In 1993, as part of an effort to
search for antibiotic host defense agents in the gut of various animals
including the dogfish shark (spiny dogfish) Squalus acanthias, the unique antimicrobial aminosterol squalamine (structure shown
above) was discovered. Squalamine is essentially the condensation adduct
between a C24-sulfated bile salt derivative and the polyamine, spermidine. The
steroidal portion of the squalamine structure bears the trans configuration at the A/B ring junction and is additionally
hydroxylated at C7 in the a-configuration.
It was initially shown that stomach extracts of the dogfish shark exhibited
potent antimicrobial activity. Further efforts to purify and identify the
bioactive molecule responsible for the observed activity led to the isolation
and structure determination of squalamine. The aminosterol was initially
identified as a broad-spectrum antimicrobial, as it was found to exhibit potent
activity against fungi, protozoa, and both Gram-negative and Gram-positive
bacteria. The antimicrobial activity was attributed to squalamine’s ability to
modify membrane integrity by increasing permeability. Interestingly, it was
later shown that squalamine also possessed antiangiogenic and antitumor
properties and the molecule was eventually advanced to Phase II clinical trials
for the treatment of patients with advanced nonsmall cell lung cancer.
Attempts to procure larger amounts
of squalamine from Squalus acanthias
resulted in the discovery and isolation of a related aminosterol MSI-1436, later dubbed
trodusquemine. Trodusquemine, as compared to squalamine, features an
invariant steroid skeleton but is conjugated at C3 to spermine (as opposed to spermidine), an elongated tetrabasic
polyamine. Rather unexpectedly, trodusquemine was found to induce profound
appetite suppression in vivo in
mammals (For another example of a natural
appetite-suppressant steroid, see here). As a result, it has been
speculated that trodusquemine is responsible for the sporadic feeding behavior
of the dogfish, which eats only once every two weeks. In vitro screening
against a panel of potential cellular targets revealed protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity in cell-free and cell-based assays.
PTP1B is a negative regulator of the effects of insulin and leptin signaling
through dephosphorylation of the insulin receptor (IR) and IR Substrate 1,
thereby inactivating the insulin pathway. Thus, inhibition of PTP1B maintains
the insulin and leptin pathways in active, phosphorylated states, which
triggers appetite suppression. Indeed, PTP1B expression and activity is
increased in obese and insulin-resistant humans and neuronal-specific PTP1B knockout
mice have markedly reduced weight.
The inhibitory activity of
trodusquemine at the cellular target PTP1B is significant because it has been
notoriously challenging for medicinal chemists to develop effective small
molecule inhibitors targeting the active site of this enzyme. One problem is a
high degree of active site protein sequence homology, leading to difficulties
in achieving selectivity over other off-target phosphatases. Moreover, potent
active site tyrosine phosphatase inhibitors were designed to mimic
phosphotyrosine (see examples shown above). Consequently, the inhibitor ligands
that were developed were highly charged and thus had limited membrane
permeability and drug development potential. A known inhibitor of
striatal-enriched protein tyrosine phosphatase (or STEP), a potential
Alzheimer’s target, is TC-2153, a synthetic molecule that features a rarely
encountered heterocyclic ring comprised of five contiguously attached sulfur
atoms. Current interest in a structure like TC-2153 reinforces the point that
it has been historically difficult to find small molecule phosphatase
inhibitors with conventional ‘druglike’ substructures and physicochemical
properties. Trodusquemine avoids many of these pitfalls by binding to an
inhibitory allosteric site within an
intrinsically disordered segment of the C-terminal, noncatalytic region of
PTP1B. Many of the previous high-throughput screening campaigns conducted
against PTP1B used a recombinant, truncated form of the enzyme lacking this
C-terminal segment and, therefore, failed to identify selective allosteric inhibitors such as
trodusquemine. As a selective, non-competitive inhibitor of PT1B, trodusquemine
has tremendous potential as an anti-obesity and anti-diabetic therapeutic agent.
The molecule has also been
shown to be able to cross the blood brain barrier (BBB), and thus may be
centrally active, opening a new range of potential indications. Trodusquemine has been well
tolerated in dose escalation and dose ranging clinical studies completed to
date in over 65 patients.
The antimicrobial natural product
squalamine has been synthesized by processes starting from b-stigmasterol, as well as chenodeoxycholic
acid (CDCA, see Scheme above). One interesting and critical transformation that
has been utilized to generate gram-quantities of squalamine is a regio- and
stereoselective reductive amination with a spermidine equivalent, wherein the distal
primary amine is masked as a non-nucleophilic nitrile. In this conversion, the
primary amine reacts preferentially over the internal secondary amine to
generate an intermediary imine species, upon elimination of water with
assistance from trimethyl orthoformate. Sodium borohydride is then used to
reduce the imine in a stereoselective fashion from the bottom face of the
molecule to give, predominantly, the desired b-orientation
at the C3 position (d.r. 6:1). The nitrile is then easily converted to the
requisite amine (spermidine) by catalytic hydrogenation, with both operations
performed in the presence of the apparently unreactive C24-sulfate. The
hydrogenation must be conducted under acidic conditions (TFA) to avoid
cyclization of the internal secondary amine onto the pendant nitrile to give a
cyclic amidine impurity. In this example, the semisynthetic squalamine was
purified by HPLC and isolated as the trifluoroacetate salt with a final purity
of 97%. One presumes that a similar process is currently used to manufacture
the related clinical candidate, trodusquemine.
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