, 405–

414.

9-BBN, 9-borabicyclo[3.3.1]nonane; AcOH, acetic acid; Boc₂O,

di-tert-butyl dicarbonate; DGK, diacylglycerol kinase; DIEA, N,

N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF,

dimethylformamide; DMS, dimethyl sphingosine; dppf, 1,1⁰-bis-

(diphenylphosphino)ferrocene; EGF, epidermal growth factor;

EtOAc, ethyl acetate; HDAC, histone deacetylase; LPA, lyso-

phosphatidic acid; MOE, molecular operating environment;

PDGF, platelet derived growth factor; PFK, phosphofructoki-

nase; PKC, protein kinase C; PyBOP, benzotriazol-1-yloxytripyr-

rolidinophosphonium hexaﬂuorophosphate; S1P, sphingosine

1-phosphate; S1PR, sphingosine 1-phosphate receptor; SAR,

structureꢀactivity relationship; SphK, sphingosine kinase;

VEGF, vascular endothelial growth factor; TBAB, tetra-n-buty-

lammonium bromide; ^tBuOH, tert-butanol; TEA, triethylamine;

TFA, triﬂuoroacetic acid; TFAA, triﬂuoroacetic anhydride

(15) Li, W.; Yu, C.-P.; Xia, J.-t.; Zhang, L.; Weng, G.-X.; Zheng,

H.-q.; Kong, Q.-l.; Hu, L.-j.; Zeng, M.-S.; Zeng, Y.-x.; Li, M.; Li, J.; Song,

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T. H.; Smith, E. M.; Kelsey, K. T.; Turek, L. P.; Ahlquist, P. Fundamental

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dx.doi.org/10.1021/jm2001053 |J. Med. Chem. 2011, 54, 3524–3548

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Development of amidine-based sphingosine kinase 1 nanomolar inhibitors and reduction of sphingosine 1-phosphate in human leukemia cells

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